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REGULATION OF ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR
FUNCTION AND PHARMACOLOGY BY AMINO ACID SEQUENCE IN THE
SECOND TRANSMEMBRANE DOMAIN
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
Andon N. Placzek
This dissertation is dedicated to my family.
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
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
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
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
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
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
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
Andon Nicholas Placzek
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
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
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.
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:
Es= R n [S] (1-2)
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
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
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
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
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
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
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.
timecourse vs. a7
(but not MLA)
timecourse vs. a7
I _I I I
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 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.
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
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 = a + ( ) (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 ECso were all unconstrained for the
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
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.
THE PHARMACOLOGICAL AND KINETIC EFFECTS OF THE a7 TM2 T6'F
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.
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
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
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).
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
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
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
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
Table 3-1. Intact oocyte [125I]a-Btx binding.
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).
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 T6'F mutant
a7 T6'S mutant
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.
50 nA T6'F
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
3 lM F---.M
J 20 nA
- WldFtip ,7
10 100 1000 W1
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
100 pM P -
1 mM --
I-- MusfT |FI
10 100 1000 ID'
1.2 r 12
Area 1 ---Area
c -u- Peak I --- Peak
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.
0 (low Ba2+)
-40 -30 -20 -10
10 20 30 -40 -30 -20 -10 10 20 30
SHolding Potential (mV) /. Holding Potential (mV)
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
ct7 1 20 nA L
3 pM ACh 100 pM SuCh 3 M ACh
TM2 T6'F nA I
20 nA K
1.2 -0-y6 ACh
-- TM2 T6'F
0.1 1 10 100 1000 104 0.1 1 10 100 1000
[agonist], pM [SuCh], iM
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
3 PIM ACh
200 nA I
10 10 nA 1
C D 11
F 37 Wild-type u7
Wiv Muscle (ai yb)
g t T T6'F mutant
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.
THE a7 TM2 T6'S MUTATION: A GAIN-OF-FUNCTION WITH a7-LIKE
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
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
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
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).
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
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.
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)
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)
Data represent the mean (+ s.e.m.) counts per minute per cell (CPM/Cell) for the
* 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
)arison for wild-type c7 and the T6'S mutant.
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.
30 nM ----- -
300 M -- 1
1 [imM -----
2 s A
B T6'S mutn I
30 1 s rrun
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.
30 M -
3 mM -
30 gM ACh Wild-type a7
a50 1W 150 200S
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.
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
T6'S Mutant Open Times
-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
0 50 100 IS10 20 250
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 ~~-----------
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).
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
Bursts with openings of n > 1
2 3 4 5 B 7 B 9
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.
10 100 1000
C [Choline], RM
0 T6'S mutant
0.1 1 10
-e- T6'S mutant
0.1 1 10 100 1000
0.1 1 10 100 10(
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.
--i- T6S mutant
-c- Wild-type a7
SAntagonist + 100 pM ACh
Bicuculline 5-HT Tubocurarine Hexamethonium
(10 pM) (20 IM) (1 RM) (100 AM)
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
* Wild-type a7
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
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.
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
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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.