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Regulation of Alpha7 Nicotinic Acetylcholine Receptor Function and Pharmacology by Amino Acid Sequence in the Second Tra...


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REGULATION OF ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR FUNCTION AND PHARMACOLOGY BY AMINO ACID SEQUENCE IN THE SECOND TRANSMEMBRANE DOMAIN By ANDON NICHOLAS PLACZEK A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Andon N. Placzek

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This dissertation is dedicated to my family.

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ACKNOWLEDGMENTS I thank my committee members for their support, encouragement, and professionalism. I especially thank my mentor, Dr. Papke, for his availability and for holding me to a standard of excellence. I also appreciate his patience with me when I was not always focused on the task at hand. I must also thank all of my family who believed in me, especially my wife Erin for putting up with so much and inspiring me to be a better person. Thanks also go to my daughter, Olivia for being a reason to strive for success. Finally, I thank God who has been my faithful friend and without whom, nothing in my life would be possible. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT.........................................................................................................................x CHAPTER 1 GENERAL INTRODUCTION....................................................................................1 General Features of Ionotropic Receptors....................................................................3 Ligand-Gated Ion Channels..........................................................................................5 The nAChR Gene Family.............................................................................................6 Subfamilies of nAChR..........................................................................................7 Unique Features of the nAChR.........................................................................7 Modes of nAChR Activation and Signaling............................................................9 nAChRs in Human Disease........................................................................................11 The Structure of the nAChR.......................................................................................14 The TM2 Domain and the Ion Channel Pore......................................................15 Structure-Function Studies in the nAChR TM2 Domain....................................16 2 METHODS.................................................................................................................20 The cDNA Clones.......................................................................................................20 Site-directed Mutagenesis...........................................................................................20 Preparation of RNA....................................................................................................20 Expression in Xenopus Oocytes..................................................................................21 Voltage-clamp Recording of Whole-oocyte Responses.............................................21 Experimental Protocols and Analysis of Data Obtained from Xenopus Oocytes.......22 Transfection and Patch-clamp Recording from GH4C1 cells....................................24 Radioligand Binding Studies......................................................................................24 Intact Oocyte Binding.................................................................................................25 v

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3 THE PHARMACOLOGICAL AND KINETIC EFFECTS OF THE TM2 T6'F MUTATION...............................................................................................................26 Introduction.................................................................................................................26 Results.........................................................................................................................27 The TM2 T6'F Mutant is Functionally Expressed in Xenopus Oocytes..............27 The TM2 T6'F Mutation Dramatically Slows the Apparent Kinetics of ACh-evoked Macroscopic Currents.................................................................29 TheT6'F Mutation Increases ACh Potency Compared to Wild-type ........30 TheT6'F Mutation Abolishes Barium Permeability and Reduces Inward Current Rectification........................................................................................31 Succinylcholine is a Selective Agonist of Muscle-type Receptors, and is also an Agonist of T6'F Mutant Receptors.........................................................32 The TM2 T6'F Mutation Abolishes Potentiation by 5-Hydroxyindole...............32 Discussion...................................................................................................................33 4 THE TM2 T6'S MUTATION: A GAIN-OF-FUNCTION WITH -LIKE PHARMACOLOGY...................................................................................................47 Introduction.................................................................................................................47 Results.........................................................................................................................49 T6'S Mutant Kinetics and Single-channel Properties.................................................49 The TM2 T6'S Mutation Slows the Macroscopic Kinetics of ACh-evoked Currents and Increases the Overall Net Charge Carried Upon Activation......49 The T6'S Mutation Does not Increase Single-channel Conductance..............50 The T6'S Mutation Produces a Significant Increase in Average Channel Open Time.................................................................................................................51 T6'S Mutant Burst Activity.................................................................................52 The Pharmacology Of The T6'S Mutant.....................................................................52 The T6'S Mutant Shows a Slight Decrease in Ach Potency and a Decreased Discrepancy Between Area and Peak CRCs....................................................53 The Agonist Selectivity Profile of the T6'S Mutant is Similar to Wild-Type .....................................................................................................................53 Antagonists of the Wild-Type Receptor are also Antagonists of the T6'S Mutant..............................................................................................................54 5-Hydroxyindole Potentiation is Significantly Diminished in the T6'S Mutant Compared to Wild-Type .............................................................................54 Discussion...................................................................................................................55 Kinetic Effects of the T6'S Mutation..........................................................................55 Pharmacological effects of the T6'S mutation............................................................58 5 GENERAL DISCUSSION.........................................................................................77 LIST OF REFERENCES...................................................................................................80 BIOGRAPHICAL SKETCH.............................................................................................89 vi

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LIST OF TABLES Table page 1-1. Major subfamilies of nicotinic acetylcholine receptors.............................................19 3-1. Intact oocyte [ 125 I]-Btx binding...............................................................................38 3-2. Curve-fit values for wild-type and T6'F mutant responses to ACh......................39 4-1. Intact oocyte [ 3 H] MLA binding...............................................................................63 4-2. Agonist profile comparison for wild-type and the T6'S mutant...........................64 vii

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LIST OF FIGURES Figure page 3-1. Wild-type and mutant nAChRs.............................................................................40 3-2. Electrophysiological responses from wild-type and mutant nAChRs..................41 3-3. Macroscopic response kinetics of wild-type 7, muscle, and the TM2 T6'F mutant nAChRs expressed in oocytes..................................................................................42 3-4. ACh concentration-response functions for wild-type and the TM2 T6'F mutant nAChR.................................................................................................................43 3-5. Wild-type and mutant nAChR current-voltage relationships and divalent ion permeability in Xenopus oocytes..............................................................................44 3-6. Wild-type and mutant nAChR responses to the muscle receptor selective agonist succinylcholine.........................................................................................................45 3-7. Wild-type and mutant nAChR potentiation by 5-Hydroxyindole.............................46 4-1. The time course and concentration-dependence of nAChR macroscopic kinetics are altered by the T6'S mutation.................................................................65 4-2. Macroscopic currents evoked by ACh in oocytes expressing the T6'S mutant carry more net charge than wild-type currents....................................................66 4-3. Single channel currents recorded from GH4C1 cells expressing the T6'S mutant....67 4-4. The T6'S mutant has a slightly lower single-channel conductance compared to wild-type ..............................................................................................................68 4-5. T6'S single-channel open times are fit by two exponentials and indicate a prolonged average open time compared to wild-type .........................................69 4-6. T6'S mutant burst activity..........................................................................................70 4-7. T6'S mutant burst durations and number of intraburst openings...............................71 4-8. Peak and area CRCs for wild-type and T6'S mutant nAChRs..................................72 4-9. Selective agonists of the wild-type nAChR..........................................................73 viii

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4-10. Antagonists of wild-type are also antagonists of the T6'S mutant......................74 4-11. The T6'S mutant shows minimal potentiation by 5HI.............................................75 4-12. Simplified kinetic scheme for the nAChR..........................................................76 ix

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy REGULATION OF ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR FUNCTION AND PHARMACOLOGY BY AMINO ACID SEQUENCE IN THE SECOND TRANSMEMBRANE DOMAIN By Andon Nicholas Placzek May, 2005 Chair: Roger L. Papke Major Department: Pharmacology and Therapeutics Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels with clearly defined physiological roles at the neuromuscular junction and in peripheral ganglia, and more mysterious roles in the mammalian brain, and non-neuronal tissues. The larger family of nicotinic receptors can be broadly categorized into three major subgroups based on subunit composition, anatomical distribution, and functional and pharmacological differences. These are the muscle-type receptors, the heteromeric neuronal receptors, and the homomeric receptors, typified by receptors composed of the subunit. Our studies demonstrate that a conferring of a functional phenotype can be accomplished by systematic substitution of amino acid sequence from the beta subunits of either muscle-type () or neuronal () nAChRs into homomeric receptors composed of mutant nAChR subunits. Specifically, the TM2 T6'F mutant shows properties similar to the muscle-type nAChR with regard to divalent ion permeability, x

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current rectification, agonist concentration-dependent kinetics, sensitivity to succinycholine, and a lack of potentiation by 5-hydroxyindole. While a variety of muscle receptor-like properties are observed in the T6'F mutant, the TM2 T6'S mutant, which has amino acid sequence identical to the neuronal subunit at this position, demonstrates significant kinetic similarities to neuronal nAChRs, but largely retains the pharmacology of the wild-type receptor. At the single-channel level, the T6'S mutant has a unitary conductance similar to that reported for wild-type but a vastly longer average open duration. Furthermore, channel burst activity indicates a significantly greater likelihood of channel opening in the sustained presence of agonist relative to wild-type. The significant impact of these TM2 6' substitutions on a variety of functional aspects of the mutant receptors suggests that amino acid sequence at this position contributes to several important features that distinguish the major nAChR subgroups from one another. Furthermore, the TM2 T6'S mutant shows a kinetic gain of function in the absence of significant pharmacological differences from the wild-type receptor. Thus it may provide the ability to observe agonist-evoked signals using contemporary high-throughput drug screening methods (where the wild-type receptor would be inhibited), implicating it as a potential tool for identifying-selective compounds. xi

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CHAPTER 1 GENERAL INTRODUCTION The discovery of chemical neurotransmission as a major form of intercellular communication in the nervous system was an essential achievement allowing several scientific disciplines to progress, and ultimately setting the stage for important insights into both normal and abnormal human physiology. Santiago Ramn y Cajal (1911) first put forth the idea that the nervous system was composed of contiguous individual units rather than a continuum of tissue, and the term synapse was first introduced by Charles Sherrington (1947) who postulated that communication between cells in the nervous system had a chemical nature. The first real evidence of chemical release due to nerve stimulation comes from the work of Otto Loewi (1957), who is credited with the first discovery of the neurotransmitter substance, acetylcholine (ACh). Through his pioneering work with nicotine and curare, Langley (1905) provided evidence for a "receptive substance" that formed a molecular target for chemical substances on the cell surface. The identification and characterization of the molecules that were the recipients of these chemical signals provided the opportunity to gain an even greater understanding of the means by which neurons (or other cellular targets, such as muscle cells) gathered and processed transmitted information. Our current concept of how synapses function has improved upon these early ideas and we now have a much better picture of the organization and mechanisms of synaptic activity, although the picture is by no means complete. Despite the fact that most of the major neurotransmitter systems have been identified and mapped, we are still in the 1

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2 process of determining the physiological roles for many of these systems. Dysfunction within these systems in a variety of diseases that affect the nervous system lends further significance to these areas of investigation. As always, new discoveries continue to force us to reconsider our notions of how certain receptors function, or even how some families of receptors that were originally identified as important synaptic components, may in fact have important roles outside of those classically associated with the chemical synapse. There are two major classes of synaptic receptor molecules: metabotropic receptors and ionotropic receptors. Metabotropic receptors are coupled to intracellular G-proteins and are involved in a variety of intracellular signaling pathways, depending on the specific G-protein the receptor is associated with. These receptors typically have a seven transmembrane domain structure and show a high degree of diversity in signaling that is regulated by the specific G-protein complexes and the downstream targets that these receptors are associated with. The other major class of receptors is the ionotropic receptor, so called because its method of signal transduction comes in the form of permitting the flow of charged particles across the cell membrane. These receptors are gated by a variety of stimuli, including mechanical stimuli, chemical ligands, and changes in membrane voltage. Unlike transporter proteins which use active transport mechanisms or require coupling to existing ionic gradients to accomplish ion transport across the membrane, ionotropic receptors passively permit ion flux by exploiting the existing driving force on those ions. There are a great variety of known physiological roles for ionotropic receptors. The sensations of touch, hearing and proprioception are mediated by ionotropic receptors that respond to mechanical stimuli. Receptors that are controlled by changes in electrical

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3 potential across the cell membrane are known as voltage-gated ion channels and are necessary for the conduction of the action potential, and also for the coupling of cellular excitation to secretion or contraction. The ionotropic receptors that respond to chemical signals are involved in a diversity of physiological processes, and are primarily thought to have evolved as participants in synaptic transmission. General Features of Ionotropic Receptors Ionotropic receptors are multimeric protein complexes composed of individual membrane-spanning subunits that, when assembled, create an aqueous pore through the cell membrane. These receptors permit the flow of charged particles through this central pore when activated, and depending on the specific ions conducted, the effect of activation can be limited to changes in membrane voltage, resting conductance, or may also result in second messenger signaling events (particularly when channel activation leads to changes in intracellular calcium concentration). Important concepts related to ion channel function can be described using fundamental principles of electricity. The most fundamental of these is Ohms Law, E = IR (1-1) where E represents voltage, I is current, and R is resistance. When applying this equation to a cell, the cell membrane functions as a capacitor. It is across this capacitor that an electrochemical potential exists. Ion channels permit current to flow across the cell membrane by entering the activated, open state, and when they are closed, resistance across the cell membrane approaches infinity. Using an extension of this fundamental equation, the driving force on a particular ion can be predicted. The Nernst equation can be used to determine the membrane potential at which there is no net flow of ions, also known as the equilibrium potential:

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4 ESRTzSF lnS2S1 (1-2) Here, S is the ionic species in question, E S is the equilibrium potential for that ionic species, R is the ideal gas constant, T is temperature, z S is the ionic valence, and F is the Faraday constant. Through both active transport and passive processes an ionic gradient that serves as a source of both an electrical potential difference and a concentration gradient for specific ions. Once the equilibrium potential has been determined, the driving force on a particular ion is then simply the difference between this value and the membrane potential, or Em-Eeq The cell then makes use of this driving force with a variety of ion channels that display varying degrees of ionic selectivity. Incorporating these ion channels into the framework, Ohm's law can be further extended to give an electrical representation of a class of channels in a non-excitable cell with the formula: I = (E M E R ) NP O (1-3) where I represents current, E M represents the voltage across the cell membrane, E R is the reversal potential, N is the number of channels present, P O is the probability of channel opening, and represents the unitary conductance for that particular channel. One of the most significant technical advances in the field of physiology that permitted researchers to study ion channels in greater detail was development of the voltage-clamp method (Cole, 1949; Marmont, 1949; Hodgkin and Huxley, 1952). This permits the investigator to hold the cell's membrane potential constant while measuring the amount of current necessary to do so, giving a direct indication of the amount of current flow through ion channels at a defined membrane voltage.

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5 Ligand-Gated Ion Channels As previously mentioned, ionotropic receptors can be activated by different classes of stimuli, including mechanical, electrical, and chemical. Those which respond to chemical signals are referred to as ligand-gated ion channels (LGICs). These types of channels are known to function in at least three different modalities. The first involves rapid activation at a postsynaptic structure in response to a relatively high concentration of quantally released agonist (approaching approximately 0.5 1 mM (Salpeter, 1987)). This mechanism is typified by the receptors expressed at the vertebrate neuromuscular junction. In the second modality, some LGICs can act as local modulators of synaptic function. An example of this comes in the form of receptors that are expressed presynaptically, and through their activation and subsequent calcium signal, facilitate the release of neurotransmitter. The third modality involves paracrine-like, volume transmission, where ligands originate from sources that are relatively distant from their target and bind to the LGIC, activating (and in some cases desensitizing) the receptor (Descarries, 1997). There is also the possibility that these receptors are fulfilling important physiological roles that are not neatly encapsulated within these three modalities. Much of what we now know and hypothesize about ligand-gated ion channels has been greatly influenced by the study of acetylcholine receptors (AChRs), largely because their importance in the motor systems of vertebrates provides a readily accessible experimental system (Fatt and Katz, 1951; Katz, 1966). Acetylcholine receptors consist of two major subtypes. Those which are activated by muscarine and are coupled to intracellular G-proteins are referred to as muscarinic receptors (mAChRs). The other major subtype is activated by the plant alkaloid nicotine, and functions as a ligand-gated

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6 ion channel. These are known as nicotinic acetylcholine receptors (nAChRs). Nicotinic receptors are widely expressed both in the peripheral (Skok, 2002) and central (Dani, 2001) nervous systems of mammals, and are well known to be the postsynaptic target in neuromuscular transmission (Katz, 1966) The nAChR Gene Family The family of nAChR subunit genes consists of seventeen members, and is a member of the same ion channel superfamily as glycine receptors, 5-HT3 receptors, and ionotropic GABA receptors (Lester et al., 2004). The nAChRs expressed on the electroplax organ of the electric ray, Torpedo Californica were the first to be cloned using the techniques of modern molecular biology (Noda et al., 1982). This particular receptor has been instrumental in the development of models of the three dimensional structure of the nAChR, using high-resolution electron microscopy (Unwin, 1989; Miyazawa et al., 2003). The Torpedo-type receptors are close structural homologues of the nAChRs of the mammalian neuromuscular junction, which are now known to be composed of , and or (depending on developmental stage (Brisson and Unwin, 1985) subunits in a 2:1:1:1 ratio, with ligand-binding occurring at the and (or -) subunit interfaces. The neuronal nicotinic receptors can be divided into heteromeric and homomeric subtypes. The heteromeric subtypes are composed of neuronal alpha (-) and beta (-) subunits and bind nicotine with high affinity (Lindstrom et al., 1995). Homomeric nAChRs are composed of alpha subunits (-) and the predominant mammalian subtype, is inhibited by the snake toxin, -bungarotoxin (-Btx, McGehee and Role, 1995). Although these are generally referred to as neuronal nAChRs, recent studies show that they are also expressed in non-neuronal tissues including microglia (Shytle et al., 2004) and peripheral macrophages (Wang et al., 2003).

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7 Subfamilies of nAChR The hypothetical evolutionary history of nicotinic receptor genes has given rise to an organizational framework based on phylogeny (Le Novere et al., 2003). Using this framework, several nAChR subfamilies can be identified, and these differences give a good representation of many of the known physiological differences these receptors exhibit, as well as differences in their patterns of tissue distribution. Previous studies have demonstrated discrete patterns of nAChR subunit expression within the mammalian brain (Clarke et al., 1985) with high levels of the subtype being expressed in rat hippocampus. Unique Features of the nAChR Receptors containing the nAChR subunit have been studied extensively since their discovery in a variety of experimental preparations. Although the majority of in vitro experimental systems focus on what are presumably homomeric receptors, there has been some suggestion that subunits can co-assemble with nonnAChR subunits in vivo (Khiroug et al., 2002). Furthermore, an splice variant with unusual functional properties has been identified in rodent cardiac ganglion (Severance et al., 2004), suggesting the possibility of splice variants in humans that may have different characteristics from receptors studied in vitro. Another potentially complicating discovery is the existence of an partial gene duplication in humans (Gault et al., 1998). This also suggests the possibility of co-assembly of wild-type subunits with a different gene product that may have functional consequences. Despite these potential complications, there has generally been good agreement between data generated using the nAChRs reconstituted in heterologous expression systems (e.g., Xenopus oocytes) and the native receptors.

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8 One particularly striking feature that distinguishes wild-type nAChRs from other nAChRs, and indeed from many other ligand-gated ion channels, is the degree to which the receptors macroscopic kinetics are impacted by agonist concentration (Papke and Thinschmidt, 1998). That is, application of higher agonist concentrations produce responses that reach their maximum, and decay to baseline far more rapidly than those produced by relatively lower agonist concentrations (see Figure 3-2A). In the case of this phenomenon is especially pronounced and it is evident that the rate-limiting process is always agonist application speed. Another potentially important feature of nAChRs is their high degree of calcium permeability. The Ca2+:Na+ permeability ratio for nAChRs has been reported to be approximately 10 (Sands and Barish, 1991) and has even been claimed to be as high as 20 or more (Role and Berg, 1996). This high level of permeability has significant implications for cell signaling. Several laboratories have shown that activation of nAChRs can be cytoprotective (Jonnala et al., 2003; Martin et al., 1994; Stevens et al., 2003). However, an optimal range of concentrations of -selective agonist corresponds to enhanced neurite survival in differentiated PC12 cells and this same range of concentrations produces an activation of protein kinase C (PKC) with a similar bell-shaped curve (Li et al., 1999). Furthermore, high concentrations of the -selective agonist GTS-21 rapidly delivered to PC12 cells produce significant cytotoxicity, whereas gradual exposure to the same concentration of GTS-21 through a gel slab drug delivery system is not (Papke et al., 2000a). In addition to directly mediating calcium influx, nAChRs have been shown to induce secondary changes in intracellular calcium concentration. Activation of -type

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9 receptors under non-voltage clamped conditions causes a transient depolarization and subsequent calcium influx through voltage-gated calcium channels expressed at the cell surface (Quik et al., 1997). Also, an increase in intracellular calcium itself can cause release of calcium from membrane-delimited stores via calcium-induced calcium release. Recent data suggests that this type of calcium-induced calcium release can be evoked by activating based on the ability of the IP3 receptor blocker, xestospongin C to partially inhibit calcium signals evoked by agonist (Dajas-Bailador et al., 2002). The current data also suggest that a significant release of calcium from intracellular stores requires the intermediate step of voltage-gated calcium channel activation. It is unclear at this point which of these mechanisms is of primary significance for promoting cell survival during activation, or if all of the aforementioned sources of calcium are required. These ancillary mechanisms of increasing intracellular calcium concentrations raise the interesting possibility that a cell can tune the magnitude (and ultimately the effect) of calcium-dependent processes elicited by activation by regulating expression of proteins involved in these secondary pathways. Modes of nAChR Activation and Signaling The physiological significance of nicotinic receptors expressed at the neuromuscular junction has been understood for decades, beginning with the early work of Fatt and Katz (1951) who described quantal postsynaptic responses at the motor end-plate. In the case of both the nAChRs of the neuromuscular junction, and those expressed in peripheral ganglia, fast synaptic transmission is the predominant functional role. Acetycholine is synthesized, packaged, and released in a quantal fashion by presynaptic cholinergic neurons, after which it traverses the synapse and binds to

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10 postsynaptic nAChRs, causing them to open, thus depolarizing the postsynaptic membrane. One feature of the -type nAChR that lends itself to signaling via volume transmission is its activation by the endogenous ligand, choline (Papke et al., 1996). Choline is a breakdown product of acetylcholinesterase (AChE) activity, and ambient levels of choline near cells that synthesize and release acetylcholine can by dynamically regulated by the activity of AChE and uptake by choline transport proteins. In hypothalamic tuberomammilary neurons that express high levels of nAChR, spontaneous activity can be modulated by bath-applied choline (Uteshev et al., 2003.), suggesting that even subtle variations in ambient choline levels may have an impact on the activity of a major source of brain histamine. Choline levels are also reported to change under pathophysiological conditions, such as ischemia or brain injury, where concentrations are elevated above normal (Scremin and Jenden, 1991). This may suggest a possible role for -type receptors in natural cytoprotective mechanisms. While there is some evidence for classical fast synaptic transmission mediated by receptors (Frazier et al., 1998; Hatton and Yang, 2002), this type of signaling modality has historically been either very difficult to demonstrate, or confined to specific regions of the nervous system. Because receptor localization studies have shown both widespread nAChR expression in mammalian brain (Clarke et al., 1985), and that this expression appears to be primarily extrasynaptic or perisynaptic (Fabian-Fine et al., 2001), it is intriguing to speculate about an alternative role for nAChRs that does not strictly fit the mold of fast synaptic transmission, typically associated with ligand-gated ion channel function.

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11 nAChRs in Human Disease In recent years neuronal nicotinic receptors have emerged as potential therapeutic targets in a variety of CNS disorders including Alzheimer's disease (AD, O'Neill et al.,2002; Shimohama and Kihara., 2001) schizophrenia (Freedman et al., 2000), and Tourette's syndrome(Mihailescu and Drucker-Colin, 2000). It remains unclear in each of these cases the exact mechanisms by which modulators of nicotinic receptor function exert their therapeutic effects. One possible reason for this is the paradoxical pharmacology of nicotinic agonists. For example, while transdermal nicotine has been used to augment neuroleptic treatment of Tourette's syndrome (Silver et al., 1996), nicotine itself has been shown to demonstrate a mixed agonist/antagonist pharmacology, and it is uncertain which of these pharmacological properties are essential in determining therapeutic efficacy. The human brain cholinergic system has been known for decades to have a role in the progression of Alzheimer's disease. The interest in this pathway came from early pathological studies of postmortem AD brain tissue. These findings suggested an early degeneration of the primary centers of cholinergic projections in the basal forebrain (Whitehouse, 1981) that correlated with memory loss. The so-called "cholinergic hypothesis" of AD (Bartus et al., 1982) gave rise to medications which, until recently were the only medications approved by the federal government to treat this disease. These drugs inhibit acetylcholinesterases (AChE) and have been shown to provide modest cognitive improvement for patients with mild to moderate AD (Robbins et al., 1997). The basic mechanism underlying the therapeutic effects of these drugs is an increase in the availability of ACh through the prevention of its breakdown by AChE,

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12 presumably compensating for less available ACh due to cholinergic cell loss or dysfunction. Unfortunately, most studies show that the relief these drugs provide is primarily symptomatic (Barner and Gray, 1998), and the disease continues to progress until the drugs are no longer effective. Despite the fact that the cholinergic hypothesis has not led us to treatments that or halt or significantly slow the progression of AD, it has led to some interesting studies using animal models that help to clarify the role that the cholinergic system may play in cognition. For example, several studies using lesions of the cholinergic projections from the basal forebrain have shown that interruption of this pathway significantly impairs memory related behaviors in a variety of tasks (McGaughy et al., 2000) and that administration of cholinergic agents can help to correct some of these deficits. In fact, cholinergic agents have long been known to enhance cognitive performance in the absence of any impairment in both animals and humans (Robbins et al., 1997), although the mechanism of these improvements in either lesioned or normal subjects remains unknown. Even though the cholinergic hypothesis has fallen out of favor among many researchers in the field of Alzheimer's disease, evidence for a potential involvement of cholinergic signaling continues to emerge. Studies of postmortem brain tissue from AD patients also show that subunits co-localize with the congophilic senile plaques that are a pathological hallmark of AD (Nagele et al., 2002). If it is true that can inhibit signaling in vivo, then the potential for inhibition of an endogenous cytoprotective mechanism exists that may contribute to the Alzheimers disease process.

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13 The recent findings that show a high-affinity (Wang et al., 2000) functional interaction (Liu et al., 2001; Petit et al., 2001) between the nAChR subtype and the Alzheimer's -amyloid (A) peptide are interesting for several reasons. Beta-amyloid is the principle component of the senile plaques found in the postmortem brain tissue of patients diagnosed with AD, and is recognized as a potentially pathogenic molecule when its levels are abnormally high (Hardy and Selkoe, 2002). The high affinity of the binding (in the picomolar concentration range) and the high potency of the functional inhibition of nAChR activity (in the nanomolar concentration range) suggests that interaction between these two molecules may occur early in the disease process, since it is the higher (micromolar) concentrations of A peptide that promote the rapid aggregation and cell death that are primarily associated with the later stages of the disease (Hardy and Selkoe, 2002). Another potentially interesting aspect of this relationship between A and is the fact that there is little evidence for a major role for nAChRs in fast synaptic transmission. This suggests that any functional interaction between A and may be occurring outside the realm of classical postsynaptic signaling. This may not only point to how may be functioning normally in the mammalian brain, but how this function may be disrupted in AD, and how this interference in normal activity may be prevented. Another potential role for nAChR in disease is suggested by recent evidence showing an important contribution by signaling to the inhibition of peripheral inflammation. This signifies as a potential therapeutic in the treatment of septic shock (Wang et al., 2003) and possibly other disorders that involve peripheral inflammation. This finding may also point to role for receptors in modulating central inflammatory processes. Chronic inflammation has been of interest to researchers in AD for several

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14 reasons. In both AD patients and animal models of AD there are reports of an upregulation of several markers on inflammation and activated microglia (McGeer and McGeer, 2001). Furthermore, in vitro and in vivo studies show that the A peptide is able to induce microglial activation and the subsequent release of pro-inflammatory cytokines that are potentially detrimental to surrounding neurons (Meda et al., 1995). Taken together with the evidence for anti-inflammatory function of nAChRs in peripheral monocytes, this suggests the possibility that a central anti-inflammatory action of may be impaired in AD, possibly through chronic inhibition by the A peptide. The other major illness that receptors have been implicated in is schizophrenia. It has been known for some time that individuals diagnosed with schizophrenia are significantly more likely to smoke than non-schizophrenics (Adler et al., 1998). This has been interpreted by some as self-medication, and has stimulated interest in the study of a possible mechanistic role for nicotinic receptors in schizophrenia. Where -type nAChRs have been implicated is in the phenomenon of auditory gating. Studies using transcranial recording methods have shown that schizophrenics have an impairment of auditory gating, as evidenced by an increase in the so-called P50 latency (Leonard et al., 1996). The implications for symptoms of psychosis are in an inability of the patient to sufficiently filter external auditory stimuli, resulting in hallucinations that result from the brain's attempt to integrate the unfiltered stimuli. The Structure of the nAChR Nicotinic receptors are believed to have a pentameric quaternary structure and demonstrate a high degree of functional diversity based on variations in subunit composition (Papke, 1993; Le Novere et al., 2002). All individual nAChR subunits have a similar secondary structure and transmembrane topology (Lindstrom, 2000) with a large

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15 extracellular N-terminal domain contributing to ligand-binding, followed by three hydrophobic membrane-spanning regions, a large intracellular loop, a fourth transmembrane region, and finally a relatively short extracellular C-terminus. The intracellular loop has been shown to have several phosphorylation sites in heteromeric (Downing & Role, 1987; Swope et al., 1992) and homomeric receptors (Moss et al., 1996), although the functional significance of subunit phosphorylation remains unclear. The TM2 Domain and the Ion Channel Pore The second transmembrane (TM2) domain of the nicotinic receptor subunit is generally considered to be the pore-forming region of the nAChR. Early studies using chimeric subunits constructed from Torpedo electric organ nAChRs and calf muscle-type receptors demonstrated that the amino acid sequence proximal to and including the delta subunit TM2 domain was responsible for differences in single channel conductance (Imoto et al., 1986). Furthermore, rings of charged residues that border the TM2 domain of Torpedo nAChR subunits have been shown to regulate cation permeation and the inwardly rectifying voltage-dependent reduction in current produced by the divalent cation, Mg2+ (Imoto et al., 1988). Pharmacological evidence in the form of photoaffinity labeling studies shows that the open-channel blocker, chlorpromazine, binds to the TM2 domain of every subunit of the Torpedo nAChR, indicating that this portion of each subunit contributes to the pore-lining region of the receptor (Oswald and Changeux, 1981; Revah et al., 1990). Mutations in the TM2 domain have also been shown to regulate voltage-dependent channel block by local anesthetics, such as the lidocaine derivative, QX-222 (Leonard et al., 1988; Charnet et al., 1990). These same studies indicated specific amino acids that

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16 were responsible for binding to QX-222, showing their likely position within the ion channel pore-lining region. It was the 6 and 10 residues (according to the numbering system proposed by Miller (1989)) that were shown to be directly responsible for interaction with, and blockade by QX-222 (Charnet et al., 1990). These positions within the TM2 domain are in direct proximity to one another based on the periodicity of the amino acid sequence of the alpha helical TM2 domain (see Figure 3-1). Structure-Function Studies in the nAChR TM2 Domain In addition to the well-established relationship between TM2 domain amino acid sequence and both channel conductance and interaction with drugs that target the pore, mutations in the TM2 domain have been shown to affect nAChR kinetics. The initial observation was made with a mutation of amino acid 247 in the chick brain nAChR (Revah et al., 1991). The substitution of a threonine residue for a leucine (L247T or L9'T) produced a significant slowing of the response kinetics under macroscopic voltage clamp conditions, an effect that was interpreted as a change in receptor desensitization. In addition to this effect on response kinetics, subsequent studies of this same mutation demonstrated an effect on several aspects of the mutant receptors pharmacology. Several antagonists of the wild-type receptor were found to be agonists of the L9'T mutant, suggesting that mutation converted what was a liganded, closed state in the wild-type nAChR, into a liganded opened state (Bertrand et al., 1992; Fucile et al., 2002; Palma, 1996; Palma et al., 1999). These effects on receptor pharmacology suggested that this mutation could impact the relationship between ligand-binding and channel gating and/or processes that resemble classical desensitization. Other studies with nontype nAChRs also indicate the importance of amino acid sequence in the TM2 domain as a determinant of receptor pharmacology. Heteromeric

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17 receptors in which a chimeric subunit with the subunit sequence at the TM2 domain show a reduced potency of blockade by mecamylamine when compared to wild-type receptors expressed in Xenopus oocytes (Webster et al., 1999). This same reduction in inhibitory potency was accompanied by a faster recovery from block by both mecamylamine and nicotine. Substitution of the TM2 6' phenylalanine of the muscle subunit for the neuronal subunit serine residue increased sensitivity to nicotine. The same study showed that these effects could be more specifically attributed to amino acid sequence at both the 6 and 10 positions of the TM2 domain (see Figure 3-1). Similar to the findings with the L9'T mutant nAChR, these studies suggested that TM2 domain amino acid sequence could have a significant degree of control over receptor pharmacology for drugs that dont necessarily exert their effects by directly interacting with the pore-forming region of the receptor. Furthermore, the effects of substituting specific amino acids from subunits of members one major subfamily of nAChR to another, and the subsequent change in pharmacology suggested that pharmacological phenotype could be conferred from one major receptor subtype to another, simply by substituting important amino acids in the TM2 domain. Given the known differences between the three major subfamilies of nAChR (i.e., muscle type, neuronal high-affinity nicotine binding, and homomeric ) it is useful to identify structural elements of the receptor complex that functionally distinguish the members of each group from those of the others. This may provide essential clues to the normal roles for those neuronal receptors that are less clearly understood, and how these receptors may be exploited in the design of therapeutic compounds. At the core of all receptor-based therapeutics is the need to understand how agonist binding is coupled to

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18 receptor activation, and how that process can be perturbed by exogenous drugs. These processes differ significantly among the three major subgroups of nAChR, and the data presented here support the hypothesis that the key to understanding these differences can be found by studying specific sequence in the respective pore forming domains of these receptors. By determining how amino acid sequence in the pore-forming domain regulates a multitude of processes, including the efficacy of specific agonists, selective permeation of divalent cations, and receptor kinetics, we may gain insights into how agonists and both competitive and noncompetitive antagonists may be developed that will be selective for functionally distinct receptor subtypes.

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19 Table 1-1. Major subfamilies of nicotinic acetylcholine receptors. -Btx sensitive High-affinity nicotine-binding Muscle-type Non-competitive antagonists BTMPS (intermediate sensitivity) Mecamylamine, BTMPS (high sensitivity) BTMPS (low sensitivity) Ca2+:Na+ permeability ratio > 10 2.0 0.2 Inward rectification Rectifying Rectifying Non-rectifying Selective agonists GTS-21, 4OH-GTS-21, Choline, AR-R17779, Tropisetron Metanicotine, Epibatidine, Succinylcholine Competitive antagonists -Btx, MLA DHE -Btx, (but not MLA) Macroscopic kinetics Rapidly desensitizing, Fast time-to-peak, Fast decay Relatively slow timecourse vs. 7 Relatively slow timecourse vs. 7

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CHAPTER 2 METHODS The cDNA Clones These experiments used the rat neuronal nAChR and the mouse muscle cDNA clones, which were obtained from Dr. Jim Boulter (UCLA). The sequences of the TM2 domains of the relevant subunits are shown in Figure 3-1. Adopting the terminology proposed by Miller (1989), the 20 residues in the putative second transmembrane sequence are identified as 1' through 20'. Site-directed Mutagenesis Site-directed mutagenesis was performed using QuickChange (TM) kits (Strategene, LaJolla, CA). In brief, two complimentary oligonucleotides were synthesized which contained the desired mutation flanked by 10-15 bases of unmodified nucleotide sequence. Using a thermal cycler, Pfu DNA polymerase extended the sequence around the whole vector, generating a plasmid with staggered nicks. Each cycle built only off the parent strands, and therefore there was no amplification of misincorporations. After 12-16 cycles, the product was treated with Dpn I, which digested the methylated parent DNA into numerous small pieces. The product was then transformed into E. coli cells, which repaired the nicks. Preparation of RNA After linearization and purification of cloned cDNAs, RNA transcripts were prepared in vitro using the appropriate mMessage mMachine kit from Ambion Inc. (Austin, TX). 20

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21 Expression in Xenopus Oocytes Mature (>9 cm) female Xenopus laevis African frogs (Nasco, Ft. Atkinson, WI) were used as a source of oocytes. Prior to surgery, frogs were anesthetized by placing the animal in a 1.5 g/L solution of MS222 (3-aminobenzoic acid ethyl ester). Oocytes were removed from an incision made in the abdomen. In order to remove the follicular cell layer, harvested oocytes were treated with collagenase from Worthington Biochemical Corporation (Freehold, NJ) for 2 hours at room temperature in calcium-free Barth's solution (88 mM NaCl, 10 mM HEPES pH 7.6, 0.33 mM MgS04, 0.1 mg/ml gentamicin sulfate). Subsequently, stage 5 oocytes were isolated and injected with 50 nl each of a mixture of the appropriate subunit cRNAs following harvest. Recordings were made 3 to 21 days after injection depending on the cRNAs being tested. In order to increase the magnitude of the functional responses from oocytes injected with the T6'F mutant, approximately 5 times (30 ng) the amount of mutant mRNA was injected compared to wild-type (approximately 6-7 ng). Since all data were normalized using each cell as its own control, absolute differences in response magnitude did not affect comparisons between receptor subtypes. Voltage-clamp Recording of Whole-oocyte Responses Data were obtained by means of two-electrode voltage-clamp recording. Recordings were made at room temperature (21-24 deg. C) in Frog Ringer's solution (115 mM NaCl, 10 mM HEPES, 2.5 mM KCl, and 1.8 mM CaCl 2 pH 7.3) with 1 M atropine to inhibit muscarinic acetylcholine receptor responses. This extracellular solution was used for all experiments unless otherwise noted. Voltage electrodes were filled with 3M KCl, and current electrodes were filled with 250 mM CsCl, 250 mM CsF, and 100 mM EGTA (pH 7.3).

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22 Bath solution and drug applications were applied through a linear perfusion system to oocytes placed in a Lucite chamber with a total volume of 0.5 ml. Drug delivery involved pre-loading a 1.8 ml length of tubing at the terminus of the perfusion system, while a Mariotte flask filled with Ringer's solution was used to maintain constant perfusion. Applications of drug solutions were then synchronized with acquisition. Current responses were recorded using a PC interfaced to either a Warner OC-725C (Warner Instruments, Hamden, CT) or a GeneClamp 500 amplifier via a Digidata 1200 digitizer (Axon Instruments, Union City, CA). In addition, some oocyte recordings were made using a beta version of the OpusXpress 6000A (Axon Instruments, Union City, CA). OpusXpress is an integrated system that provides automated impalement and voltage clamp, which in our case permitted the study of four oocytes in parallel. Cells were automatically perfused with bath solution, and agonist solutions were delivered from a 96-well compound plate. In experiments using the OpusXpress system, the voltage and current electrodes were filled with 3 M KCl. In all experiments, bath flow rates were set at 2 ml/minute. Experimental Protocols and Analysis of Data Obtained from Xenopus Oocytes Current responses to drug application were studied under two-electrode voltage clamp at a holding potential of -50 mV unless otherwise noted (-60 mV for the OpusXpress system). Holding currents immediately prior to agonist application were subtracted from measurements of the peak response to agonist. All drug applications were separated by a wash period of 5 minutes unless otherwise noted. At the start of recording, all oocytes received two initial control applications of ACh. Subsequent drug applications were normalized to the second ACh application in order to control for the level of channel expression in each oocyte. Means and standard errors (SEM) were

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23 calculated from the normalized responses of at least four oocytes for each experimental concentration. For concentration-response relations, data were plotted using Kaleidagraph 3.0.2 (Abelbeck Software; Reading, PA), and curves were generated using the Hill equation: Response = Imax[agonist]n[agonist]n(EC50)n (Eq. 2-1) where Imax denotes the maximal response for a particular agonist/subunit combination, and n represents the Hill coefficient. Imax, n, and the EC 50 were all unconstrained for the fitting procedures. For experiments measuring barium permeability, oocytes were perfused with barium Ringers (low barium: 90.7 mM NaCl, 2.5 mM KCl, 10 mM HEPES pH 7.3, 1.8 mM BaCl2, 48.6 mM sucrose; high barium: 90.7 mM NaCl, 2.5 mM KCl, 10 mM HEPES pH 7.3, 18 mM BaCl2). Shifts in reversal potential were measured by changing the holding potential from -40 mV to +30 mV by 10 mV increments. Calculations of barium sodium permeability ratios using the extended GHK equation were performed using the Clampfit analysis portion of the pClamp software suite (Axon Instruments, Union City, CA). Barium was used instead of calcium to minimize the contribution of endogenous calcium-activated chloride channels (Sands et al., 1993). Calculations of peak amplitudes and net charge were made using pClamp either during acquisition or during subsequent Clampfit analysis. Note that measurement of net charge has been shown to be a more accurate indicator of fast responses than measurement of peak response. An appropriate method using analysis of the area under the curve of agonist-evoked currents in oocytes has been previously published (Papke and Papke, 2002). Baseline was defined for Clampfit statistics based on 20 s before drug

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24 application, the analysis region for peak and net charge analysis went from 5 s before the initiation of drug application and extended at least 135 s following. Area analysis data is provided for all receptor subtypes examined in this paper for comparison to wild-type. Transfection and Patch-clamp Recording from GH4C1 cells GH4C1 cells were cultured in F10 medium (Gibco, Carlsbad, CA) at 37 C, 5% CO 2 Cells were transiently transfected using Fugene (Roche, Indianapolis, IN), according to the manufacturer instructions. One microgram of wild-type or T6'F mutant 7 cDNA (pTR-UF22, University of Florida, Gainesville, FL) was added to each 35-mm Petri dish, together with 0.5 or 1 g of the cDNA encoding the red fluorescent DsRed protein (BD Biosciences Clontech, Palo Alto, CA). Cells were used 48-72 hours after transfection. Typical transfection efficiency was 10-25% using this method. Single-channel currents were recorded in the cell-attached patch configuration using an Axopatch 200A amplifier (Axon Instruments, Union City, CA) at room temperature. Cells were bathed in a solution containing 140 mM NaCl, 2.8 mM KCl, 1 mM CaCl 2 1 mM MgCl 2 10 mM glucose, 10 mM HEPES/ NaOH (pH 7.3). Patch electrodes (tip resistances, 5-7 Mafter fire polishingwere coated with Sylgard (Dow Corning, Midland, MI) and filled with the same extracellular solution plus 1 M Atropine and 30 M ACh.. Currents were filtered at 10 kHz and digitized at 50 kHz using pClamp 8 (Axon Instruments Union City, CA). Analysis was conducted using pClamp 9. Radioligand Binding Studies GH4C1 cells were harvested from 60mm culture dishes using a sterile cell scraper and assayed for nicotine-displaceable, high-affinity [ 3 H]methyllycaconitine (MLA) binding using a modification of the procedure of Davies and colleagues (1999). Cells were suspended in 20 volumes of ice cold Krebs Ringer HEPES buffer (KRH; 118 mM

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25 NaCl, 5 mM KCl, 10 mM glucose, 1 mM MgCl 2 2.5 mM CaCl 2 and 20 mM HEPES; pH 7.5). After two 1-ml washes with KRH at 20,000g, the membranes were incubated in 0.5 ml KRH with 1, 3, 10 or 20 nM [ 3 H]MLA (Tocris, Ellisville, MO) for 60 min at 4C with or without 5 mM nicotine. Tissues were washed three times with 5 ml cold KRH by filtration through Whatman GF/C filters that had been preincubated for 2 hours in blotto (KRH with 0.5% dry milk and 0.002% sodium azide). They were then assayed for radioactivity using liquid scintillation counting. Inhibition curves generated under two-electrode voltage-clamp with oocytes expressing either wild-type or the TM2 T6'F mutant showed a less than 3-fold difference in MLA potency between the two (data not shown). Intact Oocyte Binding [ 125 I] -Bungarotoxin binding in intact oocytes was performed similar to the method described by Chang and Weiss (2002). In brief, whole Xenopus oocytes that were either uninjected, or had been injected with mRNAs encoding either wild-type or the TM2 T6'F mutant were placed in a single well of a 96-well plate containing either 20 nM [ 125 I] -Btx alone, or 20 nM [ 125 I] -Btx with 5 mM nicotine. After four 4s washes in 2.5 ml KRH, total radioactivity was measured using an automated gamma particle counter as counts per minute (CPM). Nicotine displaceable binding was calculated for at least 4 cells in each condition.

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CHAPTER 3 THE PHARMACOLOGICAL AND KINETIC EFFECTS OF THE TM2 T6'F MUTATION Introduction According to the same broad classification scheme described previously, two of the major nicotinic receptor subfamilies include the muscle-type and the -type nAChRs. Both muscle-type nAChR and homomeric receptors are widely expressed in mammals, bind -Btx with high affinity, and share many structural features. However, several important functional and pharmacological properties distinguish these two major nAChR subtypes. For example, receptors have high permeability to divalent cations, show inward rectifying current-voltage relationships, and have highly agonist concentration-dependent macroscopic response kinetics (Decker and Dani, 1990; Sands et al., 1993; Segula et al., 1993; Vernino et al., 1994; see Table 1-1 for comparison). During the course of the evolution of the various subunits of nAChR and the functional radiation of receptor subtypes into various tissues where each subtype performs highly specialized functions, there has emerged a great deal of sequence divergence. Less than 40% amino acid sequence identity exists between the alpha subunit of muscle type receptors () and the -type receptor found in the brain. It has generally been assumed that the functional differences that exist among these receptor subtypes are emergent properties of the collective sequence differences. Despite this general assumption, it is understood that specific residues, conserved across multiple subtypes, can be key to features common to all those subtypes. Examples of this type of 26

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27 important conserved sequence are in the pore-forming second transmembrane domain, and in this domain the sequence identity between and is increased to around 60%. Previous work has indicated that amino acid residues in the TM2 domain of the beta subunit of heteromeric nAChRs are important regulators of nAChR pharmacology. Specifically, substitution of the TM2 6' (according to the numbering scheme proposed by Miller ((1989) see Figure 3-1), phenylalanine of the muscle subunit for the neuronal subunit serine residue increased sensitivity to nicotine. This same study also showed that substitution of subunit amino acid sequence at the TM2 6' and 10' positions of the neuronal subunit reduced inhibition by the ganglionic blocker mecamylamine (Webster et al., 1999). This chapter describes a testing of the hypothesis that homologous substitution of amino acid sequence from the muscle subunit into the subunit would confer specific properties of muscle-type receptors to mutant nAChRs. Using site-directed mutagenesis and heterologous expression of mutant receptors in Xenopus oocytes, the effect of the T6'F mutation was examined with regard to ACh potency, ACh response kinetics, barium permeability, the sensitivity to the muscle receptor selective agonist succinylcholine, and the potentiating factor 5-hydroxyindole. Results The TM2 T6'F Mutant is Functionally Expressed in Xenopus Oocytes Injection of mRNAs encoding either wild-type or mutant subunits in Xenopus oocytes produced functional receptors that permitted us to study the receptors under two-electrode voltage clamp. Raw data traces from oocytes expressing the TM2 T6'F point mutation or wild-type are shown in Figure X. Differences between mutant and wild-type receptors were seen both in the macroscopic response kinetics (see inset, Figure

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28 3-2) and in the absolute amplitude of the response to ACh. There was consistently a relatively small peak for the T6'F response compared to the wild-type response. Quantification of differences in absolute amplitude is complicated by variations in the degree of receptor expression from cell to cell. However, variations in current amplitude similar to that shown in Figure 3-2 were consistently observed, even after oocytes expressing the mutant receptor were kept for several weeks, presumably allowing receptor expression to accumulate. Due to this difference in functional expression, oocytes expressing mutant receptors were typically used 11-21 days after injection, while oocytes expressing wild-type receptors were used 5-14 days after injection. The average amplitude of control responses (3M ACh) of mutant receptor responses recorded 11-21 days after injection was 64.5 12 nA (n= 12), while the average amplitude of control responses (300 M ACh) of wild-type receptor responses recorded 5-14 days after injection was 335.9 60 nA (n= 12). These concentrations of ACh were chosen for controls because the normalized area under the curve of these responses was at the upper end of the concentration-response function, and they are therefore roughly saturating in terms of net charge. It is important to note that the 100-fold difference in control ACh concentrations is due to the increase in ACh potency observed in the T6'F mutant (see Figure 3-4). To determine whether differences in current magnitude between wild-type and the T6'F mutant were due to a relatively low mutant receptor expression at the cell surface, or due to an effect of the mutation on single channel properties (i.e., the probability of channel opening or single-channel conductance), radioligand binding experiments were performed with both the transfected GH4C1 cells and intact oocytes.

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29 In the case of the transfected GH4C1 cells, [ 3 H]MLA binding experiments conducted 48h post-transfection indicated an approximate B max of 600 fmol/mg of protein in cells transfected with wild-type but no significant nicotine-displaceable binding was observed in cells transfected with the T6'F mutant relative to non-transfected cells (data not shown). However, single oocyte [ 125 I]-Btx binding studies indicated significant specific binding in oocytes injected with either the wild-type or the T6'F mutant (Table 3-1): These same experiments also showed no significant difference in [ 125 I]-Btx binding between intact oocytes expressing either wild-type or the T6'F mutant. Thus it is likely that poor (or possibly slow) expression of the TM2 T6'F mutant receptors prevented their study in transfected GH4C1 cells, but that alterations in receptor number alone were insufficient to explain the differences in current magnitude between wild-type and mutant receptors expressed in Xenopus oocytes. The TM2 T6'F Mutation Dramatically Slows the Apparent Kinetics of ACh-evoked Macroscopic Currents The wild-type receptor response has a characteristically rapid time course when high concentrations of ACh are applied, with the decay phase occurring well before the bath solution exchange is complete (Papke and Thinschmidt, 1998). As shown in Figure 3-3, the TM2 T6'F mutation produced a dramatic change in the apparent kinetics of ACh responses. Figure 3-3C shows the relative lack of effect of increasing concentrations of ACh on duration of the macroscopic responses of the T6'F mutants, distinct from both wild-type (Figure 3-3A) and the muscle-type nAChR (Figure 3-3B). This would be consistent with a change in receptor desensitization. Quantitative analysis is shown in Figures 3-3D & E. This shows the detailed effects of this point mutation on both the time required to reach the peak response and the return to baseline. Here it is

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30 evident that both the rise times and the decay times of the T6'F mutant showed little sensitivity to changes in ACh concentration (for the concentrations examined), which is different from the characteristically concentration-dependent kinetics of the wild-type response to ACh. The rise times of wild-type responses differed from those of both the T6'F mutant and muscle nicotinic receptors at ACh concentrations equal to and below 30 M. The rise times of muscle receptors were also relatively insensitive to changing ACh concentrations compared to wild-type In the case of the decay times, muscle receptors display a concentration dependence, but it is the opposite of that seen with wild-type with a general slowing of the decay of the muscle-type macroscopic response with increasing agonist concentration. Thus the macroscopic responses of the mutant and wild-type receptors and the muscle-type receptors are each uniquely affected by the process of agonist wash-in and wash-out, presumably due to differences in their intrinsic desensitization rates and the influence of agonist binding on those rates (as proposed for wild-type ). The kinetics of solution exchange in the oocyte bath perfusion system have been quantified previously (Papke and Thinschmidt, 1998; Papke and Papke, 2002), allowing the evaluation of the kinetics of the macroscopic responses in the context of how rapidly agonist is washed in and out of the chamber. Based on these observations, it appears that the decay of the T6'F mutant responses follows the solution exchange rates much more closely than wild-type suggesting that agonist washout may predominate in this phase of the response. TheT6'F Mutation Increases ACh Potency Compared to Wild-type Concentration-response functions for wild-type and TM2 T6'F mutant are shown in Figure 3-4. A dramatic increase (> 10 fold) in ACh potency was seen for the net charge analysis of the T6'F mutant (Figure 3-4, Table 3-2), compared to wild-type

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31 Note that both net charge (area under the curve) and peak CRCs are shown to illustrate the differences in apparent ACh potency as a function of the analysis method used. Comparison of these analyses shows that, as previously reported (Papke and Papke, 2002), the net charge CRC analysis of wild-type receptor showed a large difference in apparent potency compared to peak current analysis. In contrast, the T6'F mutant showed less difference between the two methods, due to a loss of 's characteristic concentration-dependent fast desensitization. TheT6'F Mutation Abolishes Barium Permeability and Reduces Inward Current Rectification Barium is frequently used as a charge carrier to evaluate divalent ion permeability because the relative permeability of barium to monovalent cations is generally indicative of calcium permeability, and the use of barium decreases the secondary signal transduction frequently associated with calcium influx. Current-voltage relationships obtained in either high or low extracellular barium, showed the characteristic wild-type receptor inward rectification and permeability to this divalent ion (Figure 3-5; Decker and Dani, 1990; Sands et al., 1993; Seguela et al., 1993; Vernino et al., 1994). The extended GHK equation gave an estimated barium-to-sodium permeability ratio of approximately 4:1. This ratio is substantially lower than that previously reported (Sands et al., 1993), however this apparent discrepancy may be due to the use of EGTA in the current electrode solution. By contrast, the T6'F mutant receptor showed significantly less current rectification (Figure 3-5B) as indicated by chord conductances measured between -40 and +30 mV holding potentials (p = 0.003 compared to wild-type unpaired t test), and a complete lack of a shift in reversal potential with varying

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32 extracellular barium concentrations (Figure 3-5B). The GHK equation applied to these results gives a barium-to-sodium permeability ratio that is near zero. Succinylcholine is a Selective Agonist of Muscle-type Receptors, and is also an Agonist of T6'F Mutant Receptors As shown in Figure 3-6A succinylcholine is an effective activator of muscle-type receptors, but it is relatively ineffective in activating wild-type There is a dramatic increase in sensitivity (both efficacy and potency) of the mutant receptor to succinylcholine, compared to wild-type. Figure 3-6B shows the concentration-response relationships for both ACh and succinylcholine applied to wild-type murine muscle receptors (). While succinylcholine is a partial agonist of muscle-type receptors, it produces no activation of the heteromeric neuronal nAChR, at concentrations up to 1 mM (data not shown). The effect of the T6'F mutation (Figure 3-6C) can be seen to dramatically increase both the potency and the maximum response to succinylcholine relative to wild-type which responds only to concentrations of succinylcholine that exceed 1 mM. Whereas succinylcholine is a weak partial agonist of wild-type receptors, it appears to be a full agonist of the T6'F mutant receptors. The TM2 T6'F Mutation Abolishes Potentiation by 5-Hydroxyindole Responses of nAChRs to ACh can be enhanced by 5-hydroxyindole in oocytes expressing (Gurley et al., 2000; Zwart et al., 2002). This effect is demonstrated in Figure 3-7A in oocytes expressing wild-type exposed to a co-application of 300 M ACh and 1 mM 5HI. As previously reported (Gurley et al., 2000), the 5HI effect did not impact the macroscopic kinetics of the inward current (Figure 3-7A, inset). In whole-cell voltage-clamp experiments, 1mM 5HI also potentiates wild-type expressed in transfected GH4C1 cells by 540 75% (mean SEM, n= 7; data not shown). In

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33 contrast, oocytes expressing T6'F mutant receptors show a total lack of enhancement of ACh evoked currents by 1 mM 5HI (Figure 3-7B). Comparing this lack of potentiation of T6'F mutant responses to the muscle-type nAChR shows an identical insensitivity to the same concentration of 5HI (Figure 3-7C). Discussion While examples of single point mutations producing dramatic changes in response kinetics, calcium permeability, and pharmacology of nicotinic receptors have been previously shown (Revah et al., 1991; Palma, 1996), the findings presented here are unusual in that several properties that functionally distinguish muscle-type nAChRs from members of the other major subgroups of nAChRs can be conferred to mutant receptors by a single residue. Not only does this observation suggest the importance of amino acid sequence at this particular site for the maintenance of -like properties in the wild-type receptor, but that there is a high degree of functional significance to the structure at TM2 6' position allowing a point mutation to overcome other distinguishing structural elements of the subtype. The relatively low magnitude of T6'F mutant receptor response to agonist compared to wild-type is likely the result of a combination of factors. In the oocyte system, the loss of calcium permeability alone would diminish the functional amplification of the nAChR-mediated current by calcium-dependent chloride current (Barish, 1983). Also, in the muscle receptors, the beta subunit places a single phenylalanine at a site where it aligns with hydrophilic residues in the other subunits, while in the T6'F mutant receptor there is likely to be a complete hydrophobic ring at the same site in the channel. The positioning of a hydrophobic phenylalanine in the ion permeation pathway could therefore have profound effects on single channel properties.

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34 Based on the results of our radioligand binding experiments, we can conclude that, at least in the case of the oocytes, there are comparable numbers of wild-type and T6'F mutant receptors expressed at the cell surface, and that other factors may contribute to the mutant's relatively low response magnitude. The variety of effects that we observed due to the mutation of the TM2 6' amino acid were intriguing, given the fact that this site is in the putative pore-lining region. The T6'F mutation converts from a receptor having a high barium permeability to a receptor that is relatively impermeant to barium. This observation is not surprising due to the likelihood of steric or hydrophobic interference (or both) to divalent ion flux through the pore related to the ring of phenylalanine residues present at the TM2 6' position of the mutant receptor. Furthermore, others have reported that amino acids in the TM2 domain of several different ligand-gated ion channels (including ) are essential determinants of ionic selectivity (Galzi et al., 1992; Keramidas et al., 2000; Gunthorpe and Lummis, 2001). What is more surprising about the findings presented here are the effects that this mutation has had on receptor properties that may be linked to conformational changes far removed from the pore-lining region (i.e., changes in succinylcholine pharmacology). It is thus likely that the T6'F mutation is indirectly impacting agonist binding, or that it is affecting the coupling of agonist binding and subsequent conformational effects, such as channel gating. It is important to consider the findings presented here in the context of previous reports that indicate other contributing factors shown to regulate divalent ion permeability in muscle-type receptors. For instance, the muscle gamma subunit has been shown to be an essential determinant of divalent cation permeability (Francis and Papke,

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35 1996); however, one limitation of the experimental approach used in that study was the inability to evaluate the significance of the muscle subunit. Thus, the gamma subunit is necessary, but may not be sufficient, to produce the divalent ion permeability profile characteristic of muscle nAChRs. The results presented here imply that the subunit, and particularly the TM2 6' phenylalanine may cooperate with the gamma subunit in determining this feature. It may be significant however, that the TM2 T6'F mutant has five phenylalanine residues at this position compared to the muscle receptor's one, thus potentially amplifying the effect that this amino acid sequence has on wild-type receptor function. The slowing of the macroscopic kinetics of the 6' mutant receptor responses to ACh is reminiscent of the muscle-type receptors, with the decay phase of mutant receptor response kinetics showing a relative lack of sensitivity to changes in agonist concentration. This property is more characteristic of beta subunit-containing receptors than wild-type (Papke and Thinschmidt, 1998). The T6'F mutant response kinetics also show a nearly total lack of effect of ACh concentration on the rise rates of the macroscopic response. Again, this result appears to reflect the conferring of a nonreceptor-like property on the mutant. It is interesting to note that while the widely studied L9'T (L247T) mutation appears to remove a great deal of the fast desensitization associated with the wild-type receptor (Revah et al., 1991), the amino acid substitution is nearly opposite in nature to the one reported here. While the L9'T represents the replacement of a larger, hydrophobic residue with a smaller hydrophilic one, the reverse is true for the T6'F mutation. This suggests that this aspect of the -type response may not be strictly regulated by size or hydrophobicity. Note that the 9' leucine residue has

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36 been proposed to contribute to a ring of hydrophobic residues, while the 6' threonine is more likely to contribute to a ring of polar residues approximately one level lower on the putative transmembrane helix, closer to the hypothetical selectivity filter of the channel (Corringer et al., 2000). In this regard, it is conceivable that substituted amino acids with different properties could be accomplishing a similar effect either through different mechanisms, or through similar mechanisms at different sites in the receptor. The conversion of succinylcholine from a very weak partial agonist of wild-type receptors to a relatively potent full agonist of the T6'F mutant was an indication of the pharmacological constraints that these residues appear to place on their corresponding wild-type receptors. The fact that a significant degree of succinylcholine selectivity can be attributed to a residue in the pore lining region is perhaps less obvious, and is suggestive of more global effects of this amino acid on the overall structure of the receptor in both the open and closed states. Although the mechanism of potentiation of responses by 5-hydroxyindole is unknown, previous reports (Gurley et al., 2000) combined with our results from experiments with the muscle-type nAChR, suggest that this effect may be unique to The total absence of potentiation by 5HI for both the muscle and the T6'F mutant, compared to the expected large enhancement of ACh-evoked responses from wild-type appears to be further confirmation of the ability of this amino acid substitution to imbue the mutant receptor with a muscle receptor phenotype. Without a clearer understanding of how 5HI potentiates wild-type responses, it is difficult to suggest an explanation for its absence in the T6'F mutant. It is possible that amino acid sequence at the TM2 6' position is an essential factor in the coupling of agonist evoked channel

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37 opening to an allosteric alteration produced by 5HI. Alternatively, the T6'F mutation may prevent 5HI from relieving a constitutive inhibition of wild-type a mechanism that has been proposed for a similar potentiation of by bovine serum albumin (Butt et al., 2002). The data presented here suggest that the functional changes associated with the emergence of a specialized receptor involved in muscular contraction can be attributed, at least in part, to sequence difference at a single site in what is ultimately a structural subunit of the muscle receptor complex. It is potentially significant that the muscle beta subunit is the only known nAChR gene product that contains a phenylalanine residue at this site (LeNovere and Changeux, 2001). The effects of this mutation extend beyond those that are commonly associated with amino acid structure in the pore-lining region of the receptor, and indicate that nAChR function and pharmacology can be broadly and dramatically altered by this single amino acid change. This suggests that the evolution of functional specialization in this superfamily of ligand-gated ion channels may involve something analogous to punctate equilibrium, where single small changes may produce branch points of functional significance and point to the origins of the families of receptor subtypes.

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38 Table 3-1. Intact oocyte [ 125 I]-Btx binding. CPM/Cell Rat wild-type 20 nM -Btx alone 764.32 529.4 (n = 4)* 20 nM -Btx + 5 mM nicotine 72.15 60.5 (n=4) Rat T6'F mutant 20 nM -Btx alone 1074.98 418.8 (n = 4)* 20 nM -Btx + 5 mM nicotine 261.55 137.5 (n=3) Data represent the mean ( s.e.m.) counts per minute per cell (CPM/Cell) for the indicated treatments (* p 0.05 by Student's t compared to the same receptor subtype in the presence of 20 nM -Btx with 5 mM nicotine).

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39 Table 3-2. Curve-fit values for wild-type and T6'F mutant responses to ACh. Normalized I max Hill Coefficient EC 50 Rat wild-type ACh peak a N/A N/A N/A ACh net charge 1.05 0.02 1.5 0.1 16.5 1.0 M Rat T6'F mutant ACh peak 0.89 0.04 1.5 0.4 1.7 0.4 M ACh net charge 0.96 0.04 2.4 1.1 1.1 0.2 M Data represent curve values generated by the Hill equation (see methods) for macroscopic responses from oocytes expressing either indicated receptor. a The use of peak responses has been shown to provide inaccurate estimates of these parameters (Papke & Papke, 2002) and curve-fitting was thus restricted to net charge analysis for wild-type

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40 Figure 3-1. Wild-type and mutant nAChRs. Amino acid sequences for wild-type and mutant nAChR TM2 domains. The numbering of specific residues of the second membrane-spanning region is according to that proposed by Miller (1989). The corresponding sequences for wild-type muscle and neuronal beta subunits are also given as a reference. The substituted residues are highlighted at the 6' position.

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41 Figure 3-2. Electrophysiological responses from wild-type and mutant nAChRs. ACh-evoked current responses for wild-type and the TM2 T6'F mutant nAChR expressed in oocytes. The agonist application bar at the top indicates the timing and duration of agonist application for each receptor (wild-type = 30 M ACh, T6'F = 3 M ACh). The inset shows the same traces scaled to one another for the purpose of comparison (wild-type = 100 %, T6'F = 476 %).

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42 Figure 3-3. Macroscopic response kinetics of wild-type 7, muscle, and the TM2 T6'F mutant nAChRs expressed in oocytes. A) Wild-type rat 7 current traces at the indicated concentration of ACh. B) Wild-type muscle () current traces at the indicated concentration of ACh. C) Rat 7 TM2 T6'F current traces at the indicated concentration of ACh. D & E) Rise times (10 90 %) and decay times (90 70 %) for macroscopic responses to ACh applied to each of the indicated receptors at the concentrations shown. The kinetics of muscle receptor responses as a function of increasing ACh concentration are also plotted for comparison. Means and standard errors are given for at least four cells.

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43 Figure 3-4. ACh concentration-response functions for wild-type and the TM2 T6'F mutant nAChR. Data were normalized to the maximal response for either wild-type rat (A) or TM2 T6'F (B). Peak and net charge analysis (area under the curve) are presented showing the relative sensitivity to increasing concentrations of ACh. Means and standard errors represent data acquired from at least four cells.

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44 Figure 3-5. Wild-type and mutant nAChR current-voltage relationships and divalent ion permeability in Xenopus oocytes. Current-voltage relations were examined in wild-type 7 and T6'F mutant receptors using either high (18 mM) extracellular barium or low (1.8 mM) osmotically balanced extracellular barium. Holding potentials were incrementally adjusted by 10 mV from -40 mV to +30 mV and peak responses were normalized to control responses held at -50 mV. A) The wild-type 7 I-V shows its characteristic inward rectification, as well as positive shift in reversal potential with increased extracellular barium. B) The TM2 T6'F mutant I-V shows less inward rectification, and the lack of a shift in reversal potential in high extracellular barium indicates a loss of divalent ion permeation.

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45 Figure 3-6. Wild-type and mutant nAChR responses to the muscle receptor selective agonist succinylcholine. A) Representative current traces for oocytes expressing the indicated nAChR subtype. The response to the indicated concentration of succinylcholine (SuCh) preceeded and followed by control applications of ACh. B) Net charge concentration-response relationships for wild-type muscle () exposed to either ACh or SuCh. C) Net charge concentration-response relationships for wild-type and the T6'F mutant 7 exposed to SuCh (see text for discussion of the use of net charge analysis for these experiments).

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46 Figure 3-7. Wild-type and mutant nAChR potentiation by 5-Hydroxyindole. A) Representative TEVC current traces from Xenopus oocytes expressing wild-type 7. Agonist application bars represent a control application of 300 M ACh in the first trace, and pre-incubation with 1 mM 5HI followed by co-applied 300 M ACh. The inset shows the same two traces scaled to one another, showing the lack of an effect of 5HI potentiation on macroscopic response kinetics. B) Representative current traces from Xenopus oocytes expressing the TM2 T6'F mutant receptor. The experimental design was similar to that described for the wild-type receptor above, except for the use of 3 M ACh alone or co-applied with 1 mM 5HI. C) Current responses representing a similar experimental design to that described above for oocytes expressing muscle-type nAChRs using 30 M ACh alone or co-applied with 1 mM 5HI. D) The effect of co-application of 1 mM 5HI on ACh responses normalized to the previous control application of ACh in Xenopus oocytes expressing the indicated wild-type or mutant nAChR subtype. Means and standard errors represent data from at least four cells.

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CHAPTER 4 THE TM2 T6'S MUTATION: A GAIN-OF-FUNCTION WITH -LIKE PHARMACOLOGY Introduction A potentially important feature of -type nicotinic receptors that sets them apart from other ligand-gated ion channels is the high degree of concentration-dependence to the kinetics of agonist-evoked responses. That is, with increasing concentrations of agonist, the macroscopic responses of nAChRs become more and more transient (Papke and Thinschmidt, 1998). The fact that nAChRs have been reported to have a very high permeability to calcium (Decker and Dani, 1990; Sands et al., 1993) suggests that this agonist concentration-dependent limitation of the channel's activity is part of a built-in safety mechanism designed to prevent excitotoxic injury in cells with sufficient levels of receptors expressed. This high degree of agonist concentration-dependent limitation on response magnitude has in many cases been attributed (Revah et al., 1991) to classical Katz and Thesleff (1957) type desensitization. This is a reasonable interpretation, but evidence for other mechanisms of agonist-dependent inhibition such as open channel block may also contribute to this property. Since macroscopic currents constitute the ensemble activity of channels in a variety of discrete states, the only way to actually to determine the degree to which classical desensitization contributes to agonist-dependent limitation of a response is at the single channel level. The T6'F substitution described in the previous chapter has the effect of producing a mutant receptor with several muscle nAChR-like properties. One major effect of 47

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48 this mutation is the reduction in the concentration-dependence of agonist-evoked responses that is so characteristic of wild-type (Figure 3-3). In addition to this change in kinetics, the T6'F mutant also displays some pharmacological properties that are like the muscle-type nAChR. In an effort to test if an analogous effect could be produced with a similar single amino acid substitution in the TM2 domain, an T6'S mutant was constructed. In this case the wild-type threonine is exchanged for the serine residue present at the homologous position of the neuronal / subunit (Figure 3-1). The hypothesis being tested was similar to that which was addressed by the characterization of the T6'F mutant. Specifically, a serine-for-threonine substitution at the 6' position of the TM2 domain would make the mutant receptor more like a neuronal beta subunit-containing receptor. As in the case of the T6'F mutant, much of the rationale for the hypothesized effects of the T6'S mutant are derived from previously published observations with heteromeric nAChRs. The effects reported by Webster and colleagues (1999), showed that sensitivity to the ganglionic blocker mecamylamine was increased in muscle-type receptors with chimeric () subunits, and that this effect could be attributed largely to the sequence differences at the TM2 6' and 10' positions. This suggested that a similar effect may be observed with the T6'S mutant, particularly for drugs that interact with the pore-forming region of the receptor. The result is a mutant receptor that retains much of the pharmacology of wild-type but with larger macroscopic responses and a dramatic lessening of agonist concentration-dependent limitation on response duration. The kinetic effects of this mutation are examined at the single-channel level and indicate that channel open time

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49 and burst properties are affected, rather than receptor expression, or single-channel conductance. Finally, although the T6'S mutant displays a pharmacological profile that is very similar to wild-type a gain-of-function mutant that retains much of the pharmacology of the wild-type receptor has significant implications for drug development. Results T6'S Mutant Kinetics and Single-channel Properties One of the major predictions generated by the hypothesis behind the creation of the T6'S mutant, is that this substitution would produce a receptor with response kinetics more like the high-affinity nicotine binding receptor (see Table 1-1 for comparison). In order to test this prediction, first an analysis of macroscopic responses from oocytes was performed, followed by single-channel studies in mammalian cells aimed at identifying changes in the properties of unitary events. The TM2 T6'S Mutation Slows the Macroscopic Kinetics of ACh-evoked Currents and Increases the Overall Net Charge Carried Upon Activation The TM2 T6'S mutant shows a significant change in response kinetics to ACh (Figure 4-1), with a general slowing of the macroscopic response for the mutant versus wild-type Figure 4-1 also shows the decreased effect of increasing concentrations of ACh on the macroscopic decay rate of the T6'S mutant responses (Figure 4-1B) compared to wild-type (Figure 4-1A) and its characteristically strong concentration-dependent kinetics. An analysis of the rise times (Figure 4-1C) of these responses showed that the T6'S mutant actually shows a slowing of the response with increasing agonist concentration before the eventual increase in rise time dominates at the highest concentrations. This is particularly true for ACh concentrations around 30 M. The

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50 decay rates of the T6'S mutant responses, much like the T6'F mutant, show little effect of high agonist concentrations, whereas wild-type responses become increasingly brief (Figure 4-1D). A comparison of 3 [H]MLA binding between intact oocytes expressing either wild-type or the T6'S mutant showed that the average difference in nicotine displaceable binding was not statistically significant (Table 4-1). Despite this similarity in receptor binding, the ACh evoked currents in cells from the same injection set differed substantially in the amount of charge carried. Figure 4-2A shows averaged currents from these oocytes with 30 M ACh applied, and below in figure 4-2B, the same currents are expressed showing the cumulative net charge for both the wild-type and T6'S mutant. The total area under the curve for the averaged wild-type current is 11.5% of that of the T6'S mutant, a difference that cannot be attributed to differences in receptor expression. The T6'S Mutation Does not Increase Single-channel Conductance Having observed this difference in macroscopic kinetics and the corresponding increase in net charge, single channel patch-clamp experiments were conducted in order to identify the specific channel properties affected by the mutation. One aspect of the receptor that may have been altered with significant effect on the amount of charge carried by the mutant receptor is the unitary conductance. Single channel currents were recorded from transiently transfected GH4C1 cells in the cell-attached patch configuration. Figure 4-3 shows a representative recording from a cell exposed to 30 M ACh in the patch pipette. Control cells (untransfected, n=6) showed no channel openings in the presence of ACh. A current-voltage relationship was established for the T6'S mutant in order to quantify the single-channel conductance. Figure 4-4 shows a representative plotted I-V relation. Linear regression analysis gave an average slope

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51 conductance of 61.7 5.8 pS. This is slightly less than the wild-type single-channel conductance of 91.5 8.5 pS reported by Mike and colleagues (2000). Exact comparison to the previously reported value for the wild-type receptor may not be possible due to differences in experimental solutions (e.g., no extracellular magnesium and lower calcium for the previously published experiments), but the value for the T6'S mutant is at least not greater than that reported for wild-type This suggests that the difference in response magnitude between mutant and wild-type is not attributable to a change in unitary conductance. The T6'S Mutation Produces a Significant Increase in Average Channel Open Time Since a change in the T6'S single channel conductance was clearly not contributing to the increase in net charge evident in macroscopic mutant responses, alterations in channel open times were then examined. Analysis of channel dwell time distributions (Figure 4-5A) shows that the T6'S mutant channel open times are best fit by two exponentials with the average shorter open time being 580 110 s and the longer open time being 4.308 0.856 ms. Channel open times for native wild-type receptors expressed in tuberomammillary nucleus neurons have been reported to be fit by a single exponential and had an average of 83 s (Victor Uteshev, personal communication). Since T6'S mutant channel open times were fit by two exponential components, a weighted average was used to give an estimate of the total average open time for a macroscopic current (2.470 ms). Comparing this to the previously published mean channel open time of 100 s (Mike et al., 2000), gives an approximate 24.7 fold increase in the channel open time. Taking into consideration the differences in single-channel conductance for the T6'S mutant versus that reported for the wild-type receptor (33% less

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52 for the mutant), the cumulative charge carried by the wild-type receptor shown in Figure 4-2B was scaled to show the potential contribution of these values to the differences observed in macroscopic currents (Figure 4-5B). T6'S Mutant Burst Activity Another difference in single channel activity demonstrating effects of the T6'S mutation is the nature of channel bursting. Figure 4-6A shows raw single channel events where bursts of openings were observed under steady-state conditions. Figure 4-6B shows the closed time distribution for the T6'S mutant from patches exposed to 30 M ACh. The requirement for multiple exponentials to fit the distribution is consistent with channel bursting. Using the method of Colquhoun and Sakmann (1985) a critical time value of 759 s was determined as a threshold for intraburst closures. The wild-type nAChR has been shown to have little or no burst activity under steady-state conditions (Victor Uteshev, personal communication), or in other words, bursts that consist of single openings. In such a case the average channel open time approximates the average burst duration. The average burst duration distributions (Figure 4-7A) for the T6'S mutant were best fit by two exponential components (556 104 s and 5.522 1.355 ms). Histograms of the number of bursts with more than one opening were plotted and fitted by a Poisson distribution (Figure 4-7B), providing a prediction of the probability of channel reopening. In this case, the T6'S mutant had a 42.1 6% probability of opening more than once, and as previously stated, bursts with more than one opening are rarely, if ever observed in the wild-type receptor under steady-state conditions. The Pharmacology Of The T6'S Mutant The other general prediction derived from the rationale for making the T6'S mutant was that the pharmacology would be more like a neuronal beta-subunit containing

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53 nAChR (see table 1-1). However, with one large exception in the case of potentiation by 5HI, the pharmacological profile of the T6'S mutant is generally more like the wild-type than a heteromeric neuronal nAChR. In the context of the previously described changes in kinetics, this presents an advantage to those interested in identifying drugs that target the wild-type receptor. Specifically, a receptor that has the same pharmacological features of wild-type but lacks the characteristic response limitation in the sustained presence of agonist, may serve as a useful facsimile of the wild-type receptor. The T6'S Mutant Shows a Slight Decrease in Ach Potency and a Decreased Discrepancy Between Area and Peak CRCs The concentration-response relationship for ACh applied to either the T6'S mutant or wild-type are shown in Figure 4-8, showing a slight decrease in the apparent ACh potency for the T6'S mutant compared to wild-type (see Table 4-2). As previously shown in Chapter 3, wild-type nAChRs have significantly different peak amplitude and net charge CRCs (Figure 3-4). This is indicative of the strong agonist concentration-dependence to the time course of their macroscopic responses. In a manner similar to that shown for the T6'F mutant, the T6'S mutant shows less of a difference between the two methods (Figure 4-8B), again indicating that response kinetics are far less sensitive to agonist concentration. The Agonist Selectivity Profile of the T6'S Mutant is Similar to Wild-Type Concentration response relationships for a series of nicotinic receptor agonists indicates that the T6'S mutant has a pharmacology that is similar to wild-type with regard to both potency and efficacy of the agonists examined. Figure 4-9 compares the effects of several selective agonists on oocytes expressing either the T6'S mutant or wild-type rat Although some differences in potency and efficacy are evident (with

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54 the exception of the partial agonist Tropisetron, which is nearly identical between the two receptor types (Figure 4-9D)), all agonists activate the mutant receptor. Furthermore, the potency differences observed are similar to that seen with ACh (Figure 4-8), suggesting a general trend in modest potency reduction for agonists. The similarity in agonist response profiles also extends to agonists that not selective for type nAChRs. The subunit selective agonist cytisine (Papke and Heinemann, 1994) and the selective agonist metanicotine (Papke et al., 2000b) display agonist activity at the T6'S mutant (Table 4-2). Again, there is a slight reduction in potency and efficacy, particularly in the case of cytisine, but these shifts are consistent with the general trend observed for most of the agonists examined (Table 4-2). Antagonists of the Wild-Type Receptor are also Antagonists of the T6'S Mutant Another feature of the T6'S mutant that has proven to be similar to wild-type is its sensitivity to nAChR antagonists. A somewhat peculiar feature of the L9'T mutant is that several drugs that function as antagonists of wild-type have been shown to activate this mutant (Bertrand et al., 1992; Palma, 1996; Palma et al., 1998; Tonini et al., 2003). By contrast, each of these drugs inhibit the T6'S mutant (Figure 4-10). Furthermore, for the concentrations tested, no agonist activity of these same drugs was observed (not shown). 5-Hydroxyindole Potentiation is Significantly Diminished in the T6'S Mutant Compared to Wild-Type As previously described in Chapter 3, 5HI has been reported to be an allosteric potentiator of wild-type Figure 3-7 shows this effect in oocytes where 1 mM 5HI produces a 9-fold potentiation of wild-type responses to ACh. By contrast, the T6'S mutant shows relatively very little potentiation by 5HI (Figure 4-11). While this

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55 potentiation is significant at 26 % (p < 0.05 compared to control, Student's t) it is also significantly and dramatically lower that the wild-type receptor's average potentiation of 904 % (p < 0.0001, compared to 5HI potentiated wild-type Student's t). Discussion Kinetic Effects of the T6'S Mutation The mutant receptor described in this chapter displays a profound change in response kinetics, with a significant reduction in response limitation in the presence of higher concentrations of agonist. Based on the results of receptor binding experiments, the overall increase in response magnitude observed in the T6'S mutant is apparently not due to an increase in receptor expression. This however, is not particularly surprising, since an increase in receptor number would not be a likely cause of altered response kinetics. On the other hand, the lack of an increase in single-channel conductance is perhaps more unexpected. This is particularly the case since the mutation represents the substitution of a smaller serine residue for the larger wild-type threonine within the pore-forming domain. This could conceivably result in a larger pore diameter, and thus increased single channel conductance. However, the impact of the T6'S mutation on the pore diameter is either too subtle, or compensated for by other changes, such as changes in charge distribution of the amino acids lining the pore, impacting their interaction with permeant ions. It is interesting to note that the well-studied L9'T mutation has a similar effect in that there is a gain-of-function, without a significant increase in unitary conductance (Palma et al., 1999). By contrast, an examination of the mean channel open time indicates that the T6'S mutant has far longer open durations than observed for wild-type (Mike et al., 2000; Victor Uteshev, personal communication). This almost certainly contributes to the

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56 ability to record responses from cells expressing the T6'S mutant under cell-attached patch conditions. While it is possible that wild-type receptors may also activate under similar conditions, channel openings under steady-state conditions of exposure to even low concentrations of agonist are so infrequent as to render such studies impractical, if not impossible. Since no estimates were made of the number of T6'S mutant channels present in each patch, the absolute P O could not be determined, however, it is likely to differ from wild-type since receptor expression was found to be comparable between the two in oocytes. Burst analysis indicates that there is a significantly higher likelihood of channel reopening for the T6'S mutant compared to wild-type. It should be mentioned that this increase in channel re-opening probability also reflects a general increase in overall open probability. While no effort was made in these studies to generate specific kinetic models that fit the experimental data, this basic observation of increased open probability provides information regarding transitions between kinetic states for the T6'S mutant. The kinetic scheme shown in Figure 4-12 is a simplified, slightly modified version of the fractional occupancy model proposed for wild-type by Papke and colleagues (2000a). In this model are several transition rates that are dependent on agonist concentration, all of which serve to drive the channel into desensitized, non-conducting states as agonist concentration increases. This notion is consistent with previously published observations regarding kinetics (Papke and Thinschmidt, 1998; Papke and Papke, 2002) and with the macroscopic data shown in Figure 4-1. In the interpretation of the kinetic effects of the T6'S mutation, there are several possible explanations for the reduction in concentration-dependent response limitation. One possibility is that one or more states

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57 that are closed in the wild-type receptor have been converted to an open state. For example, the states labeled A 2 R* and A 3 R* may be open states for wild-type whereas the states labeled A 4 R* and A 5 R* may be closed. This is consistent with a receptor that is more likely to be in an open state when its five putative agonist binding sites are less than fully occupied. However, in the case of the T6'S mutant, one or both of the normally non-conducting A 4 R* and A 5 R* states may have been converted to open states. It is also conceivable that one or more of the desensitized states (A n D) has been converted to an open state. Another possible interpretation is the that the T6'S mutation has induced a shift in the desensitization rates, such that transitions into the open state are generally more favored than in the wild-type receptor. When applying the above scheme to wild-type one can see that when desensitization rate constants (d+) are higher than the rate constants for the non-desensitizing transitions out of the open state (), the desensitized state becomes absorbing, the number of openings per burst is close to unity, and the probability of channel reopening approaches zero. However, in the case of the T6'S mutant, the probability of channel reopening is significantly non-zero. This indicates that the transition rates between the bound, non-desensitized states and the open states are sufficiently high relative to the desensitization rate constants to make multiple opening bursts more likely. Thus, the T6'S mutation appears to have had some effect on the rate of desensitization, or at least on the rate of desensitization relative to other transition rates. Although it may seem as though the T6'S mutation has the effect of reducing agonist potency, this effect should not be interpreted outside of the context of the kinetic

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58 effects. This is because the apparent reduction in potency may in fact be due to a broadening of the concentration-response functions for agonists. That is, at the higher agonist concentrations that impose limitations on the responses of the wild-type receptor, the T6'S mutant is still activated. This essentially stretches out the agonist CRCs for the mutant producing an apparent reduction in potency. Of course, the data presented here can not rule out direct effects on agonist potency, but the fact that T6'S mutant responses are occurring at what are normally inhibiting agonist concentrations for wild-type must also be considered. Pharmacological effects of the T6'S mutation While the data presented here do not lend support to the original hypothesis of converting a mutant receptor into neuronal beta subunit containing receptor by means of a single amino acid substitution (similar to what was observed with the T6'F mutant), the findings are interesting nonetheless. Other gain-of function mutations have been produced in but there is typically very unusual pharmacology that accompanies this effect. For example, the pharmacology of the L9'T mutant has been extensively studied, and has been shown to differ greatly from that of the wild-type nAChR (Bertrand et al., 1992; Palma, 1996; Palma et al., 1998; Tonini et al., 2003). The studies that have been published regarding the altered pharmacology of this mutant receptor have been somewhat intriguing and seem to show a strong relationship between amino acid sequence at this position of the TM2 domain, and receptor gating. There are several drugs that have been reported to be antagonists of the wild-type receptor that are agonists of the L9'T mutant. This, coupled with the significant gain-of-function that this mutation has on the receptor's response to agonist has led to the suggestion that the mutation converts a liganded, closed state to a liganded, open one (Bertrand et al., 1992).

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59 While these observations are interesting, and may have something to tell us about the structural elements involved in gating of the type nAChR, they are less useful when looking for tools to aid in drug screening for potential therapeutics that target In the effort to find drugs that have a selective effect on function, it would certainly be more desirable to have pharmacological fidelity in a screening system. The main problem with this is that some of the unique attributes of the type receptor make this a very difficult proposition. The rapid desensitization of the wild-type nAChR creates significant difficulty when attempting to study this receptor subtype in some experimental systems. This is thought to be because small amounts of agonist leakage from the application apparatus can cause pre-desensitization of the receptors, thus making it impossible to record from them. The use of the Xenopus oocyte expression system circumvents this problem to some degree by providing a large cell with sufficient tolerance for application systems that are relatively slow and less likely to produce leakage. In addition, the presence of calcium-dependent chloride currents in the oocyte produces a secondary amplification of -mediated currents at holding potentials that are more negative than the chloride reversal potential (Miledi and Parker, 1984). However, mammalian expression systems are hampered in this regard, and are notoriously sensitive to pre-desensitization by agonist. Careful control of agonist application can help to overcome some of these problems (Kabakov and Papke, 1998), but the translation of these techniques to high-throughput drug screening assays has been problematic. Consequently, some efforts in the area of drug discovery have attempted to make use of gain-of-function mutant receptors. In particular, the L9'T mutant receptor has been applied to this problem because of its slower macroscopic decay rates in the presence of

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60 agonist (Revah et al., 1991). While this kinetic feature may make it useful for identifying responses to agonist when solution control is not ideal, the problem of pharmacology presents itself in the sense that what is an agonist of the L9'T mutant, may not be an agonist of the wild-type receptor. By contrast, the T6'S mutation produces a similar gain-of-function, but has a pharmacology that is far more like the wild-type receptor than the L9'T mutant, at least with regard to the drugs tested here (Table 4-2). In particular, several of the antagonists that are converted to agonists in the case of the L9'T mutant remain antagonists of the T6'S mutant. This has obvious practical significance when screening candidate compounds in the interest of finding drugs that have a selective, functional interaction with the wild-type nAChR. A structural comparison of these two mutant receptors may shed some light on the molecular basis of these pharmacological differences. Both the TM2 6' and 9' positions line the putative pore of the ion channel and are nearly one full rotation around the alpha helix in terms of their periodicity. Both positions are located near the narrowing of the channel pore, thought to be associated with channel gating (Corringer et al., 2000). Where the two mutations differ however, is in the nature of their respective substitutions. The L9'T mutation consists of the replacement of a fairly large, hydrophobic residue with a smaller, more polar one. The T6'S mutation on the other hand, is somewhat more conservative, consisting of the replacement of a residue with both hydrophobic and polar properties with a slightly smaller residue that has similarly mixed properties. Thus, it is possible that the two mutations share the effect of converting a kinetic state of the receptor that is normally a desensitized state, into an activated state by virtue of the fact that the smaller side chains of their substituted residues allow ionic conductance that

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61 would normally be blocked by the larger residues of the wild-type receptor. Where the two mutations may differ then, could be due to the fact that when antagonists are bound to the L9'T mutant, the transition to what would normally be a non-conducting state in wild-type is an activated one in the mutant. However, in the case of the T6'S mutant, the change may not be sufficiently dramatic to produce such a conducting state. This interpretation is intriguing since, as the comparison of the T6'S and L9'T mutations suggests, the binding of antagonists may have structural significance that may only be revealed once a mutation has been introduced. An alternative interpretation would involve changes to receptor structure that would simply cause antagonists to promote the transition to the same activated state that agonists produce. The previously described hypothesis would require an unobservable transition into a resting, desensitized state that is converted to an activated state. However, since we typically think of antagonists binding to the receptor in the resting, closed state, the L9'T mutation may convert antagonists to agonists by changing the structure of the receptor so that a transition from a state where antagonist is bound, and the receptor is closed, is less energetically favored that an open, activated state. One pharmacological aspect of the T6'S mutant that does differ significantly from wild-type is in the lack of potentiation by 5HI. Since the mechanism of 5HI potentiation of wild-type remains unknown, this reason for this difference is unclear. It is possible that the effect of 5HI on the wild-type receptor is functionally similar to the effect of the T6'S mutant, in essence suggesting that the T6'S mutant is in a perpetually potentiated state. This explanation is problematic however. Potentiation by 5HI is unusual by itself, in that there is little or no effect on the macroscopic kinetics of

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62 potentiated responses compared to unpotentiated responses (Gurley et al., 2000). The fact that the T6'S mutation appears to have significant effects on the kinetics of response to agonist suggests that the mechanisms of response amplification for 5HI potentiation may be different. In conclusion, the data presented in this chapter show that the T6'S mutation produces kinetic effects that are distinctly unlike wild-type lending further support to the idea that mutations in this region of the receptor can have profound effects distinguishing feature of the nAChR. Furthermore, the relative lack of significant changes in pharmacology suggest that this mutant receptor may prove useful in the identification of compounds that selectively affect

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63 Table 4-1. Intact oocyte [ 3 H] MLA binding. CPM/Cell Rat wild-type 20 nM MLA alone 143.58 3.9 (n = 5) 20 nM MLA+ 5 mM nicotine 100.43 11.5 (n = 4) Rat T6'S mutant 20 nM MLA alone 168.68 9.9 (n = 5) 20 nM MLA+ 5 mM nicotine 105.94 5.9 (n = 5) Data represent the mean ( s.e.m.) counts per minute per cell (CPM/Cell) for the indicated treatments. p 0.05 by Student's t compared to the same receptor subtype in the presence of 20 nM MLA with 5 mM nicotine.

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64 Table 4-2. Agonist profile comparison for wild-type and the T6'S mutant. Agonist Wild-type T6'S ACh EC 50 30 M 100% agonist 100 M 100% agonist Choline 300 M 100% agonist 2 mM 95% agonist GTS-21 5 M 32% agonist 3M 12% agonist 4OH-GTS-21 1.4 M 46% agonist 3.3 M 20% agonist AR-R17779 10 M 78% agonist 30 M 90% agonist Tropisetron 0.3 M 38% agonist 0.9 M 30 % agonist RJR-2403 240 M 16 % agonist 400 M 18% agonist Cytisine 13 M 73% agonist 43 M 80% agonist EC50 values and maximum efficacy relative to ACh for each of the indicated agonists. Potency and efficacy values derived from peak CRC analysis published in Papke et al., 2000. Potency and efficacy values derived from Papke et al., 2004. Potency and efficacy values derived from Papke et al., 2005.

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65 Figure 4-1. The time course and concentration-dependence of nAChR macroscopic kinetics are altered by the T6'S mutation. Two-electrode voltage clamp responses from oocytes expressing either wild-type (A), or the T6'S mutant (B) show the relative effect of increasing concentrations of ACh. The T6'S mutant responses are slower and the effect of higher concentrations of agonist on the macroscopic kinetics reaches a maximum, whereas for wild-type no maximum is achieved. The T6'S mutant shows a slowing of the rise time near 30 M before the higher agonist concentrations dominate (C). The decay times (D) also show that the T6'S mutant is insensitive to higher agonist concentrations while the wild-type becomes increasingly rapid. Note that the wild-type data are also found in Figure 3-3A.

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66 Figure 4-2. Macroscopic currents evoked by ACh in oocytes expressing the T6'S mutant carry more net charge than wild-type currents. A) Currents from oocytes expressing either wild-type or the T6'S mutant. Each current represents an average from six different cells, all of which received mRNA injections on the same date, approximately 72 hours prior to recording. Oocytes from the same injection batches were used to obtain the binding data shown in table 4-1. B) The same responses as those shown in panel A, but indicating the cumulative charge for each receptor type illustrating the difference in total charge carried.

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67 Figure 4-3. Single channel currents recorded from GH4C1 cells expressing the T6'S mutant. Transiently transfected GH4C1 cells were studied using the cell-attached patch configuration. The representative trace shown above is from a cell with 30 M ACh in the patch pipette, at a holding potential of +50 mV (-50 mV relative to the cell interior, applied to the existing membrane potential). No currents were observed in untransfected control cells (n=6).

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68 Figure 4-4. The T6'S mutant has a slightly lower single-channel conductance compared to wild-type Single-channel I-Vs were generated and indicated a slope conductance of 61.7 5.8 pS, slightly lower than the conductance of 91.5 8.5 pS reported for the wild-type receptor (Mike et al., 2000). The data shown here are representative of those used to produce an average conductance value (n=5).

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69 Figure 4-5. T6'S single-channel open times are fit by two exponentials and indicate a prolonged average open time compared to wild-type A) Representative open time distribution for a 30 min. acquisition period in the sustained presence of 30 M ACh. B) The same cumulative charge distributions shown in Figure 4-1B, including a scaled version of the wild-type current. A scaling factor was applied that incorporates both the difference in mean channel open time, and the difference in single channel conductance between the T6'S mutant and the wild-type.

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70 Figure 4-6. T6'S mutant burst activity. A) Raw data traces recorded in the presence of 30 M ACh showing channel bursts. B) Closed time distribution for the T6'S mutant showing a fit by multiple exponentials. The existence of multiple closed times is an indicator of channel bursting. To identify bursts, the critical time threshold (t crit ) for identifying closures within bursts was determined using the method described by Colquhoun and Sakmann (1985).

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71 Figure 4-7. T6'S mutant burst durations and number of intraburst openings. A) The average burst duration distribution was best fit by two exponential components. Since wild-type receptors show little burst activity under steady state conditions, this represents a significant effect of the T6'S mutation on receptor kinetics. B) The number of bursts with intraburst openings greater than unity, indicating a significant probability of the mutant channel re-opening.

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72 Figure 4-8. Peak and area CRCs for wild-type and T6'S mutant nAChRs. A slight reduction in ACh potency (see Table 4-2) was observed for the T6'S mutant versus the wild-type when comparing net charge CRC analyses. The decreased discrepancy between peak and area analysis can be seen for the T6'S mutant, illustrating the reduction in concentration-dependent synchronization of channel activation. Each data point represents the mean normalized response ( s.e.m.) obtained from at least four oocytes.

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73 Figure 4-9. Selective agonists of the wild-type nAChR. Several selective agonists including choline (A), GTS-21 (B), AR-R17779 (C), and tropesitron (D)retain their agonist activity for the T6'S mutant. Concentration-response functions for the indicated drug for either wild-type or the T6'S mutant. Some agonists show potency and efficacy differences (see Table 4-2), but these are relatively moderate and consistent with the potency shifts seen with nonselective and nonselective agonists, including ACh. The wild-type rat CRCs for AR-R17779 and tropisetron are both used with permission from Papke et al., 2004 and Papke et al., 2005, respectively.

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74 Figure 4-10. Antagonists of wild-type are also antagonists of the T6'S mutant. Several known antagonists of the wild-type receptor have been reported to function as agonists of the L9'T mutant (see text). These same antagonists retain their inhibitory properties when applied to the T6'S mutant. Each mean and s.e.m. represents data obtained from at least four oocytes.

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75 Figure 4-11. The T6'S mutant shows minimal potentiation by 5HI. A) The allosteric potentiator, 5HI has a small effect on the T6'S mutant, but this is substantially lower than that observed for wild-type B) The T6'S mutant potentiaion was statistically different from control (p < 0.05) and from the potentiated wild-type (p < 0.0001), by t test. Each value represents the mean and s.e.m. for at least 4 oocytes.

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76 Figure 4-12. Simplified kinetic scheme for the nAChR. A Markovian kinetic model allowing for greater open probability with partial agonist occupancy for the wild-type receptor. Note the agonist concentration-dependency for both the forward binding and forward desensitization constants. The effect of the TM2 T6'S mutation may be to alter the rate constants moving in and out of desensitized states, or it may result in the conversion of a bound, closed state to an open one, or some combination of these effects.

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CHAPTER 5 GENERAL DISCUSSION The diversity of nicotinic receptors provides a rich and complex substrate for investigation, and their importance in a variety of physiological processes and pathological conditions makes their study that much more significant. Despite the fact that nAChRs are among the best characterized of the ligand-gated ion channels, we still suffer from significant gaps in our knowledge of exactly what their normal functions are, how they may be involved in a variety of disease processes, and how drugs that target nicotinic receptors may be used to treat these illnesses. An understanding of the structural underpinnings of the various properties which distinguish the different major subfamilies of nAChR will help to narrow some of these gaps. Both the number and variety of the effects of the T6'F mutation were quite remarkable. The effects ranged from those with possibly more obvious explanations (i.e., the loss of divalent ion permeability) to those which are likely to have more complex mechanisms (i.e., succinylcholine agonist effects). These wide ranging effects are taken to support the notion that amino acid sequence at this position plays a prominent role in the phenotypic qualities of the members of the major nAChR subgroups. The studies described here are aimed at helping to clarify some of the structural features that help to define and distinguish representatives of the major subfamilies of nAChRs. As always, there are limitations that should be considered when attempting to interpret these findings. The effect of both the T6'F and T6'S are interpreted as the result of single amino acid substitutions. While it is possible that the presence of the mutation 77

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78 in a single subunit is sufficient to produce some or all of the effects described here, all of the experiments were presumed to have been conducted with homopentameric mutant receptors. It may be possible for future experiments to address this issue by co-expression of the mutant subunits with wild-type. Other groups have conducted similar studies and shown intermediate effects of mutant receptors (Palma et al., 1997), so it may be that partial effects could be observed for the 6' mutants under similar conditions. Another potential limitation specifically related to the T6'F mutant, is the fact that it was never possible to express the channel in a mammalian cell. Although the Xenopus oocyte system is generally quite good in terms of its pharmacological fidelity when compared to mammalian expression systems, there are some qualities of the cells that are somewhat different, including the endogenous calcium-activated chloride currents (Miledi and Parker, 1984). However, given the apparent reduction in divalent ion permeability for the T6'F mutant, this is unlikely to factor into species differences in expression systems. It may also be possible for future studies with the T6'F mutant to explore the single-channel properties of the receptor, given the well-established methods for using this approach with oocyte membranes. There are some potentially interesting future studies that are implicated by the findings described here. One such avenue involves characterization of the effect of the T6'S mutation of toxicity/cytoprotection. It is known that activation of receptors within an optimal timing/concentration range is cytoprotective in vitro (Li et al., 1999). It would be intriguing to see the effect of prolonged activation of the T6'S mutant with what would normally be cytoprotective agonist concentrations. It is predicted that these signals would become cytotoxic in this case, either directly though excessive calcium

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79 influx through the mutant receptor, or via secondary activation of calcium pathways. In these cases one would predict that a significant shift in the potency of cytoprotective stimuli would occur, if cytoprotection via channel activation were possible at all. Furthermore, the T6'S mutant is likely to have continued usefulness as a screening tool for those working to identify agonists that are selective for nAChRs. In this case it may be very advantageous to develop a cell line that stably expresses the mutant receptor. The T6'S mutant receptor can become activated in the sustained presence of agonist (unlike wild-type ), yet reproduces wild-type pharmacology with great fidelity. Given the nature of high throughput drug screening methods and their relatively poor control of agonist solution switching, this would permit more a practical application of the unique qualities of the T6'S mutant to these kinds of drug development methods. These studies have helped to shed some mechanistic light on why members of different subfamilies of nicotinic receptors are functionally unique. Ideally, they will add to a background of information that can be used to further our understanding of the specific roles these receptors occupy in their respective physiological contexts. As the importance of these receptors in various disease process becomes more clear, this information will also aid in the creation of medicines designed to alleviate human suffering.

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87 Sands SB, Costa ACS and Patrick JW (1993) Barium permeability of neuronal nicotinic acetylcholine receptor alpha 7 expressed in Xenopus oocytes. Biophys. J. 65:2614-2621. Scremin OU and Jenden DJ (1991) Time-dependent changes in cerebral choline and acetylcholine induced by transient global ischemia in rats. Stroke 22:643-647. Seguela P, Wadiche J, Dinely-Miller K, Dani JA and Patrick JW (1993) Molecular cloning, functional properties and distribution of rat brain alpha 7: a nicotinic cation channel highly permeable to calcium. J. Neurosci. 13(2):596-604. Severance EG, Zhang H, Cruz Y, Pakhlevaniants S, Hadley SH, Amin J, Wecker L, Reed C and Cuevas J (2004) The alpha7 nicotinic acetylcholine receptor subunit existsin two isoforms that contribute to functional ligand-gated ion channels. Mol. Pharmacol. 66:420-429. Sherrington CS (1947) The integrative action of the nervous system. Cambridge University Press, Cambridge. Shimohama S and Kihara T (2001) Nicotinic receptor-mediated protection against beta-amyloid neurotoxicity. Biol. Psychiatry 49:233-239. Shytle RD, Mori T, Townsend K, Vendrame M, Sun N, Zeng J, Ehrhart J, Silver AA, Sanberg PR and Tan J (2004) Cholinergic modulation of microglial activation by alpha 7 nicotinic receptors. J. Neurochem. 89:337-343. Silver AA, Shytle RD, Philipp MK and Sanberg PR (1996) Case Study: Long-Term Potentiation of Neuroleptics with Transdermal Nicotine in Tourette's Syndrome. J. Am. Acad. Child Adolesc. Psychiatry 35:1631-1636. Skok VI (2002) Nicotinic acetylcholine receptors in autonomic ganglia. Auton. Neurosci. 97:1-11. Stevens TR, Krueger SR, Fitzsimonds RM and Picciotto MR (2003) Neuroprotection by nicotine in mouse primary cortical cultures involves activation of calcineurin and L-type calcium channel inactivation. J. Neurosci. 23:10093-10099. Swope SL, Moss SJ, Blackstone CD and Huganir RL (1992) Phosphorylation of ligand-gated ion channels: a possible mode of synaptic plasticity. Faseb. J. 6:2514-2523. Tonini R, Palma E, Miledi R and Eusebi F (2003) Properties of neuronal alpha7 mutant nicotinic acetylcholine receptors gated by bicuculline. Neuropharmacology 44:765-771. Unwin N (1989) The structure of ion channels in membranes of excitable cells. Neuron 3:665-676.

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88 Uteshev VV, Meyer EM and Papke RL (2003) Regulation of neuronal function by choline and 4OH-GTS-21 through alpha7 nicotinic receptors. J. Neurophysiol. 89:33-46. Vernino S, Rogers M, Radcliffe KA and Dani JA (1994) Quantitative measurement of calcium flux through muscle and neuronal nicotinic acetylcholine receptors. J. Neurosci. 14:5514-5524. Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S, Li JH, Yang H, Ulloa L, Al-Abed Y, Czura CJ and Tracey KJ (2003) Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature 421:384-388. Wang HY, Lee DH, D'Andrea MR, Peterson PA, Shank RP and Reitz AB (2000) beta-Amyloid (1-42) binds to a1pha7 nicotinic acetylcholine receptor with high affinity. Implications for Alzheimer's disease pathology. J. Biol. Chem. 275:5626-5632. Webster JC, Francis MM, Porter JK, Robinson G, Stokes C, Horenstein B and Papke RL (1999) Antagonist activities of mecamylamine and nicotine show reciprocal dependence on beta subunit sequence in the second transmembrane domain. Br. J. Pharmacol. 127:1337-1348. Whitehouse PJ, Price, D.L., Clark, A.W., Coyle, J.T., and Delong M. (1981) Alzheimer's disease: evidence for a selective loss of cholinergic neurons in the nucleus basalis. Ann. Neurol. 10: 122-126. Zwart R, De Filippi G, Broad LM, McPhie GI, Pearson KH, Baldwinson T and Sher E (2002) 5-Hydroxyindole potentiates human alpha 7 nicotinic receptor-mediated responses and enhances acetylcholine-induced glutamate release in cerebellar slices. Neuropharmacology 43:374-384.

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BIOGRAPHICAL SKETCH Andon Placzek was born January 3, 1971 in Sarnia, Ontario, Canada. He moved to Venice, Florida, where he attended school and eventually graduated from Venice High School in 1989. Four years later he returned to school at Manatee Community College in Bradenton, Florida, and later went on to the University of South Florida in Tampa where he eventually received a Bachelor of Arts degree in Psychology in 1998. It was here that he developed an interest in physiological psychology and began to seek out laboratory research positions that were increasingly oriented toward cellular and molecular neuroscience. After beginning graduate school at USF in the College of Medicine he eventually transferred to the University of Florida in Gainesville, and continued his studies in the Laboratory of Dr. Roger Papke where he was able to pursue his interest in ligand-gated ion channels. 89


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REGULATION OF ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR
FUNCTION AND PHARMACOLOGY BY AMINO ACID SEQUENCE IN THE
SECOND TRANSMEMBRANE DOMAIN















By

ANDON NICHOLAS PLACZEK


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Andon N. Placzek

































This dissertation is dedicated to my family.















ACKNOWLEDGMENTS

I thank my committee members for their support, encouragement, and

professionalism. I especially thank my mentor, Dr. Papke, for his availability and for

holding me to a standard of excellence. I also appreciate his patience with me when I

was not always focused on the task at hand.

I must also thank all of my family who believed in me, especially my wife Erin for

putting up with so much and inspiring me to be a better person. Thanks also go to my

daughter, Olivia for being a reason to strive for success. Finally, I thank God who has

been my faithful friend and without whom, nothing in my life would be possible.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES ............. ................... ........... .. .. ............... .. vii

LIST O F FIG U R E S ......................................................... ......... .. ............. viii

A B ST R A C T .......... ..... ...................................................................................... x

CHAPTER

1 GENERAL INTRODUCTION ...................................................... .....................

General Features of Ionotropic Receptors ........... ..............................................3
L igand-G ated Ion C hannels......................................... .......................................
T he nA C hR G ene F am ily ............................................................. ....................... 6
Subfam ilies of nA C hR ...................................................................................... .7
Unique Features of the a7 nAChR ..................... ................... ...............7
Modes of a7 nAChR Activation and Signaling ........................ ............ ......9
nAChRs in Human Disease .................. ... ......................... 11
The Structure of the nA ChR ............................................... ... ............. .. ............... 14
The TM2 Domain and the Ion Channel Pore ............................................... 15
Structure-Function Studies in the nAChR TM2 Domain..............................16

2 M E T H O D S ......................................................... ................ 2 0

The cDNA Clones.................... ............... ...................20
Site-directed M utagenesis................................................. .............................. 20
P reparation of R N A .................................................................. .................... .... ..20
Expression in X enopus O ocytes.............................................. ............... ... 21
Voltage-clamp Recording of Whole-oocyte Responses..........................................21
Experimental Protocols and Analysis of Data Obtained from Xenopus Oocytes.......22
Transfection and Patch-clamp Recording from GH4C 1 cells .............. .....................24
R adioligand B finding Studies ........................................................... .....................24
Intact Oocyte Binding....... ...... ....... ............ ......... ............. .. ............ 25







v









3 THE PHARMACOLOGICAL AND KINETIC EFFECTS OF THE a7 TM2 T6'F
M U T A T IO N ............. ............. .... ................ .............................. 2 6

In tro d u ctio n ............. ...... ...... ... ................. ................................ 2 6
R results ................ ......... ......... ...................... ..... ..27
The TM2 T6'F Mutant is Functionally Expressed in Xenopus Oocytes .............27
The a7 TM2 T6'F Mutation Dramatically Slows the Apparent Kinetics of
A Ch-evoked M acroscopic Currents .................... .... ................ .............. 29
The a7 T6'F Mutation Increases ACh Potency Compared to Wild-type a7 ........30
The a7 T6'F Mutation Abolishes Barium Permeability and Reduces Inward
C current R ectification .................. ............... ... ........ ...... .. .......... .. ...3 1
Succinylcholine is a Selective Agonist of Muscle-type Receptors, and is also
an Agonist of a7 T6'F Mutant Receptors ........................ ..................32
The TM2 T6'F Mutation Abolishes Potentiation by 5-Hydroxyindole...............32
D iscu ssio n .........................................................................................3 3

4 THE a7 TM2 T6'S MUTATION: A GAIN-OF-FUNCTION WITH a7-LIKE
P H A R M A C O L O G Y .......................................................... ...................................47

In tro du ctio n ....................................... ................... ............................ 4 7
R results .................... .... .. ..................... ....... ......... ................. 49
T6'S M utant Kinetics and Single-channel Properties ..............................................49
The TM2 T6'S Mutation Slows the Macroscopic Kinetics of ACh-evoked
Currents and Increases the Overall Net Charge Carried Upon Activation ......49
The T6'S Mutation Does not Increase a7 Single-channel Conductance..............50
The T6'S Mutation Produces a Significant Increase in Average Channel Open
T im e ............ ......... .... ................... ................ ............... 5 1
T6'S M utant Burst A activity ........................................ ........................... 52
The Pharmacology Of The T6'S M utant...................................... ......... ............... 52
The T6'S Mutant Shows a Slight Decrease in Ach Potency and a Decreased
Discrepancy Between Area and Peak CRCs................ .............................. 53
The Agonist Selectivity Profile of the T6'S Mutant is Similar to Wild-Type
a 7 ................................. ... ... ............ ...... ... .. ................. 5 3
Antagonists of the Wild-Type Receptor are also Antagonists of the T6'S
M utant ............. ............ ..... ...... ...... .... ............. ............. .... ....... 54
5-Hydroxyindole Potentiation is Significantly Diminished in the T6'S Mutant
Com pared to W ild-Type 7...................................... .......................... 54
Discussion ............................ ...... ... ........................... 55
Kinetic Effects of the T6'S Mutation................................................55
Pharmacological effects of the T6'S mutation................................. ...... ............ ... 58

5 G EN ER AL D ISCU SSION ......... ................. .........................................................77

LIST OF REFEREN CE S .. ....... ................................ ........................... ............... 80

B IO G R A PH IC A L SK E TCH ..................................................................... ..................89
















LIST OF TABLES


Table page

1-1. Major subfamilies of nicotinic acetylcholine receptors........................ ...............19

3-1. Intact oocyte [125I]a-Btx binding. ......................... ......... ................................ 38

3-2. Curve-fit values for wild-type and T6'F mutant a7 responses to ACh....................39

4-1. Intact oocyte [3H] M LA binding. ........................................ .......................... 63

4-2. Agonist profile comparison for wild-type a7 and the T6'S mutant...........................64
















LIST OF FIGURES


Figure page

3-1. W ild-type and mutant a7 nAChRs .............................. .... ............................... 40

3-2. Electrophysiological responses from wild-type and mutant a7 nAChRs ................41

3-3. Macroscopic response kinetics of wild-type a7, muscle, and the TM2 T6'F mutant
nA ChR s expressed in oocytes. ........................................................................... 42

3-4. ACh concentration-response functions for wild-type and the TM2 T6'F mutant
a7 nA C hR ...........................................................................43

3-5. Wild-type and mutant a7 nAChR current-voltage relationships and divalent ion
perm ability in Xenopus oocytes........................................ .......................... 44

3-6. Wild-type and mutant nAChR responses to the muscle receptor selective agonist
succinylcholine............................................................................................ 45

3-7. Wild-type and mutant nAChR potentiation by 5-Hydroxyindole ...........................46

4-1. The time course and concentration-dependence of a7 nAChR macroscopic
kinetics are altered by the T6'S mutation. ..................................... ............... 65

4-2. Macroscopic currents evoked by ACh in oocytes expressing the T6'S mutant
carry more net charge than wild-type a7 currents. ................................................66

4-3. Single channel currents recorded from GH4C1 cells expressing the T6'S mutant....67

4-4. The T6'S mutant has a slightly lower single-channel conductance compared to
w ild -ty p e a 7........................................................................... 6 8

4-5. T6'S single-channel open times are fit by two exponentials and indicate a
prolonged average open time compared to wild-type 7. ......................................69

4-6. T 6'S m utant burst activity ............................................................... .....................70

4-7. T6'S mutant burst durations and number of intraburst openings.............................71

4-8. Peak and area CRCs for wild-type and T6'S mutant nAChRs. ................................72

4-9. Selective agonists of the wild-type a7 nAChR ........... .... ............... ................... 73









4-10. Antagonists of wild-type a7 are also antagonists of the T6'S mutant....................74

4-11. The T6'S mutant shows minimal potentiation by 5HI.........................................75

4-12. Simplified kinetic scheme for the a7 nAChR.................. ................ ............... 76















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

REGULATION OF ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR
FUNCTION AND PHARMACOLOGY BY AMINO ACID SEQUENCE IN THE
SECOND TRANSMEMBRANE DOMAIN

By

Andon Nicholas Placzek

May, 2005

Chair: Roger L. Papke
Major Department: Pharmacology and Therapeutics

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion

channels with clearly defined physiological roles at the neuromuscular junction and in

peripheral ganglia, and more mysterious roles in the mammalian brain, and non-neuronal

tissues. The larger family of nicotinic receptors can be broadly categorized into three

major subgroups based on subunit composition, anatomical distribution, and functional

and pharmacological differences. These are the muscle-type receptors, the heteromeric

neuronal receptors, and the homomeric receptors, typified by receptors composed of the

a7 subunit.

Our studies demonstrate that a conferring of a functional phenotype can be

accomplished by systematic substitution of amino acid sequence from the beta subunits

of either muscle-type (pl) or neuronal (p2/p4) nAChRs into homomeric receptors

composed of mutant a7 nAChR subunits. Specifically, the a7 TM2 T6'F mutant shows

properties similar to the muscle-type nAChR with regard to divalent ion permeability,









current rectification, agonist concentration-dependent kinetics, sensitivity to

succinycholine, and a lack of potentiation by 5-hydroxyindole. While a variety of muscle

receptor-like properties are observed in the T6'F mutant, the a7 TM2 T6'S mutant, which

has amino acid sequence identical to the neuronal p2/p4 subunit at this position,

demonstrates significant kinetic similarities to neuronal nAChRs, but largely retains the

pharmacology of the wild-type a7 receptor. At the single-channel level, the T6'S mutant

has a unitary conductance similar to that reported for wild-type a7, but a vastly longer

average open duration. Furthermore, channel burst activity indicates a significantly

greater likelihood of channel opening in the sustained presence of agonist relative to

wild-type.

The significant impact of these TM2 6' substitutions on a variety of functional

aspects of the mutant a7 receptors suggests that amino acid sequence at this position

contributes to several important features that distinguish the major nAChR subgroups

from one another. Furthermore, the a7 TM2 T6'S mutant shows a kinetic gain of function

in the absence of significant pharmacological differences from the wild-type a7 receptor.

Thus it may provide the ability to observe agonist-evoked signals using contemporary

high-throughput drug screening methods (where the wild-type receptor would be

inhibited), implicating it as a potential tool for identifying a7-selective compounds.














CHAPTER 1
GENERAL INTRODUCTION

The discovery of chemical neurotransmission as a major form of intercellular

communication in the nervous system was an essential achievement allowing several

scientific disciplines to progress, and ultimately setting the stage for important insights

into both normal and abnormal human physiology. Santiago Ramon y Cajal (1911) first

put forth the idea that the nervous system was composed of contiguous individual units

rather than a continuum of tissue, and the term "synapse" was first introduced by Charles

Sherrington (1947) who postulated that communication between cells in the nervous

system had a chemical nature. The first real evidence of chemical release due to nerve

stimulation comes from the work of Otto Loewi (1957), who is credited with the first

discovery of the neurotransmitter substance, acetylcholine (ACh). Through his

pioneering work with nicotine and curare, Langley (1905) provided evidence for a

"receptive substance" that formed a molecular target for chemical substances on the cell

surface. The identification and characterization of the molecules that were the recipients

of these chemical signals provided the opportunity to gain an even greater understanding

of the means by which neurons (or other cellular targets, such as muscle cells) gathered

and processed transmitted information.

Our current concept of how synapses function has improved upon these early ideas

and we now have a much better picture of the organization and mechanisms of synaptic

activity, although the picture is by no means complete. Despite the fact that most of the

major neurotransmitter systems have been identified and mapped, we are still in the









process of determining the physiological roles for many of these systems. Dysfunction

within these systems in a variety of diseases that affect the nervous system lends further

significance to these areas of investigation. As always, new discoveries continue to force

us to reconsider our notions of how certain receptors function, or even how some families

of receptors that were originally identified as important synaptic components, may in fact

have important roles outside of those classically associated with the chemical synapse.

There are two major classes of synaptic receptor molecules: metabotropic receptors

and ionotropic receptors. Metabotropic receptors are coupled to intracellular G-proteins

and are involved in a variety of intracellular signaling pathways, depending on the

specific G-protein the receptor is associated with. These receptors typically have a seven

transmembrane domain structure and show a high degree of diversity in signaling that is

regulated by the specific G-protein complexes and the downstream targets that these

receptors are associated with. The other major class of receptors is the ionotropic

receptor, so called because its method of signal transduction comes in the form of

permitting the flow of charged particles across the cell membrane. These receptors are

gated by a variety of stimuli, including mechanical stimuli, chemical ligands, and

changes in membrane voltage. Unlike transporter proteins which use active transport

mechanisms or require coupling to existing ionic gradients to accomplish ion transport

across the membrane, ionotropic receptors passively permit ion flux by exploiting the

existing driving force on those ions.

There are a great variety of known physiological roles for ionotropic receptors.

The sensations of touch, hearing and proprioception are mediated by ionotropic receptors

that respond to mechanical stimuli. Receptors that are controlled by changes in electrical









potential across the cell membrane are known as voltage-gated ion channels and are

necessary for the conduction of the action potential, and also for the coupling of cellular

excitation to secretion or contraction. The ionotropic receptors that respond to chemical

signals are involved in a diversity of physiological processes, and are primarily thought to

have evolved as participants in synaptic transmission.

General Features of Ionotropic Receptors

Ionotropic receptors are multimeric protein complexes composed of individual

membrane-spanning subunits that, when assembled, create an aqueous pore through the

cell membrane. These receptors permit the flow of charged particles through this central

pore when activated, and depending on the specific ions conducted, the effect of

activation can be limited to changes in membrane voltage, resting conductance, or may

also result in second messenger signaling events (particularly when channel activation

leads to changes in intracellular calcium concentration).

Important concepts related to ion channel function can be described using

fundamental principles of electricity. The most fundamental of these is Ohm's Law,

E= IR (1-1)

where E represents voltage, I is current, and R is resistance. When applying this equation

to a cell, the cell membrane functions as a capacitor. It is across this capacitor that an

electrochemical potential exists. Ion channels permit current to flow across the cell

membrane by entering the activated, open state, and when they are closed, resistance

across the cell membrane approaches infinity.

Using an extension of this fundamental equation, the driving force on a particular

ion can be predicted. The Nernst equation can be used to determine the membrane

potential at which there is no net flow of ions, also known as the equilibrium potential:









RT [S]2
Es= R n [S] (1-2)
F [S],

Here, S is the ionic species in question, Es is the equilibrium potential for that ionic

species, R is the ideal gas constant, T is temperature, zs is the ionic valence, and F is the

Faraday constant. Through both active transport and passive processes an ionic gradient

that serves as a source of both an electrical potential difference and a concentration

gradient for specific ions. Once the equilibrium potential has been determined, the

driving force on a particular ion is then simply the difference between this value and the

membrane potential, or Em Eeq.

The cell then makes use of this driving force with a variety of ion channels that

display varying degrees of ionic selectivity. Incorporating these ion channels into the

framework, Ohm's law can be further extended to give an electrical representation of a

class of channels in a non-excitable cell with the formula:

I = (EM ER) NPOY (1-3)

where I represents current, EM represents the voltage across the cell membrane, ER is the

reversal potential, N is the number of channels present, Po is the probability of channel

opening, and y represents the unitary conductance for that particular channel.

One of the most significant technical advances in the field of physiology that

permitted researchers to study ion channels in greater detail was development of the

voltage-clamp method (Cole, 1949; Marmont, 1949; Hodgkin and Huxley, 1952). This

permits the investigator to hold the cell's membrane potential constant while measuring

the amount of current necessary to do so, giving a direct indication of the amount of

current flow through ion channels at a defined membrane voltage.









Ligand-Gated Ion Channels

As previously mentioned, ionotropic receptors can be activated by different classes

of stimuli, including mechanical, electrical, and chemical. Those which respond to

chemical signals are referred to as ligand-gated ion channels (LGICs). These types of

channels are known to function in at least three different modalities. The first involves

rapid activation at a postsynaptic structure in response to a relatively high concentration

of quantally released agonist (approaching approximately 0.5 1 mM (Salpeter, 1987)).

This mechanism is typified by the receptors expressed at the vertebrate neuromuscular

junction. In the second modality, some LGICs can act as local modulators of synaptic

function. An example of this comes in the form of receptors that are expressed

presynaptically, and through their activation and subsequent calcium signal, facilitate the

release of neurotransmitter. The third modality involves paracrine-like, volume

transmission, where ligands originate from sources that are relatively distant from their

target and bind to the LGIC, activating (and in some cases desensitizing) the receptor

(Descarries, 1997). There is also the possibility that these receptors are fulfilling

important physiological roles that are not neatly encapsulated within these three

modalities.

Much of what we now know and hypothesize about ligand-gated ion channels has

been greatly influenced by the study of acetylcholine receptors (AChRs), largely because

their importance in the motor systems of vertebrates provides a readily accessible

experimental system (Fatt and Katz, 1951; Katz, 1966). Acetylcholine receptors consist

of two major subtypes. Those which are activated by muscarine and are coupled to

intracellular G-proteins are referred to as muscarinic receptors (mAChRs). The other

major subtype is activated by the plant alkaloid nicotine, and functions as a ligand-gated









ion channel. These are known as nicotinic acetylcholine receptors (nAChRs). Nicotinic

receptors are widely expressed both in the peripheral (Skok, 2002) and central (Dani,

2001) nervous systems of mammals, and are well known to be the postsynaptic target in

neuromuscular transmission (Katz, 1966)

The nAChR Gene Family

The family of nAChR subunit genes consists of seventeen members, and is a

member of the same ion channel superfamily as glycine receptors, 5-HT3 receptors, and

ionotropic GABA receptors (Lester et al., 2004). The nAChRs expressed on the

electroplax organ of the electric ray, Torpedo Californica were the first to be cloned

using the techniques of modern molecular biology (Noda et al., 1982). This particular

receptor has been instrumental in the development of models of the three dimensional

structure of the nAChR, using high-resolution electron microscopy (Unwin, 1989;

Miyazawa et al., 2003). The Torpedo-type receptors are close structural homologues of

the nAChRs of the mammalian neuromuscular junction, which are now known to be

composed of al, pi, 6, and y or e (depending on developmental stage (Brisson and Unwin,

1985) subunits in a 2:1:1:1 ratio, with ligand-binding occurring at the a-6 and a-y (or a-e)

subunit interfaces. The neuronal nicotinic receptors can be divided into heteromeric and

homomeric subtypes. The heteromeric subtypes are composed of neuronal alpha (a2-a6)

and beta (p2-p4) subunits and bind nicotine with high affinity (Lindstrom et al., 1995).

Homomeric nAChRs are composed of alpha subunits (a7-al0) and the predominant

mammalian subtype, a7, is inhibited by the snake toxin, a-bungarotoxin (a-Btx,

McGehee and Role, 1995). Although these are generally referred to as neuronal

nAChRs, recent studies show that they are also expressed in non-neuronal tissues

including microglia (Shytle et al., 2004) and peripheral macrophages (Wang et al., 2003).









Subfamilies of nAChR

The hypothetical evolutionary history of nicotinic receptor genes has given rise to

an organizational framework based on phylogeny (Le Novere et al., 2003). Using this

framework, several nAChR subfamilies can be identified, and these differences give a

good representation of many of the known physiological differences these receptors

exhibit, as well as differences in their patterns of tissue distribution. Previous studies

have demonstrated discrete patterns of nAChR subunit expression within the mammalian

brain (Clarke et al., 1985) with high levels of the a7 subtype being expressed in rat

hippocampus.

Unique Features of the a7 nAChR

Receptors containing the a7 nAChR subunit have been studied extensively since

their discovery in a variety of experimental preparations. Although the majority of in

vitro experimental systems focus on what are presumably homomeric a7 receptors, there

has been some suggestion that a7 subunits can co-assemble with non-a7 nAChR subunits

in vivo (Khiroug et al., 2002). Furthermore, an a7 splice variant with unusual functional

properties has been identified in rodent cardiac ganglion (Severance et al., 2004),

suggesting the possibility of splice variants in humans that may have different

characteristics from a7 receptors studied in vitro. Another potentially complicating

discovery is the existence of an a7 partial gene duplication in humans (Gault et al., 1998).

This also suggests the possibility of co-assembly of wild-type a7 subunits with a different

gene product that may have functional consequences. Despite these potential

complications, there has generally been good agreement between data generated using the

a7 nAChRs reconstituted in heterologous expression systems (e.g., Xenopus oocytes) and

the native receptors.









One particularly striking feature that distinguishes wild-type a7 nAChRs from other

nAChRs, and indeed from many other ligand-gated ion channels, is the degree to which

the receptor's macroscopic kinetics are impacted by agonist concentration (Papke and

Thinschmidt, 1998). That is, application of higher agonist concentrations produce

responses that reach their maximum, and decay to baseline far more rapidly than those

produced by relatively lower agonist concentrations (see Figure 3-2A). In the case of a7,

this phenomenon is especially pronounced and it is evident that the rate-limiting process

is always agonist application speed.

Another potentially important feature of a7 nAChRs is their high degree of calcium

permeability. The Ca2+:Na+ permeability ratio for a7 nAChRs has been reported to be

approximately 10 (Sands and Barish, 1991) and has even been claimed to be as high as

20 or more (Role and Berg, 1996). This high level of permeability has significant

implications for cell signaling. Several laboratories have shown that activation of a7

nAChRs can be cytoprotective (Jonnala et al., 2003; Martin et al., 1994; Stevens et al.,

2003). However, an optimal range of concentrations of a7-selective agonist corresponds

to enhanced neurite survival in differentiated PC12 cells and this same range of

concentrations produces an activation of protein kinase C (PKC) with a similar bell-

shaped curve (Li et al., 1999). Furthermore, high concentrations of the a7-selective

agonist GTS-21 rapidly delivered to PC12 cells produce significant cytotoxicity, whereas

gradual exposure to the same concentration of GTS-21 through a gel slab drug delivery

system is not (Papke et al., 2000a).

In addition to directly mediating calcium influx, a7 nAChRs have been shown to

induce secondary changes in intracellular calcium concentration. Activation of a7-type









receptors under non-voltage clamped conditions causes a transient depolarization and

subsequent calcium influx through voltage-gated calcium channels expressed at the cell

surface (Quik et al., 1997). Also, an increase in intracellular calcium itself can cause

release of calcium from membrane-delimited stores via calcium-induced calcium release.

Recent data suggests that this type of calcium-induced calcium release can be evoked by

activating a7 based on the ability of the IP3 receptor blocker, xestospongin C to partially

inhibit calcium signals evoked by a7 agonist (Dajas-Bailador et al., 2002). The current

data also suggest that a significant release of calcium from intracellular stores requires

the intermediate step of voltage-gated calcium channel activation. It is unclear at this

point which of these mechanisms is of primary significance for promoting cell survival

during a7 activation, or if all of the aforementioned sources of calcium are required.

These ancillary mechanisms of increasing intracellular calcium concentrations raise the

interesting possibility that a cell can tune the magnitude (and ultimately the effect) of

calcium-dependent processes elicited by a7 activation by regulating expression of

proteins involved in these secondary pathways.

Modes of a7 nAChR Activation and Signaling

The physiological significance of nicotinic receptors expressed at the

neuromuscular junction has been understood for decades, beginning with the early work

of Fatt and Katz (1951) who described quantal postsynaptic responses at the motor end-

plate. In the case of both the nAChRs of the neuromuscular junction, and those

expressed in peripheral ganglia, fast synaptic transmission is the predominant functional

role. Acetycholine is synthesized, packaged, and released in a quantal fashion by

presynaptic cholinergic neurons, after which it traverses the synapse and binds to









postsynaptic nAChRs, causing them to open, thus depolarizing the postsynaptic

membrane.

One feature of the a7-type nAChR that lends itself to signaling via volume

transmission is its activation by the endogenous ligand, choline (Papke et al., 1996).

Choline is a breakdown product of acetylcholinesterase (AChE) activity, and ambient

levels of choline near cells that synthesize and release acetylcholine can by dynamically

regulated by the activity of AChE and uptake by choline transport proteins. In

hypothalamic tuberomammilary neurons that express high levels of a7 nAChR,

spontaneous activity can be modulated by bath-applied choline (Uteshev et al., 2003.),

suggesting that even subtle variations in ambient choline levels may have an impact on

the activity of a major source of brain histamine. Choline levels are also reported to

change under pathophysiological conditions, such as ischemia or brain injury, where

concentrations are elevated above normal (Scremin and Jenden, 1991). This may suggest

a possible role for a7-type receptors in natural cytoprotective mechanisms.

While there is some evidence for classical fast synaptic transmission mediated by

a7 receptors (Frazier et al., 1998; Hatton and Yang, 2002), this type of signaling modality

has historically been either very difficult to demonstrate, or confined to specific regions

of the nervous system. Because receptor localization studies have shown both

widespread a7 nAChR expression in mammalian brain (Clarke et al., 1985), and that this

expression appears to be primarily extrasynaptic or perisynaptic (Fabian-Fine et al.,

2001), it is intriguing to speculate about an alternative role for a7 nAChRs that does not

strictly fit the mold of fast synaptic transmission, typically associated with ligand-gated

ion channel function.










nAChRs in Human Disease

In recent years neuronal nicotinic receptors have emerged as potential therapeutic

targets in a variety of CNS disorders including Alzheimer's disease (AD, O'Neill et

al.,2002; Shimohama and Kihara., 2001) schizophrenia (Freedman et al., 2000), and

Tourette's syndrome(Mihailescu and Drucker-Colin, 2000). It remains unclear in each of

these cases the exact mechanisms by which modulators of nicotinic receptor function

exert their therapeutic effects. One possible reason for this is the paradoxical

pharmacology of nicotinic agonists. For example, while transdermal nicotine has been

used to augment neuroleptic treatment of Tourette's syndrome (Silver et al., 1996),

nicotine itself has been shown to demonstrate a mixed agonist/antagonist pharmacology,

and it is uncertain which of these pharmacological properties are essential in determining

therapeutic efficacy.

The human brain cholinergic system has been known for decades to have a role in

the progression of Alzheimer's disease. The interest in this pathway came from early

pathological studies of postmortem AD brain tissue. These findings suggested an early

degeneration of the primary centers of cholinergic projections in the basal forebrain

(Whitehouse, 1981) that correlated with memory loss. The so-called "cholinergic

hypothesis" of AD (Bartus et al., 1982) gave rise to medications which, until recently

were the only medications approved by the federal government to treat this disease.

These drugs inhibit acetylcholinesterases (AChE) and have been shown to provide

modest cognitive improvement for patients with mild to moderate AD (Robbins et al.,

1997). The basic mechanism underlying the therapeutic effects of these drugs is an

increase in the availability of ACh through the prevention of its breakdown by AChE,









presumably compensating for less available ACh due to cholinergic cell loss or

dysfunction. Unfortunately, most studies show that the relief these drugs provide is

primarily symptomatic (Barner and Gray, 1998), and the disease continues to progress

until the drugs are no longer effective.

Despite the fact that the cholinergic hypothesis has not led us to treatments that or

halt or significantly slow the progression of AD, it has led to some interesting studies

using animal models that help to clarify the role that the cholinergic system may play in

cognition. For example, several studies using lesions of the cholinergic projections from

the basal forebrain have shown that interruption of this pathway significantly impairs

memory related behaviors in a variety of tasks (McGaughy et al., 2000) and that

administration of cholinergic agents can help to correct some of these deficits. In fact,

cholinergic agents have long been known to enhance cognitive performance in the

absence of any impairment in both animals and humans (Robbins et al., 1997), although

the mechanism of these improvements in either lesioned or normal subjects remains

unknown.

Even though the cholinergic hypothesis has fallen out of favor among many

researchers in the field of Alzheimer's disease, evidence for a potential involvement of

cholinergic signaling continues to emerge. Studies of postmortem brain tissue from AD

patients also show that a7 subunits co-localize with the congophilic senile plaques that

are a pathological hallmark of AD (Nagele et al., 2002). If it is true that Ap can inhibit a7

signaling in vivo, then the potential for inhibition of an endogenous cytoprotective

mechanism exists that may contribute to the Alzheimer's disease process.









The recent findings that show a high-affinity (Wang et al., 2000) functional

interaction (Liu et al., 2001; Petit et al., 2001) between the a7 nAChR subtype and the

Alzheimer's p-amyloid (Ap) peptide are interesting for several reasons. Beta-amyloid is

the principle component of the senile plaques found in the postmortem brain tissue of

patients diagnosed with AD, and is recognized as a potentially pathogenic molecule when

its levels are abnormally high (Hardy and Selkoe, 2002). The high affinity of the binding

(in the picomolar concentration range) and the high potency of the functional inhibition

of a7 nAChR activity (in the nanomolar concentration range) suggests that interaction

between these two molecules may occur early in the disease process, since it is the higher

(micromolar) concentrations of Ap peptide that promote the rapid aggregation and cell

death that are primarily associated with the later stages of the disease (Hardy and Selkoe,

2002). Another potentially interesting aspect of this relationship between Ap and a7 is

the fact that there is little evidence for a major role for a7 nAChRs in fast synaptic

transmission. This suggests that any functional interaction between Ap and a7 may be

occurring outside the realm of classical postsynaptic signaling. This may not only point

to how a7 may be functioning normally in the mammalian brain, but how this function

may be disrupted in AD, and how this interference in normal activity may be prevented.

Another potential role for a7 nAChR in disease is suggested by recent evidence

showing an important contribution by a7 signaling to the inhibition of peripheral

inflammation. This signifies a7 as a potential therapeutic in the treatment of septic shock

(Wang et al., 2003) and possibly other disorders that involve peripheral inflammation.

This finding may also point to role for a7 receptors in modulating central inflammatory

processes. Chronic inflammation has been of interest to researchers in AD for several









reasons. In both AD patients and animal models of AD there are reports of an

upregulation of several markers on inflammation and activated microglia (McGeer and

McGeer, 2001). Furthermore, in vitro and in vivo studies show that the Ap peptide is

able to induce microglial activation and the subsequent release of pro-inflammatory

cytokines that are potentially detrimental to surrounding neurons (Meda et al., 1995).

Taken together with the evidence for anti-inflammatory function of a7 nAChRs in

peripheral monocytes, this suggests the possibility that a central anti-inflammatory action

of a7 may be impaired in AD, possibly through chronic inhibition by the Ap peptide.

The other major illness that a7 receptors have been implicated in is schizophrenia.

It has been known for some time that individuals diagnosed with schizophrenia are

significantly more likely to smoke than non-schizophrenics (Adler et al., 1998). This has

been interpreted by some as self-medication, and has stimulated interest in the study of a

possible mechanistic role for nicotinic receptors in schizophrenia. Where a7-type

nAChRs have been implicated is in the phenomenon of auditory gating. Studies using

transcranial recording methods have shown that schizophrenics have an impairment of

auditory gating, as evidenced by an increase in the so-called P50 latency (Leonard et al.,

1996). The implications for symptoms of psychosis are in an inability of the patient to

sufficiently filter external auditory stimuli, resulting in hallucinations that result from the

brain's attempt to integrate the unfiltered stimuli.

The Structure of the nAChR

Nicotinic receptors are believed to have a pentameric quaternary structure and

demonstrate a high degree of functional diversity based on variations in subunit

composition (Papke, 1993; Le Novere et al., 2002). All individual nAChR subunits have

a similar secondary structure and transmembrane topology (Lindstrom, 2000) with a large









extracellular N-terminal domain contributing to ligand-binding, followed by three

hydrophobic membrane-spanning regions, a large intracellular loop, a fourth

transmembrane region, and finally a relatively short extracellular C-terminus. The

intracellular loop has been shown to have several phosphorylation sites in heteromeric

(Downing & Role, 1987; Swope et al., 1992) and homomeric a7 receptors (Moss et al.,

1996), although the functional significance of a7 subunit phosphorylation remains

unclear.

The TM2 Domain and the Ion Channel Pore

The second transmembrane (TM2) domain of the nicotinic receptor subunit is

generally considered to be the pore-forming region of the nAChR. Early studies using

chimeric subunits constructed from Torpedo electric organ nAChRs and calf muscle-type

receptors demonstrated that the amino acid sequence proximal to and including the delta

subunit TM2 domain was responsible for differences in single channel conductance

(Imoto et al., 1986). Furthermore, rings of charged residues that border the TM2 domain

of Torpedo nAChR subunits have been shown to regulate cation permeation and the

inwardly rectifying voltage-dependent reduction in current produced by the divalent

cation, Mg2+ (Imoto et al., 1988).

Pharmacological evidence in the form of photoaffinity labeling studies shows that

the open-channel blocker, chlorpromazine, binds to the TM2 domain of every subunit of

the Torpedo nAChR, indicating that this portion of each subunit contributes to the pore-

lining region of the receptor (Oswald and Changeux, 1981; Revah et al., 1990).

Mutations in the TM2 domain have also been shown to regulate voltage-dependent

channel block by local anesthetics, such as the lidocaine derivative, QX-222 (Leonard et

al., 1988; Charnet et al., 1990). These same studies indicated specific amino acids that









were responsible for binding to QX-222, showing their likely position within the ion

channel pore-lining region. It was the 6' and 10' residues (according to the numbering

system proposed by Miller (1989)) that were shown to be directly responsible for

interaction with, and blockade by QX-222 (Charnet et al., 1990). These positions within

the TM2 domain are in direct proximity to one another based on the periodicity of the

amino acid sequence of the alpha helical TM2 domain (see Figure 3-1).

Structure-Function Studies in the nAChR TM2 Domain

In addition to the well-established relationship between TM2 domain amino acid

sequence and both channel conductance and interaction with drugs that target the pore,

mutations in the TM2 domain have been shown to affect nAChR kinetics. The initial

observation was made with a mutation of amino acid 247 in the chick brain a7 nAChR

(Revah et al., 1991). The substitution of a threonine residue for a leucine (L247T or

L9'T) produced a significant slowing of the response kinetics under macroscopic voltage

clamp conditions, an effect that was interpreted as a change in receptor desensitization.

In addition to this effect on response kinetics, subsequent studies of this same mutation

demonstrated an effect on several aspects of the mutant receptor's pharmacology.

Several antagonists of the wild-type receptor were found to be agonists of the L9'T

mutant, suggesting that mutation converted what was a liganded, closed state in the wild-

type a7 nAChR, into a liganded opened state (Bertrand et al., 1992; Fucile et al., 2002;

Palma, 1996; Palma et al., 1999). These effects on receptor pharmacology suggested that

this mutation could impact the relationship between ligand-binding and channel gating

and/or processes that resemble classical desensitization.

Other studies with non-a7 type nAChRs also indicate the importance of amino acid

sequence in the TM2 domain as a determinant of receptor pharmacology. Heteromeric









a3p4 receptors in which a chimeric p4 subunit with the pi subunit sequence at the TM2

domain show a reduced potency of blockade by mecamylamine when compared to wild-

type a3p4 receptors expressed in Xenopus oocytes (Webster et al., 1999). This same

reduction in inhibitory potency was accompanied by a faster recovery from block by both

mecamylamine and nicotine. Substitution of the TM2 6' phenylalanine of the muscle p1

subunit for the neuronal p4 subunit serine residue increased sensitivity to nicotine. The

same study showed that these effects could be more specifically attributed to amino acid

sequence at both the 6' and 10' positions of the TM2 domain (see Figure 3-1). Similar to

the findings with the L9'T mutant a7 nAChR, these studies suggested that TM2 domain

amino acid sequence could have a significant degree of control over receptor

pharmacology for drugs that don't necessarily exert their effects by directly interacting

with the pore-forming region of the receptor. Furthermore, the effects of substituting

specific amino acids from subunits of members one major subfamily of nAChR to

another, and the subsequent change in pharmacology suggested that pharmacological

phenotype could be conferred from one major receptor subtype to another, simply by

substituting important amino acids in the TM2 domain.

Given the known differences between the three major subfamilies of nAChR (i.e.,

muscle type, neuronal high-affinity nicotine binding, and homomeric a7) it is useful to

identify structural elements of the receptor complex that functionally distinguish the

members of each group from those of the others. This may provide essential clues to the

normal roles for those neuronal receptors that are less clearly understood, and how these

receptors may be exploited in the design of therapeutic compounds. At the core of all

receptor-based therapeutics is the need to understand how agonist binding is coupled to









receptor activation, and how that process can be perturbed by exogenous drugs. These

processes differ significantly among the three major subgroups of nAChR, and the data

presented here support the hypothesis that the key to understanding these differences can

be found by studying specific sequence in the respective pore forming domains of these

receptors. By determining how amino acid sequence in the pore-forming domain

regulates a multitude of processes, including the efficacy of specific agonists, selective

permeation of divalent cations, and receptor kinetics, we may gain insights into how

agonists and both competitive and noncompetitive antagonists may be developed that will

be selective for functionally distinct receptor subtypes.










Table 1-1. Major subfamilies of nicotinic acetylcholine receptors.


Non-competitive
antagonists

Ca2+:Na+
permeability ratio
Inward rectification
Selective agonists





Competitive
antagonists
Macroscopic
kinetics


a-Btx sensitive


BTMPS (intermediate
sensitivity)

> 10

Rectifying
GTS-21,
40H-GTS-21,
Choline,
AR-R17779,
Tropisetron
a-Btx,
MLA
Rapidly desensitizing,
Fast time-to-peak,
Fast decay


High-affinity
nicotine-binding
Mecamylamine,
BTMPS (high
sensitivity)
S2.0

Rectifying
Metanicotine,
Epibatidine,



DHpE

Relatively slow
timecourse vs. a7


Muscle-type


BTMPS (low
sensitivity)

S0.2

Non-rectifying
Succinylcholine





a-Btx,
(but not MLA)
Relatively slow
timecourse vs. a7


I _I I I














CHAPTER 2
METHODS

The cDNA Clones

These experiments used the rat neuronal nAChR and the mouse muscle cDNA

clones, which were obtained from Dr. Jim Boulter (UCLA). The sequences of the TM2

domains of the relevant subunits are shown in Figure 3-1. Adopting the terminology

proposed by Miller (1989), the 20 residues in the putative second transmembrane

sequence are identified as 1' through 20'.

Site-directed Mutagenesis

Site-directed mutagenesis was performed using QuickChange (TM) kits

(Strategene, LaJolla, CA). In brief, two complimentary oligonucleotides were

synthesized which contained the desired mutation flanked by 10-15 bases of unmodified

nucleotide sequence. Using a thermal cycler, Pfu DNA polymerase extended the

sequence around the whole vector, generating a plasmid with staggered nicks. Each

cycle built only off the parent strands, and therefore there was no amplification of

misincorporations. After 12-16 cycles, the product was treated with Dpn I, which

digested the methylated parent DNA into numerous small pieces. The product was then

transformed into E. coli cells, which repaired the nicks.

Preparation of RNA

After linearization and purification of cloned cDNAs, RNA transcripts were

prepared in vitro using the appropriate mMessage mMachine kit from Ambion Inc.

(Austin, TX).









Expression in Xenopus Oocytes

Mature (>9 cm) female Xenopus laevis African frogs (Nasco, Ft. Atkinson, WI)

were used as a source of oocytes. Prior to surgery, frogs were anesthetized by placing the

animal in a 1.5 g/L solution ofMS222 (3-aminobenzoic acid ethyl ester). Oocytes were

removed from an incision made in the abdomen.

In order to remove the follicular cell layer, harvested oocytes were treated with

collagenase from Worthington Biochemical Corporation (Freehold, NJ) for 2 hours at

room temperature in calcium-free Barth's solution (88 mM NaC1, 10 mM HEPES pH 7.6,

0.33 mM MgS04, 0.1 mg/ml gentamicin sulfate). Subsequently, stage 5 oocytes were

isolated and injected with 50 nl each of a mixture of the appropriate subunit cRNAs

following harvest. Recordings were made 3 to 21 days after injection depending on the

cRNAs being tested. In order to increase the magnitude of the functional responses from

oocytes injected with the T6'F mutant, approximately 5 times (30 ng) the amount of

mutant mRNA was injected compared to wild-type a7 (approximately 6-7 ng). Since all

data were normalized using each cell as its own control, absolute differences in response

magnitude did not affect comparisons between receptor subtypes.

Voltage-clamp Recording of Whole-oocyte Responses

Data were obtained by means of two-electrode voltage-clamp recording.

Recordings were made at room temperature (21-24 deg. C) in Frog Ringer's solution (115

mM NaC1, 10 mM HEPES, 2.5 mM KC1, and 1.8 mM CaC12, pH 7.3) with 1 jiM atropine

to inhibit muscarinic acetylcholine receptor responses. This extracellular solution was

used for all experiments unless otherwise noted. Voltage electrodes were filled with 3M

KC1, and current electrodes were filled with 250 mM CsC1, 250 mM CsF, and 100 mM

EGTA (pH 7.3).









Bath solution and drug applications were applied through a linear perfusion

system to oocytes placed in a Lucite chamber with a total volume of 0.5 ml. Drug

delivery involved pre-loading a 1.8 ml length of tubing at the terminus of the perfusion

system, while a Mariotte flask filled with Ringer's solution was used to maintain constant

perfusion. Applications of drug solutions were then synchronized with acquisition.

Current responses were recorded using a PC interfaced to either a Warner OC-725C

(Warner Instruments, Hamden, CT) or a GeneClamp 500 amplifier via a Digidata 1200

digitizer (Axon Instruments, Union City, CA). In addition, some oocyte recordings were

made using a beta version of the OpusXpress 6000A (Axon Instruments, Union City,

CA). OpusXpress is an integrated system that provides automated impalement and

voltage clamp, which in our case permitted the study of four oocytes in parallel. Cells

were automatically perfused with bath solution, and agonist solutions were delivered

from a 96-well compound plate. In experiments using the OpusXpress system, the

voltage and current electrodes were filled with 3 M KC1. In all experiments, bath flow

rates were set at 2 ml/minute.

Experimental Protocols and Analysis of Data Obtained from Xenopus Oocytes

Current responses to drug application were studied under two-electrode voltage

clamp at a holding potential of -50 mV unless otherwise noted (-60 mV for the

OpusXpress system). Holding currents immediately prior to agonist application were

subtracted from measurements of the peak response to agonist. All drug applications

were separated by a wash period of 5 minutes unless otherwise noted. At the start of

recording, all oocytes received two initial control applications of ACh. Subsequent drug

applications were normalized to the second ACh application in order to control for the

level of channel expression in each oocyte. Means and standard errors (SEM) were









calculated from the normalized responses of at least four oocytes for each experimental

concentration.

For concentration-response relations, data were plotted using Kaleidagraph 3.0.2

(Abelbeck Software; Reading, PA), and curves were generated using the Hill equation:

Imax [agonist]"
Response = a + ( ) (Eq. 2-1)
[agonist]" +(EC50)"

where Imax denotes the maximal response for a particular agonist/subunit combination,

and n represents the Hill coefficient. Imax, n, and the ECso were all unconstrained for the

fitting procedures.

For experiments measuring barium permeability, oocytes were perfused with

barium Ringers (low barium: 90.7 mM NaC1, 2.5 mM KC1, 10 mM HEPES pH 7.3, 1.8

mM BaC12, 48.6 mM sucrose; high barium: 90.7 mM NaC1, 2.5 mM KC1, 10 mM

HEPES pH 7.3, 18 mM BaC12). Shifts in reversal potential were measured by changing

the holding potential from -40 mV to +30 mV by 10 mV increments. Calculations of

barium sodium permeability ratios using the extended GHK equation were performed

using the Clampfit analysis portion of the pClamp software suite (Axon Instruments,

Union City, CA). Barium was used instead of calcium to minimize the contribution of

endogenous calcium-activated chloride channels (Sands et al., 1993).

Calculations of peak amplitudes and net charge were made using pClamp either

during acquisition or during subsequent Clampfit analysis. Note that measurement of net

charge has been shown to be a more accurate indicator of fast a7 responses than

measurement of peak response. An appropriate method using analysis of the area under

the curve of agonist-evoked currents in oocytes has been previously published (Papke and

Papke, 2002). Baseline was defined for Clampfit statistics based on 20 s before drug









application, the analysis region for peak and net charge analysis went from 5 s before the

initiation of drug application and extended at least 135 s following. Area analysis data is

provided for all receptor subtypes examined in this paper for comparison to wild-type a7.

Transfection and Patch-clamp Recording from GH4C1 cells

GH4C1 cells were cultured in F10 medium (Gibco, Carlsbad, CA) at 37 C, 5%

CO2. Cells were transiently transfected using Fugene (Roche, Indianapolis, IN),

according to the manufacturer instructions. One microgram of wild-type or T6'F mutant

a7 cDNA (pTR-UF22, University of Florida, Gainesville, FL) was added to each 35-mm

Petri dish, together with 0.5 or 1 jig of the cDNA encoding the red fluorescent DsRed

protein (BD Biosciences Clontech, Palo Alto, CA). Cells were used 48-72 hours after

transfection. Typical transfection efficiency was 10-25% using this method.

Single-channel currents were recorded in the cell-attached patch configuration

using an Axopatch 200A amplifier (Axon Instruments, Union City, CA) at room

temperature. Cells were bathed in a solution containing 140 mM NaC1, 2.8 mM KC1, 1

mM CaC12, 1 mM MgC12, 10 mM glucose, 10 mM HEPES/ NaOH (pH 7.3). Patch

electrodes (tip resistances, 5-7 MQ after fire polishing) were coated with Sylgard (Dow

Corning, Midland, MI) and filled with the same extracellular solution plus 1 pM Atropine

and 30 jpM ACh.. Currents were filtered at 10 kHz and digitized at 50 kHz using pClamp

8 (Axon Instruments Union City, CA). Analysis was conducted using pClamp 9.

Radioligand Binding Studies

GH4C1 cells were harvested from 60mm culture dishes using a sterile cell scraper

and assayed for nicotine-displaceable, high-affinity [3H]methyllycaconitine (MLA)

binding using a modification of the procedure of Davies and colleagues (1999). Cells

were suspended in 20 volumes of ice cold Krebs Ringer HEPES buffer (KRH; 118 mM









NaC1, 5 mM KC1, 10 mM glucose, 1 mM MgC12, 2.5 mM CaC12, and 20 mM HEPES; pH

7.5). After two 1-ml washes with KRH at 20,000g, the membranes were incubated in 0.5

ml KRH with 1, 3, 10 or 20 nM [3H]MLA (Tocris, Ellisville, MO) for 60 min at 40C with

or without 5 mM nicotine. Tissues were washed three times with 5 ml cold KRH by

filtration through Whatman GF/C filters that had been preincubated for 2 hours in blotto

(KRH with 0.5% dry milk and 0.002% sodium azide). They were then assayed for

radioactivity using liquid scintillation counting. Inhibition curves generated under two-

electrode voltage-clamp with oocytes expressing either wild-type a7 or the TM2 T6'F

mutant showed a less than 3-fold difference in MLA potency between the two (data not

shown).

Intact Oocyte Binding

[125I] a-Bungarotoxin binding in intact oocytes was performed similar to the

method described by Chang and Weiss (2002). In brief, whole Xenopus oocytes that

were either uninjected, or had been injected with mRNAs encoding either wild-type a7 or

the TM2 T6'F mutant were placed in a single well of a 96-well plate containing either 20

nM [125I] a-Btx alone, or 20 nM [125I] a-Btx with 5 mM nicotine. After four 4s washes in

2.5 ml KRH, total radioactivity was measured using an automated gamma particle

counter as counts per minute (CPM). Nicotine displaceable binding was calculated for at

least 4 cells in each condition.














CHAPTER 3
THE PHARMACOLOGICAL AND KINETIC EFFECTS OF THE a7 TM2 T6'F
MUTATION

Introduction

According to the same broad classification scheme described previously, two of the

major nicotinic receptor subfamilies include the muscle-type and the a7-type nAChRs.

Both muscle-type nAChR and homomeric a7 receptors are widely expressed in

mammals, bind a-Btx with high affinity, and share many structural features. However,

several important functional and pharmacological properties distinguish these two major

nAChR subtypes. For example, a7 receptors have high permeability to divalent cations,

show inward rectifying current-voltage relationships, and have highly agonist

concentration-dependent macroscopic response kinetics (Decker and Dani, 1990; Sands

et al., 1993; Segula et al., 1993; Vernino et al., 1994; see Table 1-1 for comparison).

During the course of the evolution of the various subunits of nAChR and the

functional radiation of receptor subtypes into various tissues where each subtype

performs highly specialized functions, there has emerged a great deal of sequence

divergence. Less than 40% amino acid sequence identity exists between the alpha

subunit of muscle type receptors (al) and the a7-type receptor found in the brain. It has

generally been assumed that the functional differences that exist among these receptor

subtypes are emergent properties of the collective sequence differences. Despite this

general assumption, it is understood that specific residues, conserved across multiple

subtypes, can be key to features common to all those subtypes. Examples of this type of









important conserved sequence are in the pore-forming second transmembrane domain,

and in this domain the sequence identity between al and a7 is increased to around 60%.

Previous work has indicated that amino acid residues in the TM2 domain of the

beta subunit of heteromeric nAChRs are important regulators of nAChR pharmacology.

Specifically, substitution of the TM2 6' (according to the numbering scheme proposed by

Miller ((1989) see Figure 3-1), phenylalanine of the muscle p1 subunit for the neuronal 34

subunit serine residue increased sensitivity to nicotine. This same study also showed that

substitution of p1 subunit amino acid sequence at the TM2 6' and 10' positions of the

neuronal p4 subunit reduced inhibition by the ganglionic blocker mecamylamine

(Webster et al., 1999).

This chapter describes a testing of the hypothesis that homologous substitution of

amino acid sequence from the muscle pi subunit into the a7 subunit would confer specific

properties of muscle-type receptors to mutant a7 nAChRs. Using site-directed

mutagenesis and heterologous expression of mutant receptors in Xenopus oocytes, the

effect of the T6'F mutation was examined with regard to ACh potency, ACh response

kinetics, barium permeability, the sensitivity to the muscle receptor selective agonist

succinylcholine, and the a7 potentiating factor 5-hydroxyindole.

Results

The TM2 T6'F Mutant is Functionally Expressed in Xenopus Oocytes

Injection of mRNAs encoding either wild-type or mutant a7 subunits in Xenopus

oocytes produced functional receptors that permitted us to study the receptors under two-

electrode voltage clamp. Raw data traces from oocytes expressing the TM2 T6'F point

mutation or wild-type a7 are shown in Figure X. Differences between mutant and wild-

type a7 receptors were seen both in the macroscopic response kinetics (see inset, Figure









3-2) and in the absolute amplitude of the response to ACh. There was consistently a

relatively small peak for the T6'F response compared to the wild-type a7 response.

Quantification of differences in absolute amplitude is complicated by variations in the

degree of receptor expression from cell to cell. However, variations in current amplitude

similar to that shown in Figure 3-2 were consistently observed, even after oocytes

expressing the mutant receptor were kept for several weeks, presumably allowing

receptor expression to accumulate. Due to this difference in functional expression,

oocytes expressing mutant receptors were typically used 11-21 days after injection, while

oocytes expressing wild-type receptors were used 5-14 days after injection. The average

amplitude of control responses (3 pM ACh) of mutant receptor responses recorded 11-21

days after injection was 64.5 12 nA (n= 12), while the average amplitude of control

responses (300 iM ACh) of wild-type receptor responses recorded 5-14 days after

injection was 335.9 60 nA (n= 12). These concentrations of ACh were chosen for

controls because the normalized area under the curve of these responses was at the upper

end of the concentration-response function, and they are therefore roughly saturating in

terms of net charge. It is important to note that the 100-fold difference in control ACh

concentrations is due to the increase in ACh potency observed in the T6'F mutant (see

Figure 3-4).

To determine whether differences in current magnitude between wild-type a7 and

the T6'F mutant were due to a relatively low mutant receptor expression at the cell

surface, or due to an effect of the mutation on single channel properties (i.e., the

probability of channel opening or single-channel conductance), radioligand binding

experiments were performed with both the transfected GH4C 1 cells and intact oocytes.









In the case of the transfected GH4C1 cells, [3H]MLA binding experiments conducted 48h

post-transfection indicated an approximate Bmax of 600 fmol/mg of protein in cells

transfected with wild-type a7, but no significant nicotine-displaceable binding was

observed in cells transfected with the T6'F mutant relative to non-transfected cells (data

not shown). However, single oocyte [125I]a-Btx binding studies indicated significant

specific binding in oocytes injected with either the wild-type a7 or the T6'F mutant

(Table 3-1): These same experiments also showed no significant difference in [125I]a-Btx

binding between intact oocytes expressing either wild-type a7 or the T6'F mutant. Thus it

is likely that poor (or possibly slow) expression of the TM2 T6'F mutant receptors

prevented their study in transfected GH4C1 cells, but that alterations in receptor number

alone were insufficient to explain the differences in current magnitude between wild-type

and mutant receptors expressed in Xenopus oocytes.

The a7 TM2 T6'F Mutation Dramatically Slows the Apparent Kinetics of ACh-
evoked Macroscopic Currents

The wild-type a7 receptor response has a characteristically rapid time course when

high concentrations of ACh are applied, with the decay phase occurring well before the

bath solution exchange is complete (Papke and Thinschmidt, 1998). As shown in Figure

3-3, the a7 TM2 T6'F mutation produced a dramatic change in the apparent kinetics of

ACh responses. Figure 3-3C shows the relative lack of effect of increasing

concentrations of ACh on duration of the macroscopic responses of the T6'F mutants,

distinct from both wild-type a7 (Figure 3-3A) and the muscle-type nAChR (Figure 3-3B).

This would be consistent with a change in receptor desensitization. Quantitative analysis

is shown in Figures 3-3D & E. This shows the detailed effects of this point mutation on

both the time required to reach the peak response and the return to baseline. Here it is









evident that both the rise times and the decay times of the T6'F mutant showed little

sensitivity to changes in ACh concentration (for the concentrations examined), which is

different from the characteristically concentration-dependent kinetics of the wild-type a7

response to ACh. The rise times of wild-type a7 responses differed from those of both

the T6'F mutant and muscle nicotinic receptors at ACh concentrations equal to and below

30 jM. The rise times of muscle receptors were also relatively insensitive to changing

ACh concentrations compared to wild-type a7. In the case of the decay times, muscle

receptors display a concentration dependence, but it is the opposite of that seen with

wild-type a7, with a general slowing of the decay of the muscle-type macroscopic

response with increasing agonist concentration. Thus the macroscopic responses of the

mutant and wild-type a7 receptors and the muscle-type receptors are each uniquely

affected by the process of agonist wash-in and wash-out, presumably due to differences

in their intrinsic desensitization rates and the influence of agonist binding on those rates

(as proposed for wild-type a7). The kinetics of solution exchange in the oocyte bath

perfusion system have been quantified previously (Papke and Thinschmidt, 1998; Papke

and Papke, 2002), allowing the evaluation of the kinetics of the macroscopic responses in

the context of how rapidly agonist is washed in and out of the chamber. Based on these

observations, it appears that the decay of the T6'F mutant responses follows the solution

exchange rates much more closely than wild-type a7, suggesting that agonist washout

may predominate in this phase of the response.

The a7 T6'F Mutation Increases ACh Potency Compared to Wild-type a7

Concentration-response functions for wild-type a7 and TM2 T6'F mutant are shown

in Figure 3-4. A dramatic increase (> 10 fold) in ACh potency was seen for the net

charge analysis of the T6'F mutant (Figure 3-4, Table 3-2), compared to wild-type a7.









Note that both net charge (area under the curve) and peak CRCs are shown to illustrate

the differences in apparent ACh potency as a function of the analysis method used.

Comparison of these analyses shows that, as previously reported (Papke and Papke,

2002), the net charge CRC analysis of wild-type a7 receptor showed a large difference in

apparent potency compared to peak current analysis. In contrast, the T6'F mutant showed

less difference between the two methods, due to a loss of a7's characteristic

concentration-dependent fast desensitization.

The a7 T6'F Mutation Abolishes Barium Permeability and Reduces Inward Current
Rectification

Barium is frequently used as a charge carrier to evaluate divalent ion permeability

because the relative permeability of barium to monovalent cations is generally indicative

of calcium permeability, and the use of barium decreases the secondary signal

transduction frequently associated with calcium influx. Current-voltage relationships

obtained in either high or low extracellular barium, showed the characteristic wild-type

a7 receptor inward rectification and permeability to this divalent ion (Figure 3-5; Decker

and Dani, 1990; Sands et al., 1993; Seguela et al., 1993; Vemino et al., 1994). The

extended GHK equation gave an estimated barium-to-sodium permeability ratio of

approximately 4:1. This ratio is substantially lower than that previously reported (Sands

et al., 1993), however this apparent discrepancy may be due to the use of EGTA in the

current electrode solution. By contrast, the T6'F mutant receptor showed significantly

less current rectification (Figure 3-5B) as indicated by chord conductances measured

between -40 and +30 mV holding potentials (p = 0.003 compared to wild-type a7,

unpaired t test), and a complete lack of a shift in reversal potential with varying









extracellular barium concentrations (Figure 3-5B). The GHK equation applied to these

results gives a barium-to-sodium permeability ratio that is near zero.

Succinylcholine is a Selective Agonist of Muscle-type Receptors, and is also an
Agonist of a7 T6'F Mutant Receptors

As shown in Figure 3-6A succinylcholine is an effective activator of muscle-type

receptors, but it is relatively ineffective in activating wild-type a7. There is a dramatic

increase in sensitivity (both efficacy and potency) of the mutant a7 receptor to

succinylcholine, compared to wild-type. Figure 3-6B shows the concentration-response

relationships for both ACh and succinylcholine applied to wild-type murine muscle

receptors (apy6). While succinylcholine is a partial agonist of muscle-type receptors, it

produces no activation of the heteromeric neuronal nAChR, a3p4, at concentrations up to

1 mM (data not shown). The effect of the T6'F mutation (Figure 3-6C) can be seen to

dramatically increase both the potency and the maximum response to succinylcholine

relative to wild-type a7 which responds only to concentrations of succinylcholine that

exceed 1 mM. Whereas succinylcholine is a weak partial agonist of wild-type a7

receptors, it appears to be a full agonist of the T6'F mutant receptors.

The TM2 T6'F Mutation Abolishes Potentiation by 5-Hydroxyindole

Responses of a7 nAChRs to ACh can be enhanced by 5-hydroxyindole in oocytes

expressing a7 (Gurley et al., 2000; Zwart et al., 2002). This effect is demonstrated in

Figure 3-7A in oocytes expressing wild-type a7, exposed to a co-application of 300 jiM

ACh and 1 mM 5HI. As previously reported (Gurley et al., 2000), the 5HI effect did not

impact the macroscopic kinetics of the inward current (Figure 3-7A, inset). In whole-cell

voltage-clamp experiments, ImM 5HI also potentiates wild-type a7 expressed in

transfected GH4C1 cells by 540 75% (mean SEM, n= 7; data not shown). In









contrast, oocytes expressing T6'F mutant receptors show a total lack of enhancement of

ACh evoked currents by 1 mM 5HI (Figure 3-7B). Comparing this lack of potentiation

of T6'F mutant responses to the muscle-type nAChR shows an identical insensitivity to

the same concentration of 5HI (Figure 3-7C).

Discussion

While examples of single point mutations producing dramatic changes in response

kinetics, calcium permeability, and pharmacology of nicotinic receptors have been

previously shown (Revah et al., 1991; Palma, 1996), the findings presented here are

unusual in that several properties that functionally distinguish muscle-type nAChRs from

members of the other major subgroups of nAChRs can be conferred to mutant a7

receptors by a single residue. Not only does this observation suggest the importance of

amino acid sequence at this particular site for the maintenance of a7-like properties in the

wild-type receptor, but that there is a high degree of functional significance to the

structure at TM2 6' position allowing a point mutation to overcome other distinguishing

structural elements of the a7 subtype.

The relatively low magnitude of T6'F mutant receptor response to agonist

compared to wild-type a7 is likely the result of a combination of factors. In the oocyte

system, the loss of calcium permeability alone would diminish the functional

amplification of the nAChR-mediated current by calcium-dependent chloride current

(Barish, 1983). Also, in the muscle receptors, the beta subunit places a single

phenylalanine at a site where it aligns with hydrophilic residues in the other subunits,

while in the T6'F mutant a7 receptor there is likely to be a complete hydrophobic ring at

the same site in the channel. The positioning of a hydrophobic phenylalanine in the ion

permeation pathway could therefore have profound effects on single channel properties.









Based on the results of our radioligand binding experiments, we can conclude that, at

least in the case of the oocytes, there are comparable numbers of wild-type and T6'F

mutant receptors expressed at the cell surface, and that other factors may contribute to the

mutant's relatively low response magnitude.

The variety of effects that we observed due to the mutation of the TM2 6' amino

acid were intriguing, given the fact that this site is in the putative pore-lining region. The

T6'F mutation converts a7 from a receptor having a high barium permeability to a

receptor that is relatively impermeant to barium. This observation is not surprising due to

the likelihood of steric or hydrophobic interference (or both) to divalent ion flux through

the pore related to the ring of phenylalanine residues present at the TM2 6' position of the

mutant receptor. Furthermore, others have reported that amino acids in the TM2 domain

of several different ligand-gated ion channels (including a7) are essential determinants of

ionic selectivity (Galzi et al., 1992; Keramidas et al., 2000; Gunthorpe and Lummis,

2001). What is more surprising about the findings presented here are the effects that this

mutation has had on receptor properties that may be linked to conformational changes far

removed from the pore-lining region (i.e., changes in succinylcholine pharmacology). It

is thus likely that the T6'F mutation is indirectly impacting agonist binding, or that it is

affecting the coupling of agonist binding and subsequent conformational effects, such as

channel gating.

It is important to consider the findings presented here in the context of previous

reports that indicate other contributing factors shown to regulate divalent ion

permeability in muscle-type receptors. For instance, the muscle gamma subunit has been

shown to be an essential determinant of divalent cation permeability (Francis and Papke,









1996); however, one limitation of the experimental approach used in that study was the

inability to evaluate the significance of the muscle pi subunit. Thus, the gamma subunit

is necessary, but may not be sufficient, to produce the divalent ion permeability profile

characteristic of muscle nAChRs. The results presented here imply that the pi subunit,

and particularly the TM2 6' phenylalanine may cooperate with the gamma subunit in

determining this feature. It may be significant however, that the a7 TM2 T6'F mutant has

five phenylalanine residues at this position compared to the muscle receptor's one, thus

potentially amplifying the effect that this amino acid sequence has on wild-type receptor

function.

The slowing of the macroscopic kinetics of the 6' mutant receptor responses to ACh

is reminiscent of the muscle-type receptors, with the decay phase of mutant receptor

response kinetics showing a relative lack of sensitivity to changes in agonist

concentration. This property is more characteristic of beta subunit-containing receptors

than wild-type a7 (Papke and Thinschmidt, 1998). The T6'F mutant response kinetics

also show a nearly total lack of effect of ACh concentration on the rise rates of the

macroscopic response. Again, this result appears to reflect the conferring of a non-a7

receptor-like property on the mutant. It is interesting to note that while the widely

studied L9'T (L247T) mutation appears to remove a great deal of the fast desensitization

associated with the wild-type receptor (Revah et al., 1991), the amino acid substitution is

nearly opposite in nature to the one reported here. While the L9'T represents the

replacement of a larger, hydrophobic residue with a smaller hydrophilic one, the reverse

is true for the T6'F mutation. This suggests that this aspect of the a7-type response may

not be strictly regulated by size or hydrophobicity. Note that the 9' leucine residue has









been proposed to contribute to a ring of hydrophobic residues, while the 6' threonine is

more likely to contribute to a ring of polar residues approximately one level lower on the

putative transmembrane helix, closer to the hypothetical selectivity filter of the channel

(Corringer et al., 2000). In this regard, it is conceivable that substituted amino acids with

different properties could be accomplishing a similar effect either through different

mechanisms, or through similar mechanisms at different sites in the receptor.

The conversion of succinylcholine from a very weak partial agonist of wild-type a7

receptors to a relatively potent full agonist of the T6'F mutant was an indication of the

pharmacological constraints that these residues appear to place on their corresponding

wild-type receptors. The fact that a significant degree of succinylcholine selectivity can

be attributed to a residue in the pore lining region is perhaps less obvious, and is

suggestive of more global effects of this amino acid on the overall structure of the

receptor in both the open and closed states.

Although the mechanism of potentiation of a7 responses by 5-hydroxyindole is

unknown, previous reports (Gurley et al., 2000) combined with our results from

experiments with the muscle-type nAChR, suggest that this effect may be unique to a7.

The total absence of potentiation by 5HI for both the muscle and the T6'F mutant,

compared to the expected large enhancement of ACh-evoked responses from wild-type

a7 appears to be further confirmation of the ability of this amino acid substitution to

imbue the mutant receptor with a muscle receptor phenotype. Without a clearer

understanding of how 5HI potentiates wild-type a7 responses, it is difficult to suggest an

explanation for its absence in the T6'F mutant. It is possible that amino acid sequence at

the TM2 6' position is an essential factor in the coupling of agonist evoked channel









opening to an allosteric alteration produced by 5HI. Alternatively, the T6'F mutation

may prevent 5HI from relieving a constitutive inhibition of wild-type a7, a mechanism

that has been proposed for a similar potentiation of a7 by bovine serum albumin (Butt et

al., 2002).

The data presented here suggest that the functional changes associated with the

emergence of a specialized receptor involved in muscular contraction can be attributed, at

least in part, to sequence difference at a single site in what is ultimately a structural

subunit of the muscle receptor complex. It is potentially significant that the muscle beta

subunit is the only known nAChR gene product that contains a phenylalanine residue at

this site (LeNovere and Changeux, 2001). The effects of this mutation extend beyond

those that are commonly associated with amino acid structure in the pore-lining region of

the receptor, and indicate that nAChR function and pharmacology can be broadly and

dramatically altered by this single amino acid change. This suggests that the evolution of

functional specialization in this superfamily of ligand-gated ion channels may involve

something analogous to punctate equilibrium, where single small changes may produce

branch points of functional significance and point to the origins of the families of

receptor subtypes.









Table 3-1. Intact oocyte [125I]a-Btx binding.
CPM/Cell
Rat a7 wild-type
20 nM a-Btx alone 764.32 529.4 (n = 4)*
20 nM a-Btx + 5 mM nicotine 72.15 60.5 (n=4)
Rat a7 T6'F mutant
20 nM a-Btx alone 1074.98 418.8 (n = 4)*
20 nM a-Btx + 5 mM nicotine 261.55 137.5 (n=3)
Data represent the mean (+ s.e.m.) counts per minute per cell (CPM/Cell) for the
indicated treatments (* p < 0.05 by Student's t compared to the same receptor subtype in
the presence of 20 nM a-Btx with 5 mM nicotine).






39


Table 3-2. Curve-fit values for wild-type and T6'F mutant a7 responses to ACh.


Rat a7 wild-type
ACh peak a N/A N/A N/A
ACh net charge 1.05 0.02 1.5 0.1 16.5 1.0 LM
Rat a7 T6'F mutant
ACh peak 0.89 + 0.04 1.5 + 0.4 1.7 + 0.4 LM
ACh net charge 0.96 0.04 2.4 1.1 1.1 + 0.2 LM
Data represent curve values generated by the Hill equation (see methods) for macroscopic
responses from oocytes expressing either indicated receptor.
The use of peak responses has been shown to provide inaccurate estimates of these parameters
(Papke & Papke, 2002) and curve-fitting was thus restricted to net charge analysis for wild-type
a7.


Normalized Imax


Hill Coefficient


EC5o












intracellular


wild-type a7

wild-type P1
a7 T6'F mutant

wild-type P2
wild-type p4
a7 T6'S mutant


TM2 Domain


ISLGITIVLLSLTVFMLLVAEIMP


extracellular
20'
AT


MGLSIFALLTLTVFLLLLADKVPET
ISLGIFVLLSLTVFMLLVAEIMPAT


MTL
MTL
ISL


VLL
VLL
VLL


ALTVFLLL
ALTFFLLL
SLTVFMLL


Figure 3-1. Wild-type and mutant a7 nAChRs. Amino acid sequences for wild-type and
mutant a7 nAChR TM2 domains. The numbering of specific residues of the
second membrane-spanning region is according to that proposed by Miller
(1989). The corresponding sequences for wild-type muscle pi, and neuronal
beta subunits are also given as a reference. The substituted residues are
highlighted at the 6' position.


ISKIVPPT
ISKIVPPT
VAEIMPAT










TM2 T6'F
Wild-type a7-

Wild-type a7




50 nA T6'F

10s





Figure 3-2. Electrophysiological responses from wild-type and mutant a7 nAChRs.
ACh-evoked current responses for wild-type a7 and the TM2 T6'F mutant
nAChR expressed in oocytes. The agonist application bar at the top indicates
the timing and duration of agonist application for each receptor (wild-type =
30 iM ACh, T6'F = 3 jpM ACh). The inset shows the same traces scaled to
one another for the purpose of comparison (wild-type = 100 %, T6'F = 476
%).














300 nM
1 pM-^./-----------------
3 lM F---.M
10 M
30 tM
100 ILM
300 ,tM
1 mMV


300 nM
I IiM
3 &M
10 itM
30 FM
100 IM
300 I-M
1 mM
3 mM


J 20 nA
6s


- WldFtip ,7
I-e-B- (FI|
L h~Z!ZZJ~h


10 100 1000 W1
[AChj, pM


Figure 3-3. Macroscopic response kinetics of wild-type a7, muscle, and the TM2 T6'F
mutant nAChRs expressed in oocytes. A) Wild-type rat a7 current traces at
the indicated concentration of ACh. B) Wild-type muscle (apy6) current
traces at the indicated concentration of ACh. C) Rat a7 TM2 T6'F current
traces at the indicated concentration of ACh. D & E) Rise times (10 90 %)
and decay times (90 70 %) for macroscopic responses to ACh applied to
each of the indicated receptors at the concentrations shown. The kinetics of
muscle receptor responses as a function of increasing ACh concentration are
also plotted for comparison. Means and standard errors are given for at least
four cells.


10pM
30 ILM
100 pM P -

1 mM --






6s

C


I-- MusfT |FI


10 100 1000 ID'
[ACh], pM







43


A B
1.2 r 12

Area 1 ---Area
c -u- Peak I --- Peak
08 0.8


N N

0 0,
aC 0.4 C / j 0.4 -


0 0 i
0.1 1 10 100 1000 104 0.01 0.1 1 10 100 1000
[ACh], [M [ACh], [M

Figure 3-4. ACh concentration-response functions for wild-type and the TM2 T6'F
mutant a7 nAChR. Data were normalized to the maximal response for either
wild-type rat a7(A) or a7 TM2 T6'F (B). Peak and net charge analysis (area
under the curve) are presented showing the relative sensitivity to increasing
concentrations of ACh. Means and standard errors represent data acquired
from at least four cells.










A B

0 (low Ba2+)
(high Ba2+)
0.5 0.5

-40 -30 -20 -10

10 20 30 -40 -30 -20 -10 10 20 30
SHolding Potential (mV) /. Holding Potential (mV)
-0.5 -0.5


-1 -1

Figure 3-5. Wild-type and mutant a7 nAChR current-voltage relationships and divalent
ion permeability in Xenopus oocytes. Current-voltage relations were
examined in wild-type a7 and T6'F mutant receptors using either high (18
mM) extracellular barium or low (1.8 mM) osmotically balanced extracellular
barium. Holding potentials were incrementally adjusted by 10 mV from -40
mV to +30 mV and peak responses were normalized to control responses held
at -50 mV. A) The wild-type a7 I-V shows its characteristic inward
rectification, as well as positive shift in reversal potential with increased
extracellular barium. B) The TM2 T6'F mutant I-V shows less inward
rectification, and the lack of a shift in reversal potential in high extracellular
barium indicates a loss of divalent ion permeation.











10 DDiM SuCh 3 LM ACh


300 |M ACh 10 mM SuCh 300 PM ACh


Wild-type
ct7 1 20 nA L
S 10s


3 pM ACh 100 pM SuCh 3 M ACh


TM2 T6'F nA I
20 nA K
50s
C.


1.2 -0-y6 ACh



< 0.8

Co

O
0
z
0.2

0 Z-


-0-Wild-type a7
-- TM2 T6'F


0.1 1 10 100 1000 104 0.1 1 10 100 1000

[agonist], pM [SuCh], iM


104 10s


Figure 3-6. Wild-type and mutant nAChR responses to the muscle receptor selective
agonist succinylcholine. A) Representative current traces for oocytes
expressing the indicated nAChR subtype. The response to the indicated
concentration of succinylcholine (SuCh) proceeded and followed by control
applications of ACh. B) Net charge concentration-response relationships for
wild-type muscle (capy) exposed to either ACh or SuCh. C) Net charge
concentration-response relationships for wild-type and the T6'F mutant a7
exposed to SuCh (see text for discussion of the use of net charge analysis for
these experiments).


3 PIM ACh


Wild-type
muscle


200 nA I
50s







46


A B




200 nA
l0s |
10 10 nA 1
S10 s








C D 11
10.0

F 37 Wild-type u7
8.0
Wiv Muscle (ai yb)
g t T T6'F mutant
.(30) .0
20 sN
E 4.0
0
Z 3.0
2.0
1.0


Figure 3-7. Wild-type and mutant nAChR potentiation by 5-Hydroxyindole. A)
Representative TEVC current traces from Xenopus oocytes expressing wild-
type 7. Agonist application bars represent a control application of 300 M
ACh in the first trace, and pre-incubation with 1 mM 5HI followed by co-
applied 300 WiM ACh. The inset shows the same two traces scaled to one
another, showing the lack of an effect of 5HI potentiation on macroscopic
response kinetics. B) Representative current traces from Xenopus oocytes
expressing the TM2 T6'F mutant receptor. The experimental design was
similar to that described for the wild-type receptor above, except for the use of
3 WiM ACh alone or co-applied with 1 mM 5HI. C) Current responses
representing a similar experimental design to that described above for oocytes
expressing muscle-type nAChRs using 30 jiM ACh alone or co-applied with 1
mM 5HI. D) The effect of co-application of 1 mM 5HI on ACh responses
normalized to the previous control application of ACh in Xenopus oocytes
expressing the indicated wild-type or mutant nAChR subtype. Means and
standard errors represent data from at least four cells.














CHAPTER 4
THE a7 TM2 T6'S MUTATION: A GAIN-OF-FUNCTION WITH a7-LIKE
PHARMACOLOGY

Introduction

A potentially important feature of a7-type nicotinic receptors that sets them apart

from other ligand-gated ion channels is the high degree of concentration-dependence to

the kinetics of agonist-evoked responses. That is, with increasing concentrations of

agonist, the macroscopic responses of a7 nAChRs become more and more transient

(Papke and Thinschmidt, 1998). The fact that a7 nAChRs have been reported to have a

very high permeability to calcium (Decker and Dani, 1990; Sands et al., 1993) suggests

that this agonist concentration-dependent limitation of the channel's activity is part of a

built-in safety mechanism designed to prevent excitotoxic injury in cells with sufficient

levels of a7 receptors expressed. This high degree of agonist concentration-dependent

limitation on response magnitude has in many cases been attributed (Revah et al., 1991)

to classical Katz and Thesleff (1957) type desensitization. This is a reasonable

interpretation, but evidence for other mechanisms of agonist-dependent inhibition such as

open channel block may also contribute to this property. Since macroscopic currents

constitute the ensemble activity of channels in a variety of discrete states, the only way to

actually to determine the degree to which classical desensitization contributes to agonist-

dependent limitation of a response is at the single channel level.

The T6'F substitution described in the previous chapter has the effect of producing

a mutant a7 receptor with several muscle nAChR-like properties. One major effect of









this mutation is the reduction in the concentration-dependence of agonist-evoked

responses that is so characteristic of wild-type a7 (Figure 3-3). In addition to this change

in kinetics, the T6'F mutant also displays some pharmacological properties that are like

the muscle-type nAChR. In an effort to test if an analogous effect could be produced

with a similar single amino acid substitution in the a7 TM2 domain, an a7 T6'S mutant

was constructed. In this case the a7 wild-type threonine is exchanged for the serine

residue present at the homologous position of the neuronal p2/p4 subunit (Figure 3-1).

The hypothesis being tested was similar to that which was addressed by the

characterization of the a7 T6'F mutant. Specifically, a serine-for-threonine substitution at

the 6' position of the a7 TM2 domain would make the mutant receptor more like a

neuronal beta subunit-containing receptor.

As in the case of the T6'F mutant, much of the rationale for the hypothesized

effects of the T6'S mutant are derived from previously published observations with

heteromeric nAChRs. The effects reported by Webster and colleagues (1999), showed

that sensitivity to the ganglionic blocker mecamylamine was increased in muscle-type

receptors with chimeric pl(p4) subunits, and that this effect could be attributed largely to

the sequence differences at the TM2 6' and 10' positions. This suggested that a similar

effect may be observed with the T6'S mutant, particularly for drugs that interact with the

pore-forming region of the receptor.

The result is a mutant receptor that retains much of the pharmacology of wild-type

a7, but with larger macroscopic responses and a dramatic lessening of agonist

concentration-dependent limitation on response duration. The kinetic effects of this

mutation are examined at the single-channel level and indicate that channel open time









and burst properties are affected, rather than receptor expression, or single-channel

conductance. Finally, although the T6'S mutant displays a pharmacological profile that is

very similar to wild-type a7, a gain-of-function mutant that retains much of the

pharmacology of the wild-type receptor has significant implications for drug

development.

Results

T6'S Mutant Kinetics and Single-channel Properties

One of the major predictions generated by the hypothesis behind the creation of the

T6'S mutant, is that this substitution would produce a receptor with response kinetics

more like the high-affinity nicotine binding receptor (see Table 1-1 for comparison). In

order to test this prediction, first an analysis of macroscopic responses from oocytes was

performed, followed by single-channel studies in mammalian cells aimed at identifying

changes in the properties of unitary events.

The TM2 T6'S Mutation Slows the Macroscopic Kinetics of ACh-evoked Currents
and Increases the Overall Net Charge Carried Upon Activation

The a7 TM2 T6'S mutant shows a significant change in response kinetics to ACh

(Figure 4-1), with a general slowing of the macroscopic response for the mutant versus

wild-type a7. Figure 4-1 also shows the decreased effect of increasing concentrations of

ACh on the macroscopic decay rate of the T6'S mutant responses (Figure 4-1B)

compared to wild-type a7 (Figure 4-1A) and its characteristically strong concentration-

dependent kinetics. An analysis of the rise times (Figure 4-1C) of these responses

showed that the T6'S mutant actually shows a slowing of the response with increasing

agonist concentration before the eventual increase in rise time dominates at the highest

concentrations. This is particularly true for ACh concentrations around 30 jpM. The









decay rates of the T6'S mutant responses, much like the T6'F mutant, show little effect of

high agonist concentrations, whereas wild-type responses become increasingly brief

(Figure 4-1D). A comparison of 3[H]MLA binding between intact oocytes expressing

either wild-type a7 or the T6'S mutant showed that the average difference in nicotine

displaceable binding was not statistically significant (Table 4-1). Despite this similarity

in receptor binding, the ACh evoked currents in cells from the same injection set differed

substantially in the amount of charge carried. Figure 4-2A shows averaged currents from

these oocytes with 30 pNM ACh applied, and below in figure 4-2B, the same currents are

expressed showing the cumulative net charge for both the wild-type and T6'S mutant.

The total area under the curve for the averaged wild-type a7 current is 11.5% of that of

the T6'S mutant, a difference that cannot be attributed to differences in receptor

expression.

The T6'S Mutation Does not Increase a7 Single-channel Conductance

Having observed this difference in macroscopic kinetics and the corresponding

increase in net charge, single channel patch-clamp experiments were conducted in order

to identify the specific channel properties affected by the mutation. One aspect of the

receptor that may have been altered with significant effect on the amount of charge

carried by the mutant receptor is the unitary conductance. Single channel currents were

recorded from transiently transfected GH4C1 cells in the cell-attached patch

configuration. Figure 4-3 shows a representative recording from a cell exposed to 30 jpM

ACh in the patch pipette. Control cells (untransfected, n=6) showed no channel openings

in the presence of ACh. A current-voltage relationship was established for the T6'S

mutant in order to quantify the single-channel conductance. Figure 4-4 shows a

representative plotted I-V relation. Linear regression analysis gave an average slope









conductance of 61.7 5.8 pS. This is slightly less than the wild-type a7 single-channel

conductance of 91.5 8.5 pS reported by Mike and colleagues (2000). Exact comparison

to the previously reported value for the wild-type receptor may not be possible due to

differences in experimental solutions (e.g., no extracellular magnesium and lower

calcium for the previously published experiments), but the value for the T6'S mutant is at

least not greater than that reported for wild-type a7. This suggests that the difference in

response magnitude between mutant and wild-type is not attributable to a change in

unitary conductance.

The T6'S Mutation Produces a Significant Increase in Average Channel Open Time

Since a change in the T6'S single channel conductance was clearly not

contributing to the increase in net charge evident in macroscopic mutant responses,

alterations in channel open times were then examined. Analysis of channel dwell time

distributions (Figure 4-5A) shows that the T6'S mutant channel open times are best fit by

two exponentials with the average shorter open time being 580 110 |ts and the longer

open time being 4.308 0.856 ms. Channel open times for native wild-type a7 receptors

expressed in tuberomammillary nucleus neurons have been reported to be fit by a single

exponential and had an average of 83 jps (Victor Uteshev, personal communication).

Since T6'S mutant channel open times were fit by two exponential components, a

weighted average was used to give an estimate of the total average open time for a

macroscopic current (2.470 ms). Comparing this to the previously published mean

channel open time of 100 jis (Mike et al., 2000), gives an approximate 24.7 fold increase

in the channel open time. Taking into consideration the differences in single-channel

conductance for the T6'S mutant versus that reported for the wild-type receptor (33% less









for the mutant), the cumulative charge carried by the wild-type receptor shown in Figure

4-2B was scaled to show the potential contribution of these values to the differences

observed in macroscopic currents (Figure 4-5B).

T6'S Mutant Burst Activity

Another difference in single channel activity demonstrating effects of the T6'S

mutation is the nature of channel bursting. Figure 4-6A shows raw single channel events

where bursts of openings were observed under steady-state conditions. Figure 4-6B

shows the closed time distribution for the T6'S mutant from patches exposed to 30 iM

ACh. The requirement for multiple exponentials to fit the distribution is consistent with

channel bursting. Using the method of Colquhoun and Sakmann (1985) a critical time

value of 759 jis was determined as a threshold for intraburst closures. The wild-type a7

nAChR has been shown to have little or no burst activity under steady-state conditions

(Victor Uteshev, personal communication), or in other words, bursts that consist of single

openings. In such a case the average channel open time approximates the average burst

duration. The average burst duration distributions (Figure 4-7A) for the T6'S mutant

were best fit by two exponential components (556 104 jps and 5.522 1.355 ms).

Histograms of the number of bursts with more than one opening were plotted and fitted

by a Poisson distribution (Figure 4-7B), providing a prediction of the probability of

channel reopening. In this case, the T6'S mutant had a 42.1 6% probability of opening

more than once, and as previously stated, bursts with more than one opening are rarely, if

ever observed in the wild-type receptor under steady-state conditions.

The Pharmacology Of The T6'S Mutant

The other general prediction derived from the rationale for making the T6'S mutant

was that the pharmacology would be more like a neuronal beta-subunit containing









nAChR (see table 1-1). However, with one large exception in the case of potentiation by

5HI, the pharmacological profile of the T6'S mutant is generally more like the wild-type

a7 than a heteromeric neuronal nAChR. In the context of the previously described

changes in kinetics, this presents an advantage to those interested in identifying drugs that

target the wild-type receptor. Specifically, a receptor that has the same pharmacological

features of wild-type a7, but lacks the characteristic response limitation in the sustained

presence of agonist, may serve as a useful facsimile of the wild-type receptor.

The T6'S Mutant Shows a Slight Decrease in Ach Potency and a Decreased
Discrepancy Between Area and Peak CRCs

The concentration-response relationship for ACh applied to either the T6'S mutant

or wild-type a7 are shown in Figure 4-8, showing a slight decrease in the apparent ACh

potency for the T6'S mutant compared to wild-type (see Table 4-2). As previously shown

in Chapter 3, wild-type a7 nAChRs have significantly different peak amplitude and net

charge CRCs (Figure 3-4). This is indicative of the strong agonist concentration-

dependence to the time course of their macroscopic responses. In a manner similar to

that shown for the T6'F mutant, the T6'S mutant shows less of a difference between the

two methods (Figure 4-8B), again indicating that response kinetics are far less sensitive

to agonist concentration.

The Agonist Selectivity Profile of the T6'S Mutant is Similar to Wild-Type a7

Concentration response relationships for a series of nicotinic receptor agonists

indicates that the T6'S mutant has a pharmacology that is similar to wild-type a7 with

regard to both potency and efficacy of the agonists examined. Figure 4-9 compares the

effects of several a7 selective agonists on oocytes expressing either the T6'S mutant or

wild-type rat a7. Although some differences in potency and efficacy are evident (with









the exception of the partial agonist Tropisetron, which is nearly identical between the two

receptor types (Figure 4-9D)), all agonists activate the mutant receptor. Furthermore, the

potency differences observed are similar to that seen with ACh (Figure 4-8), suggesting a

general trend in modest potency reduction for agonists. The similarity in agonist

response profiles also extends to agonists that not selective for a7 type nAChRs. The 32

subunit selective agonist cytisine (Papke and Heinemann, 1994) and the a4p2 selective

agonist metanicotine (Papke et al., 2000b) display agonist activity at the T6'S mutant

(Table 4-2). Again, there is a slight reduction in potency and efficacy, particularly in the

case of cytisine, but these shifts are consistent with the general trend observed for most of

the agonists examined (Table 4-2).

Antagonists of the Wild-Type Receptor are also Antagonists of the T6'S Mutant

Another feature of the T6'S mutant that has proven to be similar to wild-type a7 is

its sensitivity to nAChR antagonists. A somewhat peculiar feature of the L9'T mutant is

that several drugs that function as antagonists of wild-type a7 have been shown to

activate this mutant (Bertrand et al., 1992; Palma, 1996; Palma et al., 1998; Tonini et al.,

2003). By contrast, each of these drugs inhibit the T6'S mutant (Figure 4-10).

Furthermore, for the concentrations tested, no agonist activity of these same drugs was

observed (not shown).

5-Hydroxyindole Potentiation is Significantly Diminished in the T6'S Mutant
Compared to Wild-Type a7

As previously described in Chapter 3, 5HI has been reported to be an allosteric

potentiator of wild-type a7. Figure 3-7 shows this effect in oocytes where 1 mM 5HI

produces a 9-fold potentiation of wild-type a7 responses to ACh. By contrast, the T6'S

mutant shows relatively very little potentiation by 5HI (Figure 4-11). While this









potentiation is significant at 26 % (p < 0.05 compared to control, Student's t) it is also

significantly and dramatically lower that the wild-type receptor's average potentiation of

904 % (p < 0.0001, compared to 5HI potentiated wild-type a7, Student's t).

Discussion

Kinetic Effects of the T6'S Mutation

The mutant a7 receptor described in this chapter displays a profound change in

response kinetics, with a significant reduction in response limitation in the presence of

higher concentrations of agonist. Based on the results of receptor binding experiments,

the overall increase in response magnitude observed in the T6'S mutant is apparently not

due to an increase in receptor expression. This however, is not particularly surprising,

since an increase in receptor number would not be a likely cause of altered response

kinetics. On the other hand, the lack of an increase in single-channel conductance is

perhaps more unexpected. This is particularly the case since the mutation represents the

substitution of a smaller serine residue for the larger wild-type threonine within the pore-

forming domain. This could conceivably result in a larger pore diameter, and thus

increased single channel conductance. However, the impact of the T6'S mutation on the

pore diameter is either too subtle, or compensated for by other changes, such as changes

in charge distribution of the amino acids lining the pore, impacting their interaction with

permeant ions. It is interesting to note that the well-studied a7 L9'T mutation has a

similar effect in that there is a gain-of-function, without a significant increase in unitary

conductance (Palma et al., 1999).

By contrast, an examination of the mean channel open time indicates that the T6'S

mutant has far longer open durations than observed for wild-type a7 (Mike et al., 2000;

Victor Uteshev, personal communication). This almost certainly contributes to the









ability to record responses from cells expressing the T6'S mutant under cell-attached

patch conditions. While it is possible that wild-type a7 receptors may also activate under

similar conditions, channel openings under steady-state conditions of exposure to even

low concentrations of agonist are so infrequent as to render such studies impractical, if

not impossible. Since no estimates were made of the number of T6'S mutant channels

present in each patch, the absolute Po could not be determined, however, it is likely to

differ from wild-type a7 since receptor expression was found to be comparable between

the two in oocytes.

Burst analysis indicates that there is a significantly higher likelihood of channel

reopening for the T6'S mutant compared to wild-type. It should be mentioned that this

increase in channel re-opening probability also reflects a general increase in overall open

probability. While no effort was made in these studies to generate specific kinetic

models that fit the experimental data, this basic observation of increased open probability

provides information regarding transitions between kinetic states for the T6'S mutant.

The kinetic scheme shown in Figure 4-12 is a simplified, slightly modified version of the

fractional occupancy model proposed for wild-type a7 by Papke and colleagues (2000a).

In this model are several transition rates that are dependent on agonist concentration, all

of which serve to drive the channel into desensitized, non-conducting states as agonist

concentration increases. This notion is consistent with previously published observations

regarding a7 kinetics (Papke and Thinschmidt, 1998; Papke and Papke, 2002) and with

the macroscopic data shown in Figure 4-1. In the interpretation of the kinetic effects of

the T6'S mutation, there are several possible explanations for the reduction in

concentration-dependent response limitation. One possibility is that one or more states









that are closed in the wild-type receptor have been converted to an open state. For

example, the states labeled A2R* and A3R* may be open states for wild-type a7, whereas

the states labeled A4R* and A5R* may be closed. This is consistent with a receptor that

is more likely to be in an open state when its five putative agonist binding sites are less

than fully occupied. However, in the case of the T6'S mutant, one or both of the

normally non-conducting A4R* and A5R* states may have been converted to open states.

It is also conceivable that one or more of the desensitized states (AnD) has been

converted to an open state.

Another possible interpretation is the that the T6'S mutation has induced a shift in

the desensitization rates, such that transitions into the open state are generally more

favored than in the wild-type receptor. When applying the above scheme to wild-type a7,

one can see that when desensitization rate constants (d+) are higher than the rate

constants for the non-desensitizing transitions out of the open state (a), the desensitized

state becomes absorbing, the number of openings per burst is close to unity, and the

probability of channel reopening approaches zero. However, in the case of the T6'S

mutant, the probability of channel reopening is significantly non-zero. This indicates that

the transition rates between the bound, non-desensitized states and the open states are

sufficiently high relative to the desensitization rate constants to make multiple opening

bursts more likely. Thus, the T6'S mutation appears to have had some effect on the rate

of desensitization, or at least on the rate of desensitization relative to other transition

rates.

Although it may seem as though the T6'S mutation has the effect of reducing

agonist potency, this effect should not be interpreted outside of the context of the kinetic









effects. This is because the apparent reduction in potency may in fact be due to a

broadening of the concentration-response functions for agonists. That is, at the higher

agonist concentrations that impose limitations on the responses of the wild-type receptor,

the T6'S mutant is still activated. This essentially stretches out the agonist CRCs for the

mutant producing an apparent reduction in potency. Of course, the data presented here

can not rule out direct effects on agonist potency, but the fact that T6'S mutant responses

are occurring at what are normally inhibiting agonist concentrations for wild-type a7

must also be considered.

Pharmacological effects of the T6'S mutation

While the data presented here do not lend support to the original hypothesis of

converting a mutant a7 receptor into neuronal beta subunit containing receptor by means

of a single amino acid substitution (similar to what was observed with the T6'F mutant),

the findings are interesting nonetheless. Other gain-of function mutations have been

produced in a7, but there is typically very unusual pharmacology that accompanies this

effect. For example, the pharmacology of the L9'T mutant has been extensively studied,

and has been shown to differ greatly from that of the wild-type a7 nAChR (Bertrand et

al., 1992; Palma, 1996; Palma et al., 1998; Tonini et al., 2003). The studies that have

been published regarding the altered pharmacology of this mutant a7 receptor have been

somewhat intriguing and seem to show a strong relationship between amino acid

sequence at this position of the TM2 domain, and receptor gating. There are several

drugs that have been reported to be antagonists of the wild-type receptor that are agonists

of the L9'T mutant. This, coupled with the significant gain-of-function that this mutation

has on the receptor's response to agonist has led to the suggestion that the mutation

converts a liganded, closed state to a liganded, open one (Bertrand et al., 1992).









While these observations are interesting, and may have something to tell us about

the structural elements involved in gating of the a7 type nAChR, they are less useful

when looking for tools to aid in drug screening for potential therapeutics that target a7.

In the effort to find drugs that have a selective effect on a7 function, it would certainly be

more desirable to have pharmacological fidelity in a screening system. The main

problem with this is that some of the unique attributes of the a7 type receptor make this a

very difficult proposition. The rapid desensitization of the wild-type a7 nAChR creates

significant difficulty when attempting to study this receptor subtype in some

experimental systems. This is thought to be because small amounts of agonist leakage

from the application apparatus can cause pre-desensitization of the receptors, thus making

it impossible to record from them. The use of the Xenopus oocyte expression system

circumvents this problem to some degree by providing a large cell with sufficient

tolerance for application systems that are relatively slow and less likely to produce

leakage. In addition, the presence of calcium-dependent chloride currents in the oocyte

produces a secondary amplification of a7-mediated currents at holding potentials that are

more negative than the chloride reversal potential (Miledi and Parker, 1984). However,

mammalian expression systems are hampered in this regard, and are notoriously sensitive

to pre-desensitization by agonist. Careful control of agonist application can help to

overcome some of these problems (Kabakov and Papke, 1998), but the translation of

these techniques to high-throughput drug screening assays has been problematic.

Consequently, some efforts in the area of drug discovery have attempted to make use of

gain-of-function mutant a7 receptors. In particular, the L9'T mutant receptor has been

applied to this problem because of its slower macroscopic decay rates in the presence of









agonist (Revah et al., 1991). While this kinetic feature may make it useful for identifying

responses to agonist when solution control is not ideal, the problem of pharmacology

presents itself in the sense that what is an agonist of the L9'T mutant, may not be an

agonist of the wild-type a7 receptor. By contrast, the T6'S mutation produces a similar

gain-of-function, but has a pharmacology that is far more like the wild-type a7 receptor

than the L9'T mutant, at least with regard to the drugs tested here (Table 4-2). In

particular, several of the antagonists that are converted to agonists in the case of the L9'T

mutant remain antagonists of the T6'S mutant. This has obvious practical significance

when screening candidate compounds in the interest of finding drugs that have a

selective, functional interaction with the wild-type a7 nAChR.

A structural comparison of these two mutant receptors may shed some light on the

molecular basis of these pharmacological differences. Both the TM2 6' and 9' positions

line the putative pore of the a7 ion channel and are nearly one full rotation around the

alpha helix in terms of their periodicity. Both positions are located near the narrowing of

the channel pore, thought to be associated with channel gating (Corringer et al., 2000).

Where the two mutations differ however, is in the nature of their respective substitutions.

The L9'T mutation consists of the replacement of a fairly large, hydrophobic residue with

a smaller, more polar one. The T6'S mutation on the other hand, is somewhat more

conservative, consisting of the replacement of a residue with both hydrophobic and polar

properties with a slightly smaller residue that has similarly mixed properties. Thus, it is

possible that the two mutations share the effect of converting a kinetic state of the

receptor that is normally a desensitized state, into an activated state by virtue of the fact

that the smaller side chains of their substituted residues allow ionic conductance that









would normally be blocked by the larger residues of the wild-type receptor. Where the

two mutations may differ then, could be due to the fact that when antagonists are bound

to the L9'T mutant, the transition to what would normally be a non-conducting state in

wild-type a7, is an activated one in the mutant. However, in the case of the T6'S mutant,

the change may not be sufficiently dramatic to produce such a conducting state. This

interpretation is intriguing since, as the comparison of the a7 T6'S and L9'T mutations

suggests, the binding of antagonists may have structural significance that may only be

revealed once a mutation has been introduced.

An alternative interpretation would involve changes to receptor structure that

would simply cause antagonists to promote the transition to the same activated state that

agonists produce. The previously described hypothesis would require an unobservable

transition into a resting, desensitized state that is converted to an activated state.

However, since we typically think of antagonists binding to the receptor in the resting,

closed state, the L9'T mutation may convert antagonists to agonists by changing the

structure of the receptor so that a transition from a state where antagonist is bound, and

the receptor is closed, is less energetically favored that an open, activated state.

One pharmacological aspect of the T6'S mutant that does differ significantly from

wild-type a7 is in the lack of potentiation by 5HI. Since the mechanism of 5HI

potentiation of wild-type a7 remains unknown, this reason for this difference is unclear.

It is possible that the effect of 5HI on the wild-type receptor is functionally similar to the

effect of the T6'S mutant, in essence suggesting that the T6'S mutant is in a perpetually

potentiated state. This explanation is problematic however. Potentiation by 5HI is

unusual by itself, in that there is little or no effect on the macroscopic kinetics of









potentiated responses compared to unpotentiated responses (Gurley et al., 2000). The

fact that the T6'S mutation appears to have significant effects on the kinetics of response

to agonist suggests that the mechanisms of response amplification for 5HI potentiation

may be different.

In conclusion, the data presented in this chapter show that the a7 T6'S mutation

produces kinetic effects that are distinctly unlike wild-type a7, lending further support to

the idea that mutations in this region of the receptor can have profound effects

distinguishing feature of the a7 nAChR. Furthermore, the relative lack of significant

changes in pharmacology suggest that this mutant receptor may prove useful in the

identification of compounds that selectively affect a7.









Table 4-1. Intact oocyte [3H] MLA binding.
CPM/Cell
Rat a7 wild-type
20 nM MLA alone 143.58 3.9 (n = 5) *
20 nM MLA+ 5 mM 100.43 11.5 (n = 4)
nicotine
Rat a7 T6'S mutant
20 nM MLA alone 168.68 9.9 (n = 5) *
20 nM MLA+ 5 mM 105.94 5.9 (n = 5)
nicotine
Data represent the mean (+ s.e.m.) counts per minute per cell (CPM/Cell) for the
indicated treatments.
* p < 0.05 by Student's t compared to the same receptor subtype in the presence of 20 nM
MLA with 5 mM nicotine.









Table 4-2. Agonist profile com
Agonist


)arison for wild-type c7 and the T6'S mutant.


Wild-type a7


T6'S


ACh EC5o 30 iM 100 jiM
100% agonist 100% agonist
Choline 300 jiM 2 mM
100% agonist 95% agonist
GTS-21 5 iM 3 iM
32% agonist 12% agonist
40H-GTS-21 1.4 iM 3.3 jiM
46% agonist 20% agonist
AR-R17779 10 iM t 30 iM
78% agonist 90% agonist
Tropisetron 0.3 iM ft 0.9 iM
38% agonist 30 % agonist
RJR-2403 240 jIM 400 jiM
16 % agonist 18% agonist
Cytisine 13 iM 43 iM
73% agonist 80% agonist
EC50 values and maximum efficacy relative to ACh for each of the indicated agonists.
* Potency and efficacy values derived from peak CRC analysis published in Papke et al., 2000.
? Potency and efficacy values derived from Papke et al., 2004.
ft Potency and efficacy values derived from Papke et al., 2005.














10 1M
30 nM ----- -
100 'M
300 IM
300 M -- 1
1 [imM -----


2 s A
6s


14
B T6'S mutn I


Wlld-IWi"1 |
30 1 s rrun

25

20

P 15

10


10 100
S [ACh],
[ACh], pM


1000 id


Figure 4-1. The time course and concentration-dependence of a7 nAChR macroscopic
kinetics are altered by the T6'S mutation. Two-electrode voltage clamp
responses from oocytes expressing either wild-type a7 (A), or the T6'S mutant
(B) show the relative effect of increasing concentrations of ACh. The T6'S
mutant responses are slower and the effect of higher concentrations of agonist
on the macroscopic kinetics reaches a maximum, whereas for wild-type a7, no
maximum is achieved. The T6'S mutant shows a slowing of the rise time near
30 IM before the higher agonist concentrations dominate (C). The decay
times (D) also show that the T6'S mutant is insensitive to higher agonist
concentrations while the wild-type becomes increasingly rapid. Note that the
wild-type data are also found in Figure 3-3A.


10 JM
30 M -
100 IM
300 IM
1 mM
3 mM -


120
20s


100
[ACh]. iM


1000 id













30 gM ACh Wild-type a7
- A


15 nA
S 10s


T6'S mutant


B

isn -


T6'S mutant





Wild-type a7
A


a50 1W 150 200S
Time (s)



Figure 4-2. Macroscopic currents evoked by ACh in oocytes expressing the T6'S mutant
carry more net charge than wild-type a7 currents. A) Currents from oocytes
expressing either wild-type a7, or the T6'S mutant. Each current represents an
average from six different cells, all of which received mRNA injections on the
same date, approximately 72 hours prior to recording. Oocytes from the same
injection batches were used to obtain the binding data shown in table 4-1. B)
The same responses as those shown in panel A, but indicating the cumulative
charge for each receptor type illustrating the difference in total charge carried.





























50 ms


Figure 4-3. Single channel currents recorded from GH4C1 cells expressing the T6'S
mutant. Transiently transfected GH4C 1 cells were studied using the cell-
attached patch configuration. The representative trace shown above is from a
cell with 30 jpM ACh in the patch pipette, at a holding potential of+50 mV (-
50 mV relative to the cell interior, applied to the existing membrane
potential). No currents were observed in untransfected control cells (n=6).















100 80 60 40 20


Holding Potential (mV)


Current Amplitude (pA)


Figure 4-4. The T6'S mutant has a slightly lower single-channel conductance compared
to wild-type a7. Single-channel I-Vs were generated and indicated a slope
conductance of 61.7 5.8 pS, slightly lower than the conductance of 91.5 +
8.5 pS reported for the wild-type a7 receptor (Mike et al., 2000). The data
shown here are representative of those used to produce an average
conductance value (n=5).


-20 -40 -60







69



A


T6'S Mutant Open Times
120

100








-0. -0. -4 2 D 02 04 0s0 0 1 1.2 14 16 1. 2

B Log Dwell Time (ms)



N Wild-type n7, scaled

-- '




r T6'S mutant

Wild-type a7

0 50 100 IS10 20 250
Time (s)

Figure 4-5. T6'S single-channel open times are fit by two exponentials and indicate a
prolonged average open time compared to wild-type a7. A) Representative
open time distribution for a 30 min. acquisition period in the sustained
presence of 30 pM ACh. B) The same cumulative charge distributions shown
in Figure 4-1B, including a scaled version of the wild-type a7 current. A
scaling factor was applied that incorporates both the difference in mean
channel open time, and the difference in single channel conductance between
the T6'S mutant and the wild-type.















-----T-^^J'~~'c l ~~-----------






10 pA
10 ms


Log Dwell Time (ms)


Figure 4-6. T6'S mutant burst activity. A) Raw data traces recorded in the presence of
30 WM ACh showing channel bursts. B) Closed time distribution for the T6'S
mutant showing a fit by multiple exponentials. The existence of multiple
closed times is an indicator of channel bursting. To identify bursts, the critical
time threshold (trit) for identifying closures within bursts was determined
using the method described by Colquhoun and Sakmann (1985).
















120




Bo

50

40

20















0


Log Duration (ms)


Number of Everni in Burst







Figure 4-7. T6'S mutant burst durations and number of intraburst openings. A) The
average burst duration distribution was best fit by two exponential
components. Since wild-type a7 receptors show little burst activity under
steady state conditions, this represents a significant effect of the T6'S mutation
on receptor kinetics. B) The number of bursts with intraburst openings
greater than unity, indicating a significant probability of the mutant channel
re-opening.


Bursts with openings of n > 1







2 3 4 5 B 7 B 9






72


A B
12 1-2




W 004-
0.8 0 2S







[ACh], KM [ACh], pM

Figure 4-8. Peak and area CRCs for wild-type and T6'S mutant nAChRs. A slight
reduction in ACh potency (see Table 4-2) was observed for the T6'S mutant
versus the wild-type a7 when comparing net charge CRC analyses. The
decreased discrepancy between peak and area analysis can be seen for the
T6'S mutant, illustrating the reduction in concentration-dependent
synchronization of channel activation. Each data point represents the mean
normalized response ( s.e.m.) obtained from at least four oocytes.












A


0

Of


a
0




z


10 100 1000
C [Choline], RM
C


0 T6'S mutant
-O- Wild-ype7




- /


0.1 1 10
'AR-R17T'9l gM


100 1000


1.2

1

a 0.8
0.6

0.4

0,2

0


-e- T6'S mutant
--Wild-type 07









0.1 1 10 100 1000
[GTS-21], iM


S/^


0.1 1 10 100 10(
[Tropisetron], pM


Figure 4-9. Selective agonists of the wild-type a7 nAChR. Several a7 selective agonists
including choline (A), GTS-21 (B), AR-R17779 (C), and tropesitron (D)retain
their agonist activity for the T6'S mutant. Concentration-response functions
for the indicated drug for either wild-type a7, or the T6'S mutant. Some
agonists show potency and efficacy differences (see Table 4-2), but these are
relatively moderate and consistent with the potency shifts seen with non-a7
selective and nonselective agonists, including ACh. The wild-type rat a7
CRCs for AR-R17779 and tropisetron are both used with permission from
Papke et al., 2004 and Papke et al., 2005, respectively.


0.6

S0.4

o 0.2
z

a


1.2

1

a 0.8
0:
0 0.6
aN
0.4
z
0,2

0


--i- T6S mutant
-c- Wild-type a7
















08

0 .7

E o6

1 0.5


E
S0.3
z
Zi


SAntagonist + 100 pM ACh


Bicuculline 5-HT Tubocurarine Hexamethonium
(10 pM) (20 IM) (1 RM) (100 AM)


Zinc
(10 nM)


Figure 4-10. Antagonists of wild-type c7 are also antagonists of the T6'S mutant.
Several known antagonists of the wild-type receptor have been reported to
function as agonists of the L9'T mutant (see text). These same antagonists
retain their inhibitory properties when applied to the T6'S mutant. Each mean
and s.e.m. represents data obtained from at least four oocytes.












1 mM 5HI + 30 [M ACh


12s


* Wild-type a7
T6'S mutant


Figure 4-11. The T6'S mutant shows minimal potentiation by 5HI. A) The allosteric
potentiator, 5HI has a small effect on the T6'S mutant, but this is substantially
lower than that observed for wild-type a7. B) The T6'S mutant potentiaion
was statistically different from control (p < 0.05) and from the potentiated
wild-type a7 (p < 0.0001), by t test. Each value represents the mean and
s.e.m. for at least 4 oocytes.


30 [M ACh
n









5[A] k + 4[A] k + 3[A k+ 2[A] k + [A]k+
A+R 4.- AR A2R A3R = A4R = A5R
k- 2k- 3k- 4k- 5k-
ii i. 2 ,3 It I4
a+ 1 d +2 1 4

di d+ d2 + A2R* A3R* A4R* A5R*

d3 d3 + d4 d4 + d d5+ d +
Sk4[A] k + 3[A]k + 2[A]k + [A]k +
A+D AD AD 3k- A3D D A4D A5D
k 2k 3k- 4k 5k-

Figure 4-12. Simplified kinetic scheme for the a7 nAChR. A Markovian kinetic model
allowing for greater open probability with partial agonist occupancy for the
wild-type a7 receptor. Note the agonist concentration-dependency for both
the forward binding and forward desensitization constants. The effect of the
TM2 T6'S mutation may be to alter the rate constants moving in and out of
desensitized states, or it may result in the conversion of a bound, closed state
to an open one, or some combination of these effects.














CHAPTER 5
GENERAL DISCUSSION

The diversity of nicotinic receptors provides a rich and complex substrate for

investigation, and their importance in a variety of physiological processes and

pathological conditions makes their study that much more significant. Despite the fact

that nAChRs are among the best characterized of the ligand-gated ion channels, we still

suffer from significant gaps in our knowledge of exactly what their normal functions are,

how they may be involved in a variety of disease processes, and how drugs that target

nicotinic receptors may be used to treat these illnesses. An understanding of the

structural underpinnings of the various properties which distinguish the different major

subfamilies of nAChR will help to narrow some of these gaps.

Both the number and variety of the effects of the T6'F mutation were quite

remarkable. The effects ranged from those with possibly more obvious explanations (i.e.,

the loss of divalent ion permeability) to those which are likely to have more complex

mechanisms (i.e., succinylcholine agonist effects). These wide ranging effects are taken

to support the notion that amino acid sequence at this position plays a prominent role in

the phenotypic qualities of the members of the major nAChR subgroups.

The studies described here are aimed at helping to clarify some of the structural

features that help to define and distinguish representatives of the major subfamilies of

nAChRs. As always, there are limitations that should be considered when attempting to

interpret these findings. The effect of both the T6'F and T6'S are interpreted as the result

of single amino acid substitutions. While it is possible that the presence of the mutation









in a single subunit is sufficient to produce some or all of the effects described here, all of

the experiments were presumed to have been conducted with homopentameric mutant

receptors. It may be possible for future experiments to address this issue by co-

expression of the mutant subunits with wild-type. Other groups have conducted similar

studies and shown intermediate effects of mutant a7 receptors (Palma et al., 1997), so it

may be that partial effects could be observed for the 6' mutants under similar conditions.

Another potential limitation specifically related to the T6'F mutant, is the fact that it

was never possible to express the channel in a mammalian cell. Although the Xenopus

oocyte system is generally quite good in terms of its pharmacological fidelity when

compared to mammalian expression systems, there are some qualities of the cells that are

somewhat different, including the endogenous calcium-activated chloride currents

(Miledi and Parker, 1984). However, given the apparent reduction in divalent ion

permeability for the T6'F mutant, this is unlikely to factor into species differences in

expression systems. It may also be possible for future studies with the T6'F mutant to

explore the single-channel properties of the receptor, given the well-established methods

for using this approach with oocyte membranes.

There are some potentially interesting future studies that are implicated by the

findings described here. One such avenue involves characterization of the effect of the

T6'S mutation of toxicity/cytoprotection. It is known that activation of a7 receptors

within an optimal timing/concentration range is cytoprotective in vitro (Li et al., 1999).

It would be intriguing to see the effect of prolonged activation of the T6'S mutant with

what would normally be cytoprotective agonist concentrations. It is predicted that these

signals would become cytotoxic in this case, either directly though excessive calcium









influx through the mutant receptor, or via secondary activation of calcium pathways. In

these cases one would predict that a significant shift in the potency of cytoprotective

stimuli would occur, if cytoprotection via channel activation were possible at all.

Furthermore, the T6'S mutant is likely to have continued usefulness as a screening

tool for those working to identify agonists that are selective for a7 nAChRs. In this case

it may be very advantageous to develop a cell line that stably expresses the mutant

receptor. The T6'S mutant receptor can become activated in the sustained presence of

agonist (unlike wild-type a7), yet reproduces wild-type a7 pharmacology with great

fidelity. Given the nature of high throughput drug screening methods and their relatively

poor control of agonist solution switching, this would permit more a practical application

of the unique qualities of the T6'S mutant to these kinds of drug development methods.

These studies have helped to shed some mechanistic light on why members of

different subfamilies of nicotinic receptors are functionally unique. Ideally, they will add

to a background of information that can be used to further our understanding of the

specific roles these receptors occupy in their respective physiological contexts. As the

importance of these receptors in various disease process becomes more clear, this

information will also aid in the creation of medicines designed to alleviate human

suffering.















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BIOGRAPHICAL SKETCH

Andon Placzek was born January 3, 1971 in Samia, Ontario, Canada. He moved to

Venice, Florida, where he attended school and eventually graduated from Venice High

School in 1989. Four years later he returned to school at Manatee Community College in

Bradenton, Florida, and later went on to the University of South Florida in Tampa where

he eventually received a Bachelor of Arts degree in Psychology in 1998. It was here that

he developed an interest in physiological psychology and began to seek out laboratory

research positions that were increasingly oriented toward cellular and molecular

neuroscience. After beginning graduate school at USF in the College of Medicine he

eventually transferred to the University of Florida in Gainesville, and continued his

studies in the Laboratory of Dr. Roger Papke where he was able to pursue his interest in

ligand-gated ion channels.