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STABILITY AND i TIONAL STUDIES OF OLIGOMERIC M2 ION CHAN i i IN
SYNTHETIC AND NATURAL LIPID ENVIR(i "MENTS
FRANK JOSEPH RAUCCI
A THESIS P. 7N'-TED TO THE GRADUATE SCHOOL
OF THE 141 *..ERSITY OF FLORIDA IN PARTIAL FULFILL I i T
OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
First, I would like to thank my committee members, .........:- Long, Arthur
r.1..;::: and Thomas DeMarse for their g,....1::. :... advice, and support. I would also
like to thank Randolf Duran and Peter Anderson for their advice .. :: the course of
this :: i: Their 1:: .1. was invaluable. 7 :. I would like to thank all the students
and post-doctoral members of the groups involved in this project for their help in data
:::and analysis. This work could not have been ,: :1: :'.1 without the help
of Kun r,. ... Jun Zhang, Frank :'.K Chris Williams, Brian Dorvel, Joy Wattawa,
and Becky Price. I would also like to thank my .: .. = ... for their love and support. I
would like to thank my friends for their -...... 1. 1 .. :..;I some ..-. i.:. i.. and
I would like to :" r; =: ly thank T. r': =: Cullem and Kaveri Marathe. And last but
certainly not least, I would like to thank my fiancee, Kalyani Marathe, for her love and
1. :.. I :: :::. ,,.:::: :: this very hectic time. I could not have, ::=i l,'",',i this work
'... -,=: .:: t of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of .. i.:.,:: of Science
STABILITY AND FUNCTIONAL STUDIES OF OLIGON.' 7" M2 ION CHAN' iS IN
SYNTHETIC AND NATURAL LIPID ENVIRCT I" TFNTS
Frank :.1. Raucci
Chair: Thomas DeMarse
..D Biomedical 1
The self-assembly properties of the ion 1: ::.:: : i=. 1.. sC ':= .:.: of
the nicotinic acetylcholine :... '. i were examined at the air-water interface. The
... ', of monolayers formed by the i.i '- alone were c ... : .. to mixtures of
M2 with a synthetic lipid, 1,2-diphyanoyl-sn-, ---..'ro-3-phosphocholine (DPhPC) and
the1 1. I and i. i= : 1. alone viaT ::. = : ,=:-Blodgett surface pressure-area isotherms and
Angle *.i:: ::..., ...'. The DPhPC monolayer remains in a .;: : 1- phase until its
a. .w..*... pressure whereas the r monolayer undergoes two distinct :1: .. transitions
. .-.: -. collapse. M2 ion channel fluctuations were studies via tip-d:: .:1 patch clamp
methodologies with DPhPC and native 1 '..1 environments. Single channel fluctuations
were recorded and evaluated to determine the stochastic : .,.. .::.,. of the ion channel
to .'. .1 voltages. The results show that the M2 ion channel is ... .... .1 and
relatively stable. The pertinence of these results to 1:.:: !. ;:::. the .,.:- i. *:! y and
function of this ion channel in synthetic membranes .i il be discussed.
The immediate goal of this project is to study the effects of :. environment
on the -..... .. iles of a synthetic ion channel incorporated into a lipid : ..*. ...... This goal
:::::: passes achieving lipid '.. : ,....; membrane resistances greater than 106'Q cm2,
quantifying channel opening response times, and measuring single-channel current
fluctuations at low channel concentrations. The selected ion channel is the pore-
forming :.. 6-subunit of the nicotinic acetylcholine receptor, which is present in many
biologically relevant systems.
The ultimate goal of this .: .: is to use surface immobilization and
encapsulation within : ..c. mer-modified, tethered .i 2. ,: = ... in order to engineer
thin films of oriented, adds ...:- ., ion, i i The channels would then be used as
small biosensors that could detect minute amounts of a :.!:.. : :... agonist in aqueous
solution. .. : i .**. microelectronic detection methods for channel function might
include ion-sensitive microelectrode arrays (MEA's), field effect transistors (FET's), or
microelectrodes as extended gates of FET's.
The fundamental principles governing ion channel behavior in synthetic
membranes elucidated from our studies could be extended beyond the initial ion
channel being characterized in this work and may thus be..: A ..-. to a variety of
biosensors incorporating biological and synthetic ion channels with myriad functions,
physiological or otherwise. The possibility of improved medical diagnostics for a
number of toxicity-causing agents that affect the ion flow across i ii: ::: membranes in
humans may be realized by integrating the ion channels with electronics. Applications
are also ., : 1 in agricultural and military settings, as both would benefit from
a valuable diagnostic that could offer ::. ,:='.. detection of excessive acetylcholine
concentrations at the nicotinic-acetylcholine receptor caused by .....:. hosphate
*. ,..::: =. or other agonist interactions. Also, such a device could possible be extended
as a potent ,'. ....: ve to tissue harvest or human and animal research in areas such as
. 1:: ....... : :-:... The potential for ::: throughput assays of the i.. :::-: of new chemical
entities on ion channels also exists for functional biosensors.
As the nicotinic acetylcholine ::*: .',: :: is ::,* .. :::. in many tissue .. including
neurons and muscle, ... .. .. 1 ....: how the channel mechanism is by the
surrounding ~* .1 environment could also have a more direct medical :: : ::. Nicotine
and several anesthetics are numbered .......... the known ,.. .....: and ...- i of
nAChR which makes this :: ,*:: an attractive model for st ::: -. ':- transduction.
Ion channels :A -..' an >..:. : :1 role in many 1- -:1. *.-:1 functions, ..:: 1::: .::: nerve
and muscle activity (Hucho and Wiese, 2001). Virtually all membranes in 1: .. cells
. .. .' a *...= : .: ::: of potential across them due to an ::: ::. : .:1 distribution of ions
in the intracellular and extracellular domains. This .. ... separation creates voltage
which is termed membrane potential. These potential differences -**: i'::, ::::. yet
distinct roles in different biolo'.-.-,1 cells. For instance, nerve cells use -- ....... .: .
.:1. called action i: ::::1 ;.:1'. that travel the length of the nerve axons to communicate
with other nerve cells at ......, tic .::... !: n ms. The value of the potential difference across
the .. membranes of any cell is :..- : by the Goldman-Hodgkin-Katz Equation,
which is a derivation of the Nernst equation. This equation .' .....- on the relative
permeabilities, Px, and the :: !, -::: : 1 [X]i, and external, [X]e, concentrations of the
respective ions present as well as the temperature and the gas constant, R, and the
Faraday i ..: :: =' F (Hille, 2001). Note that unlike the Nernst equation, the zero-current
potential in this case is in fact non-zero and represents a steady-state movement of ions
away from equilibrium.
RT i [ ..:]e + PK [K+]e + PcI [Cil]i
E = ---- In ------------------------------------------
F PNa [Na']i+ PK [K+]i + Pci [Cll]e
The1 i 1 :: 3 .: ; of plasm a m em branes .:: -. .ide a ...:.:= .. .:: ..::. .. barrier
to the movement of i.... ...-. particles from the intracellular to extracellular domains
and vice versa, thus : ::... ":::. for the establishment of these membrane .. ,.,.:I: :. In
order to 1.. .. ..1 ions between the two ionic reservoirs on either side of the plasma
membrane, the cells make use of -..' = : .. gated transmembrane proteins, known as
ion channels, to ... *i.*..- the ion currents.
Ion channels have many distinct features that make them of interest for
.:. and physiolo '. study. The first characteristic :... ... i. of ion channels
is their high ion selectivity. Channels not only show selectivity between anionic or
cationic molecules but also between monovalent and divalent ions or even for a specific
ion, such as H+(Hille, 2001). Secondly, ions channels are dynamic proteins, which
demonstrate identifiable responses under specified conditions. At resting potential,
the channels are completely closed and impermeable to ion current flow. However,
they can be opened, or gated, by either a change in the i:: .: potential or the
binding of i ..... = ligands (Hucho and Weise, 2001). Thus the two important
functional components characteristic of all ion channels are a selectivity filter that
determines which ions will be allowed to :: :- -" through the channel pore and a
gating mechanism that controls the opening of the pore. The two major classes of
ion channels are described by their gating mechanisms and thus all channels can be
classified as either voltage-gated or ligand-gated. Ligand-gated channels undergo a
conformational :: ..,. when a ; :. 1 : agonist binds to a receptor site. Voltage-gated
channels .: :::V .ii undergo a conformational change within the lipid I::. ::: i.:::- due to
a ;... .. .', usually polar si .. .....! of the structure..: ......, within the electric field
between the two lipid layers of the membrane bilayer (Hille, 2001).
,, -.otinic Acetylcholine Receptors
Channels are further classified into families and ".: ,-::: based on
functional and .-... i:. characteristics. There are three superfamilies of :. .*
gated channels: the ATP-gated purino receptors, the ion, !:. .::'. glutamate re(, :
and receptors that are similar to the nicotinic acetylcholine .. -- j.*., 2001). The
last ..: ::: which will be our primary focus, includes the nicotinic acetylcholine
receptor (nAChR), as well as glycine receptors, GABAA receptors, and some serotonin
receptors (Hucho and Weise, 2001). These c..::: :: .. *. have s:::: :: ::: sequences and
many conserved str :.: ::.: features. They all share s:::::: i.:' glycosylation patterns,
.-: L-. peptide loops created by disulfide bridges, similar distributions of prolines,
and conserved serine and threonine polar residues in the channel-forming domain
(Hucho and Weise, 2001). The most important feature shared by all of the receptors in
this superfamily is the presence of four i:: ...:.... i::..I amino acid sequences that span
the 1:1 : :::: .: i::, ::: :: ... labeled Ml through M4.
The pore of the nicotinic acetylcholine receptor (nAChR) was the first channel
pore to be characterized : : =: mutagenesis. These studies identified the residues of
the pore-lining segments and also i ::.: .,: :- :: ,d the selectivity filter function of the
channel was described. The functional form of nAChR is <::::: posed of five homologous
subunit proteins -, !::::.. : each i:::..- the four hyd :. i.::.i : transmembrane segments (Ml-
M4) that are characteristic of their superfamily. The nAChR is found in many cells,
including those responsible for signaling in neuronal and ::::: ::.:; tissues, and can be
composed of slightly different homologues. The stoichiometry of the nAChR varies
significantly depending on where the channel is found. In muscle tissue, nAChR has
a ... :: ..:.: of c2'3P'Y'6 while neuronal nAChR is a : ..:: i;'. ': ... ::"': : of a
subunits (Oblatt-Montal et al., 1993). As its name ::: :. ;* .. the nAChR is a 1 :.:::: -gated
the sequence of this s .. ....... is
different *.:'. The actual :: :. : :::: .: .i:. : :::: is small, only 23 to 25 amino acid
residues in 1. ... ...., and is modeled as an a-helical secondary structure based on electron
microsc(. studies of intact nAChR. This structure has recently been confirmed for
lone ... segments self-assembled in lipid ...: ..... i by two-dimensional solid-state N'..;'
(O=-11: etal., 1999). The '.' segments are also :::::: = 1:,: ::: in nature and they come
together at a ..-.. ,.... to form the funnel-like pore (Hucho and Weise, 2001).
The h- :'. 1:: : portions of the helices face the lipid :: 1 ::' and act to anchor the
... :.-. in the bilayer while the hyd ....... portion lines the pore region. When gated
open, there is a conformational, :: ::. .. in the orientation of the M2 segments, .
permeation of ions t= *..... the pore. The channel is selective for cations although it
does not discriminate between monvalent cations such as K' or Na' and even allows
permeation of Ca2+ to some extent (Hille, 2001). Studies have demonstrated that the
vestibules of the nAChR channel are :. '.' -: '1 : i:... :.. : .. the observation of
0.: ... -,. ..'.. .. ;'. Studies :: : -:"' nAChR samples from the electrical organ of T...'.
S.. .. and bovine calf muscle tissue have shown that the 6-s. '::: is the sole
determinator of the conductance \: .. .:=: :: of the nAChR in the :: ; = tissue ,
(Numa, 1989). Thus, when bovine a, [, and y subunits ;T: a T. .. 6-subunit
were incorporated into the bovine muscle tissue, there was a ::: : decrease in
conductance than when bovine 6 and T ... '. .... a, p, and y subunits were used.
. i::: ithe main portion of the i: I a-helices that form the ":' itself are ::.:..::. d, the
segments have, i: :: -.1 residues at either end which form -- rings of a sort when
the peptides are assembled into a pentamer. There are three : .:::- rings *:. -, : in the
full :. =..=:, one on the extracellular side of the "' T: sequence, one on the intracellular
side, and one intermediate within the actual sequence itself (Revah et .' 1991). It has
been shown by systemic mutation ...: ::. -: -= .- that channel conductance is diminished
as the net: 1:.:: : of one of these : ":::. .. becomes less ::: ..:!.:*ve (Imoto et al., 1988).
there were several ., ...:..:.. made in -.- ...... ... these structures. The solution
NMR structure differs from that of the solid-state structure in that it shows a kink in
the helix at leucine-11 (Breed et .:. 1996). This residue is thought to be important in the
: of the channel.
The.. -.:-1 ....:. nature of the individual *-- ;..1-: is I .1 A- to be the .
force behind the self-assembly of M2 helices into voltage-gating channels. Looking at
the sequence of the M2 6-subunit we ,'* ." .: see the: ........ of the individual amino
acid residues .'1: -.' an i:: =::: role in the function of the channel. The ..: ..
particularly the OH-i ..... ; serine and threonine residues, line the pore and play a
role in ion affinity and stabilization. The ::::::*. 1,::, b. .:1:::i;V: amino acid residues in
the sequence act to 1 .. -. the protein in place in the 1: 1 environment, which leads us to
......" :: : the effect of ::.1 selection on channel ]:
S:.:.... are biological molecules that are insoluble in aqueous solutions and
soluble in .... solvents. Lipids serve not only as structural .. ......... of biological
membranes but they also :: :, reserves in the form of triacy; 1'..-:v'ols
and serve as vitamins and hormones in vivo. The membranes made up by 1 .:- are
assymetrical, fluid structures ..' 1 of fusion and are selectively permeable (: ::
et -.... 2002). They enable ....:. .>:nm of cellular or intracellular functions which by
variation in '. and protein .:::::: position, including the transmembrane proteins that
form ion channels. The .. of membranes are fatty acids, ..." .......
sphingolipids, and cholesterol. Phospholipids form the major ::1 ,'* part of biolo-. i:- 1
membranes and are composed of two fatty acids plus a .'.. : .'1 .. attached to a
glycerol. T ::.. I1: :: ::'. are like I-i '.ycerides .. .'Ithat the first hydroxyl of the
S.....molecule has a polar phosphate-u .. .. ... in place of the ;.:: acid.
This means that '1:: 1:::. i have ab f.1:. 7: head and 1: -.1::: '1::- i tail. This fact
forms the basis for the formation of self-assembled .... :.'. bilayers or micelles
in aqueous environments. The head group has a ...... that is specific for different
..1 1 ,. 1'... This head group will differ between cell membranes or different
concentrations of ... .. head groups. The .' y acid tails can also differ in :th and
,= :, but most usually have one saturated and one unsaturated leg of the tail.
. : ..... ....:.1 are a s'. of phospholipid derived from .. .... instead of glycerol.
Cholesterol is a :: ": :: :: : :: :::,.:. of cell :::: : :1.: :,., '. : .. ::1y those of eukaryotes,
and intercalcates between the fatty acid 1i ..'.. thus I ........them :..:>.. and
increased lateral fluidity (P= *:::.-= -zu et al., 2002). Cholesterol also makes the membrane
less permeable to small, water soluble molecules and it has been shown to thicken
1.... *" .... .= =. .:.= upon insertion.
With ..... to the nAChR, it has been shown that the optimal channel function
... .. .... the '=. : ... of negatively- .. .... .1 .,. 1. and cholesterol (Sunshine and
McNamee, 1992). For this project, *1: 1...'tanoyl-based lipids were chosen as they work
well for '.1 =::: ..: systems, such as the ones we are using, as well as the fact that they may
allow for more highly resistive *... .... formation than natural lipids due at least in part
to the :: ..:::: methyl groups on the fatty acid chains of the synthetic !:'. .'- They
are also well-suited for the -.. ..........- that will eventually be used to increase
stability in the electronic devices. The synthetic 1 !: I.. used in all =,.= =::==::: :::... was
the diphytanoyl derivative 1,2--. .1.A tanoy! -: .-. ycero-3--........ 1I.1:.. (DPhPC)
:,= :.. by Avanti Polar r .". whose structure is .:.::: to a native in F>:i .
3. As can be seen, DPhPC is ..... .i.. different from the native 1- associated wth
nAChR. Thus we undertook a thorough study of the channel f:: :::;... characteristics of 'V."
in DPhPC in order to establish the feasibility of I ... .. .. the lipid environment for i :.
sensor d. .L::: without ,:::":; :::::: ..::: the functionality of the ion channel.
Electrical Properties of Membranes
Figure 3: Comparison of Synthetic and Native Lipid Structure. Notice that the
fatty acid chains in POPE (a) are unmethylated whereas the synthetic DPhPC (b)
is methylated contributing to the additional rigidity of the bilayers they form.
Electrical Properties of Membranes
When attempting to measure the ease of current flow across a membrane,
whether due to pores in the membrane or functional ion channels, it is important to
understand the physical preasinciplg the governing the phenomenon. The ease current
flow is referred to as conductance and is usually measured between two conducting
electrodes in a salt solution. The conductance is governed by Ohm's Law, which states
I = gE
where I is the current, g is the conductance, and E is the potential difference across the
conducting material, in our case the ion channel. The conductance can be affected in
many ways by either changing the properties of the environment or the conducting
material. Conductance is increased by increasing the salt concentration using more
mobile salt ions, or decreasing the distance between electrodes. Conductance can be
decreased by introducing a nonconducting material between the electrodes, moving the
electrodes apart, or by increasing the viscosity of the ion salt solution. In the case of the
membrane ion channel system, the lipid bilayer acts as a barrier to ion flow and thus
decreases the conductivity or reciprocally increases resistivity of the system. Another
important consideration is the fact that a ? 1 bilayer membrane has a .'.... :- effect.
It :1.:.. 's the role of a dielectic insulator between two :.1:::. !. : ., which in this case are
the separate salt solution reservoirs on either side of the membrane. It is necessary
to consider this ,:: ,:. :...' because it ",.. a central role in the determination of the
number of ions that must be transmitted for a current ..I to be observed at the
membrane (Hille, 2001). The < ..: : :: I :':., is defined as the amount of : 1:.:: : that must
move from one reservoir to the other to set up a *.. potential:
(where Q is the *.... transferred, E is the potential. *. ...... and C is the
i .! .:... .*) The rate of change of potential due to current flow, In, at the membrane is
The capacitance is relatively constant for a .. ::: membrane environment. In terms of
m em brane :: ....: .. ...the : '..::. of their1 ..i .. .... 'is given by the .; ....
C = KEoA/d,
where K is the dielectric constant of the given lipid, Eo is the permeability of free
space, A is the surface area of one of the lipid layers and d is the distance from one
side of the bilayer to the other. The ... ..:.. of cell membranes is estimated to
be ::. ::':: ,:i.:y 1.0 pF/cm 2 and the ,,: :. :..:::..:* for a ::, native! *:, bilayer is
Sh-ttiv less at 0.8 pF/cm2 (Almers 1978). From these measurements, we can estimate
the thickness of a i ,'..1 bilayer to be y: '"::: .... 1:"y23 A, assuming a dielectric constant
for hydrocarbons of 2.1 (Q -.. 2001). The general effect of membrane capacitance is to
slow down the :.'.,; '::" to any current by a time, T, that '. :::. on the resistance and
capacitance of the membrane. Thus we can .- the i : 1.. .. rate of the membrane
,.. :: a s:
dE/dt = I/C = -E/RC = -E/T
Solving this differential equation gives us an expression for the potential across the
membrane as a function of time, t. Given an initial potential, Eo0, this relationship is
E = Eo exp (-t/RC) = Eo exp (-t/x)
The time constants for the different biological membranes and lipid bilayers can vary
significantly, thus the resting membrane resistances can vary in order of magnitude
from 10 to 106 Q (Coronado and Latorre, 1983). Thus the number of ion channels open
at rest varies significantly from membrane to membrane depending on the properties
of the lipids and channels involved. Now we have all of the components to look at the
Figure 4: Circuit Model of a Membrane. The electrical properties
of a membrane can be modeled using the conductance, gi, of ions
crossing the membrane with a membrane potential, Ei in series.
These elements are then in parallel with the capacitance Cm, set up
bilayer/channel environment as a circuit-like model. If we take the ion channel to be a
conductor, with conductance, gi, for a particular ion, i, and the membrane capacitance
and electromotive force of the pore to be Cm and Ei, respectively, then the system can
be shown to behave like the following equivalent circuit in Figure 4. The driving force
of the specific ions, I, is not simply related to E, the potential across the membrane, but
instead is determined by the difference of the total potential and the potential at the
pore. Thus the current i::.. of I through the i1:.:::::!. ,/membrane system is *,= =.-..,.... as:
l = gi (E E)
While this is adeq uate to a first :::.. ::: :..= many channels have been observed to
have non-linear current-voltage relationships when open. This non-ohmic behavior can
be due to ionic gradients across the membrane or when the structure of the ion channel
itself lacks symmetry. By looking at current versus voltage (I E) :::*.... a significant
amount of information about the ion channels being observed can be gleaned. For
S if the curves are linear and pass through the origin, then it can be inferred that
either the channels are nonselective if there are multiple salt ions in solution or that
there is no g: .. 1:. :: : set up across the membrane. If the slopes of the I E curves of a
given system :..::.. = :. it corresponds to an increase in the *:: :: '.-alent conductance and
thus an increase in the number of channels in the open state (Hille, 2001). Conductances
that show a dependence on voltage are said to undergo rectification. An environment
that shows strong rectification : : :.: il has channels that are open for some groups of
potentials while remaining closed at others.
i TRIALS AND ".. i i i i )DS
The synthetic oligomeric M2 peptide corresponding to the 255-277 segment
(F-... T->TAISVLLAQAVFLLTSQR) of the 6 form of the T .. 7.', >7 .. AChR was
S.*:: .:::: :. via solid-phase methods. The 6 form was selected due to the fact that it is
well-characterized (Oblatt-Montal et .! 1993, Opella et 1999). Solid-phase chemistry
allows for a much less time-consuming synthesis as isolation and purification is not
required at every step as in solution-phase chemistry. The synthesis was carried out
with PEG-polystyrene resins under continuous flow conditions using FMOC chemistry
by the University of Florida protein core facility. The C-terminal end of the peptide
with the protected first amino acid residue in the sequence is pre-loaded onto the resin.
The next amino acid in the sequence, which was also protected, was activated with
an acid fluoride and added to the F,.. )C-deactivated segment loaded on the resin. A
peptide bond was thusly formed between the two amino acids in the chain. Then the
excess, unreacted amino acids were washed from the resin and the activation and
deprotection steps were repeated with each subsequent amino acid in the sequence
until the full peptide was formed on the resin. A schematic of this .: .. is shown in
I it was necessary to cleave the N.' 6-subunit peptide sequence from the
resin. The first step in this process was to wash the resin with .. ....: ... 1..- 25mL
DCM for each gram of crude sample resin through a small sintered .. ... funnel. The
resin was then carefully transferred to a round bottom flask where 10mL of cleavage
reagent (90% trifluoroacetic acid (TFA)/ 5% triisopropylsilane/ 5% water) for each
IntrducliDn of auino add with Resin pre-l ded wilh first
X2 aldB-ctAn protecting group amino add In asquen:e and
aide-c-ianr pin&c.ng gmrup X-
H H 0 H H 0
I I I| I | I I1
FMOC -N-C -C- OH + Adlastor Piperidlne+ FMOC-N- -C-G-O- )
I I RqRein
Acfvualin ie prtaclion
FMOC-N--C- C -Activator H2N-C-C 0---
I I I
H H 0 I, Cleavage H 0 H 0 H F
---C-C --C--C--0-( ---HNN -- N-C--- HN--C
I IAmn I II
R2 R1 Rn R2 R1
Figure 5: Schematic of Peptide Synthesis.
gram of crude resin sample was added. All spatulas and other transfer tools were
washed into the round bottom flask with the same solution to ensure as high of a yield
as possible. The flask was then 1- .. ....1 and stirred. A small stir bar was ) 1.- *1
inside the flask to ensure :: while the reaction occurs. The solution was allowed to
react for ,. ...... :. ... !y three hours.
The mixture was then filtered : : =:: ': a sintered funnel and was then
washed with ImL TFA. The total filtrate was concentrated under vacuum at 68C to
: ::: ly 2mL. The concentrated solution was slowly transferred into two Falcon
tubes..:: .. : ": ethyl ether at -20C. The solutions were then i:- : ..... d,
y:: 1::. pellets. The ethyl ether was then decanted and the :.... .... was repeated three
times. -..* ..... .: the washes, the ethyl ether was removed under '1.'- vacuum and
then 1 .:.,.. under high vacuum in a desicator for at least two hours.
Channel Purification and Production
The cleaved peptide was purified using an Akta Purifier (Amersham Pharmica Biotech)
reversed .1..: high ?p r-, liquid .-.. .:.. (RP- : ':*:. .. apparatus equipped with
a silica Cig column from Vydac (see Figure 6). This method allows for the separation of
peptides with r: :.:::.:* that. 1 by as little as one methylene :....,. on a single
amino acid (Carr, 2002). Unlike small molecules, peptides are too large to partition
in the hydrophobic j.L .. -: stationary phase. Instead, they must be .:. i ..i .. '. to the
hydrophobic surface of a column. An absorbed peptide remains on the column until
the concentration of .. i organic modifier (:: ,:.: acetonitrile) reaches the c :::, i
value needed for A.' *::: .::: This critical value is know as the Z number and was first
S.... :i .- by Geng and Regnier. The Z number has an extremely narrow region of
concentrations that define it and it is very sensitive to the conformation of the protein
being I: ..i Once desorbed, the peptide interacts very little with the hydrophobic
surface as it elutes off the column. In RP-HPLC, the peptides are !.. .1 based on
slight differences in the "hydrophobic foot" portions of their sequences and structures,
which is the only .. .:.. of the 1.. .' in contact .. :. the hydrophobic surface of the
column. The foot is* : ::..-y correlated with the Z number as it is the only ::-;:: of the
... ;:! that is involved in the interaction with the ..... modifier. In fact, the :-
location of the bf. .l: : : residues in a helical ...*'.1: are :::. ::::, in .:
the retention time of that.:- :. :.-., in the ..... modifier. The .' ........ /desorption
step occurs only once for a .. ::: .:i: .. on the column (Carr, 2002). Since ,: .. "1:"
are very sensitive to the concentration of ... modifier present, elution gradients
are usually set up to maintain ::... ::.:. : ::: The 1'::1. :, :' ..1. are sensitive to
the concentration of.... .....:. modifier used. Relatively small concentration : ....
in the acetonitrile cause large i: .:::: : in the retention time. The : ::: modifier
concentration must be maintained precisely and isocratic elution is difficult due to the
sensitivity of the '::I', ':I : to 1:: : :: :... in the concentration of the modifier solution.
Therefore, for RP-HPLC separations, gradient elution is ... r ..-. even with small
...:.... in : .:::. modifier concentration per unit time. Thus a hybrid ...
method is indicated: small .." ::- .. desorb more quickly with small i .... in modifier
concentration than small molecules, but more slowly than proteins. In a helical 4': :
the exact location of hydrophobic residues .... .' ; influences .... ...... ;. ...'
retention (Yang et al., 1992).
The hy ... .1.1. .-. surface ... which the pol. A.- adsorb is the HPLC
i1::::::: which is a stainless steel tube filled with -. ::,': I: of small diameter.
The particles used are generally silica with -i.r-j.y uniform sites and surface areas,
which have been modified for use as adsorption agents by silane : .-:: .. : ::.. to make
them hy'.- .1.....:1.. The smaller the diameter of the adsorbent j.... :1. the ..
the specificity and resolution of the peptide fractions eluted from the column. The
adsorbents used are porous in nature and the .. 1 .. :.1. 1 enter a pore to be adsorbed
and : ': ... :: .:'.. RP- HPLC has been effectively used for i:: Ii' 'i,';:,. ',' :: : ::: since
ore silica :,. .:'1- of about 300 A were .. .. ed. Now this is the size most
commonly used, but some smaller .'. can also be .- : particles of 100
A (C.--, 2002).
L :::. a linear .: ..: hydrocarbon of 18 (Cls), eight *i or four 1 .: I.
is used as the hydrophobic = P. *!.,. and small proteins of size less than 5,000
daltons are usually separated using Cis columns. In order to :: "-.1:::*.: RP-HPLC
adsorbents, hydrocarbon chlorosilanes with one reactive chlorine are added to the
silica matrix (Carr, 2002). As these have a :*:'.-1- point of attachment with the matrix,
they are called monomerically bonded i.1: : .-. If chlorosilanes -.'. :;1. multiple reactive
chlorines are used, the product is known as a j. 1 t-merically bonded '1:.: .. in which the
individual chlorosilanes are crosslinked and form a silicone }"' Aymer on top of the silica
matrix, .. : multiple 1:::'i .1: chains attached. Monomerically and 5' "..merically
bonded phases exhibit some differences in :.. :': ; ity for ....... ..1 '
even though they have similar hy.L: 1:. *. .. and separation. Mild conditions of
acidic pH and room !.. *,:::== e are ideal for ::= ::= silica-based HPLC but extreme
conditions such as high pH or h.' ::::1 .. ..:: es .. :: ii .: :'.- :' trade silica columns.
Polystyrene-divinylbenzene, a synthetic .. ~ .mer, A.= ides a very sturdy alternative
matrix for separations. Synthetic polymers provide the advantage of not degrading at
extreme 1: ::= in pH. Therefore very acidic or basic solutions can be used to clean
the apparatus. Using very ,!:,:: :. :::. to wash the column does not adversely
the column performance, as indicated by an examination in which -A... '.
chromatographed before and after -..,1:: .. :1:: both strong acid and base had similar
,1 ,: := .. retention, and resolution.
Column: ::...th does not .: :::::: iy the .: .1:.:.. :-:: and resolution of
=.: because both adsorption and I... .. 1 := 'take 1 ..... near the top of the
column (Yang et al., 1992). Short columns (of 5-15 cm) are frequently used for protein
separations. Small peptides are better.- ...= .. .1. on longer columns, and columns of 15-
25 cm are used for the separation of synthetic and natural -:'"1- ., Column: 1::: :" is
S......i .....,. to column back-pressure. While the column diameter does not affect peak
resolution, it does ": -*: ::.:. : ..!:,,-. loading. The detection sensitivity is increased when the
column diameter is reduced, because the flow rate is reduced, and therefore less solvent
is used (Rivier et a.l, 1984).
Aqueous solvents contain an ... modifier and an ion-pair .,... I or buffer.
The ::: :: solvent is used to desorb j" b -" '; ;'~=:' molecules from the adsorbent
hydrophobic surface. This is achieved by slowly ..... the concentration of solvent
until the '::,". ; '."".I."" desorb and elute. This results in the --.1'-: :i iest ... and best
resolution. The most commonly used ... modifier is acetonitrile (ACN) for several
reasons: it is very volatile and therefore easily removed from ": .. .::::: : its low' ....
minimizes back-pressure; it has little UV ..1.... at low wa' : ths; it has ....
to be consistently reliable in RP-HPLC separations (Carr 2002, Y.:: :: et al., 1992).
Low-gradient slopes are.. f- to isocratic elution, as a uniform
concentration of. .::::. modifier i, ; : ... -tably broad '. =:1 (Yang et *
1992). The slope of a 1 :21 solvent gradient is 0.5 to 1% per minute increase in -
modifier solution. To maximize resolution, very -. :,:::. -.. gradients :j:: ::::: 0.5 to 0.1% 0:
...:..1'.. can be used. Extremes in ..... .. modifier concentration are to be avoided in
order to have the best equilibration and reproducibility. Gradients should be begun at
no less then 3 to 5% ...... modifer concentration, and should not exceed 95% ..
modifier. The higher concentration of ::: ::.. modifier may remove all the water in
solution, thus .. ..1 .: column ..... more difficult (:r'., 2002).
The slope of the solvent gradient may S=q: = .= '.'.- s.. K.. Iy and resolution
between i .*.. .- because of the slight differences in the way that some of the
peptides interact .. .1: the reversed-phase surface. The purpose of the ion-i: : ..
.1 /buffer is to set the eluent pH and interacts 0.. T:. the pol' :. -: : to enhance the
... ::: (Hoeger et.,. 1987). Trifluoroacetic acid is the most commonly used ion-
....... ...... because it is volatile and can be .,. ..: removed from fractions, it has
very little UV adsorption at low wa. 1. ...ths, and it has ........ reliable in RP-HPLC
SU. ::-- concentrations of TFA at lower than 0.1% when chromato'-: :.;'1:
proteins may A.!...ade, .'" .'.*" As the solvent environment. ..... from aqueous
to non-aqueous the dielectric constant, 1B .. : .::. :: n-n electron : ... ...
which affect the 190-250 nm region of the ,..... -'- :. ......., and causes a baseline
shift (Rivier et 1984). Adjusting the we.... .: .:::.. .1: as close to 215 nm and putting 15%
less TFA in the ..... .....: modifier solvent than in the buffer solvent reduces or eliminates
the baseline drift due to TFA .... ::.:1 adsorption. Using high-i ::: TFA is also
essential, as low quality or aged TFA may cause the ... of -... peaks.
Po. :..I':1 .,, :: : ..: *:::. are not very affected by flow rate. The eluent flow
rate may affect the resolution of small peptides because their behavior in RP-HPLC
columns is between that of ,::': :'. and small molecules (Y ::::- et al., 1992, Carr, 2002).
Adsorption and sensitivity increase with lower flow rates because smaller volumes
of solvent are used. This is also why narrowbore HPLC columns increase detection
sensitivity: they are run at low flow rates. However, *...,. it increases the amount
of solvent to be removed from the purified.. 1.:::: 1 ', high-flow rates may : :: the
solubility of hydrophobic pol. .. (Rivier et.:' 1984).
RP-HPLC is used to purify mic ..: to :, ,. : .,,,, quantities of I.'i ;'I.1
for research ** For .,... ...- preparation, however, some other
constraints exist as well (Rivier et al., 1:: .J The same ion- ::. : agents commonly
used for analytical ****j. .... are not always as .. :..' for 1..... ..., chromatography.
When t ..;:::. a ..:: :." "... '::, '.:: ::. some factors that need to be considered are:
low .*. : loads yield best resolution, only small ;:....*::. are purified per run; the
of the ::::,,.. is ... ....1 in terms of ,: :: : ,. of total weight of final purified
product; and the yield is the .... ,..- of final purified product per total amount of
'. : ::. -.:::1n in : : .: :. (Y.: :::- et 1992).Higher resolution enables
maximum yield. Thus several factors must be balanced. The three measures of .,
(... ....modifier, and the other between 53-' c......- modifier. 8 mL fractions from
45% to 60% ::: ::::. modifier were collected in Falcon tubes. Then each individual
fraction was placed in a round-bottom =.,. and the ACN and any trace of TFA were
removed using the Roto-Vap under vacuum -.. :i:: a water bath at a .:::,:: ..: ::'e of
approximately 50C and Roto-Vap coolant water at 4C. Once the A. -.... were
concentrated to approximately half their ::: :-;:::.:1 volume, they were removed by glass
-... !!. and put back into the ...... .1 Falcon tubes. For each fraction, the round-
bottom flask was rinsed with deionized water and the water was .'.i --l 'tted into the
... .... .. Falcon tube to ensure maximum yield. If the same round-bottom
flask was used for :1! :. .1,. concentrations, it was rinsed three times ::: ::::: .i;::. with
methanol and deionized water. The ...... x: were then frozen at -30C. Once ..
the : :; '.. of the I:: = :::: tubes were removed, and the tubes were sealed ..,.: 1 :
Then three small puncture holes were made in each parafilm seal and the tubes and
--. w ere : :.:: :! in the glass !- : :: ., : :: container. The ... ::::::, '1: ". w ere 1" :i :
under vacuum at less than 100mTorr until only a white powder remained.
In order to identify which of the :::: ::: :.'.: ". contained the correct ..
the samples were ...... -* for mass ... 1 .. y. First, a solution of ACN with 0.1%
TFA and deionized water ... i1: 0.1% TFA in a one to one volumetric ratio was I::: -: .
A small ..... i*. of the lyophilized *- .. 1 for each major .-1. was dissolved in 90
pL of this solution to yield an -:::: :;: .. concentration of 0.1 pM -. "'!i .1:' Then the
solution was ci: .:: tenfold with the 1:1 ratio of ACN: H20 (both with 0.1% TFA). And
then the diluted fraction was : : ::, diluted with the same solvent. Next a cyano-4-
hydroxycinnamic acid (CHCA) matrix was prepared by dissolving 10 mg sa::::..i- in IX
methanol in 0.5 mL of solvent. The solution was then ..:- .. .- and methanol was then
added until the solution was : :: .:..- 1 Then the matrix solution was centrifuged and
the supernatant was carefully removed. The matrix and the diluted : :: pie were mixed
in a 10:1 ratio. Then 1 of 2 pL of solution for each sample was placed on a specific
location on a MALDI plate. The CHCA matrix was A.... on a few of the MALDI
locations alone for use as a blank. First the blanks were tested to see if good resolution
could be obtained. Then the other samples were tested on the MALDI plate, with the
higher concentration -.,. : :1:" .:. ;:... the best resolution. The results show the ..'
molecular mass of ') amu for the .. -;. collected at .: .:... -. iy 48% .
modifier .1: :::::: the PR-HPLC. The other :.:: i. : :, :1 .. had molecular masses of 2409.38
amu and "'.' ---.31 amu which represented a.. .:.- .... an amino acid residue in the
sequence, most likely valine and 1". : ., '. vely.
To verify the sequence and concentration of the -. :., l-. .1....:: .. samples
of the different ,=. :1 fractions were dissolved in ,: .':: ::.. 1y 100 pL TFE for each 1
mg of sample used. Each .. :.1. was then heated in a water bath at about 40-' .= for
approximately 2 hours. Then the solutions were diluted to 2 mL with H20. Then 100iL
of this dilute solution was further diluted to 1 mL with H20. 100 pL of this solution
was sent to the protein core facility at the University of Florida to have amino acid
analysis performed. Also, this solution was used to obtain UV .. -. 1:.: from 200-400
nm. QuartzUV cells were used because residual p ..1 *. near 200 nm were '.
for blanked solutions when methyl crylate cells were used. The results of the amino
acid analysis were in close .* :'= ::: : for the .1 c. :: ..'..:. .::, of the M 2 :... :i. 1..
The predicted peptide sequence had an average percent error of 12. ::::: .: 1 to
the known ..::..... :..... which is within the error of '.: ."" .* amino acid analysis. The
predicted concentration of the sample was 69.8 -::::,. 1 / pL L or 0.16 pg/pL.
In order to be able to determine concentrations of : solutions with :. : !,-: ease
via UV .:1--:: 1 ..::::: it was necessary to calculate extinction coefficients to be used with
the Lambert-Beer law. The law can be stated as
where A is the absorbance, I is the path length, c is the concentration, and is the
extinction coefficient. Extinction coefficients for absorbances at wa.. 1; ::.. ; of 208 nm,
"1 3:.::: and 220nm were calculated: ':== the UV .=.. =., of a known concentration of
M2 in deionized water. This concentration was determined by amino acid analysis
and the path length for the glass cells was 1 cm. Using the Lambert-Beer Law, the
concentration of all subsequent i. ,i ,,1: was determined. The ::-- :: at 208 nm
was determined to be 6.517 x 10-3 Au pL/1::.::. .1 cm or 2.810 Au pL/pg cm. At 214 nm,
the coefficient is 7 .-'" x 10-3 Au pL/pmol cm or 1.638 Au pL/pg cm. And the values
for the coefficient at 220 nm were 2.244x10-3 Au pL/pmol cm and 0.961 Au pL/pg
cm. Once concentration was determined for a particular ::::: 1: it was then :
..:. ... .. and then i ..... :.. ............ up in an equal volume of 30:70 methanol
('.. -..OH): chloroform (CHC13) solution. The synthetic DPhPC l *i *: T were also':.-:: -1-:
up in solution of the same solvent ratio and then solutions of various molar ....
ratios were .::..'.::,.,1 from these stock solutions.
Circular dichroism .. .. ..! were done .." :. .. i. in different solvents to
determine the secondary structure of '.-" in solution. Solutions of the .: :.: =- 1:1
TFE:H20, 30:70 TFA: H20, 30:70 MeOH: H20, 30:70 TFA: CH( i and 30:70 MeOH: CHC13,
each with and without M2 peptide present were prepared. :::::. : pes of 400piL were used
for each run in a cuvette with cell length 0.1 cm. Samples containing chloroform or TFA
scattered the i= =::=! light such that no useful data could be obtained as a clear spectrum
could not be..i ... =. 1 The molar ellipticity as a function of wavelength was plotted
for the M2 in solution, for : :::: pes where ,.: !:= could be obtained. Molar ellipticity
can be ::- as number of millidegrees of rotation of polarized light divided by
the product of the number of amino acid residues in the p-',. !K the concentration of
the peptide, and ten times the cell length. The results of these experiments, after I =
subtraction, showed that M2 was in an unstructured, random coil conformation in
the 30:70 )i'. )H: H20 solution. This is what would be ..- : :1 for a non-aggregating
.............. 1 ; 1. ,F 1... ;. 1., ,
I i '.
Monolayer Formation and Isotherm Data Acquisition
after Irwin I :.:: ::::: :, who in the late 1910's was the first to perform
systematic studies on ...- .- monolayers in water, T ......... films are -* .11
in i:-:: :- .......in the scientific community. The name Tl .1A. ett was added several
years later when Katherine Blodgett gave a detailed description of monolayer transfer.
I :- =:::;: films usually refer to floating monolayers, and T :... '-Blodgett films refers
to a monolayer attached to a solid substrate.
The -.!.. ::-:A-- force that causes the formation of monolayers in such a system
is surface tension (Roberts, 1990). Molecules in a liquid i ... ...... attractive forces
in all directions. The molecules on the surface of the liquid :: stronger
intermolecular *....... with other molecules of the liquid as opposed to the air molecules
at the interface. Therefore the liquid will automatically minimize the surface area
1 to the air. F., :. T a minimum surface area exposed to the air also lowers the
of the liquid molecules, which is favorable. The more polar the molecules of
the liquid, the 1....... .. the intermolecular *. ..... involved, and the 1... .... the surface
tension will be. Any contamination will :I. the surface tension, and therefore
researchers should avoid any contamination ...,w, 1980). Also, : .. the
:: :'e will lower the surface tension.
Surfactants are surface active agents and they consist of a 1.. .. .... .1.::- portion
and a t:. .:= =.1:,::.VC, non-polar p:,u: As they are contain segments soluble in
both .. and polar solvents, .. or .. .... molecules tend to
accumulate at interfaces (Laughlin, 1994). Altering the size of both the1:; 1: 1:: 1. and
hydrophobic j- -.. ... ........a wide variety of self-assembled structures, but
all of them come about due to a reduction in free, .. :. .::. .r. T .. :::: :: films are a one-
molecule-thick insoluble monolayer that come about when an ........:. substance is
spread on the surface of water (Roberts, 1990, Binks, 1991). The v ::.': ends stay on the
water side of the interface, and the ...... :olar ends point up. Usually, the hydrocarbon
chain, the ....... polar .. ...... must be at least 12 hydrocarbons in order to form a
monolayer rather than a micelle (TT -:::1 :'.. 1952). However, if the hydrocarbon chain is
too long, the molecules .. ::crystallize on the surface rather than f........ .. a monolayer.
Therefore, a delicate balance must be attained.
Water has a very 1.1' surface tension compared to other liquids, and therefore
makes a good subphase for monolayer -. ::.::... The ':: .':::e for f::::::::- a
T..... uir film is as follows: first A *. ... .,. "..- .:. solution (in a non-polar .1. -.0
with micro .. : on the surface of the si ... :,:-.. solution; as solvent dissolves, a
monolayer forms. This monolayer is considered a "gas" because the molecules are
quite far ::.== (Roberts, 1990). Barriers on the far ends of the water trough are ':. :.. I
closer together very slowly. As the surface area of the monolayer decreases, the surface
.. .... increases and the molecules repel one another. The equation
1 = y Yo
for surface pressure, H, is a two-dimensional reckoning of pressure, where y is the
surface tension in absence of a monolayer and yo is surface tension when the monolayer
is A.: .. .
The trough for making a T .:: ::: monolayer is made of Teflon so as to
:. any 1. 1 of the -: '-1. (Ad. .:::: 1976). Water is circulated in channels
underneath the Teflon trough in order to maintain the : '* :: : : ..:..::: The surface
area of the trough can be adjusted with movable barriers. Each barrier is heavy enough
to prevent to prevent any leakage of the monolayer underneath the barriers. The
=:" .. = method is used to measure the surface pressure. A schematic of this
system is ... .. in i :.::::. 7. In this method, a plate is suspended partially immersed
in the subphase. The force exerted by the surface is converted into surface pressure by
accounting for the size of the I".:!. '- (Gaines, 1966, Robert, 1990). The forces on the 1.:
are surface tension ... ::.= downward, ..'. ., ~., and buoyancy .. !:J ... 'i' 1.
by:::- .: ::::'::the surface pressure as the : area is decreased by ::: of
the monolayer. Sometimes, many repetitions are:, ..:': .1 to find isotherms that are
:. .: .::: 1*1- as small variations in the system variable or the presence of contaminants
can have a ': f: ... : effect on the monolayer behavior. Several areas of constant
slope become evident upon examination of an isotherm. The :.- .."::: are known as
S. Generally, the :11 .. behavior is determined by the physical and chemical
.* : .: of the .::::: -1:: 1 the -.:::1i 1:.:..- com position, and .. ::: : .:. :: 'e (Roberts,
1990, Binks, 1991). The !i. of the hydrocarbon chain and the magnitude of any
other forces existing between the head. :..: :*. determines the various monolayer
states. The surface-pressure-area isotherm can be condensed by ::: .. :: the chain
.. ::.. !:, which in turn increases attraction between molecules. A nomenclature system
was i,.. '-'L; -""1 by W.D. Harkins in which monolayers in the gaseous state are given
names (G) (Roberts, 1990). When compressed they will transition to the (L ) state
known as the liquid-expanded state. When compressed further the liquid transitions
to the liquid-condensed state (L2) and when condensed further it becomes the solid
state (S). However, if the monolayer is compressed any further it will collapse. The
collapse is evidenced by a rapid decrease in surface pressure or a break in the isotherm.
Several other critical points exist in the surface-pressure-area isotherm, such as A.
which is .::::::, -::. by the pronounced increase in the surface pressure as well as surface
pressures at which phase transitions occur between the other states, marked by a
1::. ::.: in slope.
The ... :... behavior of M2 and DPhPC was studied T. .. T .. uir
monolayers as described above. Surface-pressure-area isotherm data was- ....:: :: '..
.. a KSV 5000T ..... uir ...... ..1 with dimensions 44 cm x 7.35 cm and a: ......
balance (see Figure 8). The 30:70 MeOH: CHC13. i::, .:: :.:::. solution ,::... ::::.1
Brewster Angle Microscopy
When looking at the angle of incidence of non-polarized light on a flat insulating
surface, at one specific angle all of the reflected light waves are polarized into a single
Figure 9: Principle of Brewster Angle Microscopy. When incident light
reaches the subphase interface (left) it is transmitted and refracted toward the
membrane normal. However, the addition of a monolayer at the air-water
interface allows for reflected, polarized light at the specific Brewster Angle.
plane. This angle is known as Brewster's angle. It can be calculated using the following
n = sin(Oi)/sin(O,) = sin(Oi)/sin(O9o-i) = tan(Oi)
where n is the refractive index of the medium from which the light is reflected, 61 is the
angle of incidence, and 0r is the angle of refraction (see Figure 9). It is therefore
very easy to calculate the refractive index of an unknown substance. Snell's law is
inapplicable for opaque materials with a high absorption for transmitted light, so
figure 1I: Brewster Angle Microscope.
this 1. is ... i...I Also, this technique determines the amount
of,. I :::: :,.::, and can be used to establish what :: -1:: : the monolayer is in. The
.... used is shown in : -. .. 10. **. ..- / ... Micros. *.. was used in
S ::: with the I.::::::::: '-Blodgett conditions c',.. =:= i above in order to get a
:.'.. .... ..'e of the morphologies of the mixtures at the interface.
Patch Clamp Recordings
The patch: 1. *. method for 1.. .... .. ,-channel activity was first developed in
1976 by Neher and Sakmann. The ; *:'..1:: refers to either a full or p.::,:1 attachment of
membrane or lipid .:. *." .. to the tip of a .. .... that encases a microelectrode that
can be used for .I: .= .. .' small electrical ......... There are two *.* p,. of. I,::: :-.. that can
be .,: -.' :1 to cell membrane or lipid *... ..... The first is .. .....a current
through a membrane 1'.= "= and =1 ..= ::: the potential difference across the membrane.
However, more commonly used for smaller systems is a voltage i... :;, in which a
defined :. 1.'::I'.=1 difference is ,= .. ,1 across the membrane and the corresponding
current flow is measured. What is essentially created is an equivalent circuit whose
components are made up of the : -:' membrane, the::::: ...... ::: -. and the ,:::1:
solution. This equivalent circuit is shown in Y: .. .. 11. In order to observe channel
function .,. : ,i1:::::: the iT ',.1 environment, a .: :. ":: seal must be made that ..- '.:: ..... the
solution in the micropipette, which acts as a conductor, and the .. .** (I-. 2002).
This can be done either by t': = *::. = the::: pipette tip ::, :'= :,. the membrane of a
vesicle or a whole cell or by < ..... a !..1'. ... at the tip :",' a method known as
tip- .. :::. As was discussed in the introduction.,. ::: : :::" the electrical .:::: ::' ..
of .... .. the resistance observed at the tip depends on the resistance of the I'- :.
layer itself as well as on the .:l: :. ive effect of the membrane. The tip resistance is also
affected by leak .. .... which reduces the effective resistance of the patch as well
as the :'.:. 've effect of the glass p'."',. Single channel measurements were made
: the tip-O:K.- patch 1. .. method (Coronado and Latorre, 1983, Suarez-Isla,
Wan, et al, 1983). First, the micropipettes used to house the electrode were prepared
using a Flamming/Brown Micropipette Puller (Model 97, Sutter Instruments Co.) with
a heated filament. The micropipettes were pulled to defined micron-scale apertures of
approximately 1 pm2, as verified by bubble-rate test. Tip-dipping experiments were
performing in a circular Teflon trough that could be manipulated vertically. Also,
some experiments were done using a three-axis manual micro-manipulator. The
subphase solution used was composed of 500 mM KC1, 5 mM HEPES, and 1 mM CaC12,
made with milli-O water and brought to DH 7.4. Bilavers of DPhPC with incorDorated
Equivalent Circuit for an Excised Patch Configuration
Palcl Pipotte l0letrode-
R_ 11 m_ nm 0 .
Figure 11: Excised Patch Configuration. The electrical properties of the micropipette and the
membrane must be considered when making patch recordings. The membrane resistance, which
is important for determining the presence of a seal at the micropipette tip, can be expressed in
terms of the membrane capacitance and the time constant, Tmem, of the bilayer. Adjustments are
marked at the amplifier to counteract the effect of pipette resistance and capacitance.
This c........ ...... to ..... : ... .y 2 to 4 times the amount necessary to cover the
surface area of the .::: 1: .:*. in the Teflon trough exactly, to ensure that at least a single
monolayer covered the entire surface. It is estimated at these concentrations that there
are on the order of tens to hundreds of single channels present in any .. :::. tip.
RESULTS AND DISCUSSION
First let us look at surface-pressure-area isotherms of pure M26 peptide. As it is
amphipathic, we might predict that its behavior would be comparable to that of other
amphipathic molecules such as lipids. Looking at the isotherms, we see that they have a
shape that supports this proposition. Three distinct kinks occur in the ( ::: indicating
that the peptides on the surface are present in three separate phases before collapsing.
The area per molecule at these different phases can be estimated by taking the slopes
of the curve at each of the phase transitions and examining where the slopes intersect
with the x-axis, ..,. i:::. .:: measures area per molecule. From these data we are able to
determine area of onset. The onset area is the area per molecule at the beginning of a
1 '.:: : :: i.: phase. The limitation area is the area per molecule just before the next phase
transition. Figure 16 0 .; .. !. the phase transitions for M2 :: 1:. 1 at a concentration of
0.0625 mg/mL in 30:70 MeOH: Chloroform. Under these conditions, the area of onset
is 268 A2 and the film remains in this first phase, which is analogous to a gas I'...'..
(G). Once the surface pressure is increased to about 5.9 :::' ./m, we see transition to
the next phase, termed the liquid .... .. l.. .. (Li). Again, the area of this phase
is determined by the slope of the curve, following the corresponding phase transition
.... This area was determined to be 194 A2 and remained in this phase between
surface pressures 6.7 and 28 1::T "/m. The final phase is present above 30 mN/m until
S..^....." at.~ ..... ...i. y 40 m N/m Ithas an area of 136 A2, ;...... : ....to
leaky seals or formation of non-gating oligomer pores by M2. In order to show this a
gradient was set up across the membrane formed at the tip of the patch clamp pipette.
With no applied voltage across the membrane, the baseline remained flat, as shown
in Figure 22. When a voltage was applied, single-channel fluctuations could be seen,
Figure 22: Single-Channel Recording of 1:1000 M2:DPhPC with
Ionic Gradient at 0 mV When no voltage is applied across the
membrane, we see no change in the observed current, indicating that
confirming that the pentamer was forming, and functioning as a voltage-gated channel
as seen in Figure 23.
Once this was confirmed, we could continue with other electrophysiology
experiments. Experiments were carried out with the ion gradient across the membrane
the bilayer and are functioning as voltage-gated ion channels.
the bilayer and are functioning as voltage-gated ion channels.
pharmac.-. response of the conductance, mean ... time, and A.. ... *.. abilitiess in
the ..:::: of agonists, such as anesthetics (T,::::.. and Xu, 2002).
Other future work that should be considered includes .i ...:.... surface-
pressure-area isotherms for the native lipids with and without cholesterol. This would
allow for a more systematic comparison of the .. ...* ..i of the lipid environment in the
::..,.: of M2 peptide. Also, structural studies .:..::: solid-state NMR techniques
should be considered in order to better understand the dynamics of the channel in these
different environments. Such studies may help to: :,i:;: the observed increase in
mean -. :... time as the lipid environment increased in -. .-; -. ..,.
The data presented in this .: *. ::. 1:: are those used for the computation of the
values in Table 2 of the text for mean conductance, mean open time, and mean open
probabilities for the DPhPC lipid environments (9 total traces). A trace is shown for
each of the other lipid environments and more raw data can be obtained upon request.
The estimates for the gating voltages were made from data not presented here. In most
cases, the data include the filename for each data set as it i in the compiled da-
tabase, the recordings after baseline adjustment and filtering at I kHz with a Bessel
8-pole filter, and a plot of the open versus closed probabilities for each recording. Note
that in some cases there is no open versus closed graph i=: i.::: i. i. for :.. I: :::..:: data sets.
This is due to the fact that plots could not be obtained that were consistent with the raw
data, however in these cases software calculations that were consistent were obtained
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Frank Raucci was born in Pittsburgh, Pennsylv ..... in 1981. His family moved
to T::: U. :-Tequesta, HT. : :. :: where he spent the::: :j::: : :y of his childhood, in 1984. He
attended Suncoast Community High School in Riviera Beach and graduated in 1999
from the .. !:?1:, S :.=* and T: :,=.., :.:" and International Baccalaureate '.. .-:::: t Pro-
grams. His formal, : ............ .*'y education .. at the University of Florida in the
Fall term of 1999. While ::: =. *: -.:! ::. in undergraduate research projects throughout
his time at the University, he graduated .... ...., cum laude in 2003 with a Bachelor of
Arts degree in physics and a Bachelor of Science degree in chemical ::. ::": :,: ::-: He
then 1. .... work on this project as a '*-.: *.- *. *1 for his Master of Science degree in bio-
medical .::" :,,: :::* Frank .. .= be i::-=::." the Medical College of V ':":' at V.":::
ia Commonwealth U ..;. : in Richmond. He ,. be ...'. -:.:.i ..: in the MD/PhD
pro". :', := at this institution.