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Positive and negative modulatory sites for ethanol in alpha1 glycine receptors

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Positive and negative modulatory sites for ethanol in alpha1 glycine receptors

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POSITIVE AND NEGATIVE MODULATORY SITES FOR ETHANOL
IN α1 GLYCINE RECEPTORS

by

Daniel Kenneth Crawford

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(NEUROSCIENCE)

August 2007

Copyright 2007 Daniel Kenneth Crawford
ii
DEDICATION

To my friends and family, all of whom make every day worthwhile.
iii
TABLE OF CONTENTS
Dedication ii

Table of Contents iii

List of Figures v

List of Tables vi

Abbreviations vii

Abstract viii

Chapter 1: Introduction 1
Background 1
Ligand-Gated Ion Channels as Putative Ethanol Targets in the CNS 4
Investigating the Molecular Targets for Ethanol 7
A Novel Ethanol Antagonist 10
Behavioral Studies 11
Biochemical Studies 13
Electrophysiological Studies 14
Conclusion for the Introduction 16

Chapter 2: Evidence that Ethanol Acts on a Target in Loop 2 of the
Extracellular Domain of α1 Glycine Receptors
19
Abstract 19
Introduction 20
Materials and Methods 22
Results 28
Glycine Concentration Response 28
Ethanol and Hexanol Concentration Responses 29
Single PMTS Application 31
Sequential PMTS Applications (PMTS – PMTS) 33
PMTS – PMTS Concentration Response 33
PMTS – Ethanol 36
Alcohol Cutoff 38
Molecular Modeling 39
Discussion 52

iv
Chapter 3: Loop 2 Residues of the α1 Glycine Receptor Play a Role in
Agonist Activation and Chloride Ion Movement in the Pore
60
Abstract 60
Introduction 61
Materials and Methods 63
Results 68
Glycine Concentration Response 68
MTS Reagent Responses 70
Discussion 80

Chapter 4: Ethanol and Anesthetic Agonist Sensitivity of α1 Glycine
Receptors Altered by Cysteine Mutations in the Loop 2 Region
84
Abstract 84
Introduction 85
Materials and Methods 87
Results 94
Glycine Concentration Response 94
Ethanol Concentration Response 94
Isoflurane Concentration Response 95
Propofol Concentration Response 96
Single PMTS Application 97
PMTS – Ethanol 98
Discussion 109

Chapter 5: Summary and Overall Conclusions 112

References 117

v
LIST OF FIGURES
Figure 1 There is no significant difference between wildtype and
mutant α1GlyRs in sensitivity to glycine
41
Figure 2 Cysteine substitutions in α1GlyRs produce position-
specific differences in ethanol and hexanol responses
43
Figure 3 PMTS exposure in the presence of glycine irreversibly
potentiates A52C GlyRs
44
Figure 4 A second exposure of A52C GlyRs to PMTS suggests
multiple sites of PMTS action in α1GlyRs
45
Figure 5 Concurrent activation of positions 52 and 267 by PMTS is
sufficient to explain all of the effects of PMTS on α1GlyRs
47
Figure 6 PMTS binding to cysteines substituted at position 52 and/or
267 reveals position-specific negative and positive
modulation by ethanol in α1GlyRs
49
Figure 7 PMTS exposure in α1 (A52C) GlyRs reduces the alcohol
cutoff
50
Figure 8 Molecular model of an α1GlyR subunit with the alcohol
pocket highlighted
51
Figure 9 Cysteine point mutations in α1 GlyRs produce position-
specific shifts in the glycine concentration responses
76
Figure 10 The negative charge at position 53 is important for agonist
activation and chloride ion flux through the channel
77
Figure 11 Molecular model of an α1GlyR subunit 78
Figure 12 Comparison of the changes in agonist EC
50
for α1 GlyRs
and α7 nAChRs
79
Figure 13 Cysteine point mutations in α1GlyRs produce position-
specific shifts in the glycine concentration responses
102
Figure 14 Cysteine substitutions in α1GlyRs produce position-
specific differences in the ethanol responses
103
Figure 15 Cysteine substitutions in α1GlyRs produce position-
specific increases in the isoflurane responses
104
Figure 16 Propofol sensitivity in Loop 2 cysteine mutant GlyRs is
only altered in I51C GlyRs
105
Figure 17 PMTS exposure in the presence of glycine irreversibly
potentiates some, but not all, cysteine mutant GlyRs
106
Figure 18 PMTS binding in the triple mutant GlyR does not block all
of the effects of ethanol
107
Figure 19 Molecular model of an α1GlyR subunit 108

vi
LIST OF TABLES
Table 1 Summary of non-linear regression analysis results for the
glycine concentration responses in WT and mutant
α1GlyRs
40
Table 2 Summary of non-linear regression analysis results for the
glycine concentration responses in WT and mutant
α1GlyRs
73
Table 3 Summary of non-linear regression analysis results for the
glycine concentration responses in MTS-exposed WT and
E53C GlyRs
74
Table 4 Summary of non-linear regression analysis results for the
glycine concentration responses in WT and mutant
α1GlyRs
101

vii
LIST OF ABBREVIATIONS
AChBP, acetylcholine binding protein
ANOVA, Analysis of Variance
DMSO, dimethyl sulfoxide
EC
X
, effective concentration producing X% of the maximal current
GABA
A
R, γ-aminobutyric acid type-A receptor;
GlyR, glycine receptor;
nAChR, nicotinic acetylcholine receptor
LGIC, ligand-gated ion channel;
MAC, minimum alveolar concentration
MBS, Modified Barth’s Saline;
MTS, Methanethiosulfonate
nAChR, nicotinic acetylcholine receptor
PBS, Phosphate Buffered Solution;
PMTS, propyl methanethiosulfonate;
PVDF, polyvinylidene fluoride
SDS-PAGE, Sodium Dodecyl Sulphate – Polyacrylamide Gel Electrophoresis;
TM, transmembrane;
WT, wildtype;

viii
ABSTRACT
Ligand-gated ion channels of the cys-loop receptor family have received
considerable attention as putative sites of action causing the behavioral effects of
ethanol. Studies over the last decade identified several positions in the
transmembrane domain critical for ethanol modulation of glycine receptors (GlyRs).
Studies to date in the extracellular domain of α1GlyRs found that mutations at
position 52 in Loop 2 change ethanol sensitivity, alter sensitivity to an ethanol
antagonist and can eliminate subunit-dependent differences in ethanol sensitivity
between α1 and α2GlyRs. These findings suggest that the extracellular domain also
represents a target for ethanol in GlyRs. We tested this hypothesis by investigating
the effect of cysteine substitutions at positions 52 and 267 on responses to n-alcohols
and propyl methanethiosulfonate (PMTS) in α1GlyRs. We also tested the role of
Loop 2 in agonist activation and ethanol modulation by investigating the effect of
cysteine point mutations at positions 50-60. Xenopus oocytes expressing human α1
wildtype (WT) or mutant GlyRs were voltage-clamped and tested 3-10 days post-
injection. In support of the hypothesis, we found that: 1) The A52C mutation
changed ethanol sensitivity compared to WT GlyRs; 2) PMTS, a thiol agent held to
mimic ethanol action, produced irreversible alcohol-like potentiation in A52C
GlyRs; 3) PMTS binding reduced the alcohol cutoff in A52C GlyRs. Further studies
used PMTS binding to cysteines to block ethanol action at one site in order to
determine its effect at the other site. In these situations, ethanol caused negative
modulation when acting at position 52 and positive modulation when acting at
ix
position 267. Latter studies found that cysteine substitutions in Loop 2 produced
position-specific changes in glycine sensitivity consistent with β strand-like structure
of the Loop and suggest that odd-numbered positions in Loop 2 interact with other
agonist-activation elements. We then found that several Loop 2 cysteine mutations
changed ethanol sensitivity and one mutation switched the qualitative ethanol
response. Together, these findings support the hypothesis that Loop 2 residues play
an important role in agonist activation and ethanol modulation. The findings provide
new molecular models and insight into structure-function relationships for GlyRs
and possibly other ligand-gated ion channels.
1
CHAPTER 1
INTRODUCTION

Background
Alcohol abuse represents a significant problem in our society, affecting over
14 million people in the United States alone (Volpicelli J.R. 2001;NIAAA 2000).
The economic costs of alcohol-related disorders was estimated to exceed a
staggering 185 billion dollars per year in the United States alone (2000), compared to
730 million dollars spent on alcohol-related research worldwide (Rajdendram et al.
2006). Consumer expenditure on alcohol in the United States in 1999 was $116.2
billion (Foster et al. 2003). Despite the wide consumption and the issues associated
with the excessive intake of alcoholic beverages, the mechanisms for the action of
ethanol are still poorly understood.
Historically, alcohols and anesthetics were believed to act by perturbing the
lipid membrane. Over 100 years ago, H.H. Meyer (Meyer 1899;Meyer 1901) and
C.E. Overton (Overton 1901) independently found that the potency of alcohols and
general anesthetics is proportional to their partition coefficient between the aqueous
phase and the oil phase (water and olive oil, respectively) (Roth 1979;Heimburg and
Jackson 2006). K.H. Meyer refined the Meyer-Overton rule (Meyer 1937) by
proposing that anesthesia occurs when any chemically indifferent substance attains a
certain concentration within the lipid bilayer of the cell (Roth 1979). L.J. Mullins
supported the notion that anesthetic potency is related to the concentration in the
2
membrane, but suggested that the volume occupied by the anesthetic is also
important for anesthesia (Mullins 1954;Roth 1979). He predicted that anesthesia
occurs when a critical volume fraction of anesthetic exists within the membrane
phase. Mullins suggested that the permeability of ions would be depressed once a
critical volume of anesthetic within the membrane was reached, thus resulting in a
loss of excitability. Later work extended Mullins’ theory to suggest that adsorption
of an anesthetic would expand the membrane, thereby providing a basis for the
mechanism of anesthetic action (Lever et al. 1971;Miller et al. 1973;Halsey 1982).
The critical volume hypothesis states that "anesthesia occurs when the volume of a
hydrophobic region is caused to expand beyond a certain critical amount by the
adsorption of molecules of an (inert) substance. If the volume of this hydrophobic
region can be restored by changes of temperature or pressure then the anesthesia will
be removed."
Structural diversity among anesthetic agents lent support to the concept of a
nonspecific, lipid target and mechanism of action for all anesthetics, such as
perturbation of membrane lipids (Roth 1979). However, mounting evidence
suggests that alcohols and anesthetics act on proteins (either within or independent of
the membrane) and that membrane perturbation alone is not sufficient to cause
anesthesia (Hunt 1985;Buck et al. 1989;Dietrich et al. 1999). The effects of alcohols
on membrane disorder are generally measurable only at concentrations well above
the pharmacological range (Goldstein 1984). For example, at concentrations
associated with anesthesia, there would be only one ethanol molecule per
3
approximately 200 lipid molecules. Furthermore, the effects of intoxicating
concentrations of alcohols on membrane order can be mimicked by an increase in
temperature of just a few tenths of a degree Celsius (Pang et al. 1980;Franks and
Lieb 1982), which clearly does not produce behavioral signs of alcohol intoxication.
More recent studies found that general anesthetics inhibit the light-emitting
firefly luciferase reaction (a protein only preparation) and suggested that anesthetics
“act by competing with endogenous ligands for binding to specific receptors (Franks
and Lieb 1984). Subsequent work found that highly purified optical isomers of the
inhalational general anesthetic isoflurane, which are equally effective at disrupting
lipid bilayers, exhibited clear stereoselectivity in their effects on particularly
sensitive ion channels (Franks and Lieb 1991;Jones and Harrison 1993). Moreover,
there was a strong correlation between the stereoselective effects of isoflurane on
these ion channels and the potency of the anesthetic isomers in vivo (Harris et al.
1992;Lysko et al. 1994).
Collectively, these findings demonstrate that the lipid bilayer cannot account
for all of the alcohol and anesthetic effects in the CNS and that more specific sites of
action (such as membrane proteins) also play an important role. In spite of the
bodies of evidence implicating lipid membranes and proteins, alcohols and
anesthetics could have a multitude of targets, including both head and tail groups of
lipids, membrane proteins, and/or the annular lipids of proteins (Franks and Lieb
1987a;Franks and Lieb 1987b). It is unlikely that any one of these targets can
account for all of the alcohol and anesthetic effects in the CNS. Therefore, further
4
research is needed to understand the interactions at each of these targets in more
detail.

Ligand-Gated Ion Channels as Putative Ethanol Targets in the CNS
Ligand-gated ion channels (LGICs) have received considerable attention as
putative sites of action causing the behavioral effects of ethanol (Dietrich et al.
1999;Harris 1999;Davies et al. 2003). Research in this area has focused on
investigating the effects of ethanol on three large “superfamilies” of LGICs: (1) the
glutamate superfamily (Monaghan et al. 1989;Sommer and Seeburg 1992), which
includes N-methyl D-aspartate (NMDA), α-amino-3- hydroxyisoxazolepropionic
acid (AMPA), and kainate receptors (Dildy-Mayfield and Harris 1995;Lovinger et al.
1989;Ronald et al. 2001); (2) the purinergic (P2X) superfamily (Li et al. 1998;Li et
al. 2000;Xiong et al. 2000;Davies et al. 2002;Davies et al. 2005); and (3) the cys-
loop receptor superfamily (Ortells and Lunt 1995;Xiu et al. 2005), whose members
include nicotinic acetylcholine receptors (nAChRs), 5-hydroxytryptamine3 receptors
(5-HT
3
Rs), γ-aminobutyric acid type-A receptors (GABA
A
Rs) and glycine receptors
(GlyRs) (Aistrup et al. 1999;Cardoso et al. 1999;Davies and Alkana 2001a;Davies et
al. 2003;Davies et al. 2004;Godden et al. 2001;Machu and Harris 1994;Mihic et al.
1997;Ye et al. 1998;Zhou and Lovinger 1996).
Cys-loop receptor subunits share significant sequence homology and consist
of four transmembrane (TM) α-helical segments, an intracellular component for
cytosolic interactions, and a large, extracellular ligand-binding domain (Langosch et
5
al. 1988;Ortells et al. 1997;Brejc et al. 2001;Tang et al. 2002;Unwin 2005). Five
individual subunits assemble to form functional ion channels with TM2 of each
subunit facing the channel pore. Binding of neurotransmitter to residues in the
extracellular domain is believed to cause lateral twisting of the TM2 domains,
resulting in an enlargement of the channel pore as the receptor-channels convert
from their closed, non-conducting conformation to an open, conducting state
(Sansom 1995;Unwin 1995;Lynch et al. 1997). Residues within TM2 are largely
responsible for the ion selectivity of the channel (Keramidas et al. 2000).
Specifically, nAChRs and 5-HT3Rs have residues that form three negatively charged
rings and a central polar ring, whereas GABA
A
Rs and GlyRs have residues that form
two positively charged rings at the intra- and extracellular ends of the ion pore.
Collectively, these ionotropic receptors play a significant role in fast synaptic
neurotransmission within the central nervous system (CNS) and provide a potential
target for rapid modulation by a variety of agents (Laube et al. 1995;Harris et al.
1997;Beckstead et al. 2000;Cherubini and Conti 2001;Laube et al. 2002;Olsen et al.
2004;Baur et al. 2005;Hejazi et al. 2006;Lynch and Callister 2006;Moore et al.
2005).
Previous studies in vivo support the notion that cys-loop receptors play a role
in the behavioral effects of ethanol. For example, several studies have focused on
the role of GlyRs because of its pharmacological properties and its localization in the
CNS. Microdialysis infusions of glycine or strychnine into the nucleus accumbens
suggest that GlyRs are important for regulating voluntary ethanol intake and may act
6
as an entrance point into the brain reward system (Molander and Soderpalm
2005;Molander et al. 2005). Previously, glycine and D-serine (a glycine pre-cursor)
were shown to enhance ethanol-induced loss of righting reflex in mice (Williams et
al. 1995). The effect of glycine on the loss of righting reflex was blocked by
strychnine, suggesting that glycine enhances the action of ethanol by acting on
strychnine-sensitive GlyRs. Recent transgenic studies in mice also add to the
evidence that GlyRs participate in the action of ethanol in mammals (Findlay et al.
2002). Similarly, GlyRs have also been shown to mediate part of the immobility
produced by anesthetics (Quinlan et al. 2002;Zhang et al. 2001;Zhang et al. 2003).
Taken together, these findings suggest that GlyRs mediate at least a subset of
ethanol-induced behavioral effects.
Electrophysiological studies of GlyRs also support a role for GlyRs in
mediating the effects of ethanol in the CNS. Behaviorally relevant concentrations of
ethanol positively modulate GlyR function measured in synaptoneurosomes of
whole-rat brain (Engblom and Åkerman 1991), embryonic spinal neurons of mouse
and chick (Celentano et al. 1988;Aguayo and Pancetti 1994;Aguayo et al.
1996;Tapia et al. 1998;Van Zundert et al. 2000;Ziskind-Conhaim et al. 2002), freshly
dissociated rat neurons (Ye et al. 2001a;Ye et al. 2001b;Ye et al. 2002;Tao and Ye
2002;Jiang and Ye 2003;McCool et al. 2003) and brain slice preparations (Eggers et
al. 2000;Eggers and Berger 2004;Sebe et al. 2003;Darstein et al. 1997). In addition,
ethanol reliably and robustly potentiates human recombinant α1 and α2 GlyRs
measured electrophysiologically (Mascia et al. 1996a;Mascia et al. 1996b;Mihic et al.
7
1997;Valenzuela et al. 1998;Yamakura and Harris 2000;Davies et al. 2003;Davies et
al. 2004). Despite the advances in understanding the effects of ethanol in vivo and in
vitro, the precise molecular sites of action for ethanol in LGICs are not yet well
understood.

Investigating the Molecular Targets for Ethanol
Initial investigations into the molecular targets for ethanol within cys-loop
receptors utilized two receptors: α1 GlyRs and ρ1 GABA
A
Rs (Mihic et al. 1997).
Both receptors form functional homomers and have qualitatively different responses
to ethanol ( α1 GlyRs are potentiated by ethanol and ρ1 GABA
A
Rs are inhibited by
ethanol). Chimeras of these receptors were created and the site of ethanol action
determined by observing which segment of the protein could be spliced into the
corresponding location of the other receptor to change the ethanol response. This
study identified a 45 amino acid residue region containing TM2 and TM3 that was
both necessary and sufficient for ethanol modulation of receptor function. Sequence
alignment comparison of α1 GlyRs and ρ1 GABA
A
Rs revealed twelve differences at
homologous residues within this 45 amino acid residue region. Replacing the amino
acids at these positions in α1 GlyRs with the respective residues of the ρ1 GABA
A
R
identified specific TM 2 and 3 residues (S267 and A288 in the α1 GlyR) that are
critical for allosteric modulation of these receptors by alcohols and volatile
anesthetics.
8
Subsequent studies found that systematic replacement of the serine at
position 267 with the other nineteen amino acids altered ethanol potentiation of GlyR
function (Ye et al. 1998). Moreover, this work revealed an inverse correlation
between molecular volume of the amino acid substitution and the effect of ethanol,
with a crossover from ethanol potentiation to inhibition beginning at residues equal
to or larger in volume than isoleucine. Potentiation by alcohols and anesthetics was
also altered by mutations at I229 in TM1, Ala288 in TM3, and W407 and Y410 in
TM4 (Greenblatt and Meng 1999;Jenkins et al. 2001;Yamakura et al. 1999;Lobo et
al. 2004;Lobo et al. 2006). Together, these results demonstrate that the amino acid
residues in TM domains play a crucial role in determining the functional
consequences of allosteric modulation of the GlyR by alcohols and anesthetics.
Additional investigations approached studying the role of TM residues in the
actions of ethanol via the n-chain alcohol cutoff. The cutoff refers to the failure to
increase potency as a function of increasing the n-chain alcohol length and is thought
to occur when the molecular volume of the alcohol exceeds the finite volume of a
putative alcohol pocket (Wick et al. 1998;Pringle et al. 1981;Alifimoff et al. 1989).
Different LGICs exhibit different cutoff points (the carbon chain length for GlyRs
and GABA
A
Rs = 10-12, GluRs, AMPARs, and KARs = 7, ρ1 GABA
A
Rs = 7,
P2XRs = 3) (Dildy-Mayfield et al. 1996;Mascia et al. 1996a;Mihic and Harris
1996;Li et al. 1994), supporting the existence of alcohol binding pockets of variable
size on these membrane proteins (Wick et al. 1998). Therefore, to test the hypothesis
that the TM domains of α1 GlyRs and ρ1 GABA
A
Rs could form an alcohol binding
9
pocket, mutations were performed on TM2 residues and the alcohol cutoff was
observed. Substituting larger residues into position 267 of the α1 GlyR significantly
decreased the alcohol cutoff, whereas mutation of the homologous position in the ρ1
GABA
A
R to smaller residues increased the cutoff. These results support the
existence of alcohol binding pockets within each of these receptors and suggest that
the amino acid residues present within the TM domains can control the size of the
alcohol binding cavity.
Subsequent experiments tested the hypothesis that amino acids S267 in TM2
and A288 in TM3 in the α1 GlyR represent critical binding sites for alcohols and
anesthetics by using cysteine mutagenesis and anesthetic-like thiol or
methanethiosulfonate (MTS) reagents (Mascia et al. 2000). The authors suggested
that the creation of a disulfide bond following covalent binding at a single site would
change the reversible potentiating effect of the agent to an irreversible effect if the
actions of the alcohol or anesthetic result from binding at this site. Consistent with
their hypothesis, propanethiol and propyl methanethiosulfonate (PMTS) exposure to
α1 S267C GlyRs caused the expected change. Furthermore, PMTS binding to
position 267 abolished the potentiating effects of enflurane, isoflurane and octanol
suggesting competition for a single binding site among PMTS and alcohols and
anesthetics. When in the resting state of the receptor, neither propanethiol nor PMTS
were able to irreversibly enhance the function of the A288C TM3 mutant or prevent
action of isoflurane on these receptors. However, PMTS exposure in the presence of
glycine causes a conformational change sufficient to allow access to the site and
10
covalent labeling of the target with PMTS (Lobo et al. 2004). In addition, ethanol, in
the absence of agonist, causes a conformational change in the receptor that can
change accessibility of TM residues (Jung et al. 2005;Jung and Harris 2006). Finally,
PMTS occupancy of a single anesthetic binding pocket within the GlyR pentamer is
sufficient to enhance receptor function (Roberts et al. 2005). Taken together, these
results are consistent with the notion that LGICs are dynamic proteins during alcohol
modulation and channel gating. Collectively, the findings add further support for the
hypothesis that the TM domains contribute to an alcohol binding pocket and play a
role in causing alcohol modulation of receptor function.

A Novel Ethanol Antagonist
A different approach to studying the effects of ethanol on the nervous system
utilized a novel ethanol antagonist to investigate the sites and mechanisms of
ethanol. Traditionally, the site and mechanism of drug action can be identified and
compared using the appropriate receptor agonists and antagonists. However, the
physical-chemical nature of the mechanism of action for ethanol limits the utility of
neuronal receptor ligands as tools for investigating the mechanism of action for
ethanol across receptors for two reasons: (1) they cannot be used to identify the
precise site of action of ethanol within a receptor complex because they do not act at
the same sites as ethanol (Eckenhoff and Johansson 1997;Davies and Alkana
1998;Davies et al. 2003), and (2) their dependence on structure related selectivity for
11
specific protein targets precludes their use in looking for common sites and/or
mechanisms across different receptors. Therefore, it has not been possible to directly
test the hypothesis that ethanol acts by a common mechanism on different receptors.
To address these issues, increased atmospheric pressure (hyperbaric
exposure) was developed as an alternative to a traditional pharmacological ethanol
antagonist. The results summarized below indicate that pressure can be used in lieu
of a traditional pharmacological antagonist to help identify the sites of action for
ethanol within a receptor and to test for a common mechanism of action for ethanol
across different receptors (Davies et al. 1999).
Behavioral Studies: This work found that exposure to 12 times normal
atmospheric pressure (ATA) of helium-oxygen gas (heliox) antagonizes ethanol-
induced behavioral effects, including: the loss of righting reflex duration (Malcolm
and Alkana 1982), anticonvulsant effects (Davies et al. 1994), depression of
aggressive behavior (Alkana et al. 1991), and the biphasic effect of low and high
ethanol doses on locomotor activity (Syapin et al. 1988;Bejanian et al. 1993;Alkana
et al. 1995). The hyperbaric ethanol antagonism at 12 ATA was observed in a
variety of mouse genotypes (129, A/He, BALBc, C57/Bl, CFW, DBA/2, Long Sleep
and Short Sleep) (Alkana et al. 1991;Alkana et al. 1992;Alkana and Malcolm
1981;Bejanian et al. 1993;Davies and Alkana 2001b;Syapin et al. 1988) without
causing the generalized increases in CNS excitability (Davies et al. 1994;Davies et
al. 1999;Syapin et al. 1996) that called into question the specificity and utility of the
higher pressures (50–300 ATA) used previously for pressure reversal of anesthesia
12
(Brauer et al. 1979;Halsey 1982;Kendig 1984;Bowser-Riley et al. 1988;Wann and
MacDonald 1988;Franks and Lieb 1994;Little 1996;Davies et al. 2001).
The atmospheric conditions used in these behavioral experiments were
selected so that potential experimental confounds were minimized. The heliox gas
composition (1.7% O
2
, 98.3% He) is such that the partial pressure of O
2
at 12 ATA
using heliox is the same as the partial pressure of O
2
in air at normal atmospheric
conditions. Controlling for O
2
partial pressure also prevents complications that arise
from oxygen toxicity (Alkana and Malcolm 1982a). Aside from the specified O
2

levels, the heliox gas was balanced with the inert gas helium. Helium was selected
as a substitute for the nitrogen in air since, unlike nitrogen (Bennett et al. 1975),
helium does not exert depressant effects within the pressure range examined in these
studies (MacInnis et al. 1967;Membery and Link 1964). Numerous additional
studies eliminated helium-induced hypothermia, reduced brain ethanol
concentrations secondary to alterations in ethanol distribution, and an increased rate
of ethanol elimination as critical factors in mediating the antagonism (Alkana and
Malcolm 1980;Alkana and Malcolm 1981;Malcolm and Alkana 1982).
Considerable evidence indicates that the characteristics of low-level
hyperbaric ethanol antagonism are similar to those of a classical pharmacological
antagonist—i.e., the mechanism of antagonism is direct (Alkana and Malcolm
1982a;Alkana and Malcolm 1982b;Davies et al. 1994;Malcolm and Alkana
1982;Syapin et al. 1996), the degree of antagonism is directly related to pressure
(Alkana and Malcolm 1981), the antagonism causes a parallel shift to the right in the
13
ethanol dose response curve (Bejanian et al. 1993), the antagonism is surmountable
by increasing the ethanol dose (Bejanian et al. 1993) though not into lethal ranges of
ethanol (Malcolm et al. 1985), and pressure antagonizes ethanol’s chronic effects
(tolerance, dependence and precipitation of withdrawal symptoms after dependence)
in the predicted manner (Alkana et al. 1985;Alkana et al. 1987). Collectively, these
findings suggest that there is a common mechanism for the behavioral effects of
ethanol that can be antagonized by pressure (Davies et al. 1999).
Biochemical Studies: Additional studies extended hyperbaric investigation to
the biochemical level by testing the effects of pressure on ethanol-induced
potentiation of GABA
A
R function. This work measured the effects of pressure
versus ethanol potentiation of GABA-induced uptake of radiolabeled
36
Cl
-
by
synaptosomal membranes (microsacs) (Davies and Alkana 1998;Davies et al.
1999;Davies and Alkana 2001a;Davies et al. 2001). The microsacs are pre- and
post-synaptic vesicles which retain functional coupling between agonist and
allosteric modulatory sites (Davies and Alkana 2001b). Exposure to 12 ATA heliox
antagonized ethanol enhancement of GABA-activated
36
Cl
-
uptake in microsacs
without altering receptor function or responsiveness of the receptor to GABA, THIP,
bicuculline, or picrotoxin in the absence of ethanol (Syapin et al. 1996;Davies and
Alkana 1998;Davies et al. 1999). Moreover, pressure antagonism appears to be
selective for drugs that act via specific allosteric mechanisms in that pressure
antagonizes alcohols, benzodiazepines, and barbiturates, but does not antagonize the
effects of morphine or isoniazid (Alkana et al. 1995;Davies et al. 1994;Davies et al.
14
1996;Davies et al. 1999;Davies et al. 2001). These findings demonstrate the
selectivity of pressure antagonism and add critical support for a direct mechanism of
antagonism at the biochemical level. Taken in the context of the ability of pressure
to antagonize the behavioral effects of ethanol, these biochemical findings also
added a new line of support for a cause–effect link between the ethanol-induced
potentiation of GABA
A
R function and ethanol-induced behavioral changes (Davies
and Alkana 2001b).
Collectively, these behavioral and biochemical studies provide strong,
consistent evidence that increased atmospheric pressure is a direct, selective
antagonist with properties that can be used to study the sites and mechanisms of
ethanol action within and across receptors. This selectivity of pressure antagonism
cannot be based on structure-related competition at receptor binding sites because
pressure does not have a chemical structure. Rather, the selectivity of pressure
antagonism must be based on the ability of pressure to block or offset specific
physical-chemical changes induced by ethanol. Therefore, unlike typical
pharmacological antagonists, the selectivity of pressure antagonism should not be
limited by structural constraints to a single receptor. This suggests that pressure is a
unique tool that can be used to test whether a common physical chemical, pressure-
antagonism–sensitive mechanism initiates the effects of ethanol across different
ethanol sensitive receptors (e.g., GABA
A
Rs versus GlyRs).
Electrophysiological Studies: More recently, these hyperbaric investigations
were extended to two-electrode voltage clamp studies of LGICs expressed in
15
Xenopus oocytes. Initial results demonstrated that α1 GlyRs are sensitive to pressure
antagonism of ethanol at high concentrations (50-200 mM) (Davies et al. 2003). In
addition, the degree of ethanol antagonism was inversely proportional to the ethanol
concentration and increased as pressure increased. These characteristics were
consistent with the hyperbaric behavioral and biochemical studies and support the
contention that increased atmospheric pressure exhibits characteristics similar to
conventional pharmacological antagonists. Remarkably, low concentrations of
ethanol (10 and 25 mM) were completely insensitive to antagonism by pressure
(Davies et al. 2004). These concentration-dependent differences suggest that ethanol
acts at more than one site of action on α1 GlyRs: a high concentration pressure-
sensitive site and a low concentration pressure-insensitive site. In contrast to the α1
GlyR, α2 GlyRs were insensitive to pressure antagonism of ethanol at all
concentrations tested (Davies et al. 2004). Given that there is approximately 79%
sequence homology between α1 and α2 GlyRs, it was hypothesized that sensitivity
to pressure antagonism of ethanol is determined by the few locations where residues
differ between α1 and α2 GlyRs.
Interestingly, recombinant studies of GlyRs demonstrated that α1 GlyRs
were more sensitive to ethanol than α2 GlyRs (Mascia et al. 1996b). In addition, an
alanine to serine exchange at position 52, located at the beginning of Loop 2 (Brejc
et al. 2001) and one of the residues that differs between α1 and α2 GlyRs, decreased
the ethanol sensitivity of the α1 GlyR to the point where the ethanol response
resembled that of the α2 GlyR. The authors presented several hypotheses that could
16
explain this last result, one of which suggested that the subunit-dependent
differences in ethanol sensitivity might arise from multiple ethanol binding sites,
with the A52S mutation effectively eliminating one of those sites.
The A52S GlyR mutation was utilized in hyperbaric experiments to
investigate the hypothesis that position 52 is a target for ethanol and for pressure-
antagonism of ethanol. These studies not only confirmed the change in ethanol
sensitivity (making α1 GlyR ethanol sensitivity resemble α2 GlyR ethanol
sensitivity), but also showed that the A52S mutation changes sensitivity to pressure
antagonism of ethanol (making the pressure antagonism sensitive α1 GlyR now
insensitive like the α2 GlyR) (Davies et al. 2004). Preliminary studies with the more
accurate α1 versus α2 mutation, α1 A52T GlyR, also found that sensitivity to
pressure antagonism of ethanol was abolished. Moreover, the converse experiment
(substituting alanine for threonine at the homologous position in α2 GlyRs)
conferred sensitivity to pressure antagonism of ethanol to α2 GlyRs. Therefore, the
results using increased atmospheric pressure as an ethanol antagonist suggest that
position 52 in Loop 2 of the extracellular domain may play an important role in
ethanol potentiation of GlyR function.

Conclusions for the Introduction
Increasing evidence suggests that ethanol acts via multiple targets to produce
behavioral intoxication. Early experiments suggested that alcohols and anesthetic act
through perturbation of the lipid bilayer of neurons, thereby causing a disruption of
17
ion flow and impairing signal transmission in the nervous system. Later work
demonstrated that membrane perturbation is not sufficient to produce anesthesia and
that alcohols and anesthetics could act directly on proteins (within or independent of
the cell membrane).
LGICs, which mediate fast synaptic neurotransmission, have been proposed
to be an important target for the behavioral effects of ethanol. For LGICs of the cys-
loop receptor family, the TM domains have received considerable attention as a
putative site of action. Investigations constructing chimeras of LGICs with
qualitatively different ethanol responses ( α1 GlyRs and ρ1 GABA
A
Rs) suggested
that the TM domain is necessary and sufficient for the actions of ethanol.
Subsequent experiments at several key positions within the TM of α1 GlyRs (e.g.
position 267) revealed that mutations of these residues could alter ethanol
potentiation and reduce the n-chain alcohol cutoff. Cysteine substitution at position
267 and reaction with PMTS yielded irreversible alcohol-like potentiation which
provided additional evidence that the TM domain could represents a binding site for
ethanol. Collectively, these findings suggest that the TM domains contribute to an
alcohol binding pocket and play a role in causing alcohol modulation of α1 GlyR
function.
In contrast to the TM region, the extracellular domain has received relatively
little attention as a potential site of action for ethanol. The use of increased
atmospheric pressure as an ethanol antagonist has brought attention to the
extracellular domain, and in particular, position 52 of the α1 GlyR. Previously, α1
18
GlyRs were shown to be more sensitive to the effects of ethanol than are α2 GlyRs.
Switching serine for alanine at position 52 (A52S) of the α1 GlyR decreased the
ethanol sensitivity of the α1 GlyR to the point where the ethanol response resembles
that of the α2 GlyR. The A52S mutation also changed sensitivity to pressure
antagonism of ethanol (making the pressure antagonism sensitive α1 GlyR now
insensitive like the α2 GlyR). These results suggest that position 52 in the
extracellular domain may be an important target for ethanol action in α1 GlyRs.
The present studies test the hypothesis that Loop 2 of the extracellular
domain is a target for ethanol action in α1 GlyRs. This work also begins to test the
relationship between the TM domains and putative extracellular targets in mediating
the actions of ethanol on this receptor. In addition, the current investigation focused
on scanning through the Loop 2 region in order to study the role each residue plays
in agonist activation and modulation. Together, these studies aim to provide a better
understanding of the complex interaction between separate sites of ethanol action in
the extracellular and TM domains of α1 GlyRs.

19
CHAPTER 2
EVIDENCE THAT ETHANOL ACTS ON A TARGET IN LOOP 2 OF THE
EXTRACELLULAR DOMAIN IDENTIFIED OF α1 GLYCINE RECEPTORS

ABSTRACT
Considerable evidence indicates that ethanol acts on specific residues in the
transmembrane domains of glycine receptors (GlyRs). Here we tested the hypothesis
that the extracellular domain is also a target for ethanol action by investigating the
effect of cysteine substitutions at positions 52 (extracellular domain) and 267
(transmembrane domain) on responses to n-alcohols and propyl
methanethiosulfonate (PMTS) in α1GlyRs expressed in Xenopus oocytes. In support
of the hypothesis: 1) The A52C mutation changed ethanol sensitivity compared to
WT GlyRs; 2) PMTS produced irreversible alcohol-like potentiation in A52C GlyRs;
3) PMTS binding reduced the n-chain alcohol cutoff in A52C GlyRs. Further studies
used PMTS binding to cysteines at positions 52 or 267 to block ethanol action at one
site in order to determine its effect at other site(s). In these situations, ethanol caused
negative modulation when acting at position 52 and positive modulation when acting
at position 267. Collectively, these findings parallel the evidence that established the
TM domain as a target for ethanol, suggest that positions 52 and 267 are part of the
same alcohol pocket and indicate that the net effect of ethanol on GlyR function
20
reflects the summation of its positive and negative modulatory effects on different
targets.

INTRODUCTION
Glycine and GABA
A
receptors (GlyRs and GABA
A
Rs) are members of the
cys-loop superfamily of ligand-gated ion channels (LGICs) (Ortells and Lunt
1995;Xiu et al. 2005). These LGICs have received considerable attention as putative
sites of action causing the behavioral effects of ethanol (Dietrich et al. 1999;Harris
1999;Davies et al. 2003). Studies over the last decade identified several positions in
the transmembrane (TM) domain that play a critical role in ethanol modulation of
GlyRs and GABA
A
Rs (Mihic et al. 1997;Ye et al. 1998;Wick et al. 1998;Yamakura
et al. 1999;Ueno et al. 2000;Jenkins et al. 2001;Lobo et al. 2006). Chimeric studies
suggested that serine-267 (S267) and alanine-288 (A288) in TM segments 2 and 3 of
the α1GlyR subunit are important for allosteric modulation by ethanol (Mihic et al.
1997). Further work found that mutations at position 267 changed receptor
sensitivity to ethanol, altered the qualitative ethanol response from potentiation to
inhibition, and decreased the size of a putative alcohol pocket (Ye et al. 1998;Wick
et al. 1998). These findings in GlyRs suggest that the TM domains play a role in
alcohol modulation of receptor function and may form part of an alcohol pocket.
Subsequent studies tested the hypothesis that position 267 in the α1GlyR is a
binding site for alcohols and anesthetics by combining use of the anesthetic-like
21
propyl methanethiosulfonate (PMTS) with the substituted cysteine accessibility
method (Mascia et al. 2000;Karlin and Akabas 1998;Lobo et al. 2004). The authors
proposed that PMTS would covalently bind to the substituted cysteine residue and
change the normal effect of PMTS (reversible potentiation) to irreversible
potentiation if the actions of PMTS and alcohols result from binding at this site. As
predicted, exposing S267C GlyRs to PMTS caused irreversible potentiation and,
thus, supported the notion that the actions of alcohols and anesthetics in these
receptors are due to binding in the TM domains.
Recent experiments suggest that ethanol also acts on the extracellular
domain. The initial evidence came from studies demonstrating that α1GlyRs are
more sensitive to ethanol than are α2GlyRs despite high sequence homology
between α1 and α2 GlyRs. This work also found that an alanine to serine exchange
at position 52 (A52S), located at the beginning of Loop 2 (Brejc et al. 2001) and one
of the residues that differs between α1 and α2 GlyRs, could eliminate the difference
in ethanol sensitivity between α1 and α2 GlyRs (Mascia et al. 1996b). More recent
studies, which found that the A52S mutation eliminated the sensitivity of α1GlyRs
to a direct ethanol antagonist, drew further attention to the extracellular domain as a
target for ethanol action (Davies et al. 2004). Collectively, these studies suggest that
there are multiple sites of ethanol action in α1GlyRs, with one site located in the TM
domain (e.g. position 267) and another in the extracellular domain (e.g. position 52).
The present study tested the hypothesis that the extracellular domain is a
target for ethanol action in α1GlyRs. This work also began to test the relationship
22
between the extracellular and TM domains in mediating the actions of ethanol on this
receptor. We accomplished this by investigating the effect of cysteine mutations at
positions 52 and/or 267 on α1GlyR responses to glycine, alcohols, and PMTS.

MATERIALS AND METHODS
Materials. Adult female Xenopus laevis frogs were obtained from Nasco (Fort
Atkinson, WI). Glycine, alcohols and alkanediols were purchased from Sigma (St.
Louis, MO). PMTS was purchased from Toronto Research Chemicals Inc. (North
York, Toronto, Canada). 100 mM stock solutions of PMTS in dimethyl sulfoxide
(DMSO), stored in aliquots at 4 °C, were thawed and serially diluted with buffer
(DMSO ≤ 0.3%) immediately prior to testing. DMSO at 0.3%, with or without
glycine, had no appreciable effect on GlyR currents in WT or mutant receptors.
DMSO at 0.3%, with or without glycine, had no appreciable effect on GlyR currents
in WT or mutant receptors.

Mutagenesis and Expression of Human α1GlyR Subunit cDNA. Site-directed
mutagenesis in the human GlyR α1 subunit was performed on cDNA subcloned into
the pBK-CMV N/B 200 vector using the QuikChange Site-Directed Mutagenesis kit
(Stratagene, La Jolla, CA). Point mutations were verified by partial sequencing (DNA
Core Facility, University of Southern California). Xenopus laevis oocytes were isolated
and injected with 1 ng of WT or mutant A52C, S267C, or A52C-S267C α1GlyR
23
cDNA using procedures previously described (Davies et al. 2003;Davies et al. 2004).
Injected oocytes were incubated at 18 °C stored in petri dishes with incubation medium
(containing in mM: KCl 2, NaCl 96, MgCl
2
1, CaCl
2
1, HEPES 5, Theophylline 0.6,
Pyruvic Acid 2.5, 1% horse serum and 0.05 mg/mL gentamycin).

Electrophysiology. Electrophysiological measurements were made 1 to 10 days after
injection as previously described (Davies et al. 2003;Davies et al. 2004). Briefly,
oocytes expressing WT and mutant GlyRs were perfused in a 100 μl volume oocyte
bath with Modified Barth’s Saline (MBS) ± drugs at 2.0 mL/min. MBS contains in
mM: 83 NaCl, 1 KCl, 10 HEPES, 0.82 MgSO
4
, 2.4 NaHCO
3
, 0.91 CaCl
2
and 0.33
Ca(NO
3
)
2
adjusted to pH 7.5. Oocytes were impaled with two electrodes backfilled
with 3M KCl with resistances of 0.5-3 M Ω and voltage clamped (–70 mV) (Warner
Instruments, Model OC-725C, Hamden, CT). Currents were continuously recorded
with a strip-chart recorder (Barnstead/Thermolyne, Dubuque, IA).

GlyR Agonist Activation. Oocytes expressing WT or mutant α1GlyRs were
exposed to glycine (10-1,000 μM) for 30s using 5-15 min washouts between
applications to ensure complete resensitization (Mascia et al. 1996b;Mascia et al.
1996a;Davies et al. 2004). Responses were normalized to the maximal glycine
response. Pilot experiments found that WT and cysteine mutant GlyR responses
using a 1 min glycine application reached maximal response, which did not differ
appreciably from results using 30s applications. Therefore, we used the shorter
24
application time to increase efficiency and to minimize desensitization at the higher
glycine concentrations.

GlyR Agonist Modulation. We used specified effective concentrations of agonist
producing a specified percentage of the maximal current (EC
x
) in each experiment to
facilitate comparison of alcohol and PMTS effects across oocytes and receptor
subtypes, while minimizing influence by differences in receptor expression levels
(Davies et al. 2003;Davies et al. 2004). We used a glycine EC
2
for ethanol and
hexanol experiments based on prior studies in WT GlyRs, which showed that
alcohols robustly potentiate GlyR currents at low (EC
2-10
), but not high (EC
50+
)
glycine ECs (Mascia et al. 1996b;Davies et al. 2004). We used a glycine EC
10
with
PMTS in order to facilitate comparison with prior studies in the TM domain (Mascia
et al. 2000;Lobo et al. 2004). Preliminary studies from our laboratory in WT and
mutant α1GlyRs (including A52C and S267C GlyRs) found that their responses to
ethanol did not markedly differ when tested at ECs from 2-10.

Ethanol and Hexanol: Oocytes were preincubated with ethanol (25-100 mM) or
hexanol (30-300 µM) for 60s before coapplication of EC
2
glycine plus ethanol or
hexanol for 30s. Ethanol, hexanol and PMTS did not significantly affect holding
currents of WT and A52C GlyRs in the absence of glycine. In contrast, these agents
produced small direct effects in S267C and A52C-S267C GlyRs. Therefore, in the
latter mutants, we measured peak height from the shifted baseline. The
25
concentrations of ethanol tested in this study are commonly used in
electrophysiological recordings of GlyRs and GABA
A
Rs and roughly correspond to
ethanol concentrations that cause behavioral intoxication (a blood alcohol
concentration of 0.08% is approximately 17 mM) and anesthesia (the minimum
alveolar concentration or MAC for ethanol is 138 mM) (Krasowski and Harrison
1999).

Single PMTS Application. We used two protocols to determine the accessibility of
position 52 to PMTS. Protocol 1 (Open-Desensitized State): Oocytes were perfused
with 30 µM PMTS for 60s before a 30s coapplication with EC
10
glycine to test for
PMTS binding under conditions in which channels are open. Protocol 2 (Resting
State): Oocytes were perfused with 30 µM PMTS for 90s in the absence of glycine to
test for PMTS binding under conditions where the channels are closed. MBS was
perfused for at least 10 min after PMTS exposure to ensure that unbound reagent
completely washed out. The responses to EC
10
glycine and/or PMTS were expressed
as percent control of the initial glycine EC
10
response.

Sequential PMTS Applications. We extended Protocol 1 (Open-Desensitized
State) to include a second PMTS exposure to test for PMTS saturation of cysteine
residues at position 52 and/or 267. EC
10
glycine and/or PMTS (30-300 µM) were
applied as follows, with MBS washout between applications: 1) Glycine; 2) PMTS
26
preincubation/PMTS + Glycine coapplication; 3) Glycine; 4) PMTS
preincubation/PMTS + Glycine coapplication; 5) Glycine.

Sequential PMTS - Ethanol Applications. The sequential protocol described
above was modified to isolate the effects of ethanol on the extracellular and TM
domains. This was accomplished by using saturating concentrations of PMTS (30-
300 μM depending on the mutant receptor tested) for the first application and 100
mM ethanol substituted for the second PMTS application.

Alcohol Cutoff. The n-chain alcohol cutoff refers to the failure to increase potency
as a function of increasing the n-chain alcohol (n-alcohol) length and is thought to
occur when the molecular volume of the alcohol exceeds the finite volume of a
putative alcohol pocket (Wick et al. 1998;Pringle et al. 1981;Alifimoff et al. 1989).
To investigate whether position 52 is part of an alcohol pocket, we tested the effects
of octanol and decanol on A52C GlyRs, before and after PMTS exposure, using the
general procedures described for sequential applications. We also tested the
respective diols (1,8-octanediol; 1,10-decanediol) to minimize confounds from
reduced solubility that occurs as the alcohol chain length increases (Peoples and Ren
2002). We limited the concentration and range of n-alcohols used in this study based
on previous work that established alcohol cutoff in WT and S267C GlyRs (Wick et
al. 1998;Mascia et al. 2000). We counterbalanced the order of drug presentations.

27
Molecular Modeling. To help visualize a putative alcohol pocket that incorporated
the current findings, we built a model of the α1GlyR by threading the human
α1GlyR subunit primary sequence onto the backbone coordinates of a template,
essentially as previously described (Trudell and Bertaccini 2004;Trudell 2002). In
this case, the template was the cryo-electron micrograph of the nicotinic
acetylcholine receptors (nAChR) - PDB ID 2BG9 (Unwin 2005). We used the
Homology module of Insight 2005L (Accelrys, San Diego, CA). The alignment of
the extracellular domain of GlyR with nAChR was as suggested by Sixma and
coworkers (Brejc et al. 2001) and the alignment of the TM domain was as suggested
by Bertaccini (Bertaccini and Trudell 2002). Loops and gaps in the threaded
structure were generated with the same Homology module. The resulting α1GlyR
structure was refined by optimizations to a gradient of 0.1 kcal/Å in which the
backbone harmonic restraints were successively reduced from 100 to 10, 1, and 0
kcal/Å
2
using the Discover_3 module of Insight 2005L.
Cavities in a single subunit of the resulting model were identified with the
Binding Site Analysis module of Insight 2005L using a 1Å cubic grid and default
settings except for a limit on the cavity aperture of 9Å. The results are necessarily
dependent on the choice of grid spacing. In addition to the final cubic grid spacing
of 1Å, cubic grid spacings of 0.5 to 1.5 Å per side were also used. The smallest grid
spacing produced interconnected cavities that had dimensions too small to
accommodate alcohols. Larger grid spacing produced many small, but discontinuous
cavities. The ability of a cavity to accommodate an alcohol molecule is further
28
modified by the dynamic motion of the side chains and the ability of the protein
backbone to deform. Based on the current findings and previous work, we chose the
output in the Results to illustrate the general nature and relative dimensions of the
putative alcohol pocket. Although the volume of the cavity is a function of the
choice of grid spacing, the C α to C α distance between A52 and S267 is taken from
the cryo-electron micrograph of the nAChR (PDB ID 2BG9).

Data Analysis. Data for each experiment were obtained from oocytes from at least
two different frogs. Results are expressed as mean ± SEM. Where no error bars are
shown, they are smaller than the symbols. We used Prism (GraphPAD Software, San
Diego, CA) to perform statistical analyses using one- or two-way repeated measures
ANOVA and Bonferroni post-hoc analyses. Concentration response data were
analyzed using non-linear regression [I = I
max
[A]
nH
/ ([A]
nH
+ EC
50

nH
)] where I is the
peak current recorded following application of a range of agonist concentrations, [A];
I
max
is the estimated maximum current; EC
50
is the glycine concentration required for a
half-maximal response and n
H
is the Hill slope.

RESULTS
Glycine Concentration Response. We first tested the effect of cysteine mutations
at positions 52 and/or 267 on α1GlyR glycine sensitivity and maximum current
responses (Fig. 1). The wildtype GlyR (WT) EC
50
and Hill slope for glycine agree
29
with prior studies (Saul et al. 1994;Ryan et al. 1994;Mascia et al. 1996b;Davies et al.
2004). There were no significant differences between WT and A52C GlyRs in EC
50

or Hill slope (Table 1). Therefore, this mutation did not affect receptor agonist
sensitivity. Likewise, there was not a significant difference between the maximal
current amplitude (I
max
) in WT and A52C GlyRs.
In contrast, the I
max
of S267C and A52C-S267C GlyRs were significantly
lower than for WT GlyRs. Moreover, there was a non-significant leftward shift in
glycine sensitivity in S267C-containing mutant GlyRs. These changes are consistent
with prior findings with S267C (Roberts et al. 2006) and S267Q GlyRs (Findlay et
al. 2002;Findlay et al. 2003), and suggest that mutations at position 267 can affect
agonist responses by altering channel stability. The heterologous expression of
GlyRs in Xenopus oocytes via nuclear cDNA injection does not provide the ability to
measure channel kinetics or whether differences in I
max
reflect changes in receptor
expression or function. Regardless, all of the receptors appeared to respond
normally to glycine measured via two-electrode voltage clamp.

Ethanol and Hexanol Concentration Responses. We next tested the effect of
cysteine mutations on ethanol sensitivity (Fig. 2). Ethanol (25-100 mM) potentiated
WT GlyR function in a concentration dependent manner, which was similar to
previous reports (Mascia et al. 1996b;Davies et al. 2003;Davies et al. 2004). The
A52C mutation right shifted the ethanol concentration response, which supports the
notion that position 52 is important in mediating the actions of ethanol.
30
Substituting cysteine for serine at position 267 changed ethanol modulation
of glycine in α1GlyRs from potentiation to inhibition (Fig. 2C). Ethanol inhibited
S267C and A52C-S267C GlyR function in a concentration dependent manner. The
ethanol inhibition in S267C GlyRs agrees with previous findings using 200 mM
ethanol in this mutant (Ye et al. 1998) and is consistent with the notion that the
residue at position 267 is important for determining ethanol responses in GlyRs.
We tested hexanol at concentrations (30-300 µM) that were functionally
equivalent to those for ethanol. The effect of cysteine mutations on hexanol
sensitivity paralleled the effects of ethanol (Fig. 2D). Since hexanol is
approximately the same volume as PMTS, these results suggest that any differences
between ethanol and PMTS effects on GlyRs in later studies do not reflect volume
differences.
It is noteworthy that ethanol and hexanol, in the absence of glycine, produced
small outward currents in S267C (data not shown) and A52C-S267C GlyRs (Figure
2B). Prior studies found that mutation at the homologous position in GABA
A
Rs also
resulted in ethanol causing direct effects (Ueno et al. 2000). We explored possible
mechanisms for the direct effects of ethanol in α1GlyRs using strychnine and
picrotoxin on the mutant receptors in the absence of glycine (data not shown). As
expected, the GlyR antagonist strychnine (10 μM) did not alter the holding currents
in S267C GlyRs. This finding suggests that there is no obvious contamination or
receptor conformation change in S267C GlyRs that produces “pseudo-agonist”
induced activation in the absence of glycine. In contrast, the channel blocker
31
picrotoxin (100 µM) caused outward currents in S267C GlyRs that were similar to
the outward currents produced by ethanol and hexanol in the absence of glycine.
The ability of picrotoxin to produce outward currents in S267C GlyRs suggests that
there is a small tonic chloride conductance in receptors containing the cysteine
mutation at position 267. Co-application of ethanol and picrotoxin did not further
increase the outward currents elicited by picrotoxin. Taken together, these findings
suggest that the direct effects of ethanol and hexanol arise through a reduction in the
tonic chloride conductance. The decreased tonic conductance produced by ethanol
and hexanol in S267C GlyRs is likely the result of stabilizing the closed state of the
receptor and is consistent with previous studies (Findlay et al. 2002;Findlay et al.
2003).

Single PMTS Application. We tested the hypothesis that position 52 of the α1GlyR
is a target for ethanol capable of causing alcohol-like receptor modulation by using
PMTS in cysteine substituted GlyRs. We predicted that PMTS would cause
irreversible potentiation if PMTS binds to the substituted cysteine site and if the site is
capable of causing alcohol-like receptor modulation (Mascia et al. 2000;Karlin and
Akabas 1998;Lobo et al. 2004). We also evaluated whether receptor state affects the
accessibility of PMTS to cysteines substituted at position 52.
The first experiment tested for PMTS binding under conditions in which
channels are open (Protocol 1—PMTS in the presence of glycine). We found that
exposure to 30 µM PMTS with EC
10
glycine significantly potentiated glycine
32
responses in WT and A52C GlyRs (Fig. 3). Following washout, the responses to
EC
10
glycine in A52C GlyRs were significantly greater than the glycine responses
prior to PMTS exposure. In contrast, the glycine response post-washout in WT
GlyRs did not differ from the pre-PMTS response. These findings show that PMTS
causes irreversible potentiation in A52C GlyRs, but not WT GlyRs. The absence of
irreversible potentiation in WT GlyRs indicates that potentiation by PMTS in WT
GlyRs does not result from it acting on and/or binding to naturally occurring cysteine
residues.
We also tested for PMTS binding using a protocol in which channels are
closed (Protocol 2—PMTS in the absence of glycine). The results with this protocol
were not markedly different than the results using Protocol 1 when PMTS was tested
in the presence of glycine (data not shown). As with Protocol 1, there was no
irreversible potentiation in WT GlyRs. The degree of irreversible potentiation in
A52C GlyR exposed to PMTS in the absence of glycine (125.3 ± 5%, n = 5) was
significantly less than that seen with PMTS in the presence of glycine (145.9 ± 9%, n
= 5, p < 0.05 by Student’s t-test). This difference indicates that PMTS accessibility
to position 52 is greater in the open state of the receptor than in the resting state of
the receptor. Based on these findings, subsequent experiments tested PMTS in the
presence of glycine.

33
Sequential PMTS Applications
PMTS - PMTS (WT and A52C GlyRs). To investigate whether 30 µM PMTS
exposure saturated the cysteines at position 52, we tested sequential PMTS
applications in the presence of glycine in WT and A52C GlyRs (Fig. 4). The first
and second PMTS applications each produced equivalent degrees of reversible
potentiation in WT GlyRs. In contrast, the first and second PMTS applications to
A52C GlyRs produced different responses. The first PMTS application to A52C
GlyRs (Fig. 4C) produced irreversible potentiation that persisted at the same
magnitude over several washouts and glycine applications. The second PMTS
application to A52C GlyRs produced significant, reversible potentiation over and
above the irreversible potentiation from the first PMTS exposure. The complete
reversibility of the second PMTS application on A52C GlyRs suggests that the first
exposure to 30µM PMTS saturated all cysteines at position 52. The reversible
potentiation suggests that position 52 is not the only site in α1GlyRs that causes
PMTS potentiation.

PMTS - PMTS Concentration Response. To investigate the possibility that PMTS
acts on sites in the extracellular and TM domains, we tested the effects of sequential
applications of a single PMTS concentration (30-300 μM) to individual oocytes
expressing WT or mutant GlyRs (Fig. 5). This protocol also further investigated the
PMTS concentration necessary to saturate the substituted cysteines in these mutant
GlyRs.
34
WT GlyRs: The first and second PMTS applications reversibly potentiated
WT GlyR function in a concentration dependent manner (Fig. 5A). The degree of
potentiation did not differ between the first and second PMTS applications.
A52C GlyRs: The first PMTS application potentiated A52C GlyR responses
in a concentration dependent manner similar to WT (Fig. 5B). Subsequent tests with
glycine revealed that the initial responses to high PMTS concentrations reflected
both irreversible and reversible components and that the magnitude of the
irreversible component was the same for all PMTS concentrations. This lack of a
concentration response in the degree of irreversible PMTS potentiation confirms that
30 µM PMTS saturates the cysteine substitutions at position 52. In contrast to the
irreversible component, the reversible component of the PMTS response was
concentration dependent. The response to a second PMTS application produced
results similar to those seen following the first PMTS application. Together, these
findings suggest that PMTS has multiple sites of action in GlyRs—one at position 52
(irreversible component) and at least one at another target (reversible component).
S267C GlyRs: The PMTS responses of S267C GlyRs were similar overall
to those in A52C GlyRs, but there were some important distinctions (Fig. 5C). As in
A52C GlyRs, the first PMTS application potentiated S267C GlyR responses in a
concentration dependent manner. The degree of PMTS potentiation was several-fold
greater in S267C GlyRs than in WT or A52C GlyRs, which suggests that the
cysteine substitution at position 267 affects PMTS efficacy at this site. In contrast to
A52C GlyRs, the degree of the irreversible component seen after washout was
35
concentration dependent. Exposure to 300 μM PMTS caused potentiation with both
irreversible and reversible components. The response to a second PMTS application
produced results similar to those seen following the first application. The second
300 µM PMTS application did not increase the degree of irreversible potentiation,
which indicates that binding to the cysteine substitutions at position 267 reached
saturation with 300 μM PMTS. Together, these findings further support the notion
that PMTS has multiple sites of action in GlyRs—one at position 267 (irreversible
component) and at least one at other targets including position 52 (reversible
component).
A52C-S267C GlyRs: The overall responses of A52C-S267C GlyRs to
PMTS were similar to A52C and S267C GlyRs, with one major difference (Fig. 5D).
As with both single mutants, the first PMTS application potentiated A52C-S267C
GlyR responses in a concentration dependent manner. PMTS potentiation in the
double mutant, as with S267C GlyRs, was several-fold greater than in WT or A52C
GlyRs. Like S267C GlyRs, the degree of irreversible potentiation seen after washout
in the double mutant was concentration dependent. Unlike either of the single
mutations, neither the first nor second PMTS application produced reversible
potentiation at any concentration tested. The second 300 µM PMTS application did
not increase the degree of irreversible potentiation, which indicates that binding to
the cysteine substitutions at positions 52 and 267 reached saturation with 300 µM
PMTS. These findings in WT and mutant GlyRs suggest that PMTS acts at positions
52 and 267. The lack of a reversible component to the PMTS potentiation in A52C-
36
S267C GlyRs indicates that all of the effects of PMTS result from action at these two
targets and suggested that the same might hold true for ethanol.

PMTS - Ethanol. To isolate the effects of ethanol acting on positions 52 and/or
267, we tested the effects of sequential applications of PMTS followed by 100 mM
ethanol in WT and mutant GlyRs (Fig. 6). We reasoned that PMTS would bind to
and saturate all cysteine substituted residues, produce maximal irreversible
potentiation, and would effectively block further modulation through these bound
sites. Subsequent ethanol applications would be active and produce reversible
effects only if there were sites of action still available that were not bound or
sterically hindered by PMTS binding to the substituted cysteine. The nature of the
reversible response would reflect the effects of ethanol on the remaining sites.
WT GlyRs: The pattern of responses to sequential applications of PMTS
followed by 100 mM ethanol in WT GlyRs was essentially the same as found with
sequential PMTS applications. PMTS and ethanol each reversibly potentiated WT
GlyR function (Fig. 6A).
A52C GlyRs: PMTS exposure caused irreversible potentiation in A52C
GlyRs (Fig. 6B). Subsequent ethanol exposure produced significant, reversible
potentiation over and above the irreversible potentiation from the initial PMTS
exposure. These findings in A52C GlyRs indicate that PMTS binding to cysteines
substituted at position 52 does not prevent further modulation by ethanol and are
37
consistent with the notion that ethanol causes potentiation by acting on other targets
like position 267.
S267C GlyRs: PMTS exposure caused irreversible potentiation in S267C
GlyRs (Fig. 6C). Subsequent 100 mM ethanol exposure reversibly reduced the
magnitude of the irreversible potentiation from the initial PMTS exposure (negative
modulation). These findings in S267C GlyRs indicate that: 1) PMTS binding to
cysteines substituted at position 267 does not prevent further modulation by ethanol;
2) Ethanol acts at sites other than those activated by PMTS binding at position 267,
and 3) Ethanol acting on these other sites causes negative modulation of the receptor.
A52C-S267C GlyRs: PMTS exposure caused irreversible potentiation in
A52C-S267C GlyRs (Fig. 6D). Subsequent ethanol exposure caused negative
modulation. These results are similar to those with S267C GlyRs, but the magnitude
of negative modulation by ethanol in the PMTS-exposed double mutant was
significantly less than in PMTS-exposed S267C GlyRs (Fig. 6C vs. 6D, p < 0.05 by
Student’s t-test). Therefore, blocking the negative modulation by ethanol acting on
position 52 reduced the extent of negative modulation by ethanol, but did not
eliminate it. The ability of ethanol to cause negative modulation when access to
positions 52 and 267 is blocked or sterically hindered suggests that there is at least
one other target for ethanol in GlyRs that can function independently from positions
52 and 267 to produce net negative modulation.

38
Alcohol Cutoff. Prior work found that PMTS binding to cysteine substituted
residues at position 267 reduced the alcohol cutoff in GlyRs from between decanol
and dodecanol (Mascia et al. 1996a;Mascia et al. 2000) to below octanol, but did not
alter WT GlyR cutoff (Mascia et al. 2000). These findings were taken to support the
hypothesis that residues in the TM domain of α1GlyRs represent part of an alcohol
pocket. We hypothesized that position 52 in Loop 2 of the extracellular domain is
also part of an alcohol pocket. If true, then PMTS binding to cysteines substituted at
position 52 should also decrease the alcohol cutoff.
To test the notion that position 52 is part of an alcohol pocket, we studied the
effects of n-alcohols on A52C GlyRs before and after PMTS exposure (Fig. 7).
Octanol and decanol each produced significant reversible potentiation when applied
prior to PMTS in A52C GlyRs (Fig. 7A). As expected, PMTS produced irreversible
potentiation in A52C GlyRs when applied after the alcohols (data not shown).
Following PMTS exposure, octanol, but not decanol, caused significant reversible
potentiation in PMTS-exposed A52C GlyRs (Fig. 7A). This loss of decanol potency
in PMTS-exposed A52C GlyRs indicates that PMTS binding to cysteines substituted
at position 52 reduced the n-alcohol cutoff in these receptors.
We also tested alkanediols (diols) in order to investigate the possibility that
the reduced decanol potency following PMTS exposure reflected its lower solubility
compared to octanol (Peoples and Ren 2002). The responses by the diols paralleled
those observed with n-alcohols of the same length (Fig. 7B). The higher
concentrations of decanediol produced some potentiation when applied after PMTS,
39
but the maximal level of potentiation plateaued at a significantly lower level than for
decanediol applied before PMTS. Therefore, the diol findings are consistent with the
notion that PMTS binding at position 52 reduces the size of an alcohol pocket.

Molecular Modeling. The Binding Site Analysis module of Insight 2005L explored
the GlyR α1 subunit described in the Methods (Trudell and Bertaccini 2004;Trudell
2002) and found several cavities. However, not all the cavities found meet the
criteria set by the experimental evidence. The largest cavity fulfills these criteria and
is shown in Fig. 8. This pocket extends from the S267 residue in the TM domain,
through the interface between the TM and extracellular domains, and ends at residue
A52 in Loop 2 of the extracellular domain, with approximately 28 Ǻ separating the
C α atoms of A52 and S267. The ability of the A52C-S267C double mutant to gate
currents and bind PMTS is consistent with there being enough distance between
positions 52 and 267 to prevent spontaneous disulfide linkage between the cysteine
substitutions.

Table 1. Summary of non-linear regression analysis results for the glycine
concentration responses in WT and mutant α1GlyRs. EC
50
, Hill slope, and
maximal current amplitude (I
max
) are presented as mean ± SEM from 4-5 different
oocytes (as shown in Fig. 1). Statistical significance from WT α1GlyRs was
assessed using one-way ANOVA with Bonferroni post test. (* p < 0.05 versus WT).

40

Fig. 1. There is no significant difference between wildtype and mutant α1GlyRs
in sensitivity to glycine. The curves represent non-linear regression analysis of the
glycine concentration responses in WT and mutant α1GlyRs from 4-5 different
oocytes. Each data point represents the mean ± SEM.
41
42
Fig. 2. Cysteine substitutions in α1GlyRs produce position-specific differences
in ethanol and hexanol responses. Representative sequential tracings (from left to
right) for the (A.) potentiating (WT α1GlyR) or (B.) inhibitory ( α1 A52C-S267C
GlyR) effects of ethanol. A52C GlyR tracings resemble the WT GlyR and S267C
GlyR tracings resemble that shown for the A52C-S267C GlyR (data not shown).
The arrows in (B.) indicate the change in holding current in response to ethanol in
A52C-S267C GlyRs and mark the point from which peak heights were measured for
these mutant GlyRs. The horizontal bars above the tracing indicate time of glycine
(lower, solid) and ethanol (upper, slanted line interior) applications (vertical scale bar
= 100 nA; horizontal scale bar = 48s). Ethanol concentrations are (from left to right)
25, 50, and 100 mM. The dashed horizontal line represents the initial glycine EC
2

response. The EC
2
for oocytes in each receptor subtype occurred in the following
ranges (in μM): WT (15-40), A52C (20-35), S267C (10-20), and A52C-S267C (20-
25). (C.) Ethanol. Mean ± SEM percent ethanol modulation of the EC
2
glycine
response for WT and mutant α1GlyRs (n = 4 - 8). ANOVA revealed significant
main effect of mutation [F
3,58
= 61.34, p < 0.0001], a trend for the main effect of
ethanol concentration [F
3,58
= 2.41, p = 0.077] and an interaction between main
effects [F
9,58
= 12.35, p < 0.0001]. (D.) Hexanol. Mean ± SEM percent hexanol
modulation of the EC
2
glycine response for wildtype and mutant α1GlyRs (n = 4 -
6). ANOVA revealed significant main effects of mutation [F
3,58
= 61.45, p <
0.0001], hexanol concentration [F
3,58
= 11.64, p < 0.0001] and an interaction between
the main effects [F
9,58
= 13.09, p < 0.0001].

43

Fig. 3. PMTS exposure in the presence of glycine irreversibly potentiates A52C
GlyRs. Representative sequential tracings (from left to right) for (A.) WT and (B.)
A52C GlyRs. Horizontal bars above the tracing indicate time of glycine (lower,
solid) and 30 μM PMTS (upper, open) applications (vertical scale bar = 500nA;
horizontal scale bar = 48s). The dashed horizontal line represents the initial glycine
EC
10
response. (C.) Mean ± SEM percent control glycine response for WT and
A52C GlyRs (n = 5). The grey shaded box indicates GlyR responses in the presence
of PMTS. There was a significant main effect of treatment [F
2,12
= 17.98, p <
0.0001], a trend for the main effect of mutation [F
1,12
= 5.57, p = 0.056] and an
interaction between main effects [F
2,12
= 11.15, p < 0.01]. (* p < 0.05 versus control
glycine EC
10
response).

44

Fig. 4. A second exposure of A52C GlyRs to PMTS suggests multiple sites of
PMTS action in α1GlyRs. Representative sequential tracings (from left to right)
for (A.) WT and (B.) A52C GlyRs. Horizontal bars above the tracing indicate time
of glycine (lower, solid) and 30 μM PMTS (upper, open) applications (vertical scale
bar = 500nA; horizontal scale bar = 48s). The dashed horizontal line represents the
initial glycine EC
10
response. (C.) Mean ± SEM percent control glycine response
for WT and A52C GlyRs (n = 4). The grey shaded boxes indicate GlyR responses in
the presence of PMTS. There were significant main effects of treatment [F
5,35
=
17.79, p < 0.0001], mutation [F
1,35
= 6.88, p < 0.05] and an interaction between main
effects [F
5,35
= 7.49, p < 0.0001]. (* p < 0.05 versus control glycine EC
10
response, †
p < 0.05 versus residual effect of PMTS)

45
46
Fig. 5. Concurrent activation of positions 52 and 267 by PMTS is sufficient to
explain all of the effects of PMTS on α1GlyRs. Mean ± SEM percent control
glycine response for WT and mutant α1GlyRs (n = 4 - 10). Note that the graphs for
A. and B. are presented on an expanded scale to facilitate interpretation of the PMTS
concentration response in WT and A52C GlyRs. The grey shaded boxes indicate
GlyR responses in the presence of PMTS. (A.) WT GlyRs: There was a significant
main effect of treatment [F
4,48
= 24.66, p < 0.0001], but there was not a significant
main effect of PMTS concentration [F
3,48
= 2.064, p > 0.05] nor an interaction
between main effects [F
12,48
= 1.45, p > 0.05]. (B.) A52C GlyRs: There were
significant main effects of treatment [F
4,64
= 68.31, p < 0.0001], a trend for the main
effect of PMTS concentration [F
3,64
= 3.00, p = 0.061] and an interaction between
main effects [F
12,64
= 3.23, p < 0.01]. (C.) S267C GlyRs: There were significant
main effects of treatment [F
4,80
= 70.21, p < 0.0001], PMTS concentration
[F
3,80
=20.2, p<0.0001] and an interaction between main effects [F
12,80
= 14.72, p <
0.0001]. (D.) A52C-S267C GlyRs: There were significant main effects of
treatment [F
4,60
= 44.56, p < 0.0001], PMTS concentration [F
3,60
= 15.7, p < 0.0001]
and an interaction between main effects [F
12,60
= 12.91, p < 0.0001]. (* p < 0.05
versus control glycine EC
10
response, † p < 0.05 versus residual effect of PMTS;
Statistical significance between treatment groups is indicated only for 300 μM
PMTS).

47 47
48
Fig. 6. PMTS binding to cysteines substituted at position 52 and/or 267 reveals
position-specific negative and positive modulation by ethanol in α1GlyRs. Mean
± SEM percent control glycine response for (A.) WT and (B. - D.) mutant α1GlyRs
(n = 4 - 9). The shaded boxes indicate GlyR responses in the presence of PMTS
(grey) or 100 mM ethanol (yellow). There were significant main effects of treatment
[F
4,76
= 65.31, p < 0.0001], mutation [F
3,76
= 19.43, p < 0.0001] and an interaction
between main effects [F
12,76
= 18.95, p < 0.0001]. (* p < 0.05 versus control glycine
EC
10
response, † p < 0.05 versus residual effect of PMTS).

49 49

Fig. 7. PMTS exposure in α1 (A52C) GlyRs reduces the alcohol cutoff. (A.)
Alcohols and (B.) Alkanediols. Mean ± SEM percent potentiation of EC
10
glycine
response before (white bars) and after (black bars) exposure to 30 μM PMTS (n = 4 -
6). (* p < 0.05 Pre-PMTS versus Post-PMTS by Student’s t-test)

50

Fig. 8. Molecular model of an α1GlyR subunit with the alcohol pocket
highlighted. (A.) Full subunit. The backbone atoms of one α1GlyR subunit are
shown as a yellow ribbon. Residues A52 and S267 are rendered as space-filling
surfaces and atoms are colored red, black, white, and blue for oxygen, carbon,
hydrogen, and nitrogen, respectively. The largest cavity found by the Binding Site
Analysis module of Insight 2005L is shown with a red surface. (B.) Zoom view.
The area enclosed by a rectangle in (A.) is expanded to provide a view of the
interface of the two domains. The C α atoms of A52 and S267 are separated by
approximately 28 Ǻ.

51
52
DISCUSSION
The focus of the present investigation on the extracellular domain of
α1GlyRs marks a departure from prior studies which implicate the TM domain as
the initial target for ethanol action (Mihic et al. 1997;Ye et al. 1998;Wick et al.
1998;Yamakura et al. 1999;Ueno et al. 2000;Jenkins et al. 2001;Lobo et al. 2006).
The limited studies to date in the extracellular domain found that mutations at
position 52 in Loop 2 change ethanol sensitivity, alter sensitivity to an ethanol
antagonist and can eliminate the subunit-dependent differences in ethanol sensitivity
between α1 and α2 GlyRs (Mascia et al. 1996b;Davies et al. 2004). These initial
findings support the notion that ethanol also acts on the extracellular domain.
The current investigation adds two key elements to the evidence supporting
the extracellular domain as an ethanol target. First, this work employed cysteine
mutagenesis at position 52 in α1GlyRs to show that PMTS binding to this site
caused irreversible alcohol-like potentiation. These results demonstrate a cause-
effect relationship between action on a site in the extracellular domain and alcohol-
like GlyR modulation. Second, PMTS binding to cysteines substituted at position 52
in A52C GlyRs decreased the alcohol cutoff. The cutoff reduction suggests that the
extracellular domain contributes to an alcohol pocket. Collectively, these findings
with position 52 of the α1GlyR parallel findings that established the TM domain as a
target for ethanol and indicate that ethanol acts on targets in both the TM and
extracellular domains.
53
Our findings also suggest differences in the responses to ethanol at targets in
the extracellular and TM domains in α1GlyRs. Ethanol potentiated WT and A52C
GlyR function, but inhibited agonist action in S267C and A52C-S267C GlyRs. In
contrast, PMTS caused potentiation in all receptors tested. The differences between
PMTS and ethanol do not appear to reflect the differences in their volumes since
hexanol results paralleled those of ethanol. Together, these findings with PMTS and
ethanol suggest that the responses to anesthetics vary by agent (e.g., ethanol versus
PMTS), position (e.g., position 52 versus 267), and possibly domain (e.g.
extracellular versus TM).
We tested these notions by using PMTS, in combination with cysteine
mutations, to isolate PMTS and ethanol effects on putative sites of action in the
extracellular and TM domains. We reasoned that PMTS binding to a cysteine-
substituted residue would prevent further modulation at that site and, thus, would
reveal the nature of the PMTS or ethanol effects through action on other targets that
can function independently from the site to which PMTS binds. This approach is
analogous to using specific receptor antagonists to help isolate and characterize the
action of ligands at different sites.
From this perspective, our findings provide evidence that PMTS and ethanol
cause different responses in α1GlyRs when their access to position 52 and/or 267 is
blocked or sterically hindered. PMTS acting at either position 52 (extracellular
domain) or 267 (TM domain) caused reversible potentiation when the other target
was blocked. PMTS did not have an effect in the double mutant when access to both
54
positions was blocked. Together, these findings indicate that: 1) PMTS causes
potentiation when it acts position 52 or 267 and 2) PMTS acting on positions 52 and
267 accounts for all of the effects of PMTS on α1GlyR function.
In contrast to PMTS, the characteristics of the ethanol response depended on
whether the initial PMTS exposure blocked access to position 52 and/or 267 in
α1GlyRs. When position 52 was blocked, ethanol caused reversible potentiation,
which suggests that ethanol acting on other targets (e.g., position 267) produces
positive modulation. On the other hand, when position 267 was blocked, ethanol
caused reversible inhibition, which suggests that ethanol acting on other targets (e.g.,
position 52) produces negative modulation. Interestingly, when positions 52 and 267
were both blocked, ethanol caused a small, but significant, amount of reversible
negative modulation. This small amount of residual negative modulation in the
double mutant GlyR indicates that positions 52 and 267 do not account for all of the
effects of ethanol and that ethanol acting on the remaining site(s) causes negative
modulation.
The aforementioned ethanol effects in PMTS-exposed WT and mutant GlyRs
provide insight into the respective actions of ethanol on positions 52 and 267.
Ethanol produced negative modulation when its action on position 267 was blocked
by PMTS exposure in S267C GlyRs. The double mutant GlyR data indicates that
blocking ethanol access to position 52 reduced the degree of negative modulation
produced by ethanol compared to S267C GlyRs. These findings suggest that ethanol
acting on position 52 causes negative modulation. If true, then one would predict
55
that blocking ethanol action on position 52 in A52C GlyRs would increase the
degree of positive modulation versus WT GlyRs. The findings confirm this
prediction and further support the conclusion that ethanol acting on position 52
produces negative modulation. Parallel evidence supports the notion that ethanol
acting on position 267 produces positive modulation.
These ethanol findings are consistent with at least three functionally different
targets for ethanol action in GlyRs: 1) Position 267 in the TM domain (ethanol-
induced positive modulation); 2) Position 52 in Loop 2 of the extracellular domain
(ethanol-induced negative modulation) and 3) One or more other sites (net ethanol-
induced negative modulation) that may include residues near positions 52 and 267
not sterically hindered by PMTS binding. Collectively, these findings suggest that
the net ethanol effect on WT GlyRs represents the summation of positive and
negative modulatory effects on multiple targets.
Interestingly, the results indicate that the effect of PMTS acting on position
52 (positive modulation) is different than the effect of ethanol on the same site
(negative modulation). This difference is conceptually analogous to benzodiazepine
agonists and inverse agonists, which are held to act on the same sites in GABA
A
Rs
(Kucken et al. 2000).
The contention that ethanol causes opposing actions on different targets
within a receptor is supported by previous findings in the TM region of GlyRs (Ye et
al. 1998). This prior study found an inverse linear relationship between the effects of
ethanol on GlyR function and the molecular volume of amino acid substitutions at
56
position 267, with a crossover from potentiation to inhibition at isoleucine. It was
suggested that this switch in ethanol response reflected solely a crossover in the
ethanol response at position 267. Subsequent studies with the S267Q mutation
supported this hypothesis and suggested that the mutation changed ethanol from a
positive to a negative allosteric modulator (Findlay et al. 2002; Findlay et al. 2003).
The present findings suggest that the crossover in ethanol effect with mutation of
position 267 could result from changes in the summed response to ethanol action on
multiple sites. For example, progressive increases in the molecular volume of the
substitution at position 267 could reduce the degree of ethanol-induced positive
modulation at a given concentration to the point where it reveals the negative
modulation by ethanol acting on position 52 and/or other targets. Further research is
necessary to investigate these scenarios. Regardless, the present findings add a new
dimension to interpreting the relationship between structure and function in the
actions of ethanol on GlyRs and other LGICs.
Prior studies support the concept that ethanol may have multiple targets with
different responses in the same receptor protein. Propanethiol binding to cysteine
substitutions revealed changes in n-alcohol modulation consistent with an excitatory
site and an inhibitory site in the TM2 region of nAChRs (Borghese et al. 2003). In
contrast to the present findings in GlyRs, the excitatory and inhibitory sites in
nAChRs were on adjacent positions in the same domain. Similarly, recent studies
with a TM mutation suggest positive and negative modulatory sites for isoflurane in
GABA
A
Rs (Hall et al. 2005). Moreover, the present findings with ethanol parallel
57
the excitatory and inhibitory responses of GlyRs to low and high Zn
++

concentrations, which are believed to result from its action at different sites on the
receptor (Laube et al. 1995). Together, the evidence suggests that LGICs in general
may have both positive and negative modulatory targets for ethanol and that these
targets may reside in either or both the extracellular and TM domains.
It is important to consider alternative explanations for the evidence that
ethanol causes negative modulation by acting on position 52 in α1GlyRs. For
example, the proposed negative modulation could reflect conformational changes
induced by mutation or by PMTS binding to cysteine substitutions at positions 52
and/or 267. These conformational changes in the receptor could alter interactions
between these and other sites, which in turn, could change responses to allosteric
modulators in a manner not consistent with responses at specific sites in WT GlyRs.
The present and previous findings (Ye et al. 1998), which demonstrate that mutating
positions 52 and 267 can alter the ethanol response of α1GlyRs in the presence and
absence of glycine, are consistent with the latter possibility. However, the
congruence of findings would be difficult to explain simply by mutation-induced
conformational changes in receptor function. For example, the concept that ethanol
acting on position 52 causes negative modulation is supported by a combination of
findings in WT and mutant GlyRs, with different responses to ethanol and PMTS,
that are unlikely to reflect coincidental changes in receptor function. Nevertheless,
further studies are necessary to explore these and other possible alternative
explanations to the present findings.
58
The present study identifies a new question: Are the putative sites of ethanol
action in the TM and extracellular domains of α1GlyRs part of a single alcohol
pocket? We addressed this question by investigating the effects of PMTS binding to
cysteines substituted at positions 52 (extracellular domain) and/or 267 (TM domain)
on the alcohol cutoff. We postulated that at least one of these ethanol targets must be
in the pocket that determines the WT GlyR cutoff. If each site is part of a separate
pocket, then reaction of one site with PMTS should alter the WT cutoff, but reaction
of the other site should not. Previous studies found that WT α1GlyRs have a cutoff
between decanol and dodecanol and that PMTS binding to cysteines substituted at
position 267 reduced the cutoff to below octanol (Mascia et al. 1996a;Mascia et al.
2000). We found that PMTS binding to cysteines substituted at position 52 reduced
the cutoff to between octanol and decanol. PMTS binding to cysteines substituted at
both position 52 and 267 appeared to reduce the cutoff to below hexanol (sensitive to
ethanol, but not PMTS as shown in Figs. 5D and 6D). Therefore, the cutoff findings
indicate that neither positions 52 nor 267 belong to a separate pocket that can
independently account for the WT cutoff.
The present findings do not eliminate the possibility that positions 52 and 267
are in separate pockets; however there is no experimental evidence to support this
alternative. It is also possible that a pocket involving positions 52 and 267 could
extend between subunits, but this possibility is not consistent with the orientation of
position 267, in which the residue side chain faces into the subunit (Xu and Akabas
1996). Therefore, the available evidence supports the notion that position 52 of the
59
extracellular domain and position 267 in the TM domain are part of the same alcohol
pocket.
Collectively, the present study provides evidence that the extracellular and
TM domains are targets for ethanol action in α1GlyRs and that positions 52 and 267
in the extracellular and TM domains, respectively, are part of a single alcohol
pocket. Given that this pocket contains sites capable of producing ethanol effects,
we describe the pocket as an ethanol “action pocket” to distinguish it from classical
binding sites, which have higher affinity for their substrates. Taking these findings
and previous work into consideration, molecular modeling of the α1GlyR revealed a
cavity that extends approximately 28 Ǻ from the C α atoms of A52 to S267 that could
function as this alcohol action pocket. The interconnected sections of the pocket are
large enough to accommodate ethanol and to allow its passage between these
regions. This putative alcohol action pocket is consistent with the reduced electron
density seen in cryo-electron micrographs of nAChRs (Unwin 2005) and with the
intertwined loops in our previous molecular models (Trudell and Bertaccini
2004;Trudell 2002). Further study is necessary to map the role of other extracellular
domain residues (within and outside of Loop 2) in the actions of ethanol, to discover
if other alcohol action pockets exist, and to investigate whether the present findings
generalize to other LGICs.
60
CHAPTER 3
LOOP 2 RESIDUES OF THE α1 GLYCINE RECEPTOR PLAY A ROLE IN
AGONIST ACTIVATION AND CHLORIDE ION MOVEMENT IN THE
PORE

ABSTRACT
Recent work suggests that Loop 2 of the α1 glycine receptor (GlyR) plays a
role in agonist activation. Loop 2 has also been shown to line part of the channel
pore and hypothesized to contribute to chloride ion permeability. The present study
systematically tested the role of Loop 2 residues in agonist activation by
investigating the effect of cysteine point mutations at positions 50-60 on the glycine
responses in α1GlyRs expressed in Xenopus oocytes. This study also tested the
hypothesis that charged residues in Loop 2 contribute to an energy well within the
pore which influences chloride ion permeability. Cysteine substitutions at positions
51, 53, 55 and 57 increased the glycine EC
50
. These position specific changes in
glycine sensitivity are consistent with β strand-like structure of the Loop and suggest
that the odd numbered positions in this sequence interact with other agonist-
activation elements at the interface between extracellular and TM domains.
Exposure to negatively charged MTSES, but not positively charged MTSEA or
MTSET, decreased the glycine EC
50
for E53C GlyRs to resemble WT GlyR
responses. Exposure to these MTS reagents did not significantly alter the glycine
61
EC
50
for WT GlyRs. Collectively, these results support the hypothesis that residues
in the Loop 2 region play an important role in agonist activation and chloride ion
movement in α1GlyRs. These findings also provide the basis for a new molecular
model of the Loop 2 region, which offers insight into the structure-function
relationships in GlyRs and possibly other ligand-gated ion channels.

INTRODUCTION
Glycine is a major inhibitory neurotransmitter in the mammalian central
nervous system (Rajendra et al., 1997;Lynch, 2004). It reduces central nervous
system excitability via activation of a ligand-gated receptor linked to an integral
chloride channel. Strychnine-sensitive glycine receptors (GlyRs) belong to a
superfamily of ligand-gated ion channels (LGICs) known as cys-loop receptors
(Ortells and Lunt, 1995;Xiu et. al., 2005) whose members also include γ-
aminobutyric acid type-A (GABA
A
), nicotinic acetylcholine and 5-
hydroxytryptamine
3
, all of which assemble to form ion channels with a pentameric
structure. Cys-loop receptor subunits share significant sequence homology and
consist of four transmembrane (TM) α-helical segments, an intracellular component
for cytosolic interactions, and a large, extracellular ligand-binding domain (Ortells
1997;Brejc et al., 2001;Tang et al., 2002,Unwin, 2005).
Considerable evidence indicates that Loop 2 in the extracellular domain of
cys-loop receptors, as defined by Sixma and colleagues (Brejc et al., 2001), is
62
important for coupling agonist binding to channel gating (Xiu et al., 2005; Kash et
al., 2003; Absalom et al., 2003; Chakrapani et al., 2004; Reeves et al., 2005). The
importance of the α1GlyR Loop 2 region in agonist activation was first noted when
the phenotype of the spasmodic mouse was traced to a naturally occurring alanine-
to-serine exchange at position 52 that results in a significant reduction in glycine
sensitivity without affecting agonist binding characteristics (Saul et al., 1994; Ryan
et al., 1994). Moreover, a splice variant of the α2GlyR revealed that replacing
residues at positions 58 and 59 in α2GlyRs with the residues from homologous sites
in α1GlyRs (I51 and A52) increased α2GlyR glycine sensitivity to resemble that of
the α1GlyR (Miller et al., 2004). Recent work found that mutating the charged
residues in Loop 2 of the α1GlyR (positions 53 and 57) also altered GlyR glycine
sensitivity (Absalom et al., 2003). Taken together, these findings in GlyRs suggest
that Loop 2 residues are not involved in agonist binding, but that several of these
residues play a significant role in transducing agonist activation.
Recent evidence also suggests that Loop 2 lines part of the channel pore and
may influence chloride ion movement (O’mara 2003 and 2005). In these studies,
molecular modeling of the GlyR or GABA
A
R showed that the extracellular half of
the GlyR channel contains a large external vestibule and a small oval chamber, with
the latter being bounded, in part, by residues from Loop 2. Brownian dynamics
simulations then revealed that the charged residues in Loop 2 create an energy
barrier to chloride ion movement (O’mara 2005). To reduce the effective
electrostatic barrier and permit chloride ion movement deeper into the channel, these
63
acidic residues must move away from the pore or be partially neutralized due to
altered pKa. Thus, molecular modeling studies suggest that Loop 2 is important for
chloride ion movement in GlyRs and GABA
A
Rs.
This convergence of evidence led us to hypothesize that Loop 2 plays two roles
in mediating chloride ion flux through GlyRs: one in the transduction of agonist
activation to channel gating and another in influencing the presence and movement of
chloride ions within the channel. The current investigation tested the first hypothesis by
systematically scanning through the Loop 2 region to study the role each residue plays
in glycine activation. To accomplish this goal, we tested the effect of cysteine point
mutations in Loop 2 (positions 50-60) on α1GlyR agonist responses. We tested the
second hypothesis by binding methanethiosulfonate (MTS) reagents with different
charges to cysteine substitutions at position 53, an acidic residue in the pore, to
determine the effect of charge on chloride ion flux through the channel.

MATERIALS AND METHODS
Materials. Adult female Xenopus laevis frogs were obtained from Nasco (Fort
Atkinson, WI). Glycine was purchased from Sigma (St. Louis, MO). 2-Aminoethyl
Methanethiosulfonate (MTSEA), 2-Sulfonatoethyl Methanethiosulfonate (MTSES),
and 2-Trimethylammonioethyl Methanethiosulfonate (MTSET) were purchased from
Toronto Research Chemicals, Inc. (North York, Toronto, Canada).

64
Loop 2 Cysteine Mutagenesis and Expression of α1 GlyR Subunit cDNA. To
investigate the role of Loop 2 residues in ethanol modulation of α1 GlyR function, we
performed cysteine mutagenesis on residues in and adjacent to Loop 2. Loop 2 is
defined as positions 51-57 per alignment with the acetylcholine binding protein
(AChBP) as suggested by Sixma and colleagues (Brejc et al., 2001). The residues
tested in the present study extend beyond this defined range to positions 50-60 to test if
positions near, but outside of Loop 2, play a role in agonist activation or modulation.
Site-directed mutagenesis in the human GlyR α1 subunit was performed on cDNA
subcloned into the pBK-CMV N/B 200 vector using the QuikChange Site-Directed
Mutagenesis kit (Stratagene, La Jolla, CA). Point mutations were verified by partial
sequencing (DNA Core Facility, University of Southern California). Xenopus laevis
oocytes were isolated and injected with 1-10 ng of wildtype (WT) or mutant α1GlyR
cDNA using procedures previously described (Davies et. al., 2003;Davies et. al.,
2004;Crawford et al., in revision).

Electrophysiology. Electrophysiological measurements were made 2 to 10 days after
oocyte injection as previously described (Davies et. al., 2003;Davies et. al.,
2004;Crawford et al., in revision). Briefly, oocytes expressing wildtype and mutant
α1GlyRs were perfused in a 100 μl volume oocyte bath with Modified Barth’s Saline
(MBS) ± drugs at 4.0 ml/min using a peristaltic pump (Rainin Instruments, Oakland,
CA). MBS contains in mM: 83 NaCl, 1 KCl, 10 HEPES, 0.82 MgSO
4
, 2.4 NaHCO
3
,
0.91 CaCl
2
and 0.33 Ca(NO
3
)
2
adjusted to pH 7.5. Oocytes were impaled with two
65
electrodes (1.2mm ID thick-walled filamented glass capillaries) backfilled with 3 M
KCl with resistances of 0.5 -3 M Ω and voltage clamped (–70 mV) using a Warner
Instruments Model OC-725C (Hamden, CT) oocyte clamp. Currents were
continuously recorded with a strip-chart recorder (Barnstead/Thermolyne, Dubuque,
IA).

Glycine Concentration Response. Oocytes expressing wildtype or mutant α1GlyRs
were exposed to glycine for 30s, using 5-15 min washouts between applications to
ensure complete resensitization (Mascia et. al. 1996a;Mascia et. al. 1996b;Davies et
al 2004). Pilot experiments found that WT and cysteine mutant GlyR responses
using a 1 min glycine application reached maximal response, which did not differ
appreciably from results using 30s applications (data not shown). Therefore, we
used the shorter application time to increase efficiency and to minimize
desensitization at the higher glycine concentrations. Responses were normalized to
the maximal glycine response. Concentration response curves were analyzed using
non-linear regression.

MTS Reagent Protocol. Previous experiments have used MTS reagents to
investigate the role of charge in the function of ion channels (Zhang et al., 1997;
Karlin et al., 1998; Wilson et al., 2000; Yoshimura et al., 2001). We use a similar
strategy in this experiment in order to study the role of a charged Loop 2 residue on
α1GlyR function. Oocytes expressing WT or E53C GlyRs were immersed for two
66
minutes in MTSEA (1 mM), MTSES (10 mM), or MTSET (1 mM). Following the
two minute saturation exposure, oocytes were transferred to the recording chamber
and tested as described above for the Glycine Concentration Response study. MTS
solutions were prepared immediately before testing.

Cell-Surface Biotinylation and Immunoblotting. Biotinylation of surface-
expressed proteins was performed as previously described (Chen et al., 2005). Four
days after cDNA injections, oocytes (15 oocytes per group) were incubated with 1.5
mg/mL membrane-impermeable Sulfo-NHS-SS-biotin (Pierce Biotechnology,
Rockford, IL) for 30 min at room temperature. After washing once with 25 mM Tris
(pH 8.0) and twice with Phosphate Buffered Saline (PBS), oocytes were
homogenized in 500 μL of lysis buffer [40 mM Tris (pH 7.5), 110 mM NaCl, 4 mM
EDTA, 0.08% Triton X-100, 1% protease inhibitor cocktail (Vector Laboratories,
Burlingame, CA)]. The yolk and cellular debris were removed by centrifugation at
3600g for 10 min. Aliquots of the supernatant were mixed with 5X Sodium Dodecyl
Sulphate (SDS) loading buffer and stored at -20˚C to assess total receptor fraction.
The remaining supernatant was incubated with streptavidin beads (Pierce
Biotechnology) overnight at 4˚C. Beads were washed three times with lysis buffer
and the biotinylated proteins eluted using SDS loading buffer. The surface and total
proteins were separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes
were incubated overnight with rabbit anti-GlyR antibody (1:100 dilution, Chemicon
67
International, Temecula, CA), followed by incubation with the appropriate
horseradish peroxidase-conjugated secondary antibody. Protein bands were
visualized using enhanced chemiluminescence (Pierce Biotechnology). The blots
were then scanned and analyzed using Scion Image software (Scion Corporation,
Frederick, MD).

Molecular Modeling. To help visualize Loop 2 characteristics which incorporated
the current findings, we built a model of the α1GlyR by threading the human
α1GlyR subunit primary sequence onto the backbone coordinates of a template,
essentially as previously described (Crawford et al., in press; Trudell and Bertaccini,
2004; Trudell, 2002). In this case, the template was the cryo-electron micrograph of
the nicotinic acetylcholine receptors (nAChR) - PDB ID 2BG9 (Unwin, 2005). We
used the Homology module of Insight 2005L (Accelrys, San Diego, CA). The
alignment of the extracellular domain of GlyR with nAChR was as suggested by
Sixma and coworkers (Brejc et al., 2001) and the alignment of the TM domain was
as suggested by Bertaccini (Bertaccini and Trudell, 2002). Loops and gaps in the
threaded structure were generated with the same Homology module. The resulting
α1GlyR structure was refined by optimizations to a gradient of 0.1 kcal/Å in which
the backbone harmonic restraints were successively reduced from 100 to 10, 1, and 0
kcal/Å
2
using the Discover_3 module of Insight 2005L. We considered interactions
among several regions at the interface of the extracellular domain and the TM
domain: Loop 2 (residues 51-57), Loop 7 (cys-loop, residues 138-152), Loop 9
68
(residues 182-188), the pre-TM1 segment (residues 216-221), and the TM2-TM3
loop (residues 276-283).

Data Analysis. Data for each experiment were obtained from oocytes from at least
two different frogs. The n refers to the number of oocytes tested. Results are
expressed as mean ± SEM. Where no error bars are shown, they are smaller than the
symbols. We used Prism (GraphPAD Software, San Diego, CA) to perform curve
fitting and statistical analyses. Concentration response data were analyzed using non-
linear regression analysis: [I = I
max
[A]
nH
/ ([A]
nH
+ EC
50

nH
)] where I is the peak
current recorded following application of a range of agonist concentrations, [A]; I
max
is
the estimated maximum current; EC
50
is the glycine concentration required for a half-
maximal response and n
H
is the Hill slope. Data were subjected to one-way or two-way
Analysis of Variance (ANOVA) with Dunnett’s multiple comparison post test when
warranted. Statistical significance was defined as p < 0.05.

RESULTS
Glycine Concentration Response. We first tested the effect of cysteine point
mutations at positions 50-60 on the glycine sensitivity of α1GlyRs (Figure 1).
Inward Cl
-
currents were evoked in a concentration-dependent manner by glycine in
WT and all mutant GlyRs. The responses were analyzed using non-linear regression.
The glycine EC
50
and Hill slope for WT α1GlyRs agree with previous studies (Table
69
1) (Ryan et al 1994;Saul et al 1994;Mascia et al 1996b;Davies et al 2004;Crawford et
al., in revision). One-way ANOVA revealed significant differences between WT
and several cysteine mutant GlyRs in EC
50
, Hill slope (n
H
) and/or maximal current
amplitude (I
max
). Five mutations (I51C, E53C, T55C, M56C, and D57C)
significantly right shifted and one mutation (T54C) significantly left shifted the
glycine concentration response curve as compared to WT α1GlyRs. Seven
mutations (S50C, I51C, A52C, T55C, M56C, D57C, and Y58C) significantly
reduced the Hill slope. Collectively, these changes in glycine sensitivity support the
notion that many of the residues in the Loop 2 region play an important role in
agonist activation.
The significant reduction in the I
max
for M56C GlyRs, which was not
observed with any other Loop 2 cysteine mutant GlyR, complicates interpretation of
the responses from this mutation. Injecting higher concentrations of cDNA did not
alleviate the reduced I
max
. The methionine residue at this position is conserved
across GlyR and GABA
A
R subunits. Similar impairment of receptor function was
previously reported with the homologous position of the GABA
A
R (Theusch and
Czajkowski, 2005). To determine if the decreased current responses of M56C GlyRs
reflected reduced surface expression levels, we compared the GlyR protein content
of WT and M56C GlyRs via cell-surface biotinylation and immunoblotting analysis.
The findings indicate that M56C GlyR protein expression on the cell surface is
significantly decreased compared to WT GlyRs (data not shown). Therefore, the
70
M56C GlyR mutation was not studied further due to its significantly impaired
expression.

MTS Responses. Based on prior studies, which suggested that charged residues in
the ion pore influence chloride ion movement in response to agonist activation
(O’Mara et al., 2003; O’Mara et al., 2005), we tested the effect of charge at position
53 on chloride ion movement. To accomplish this, we tested the effects of changing
the charge at position 53 by binding positive (MTSEA and MTSET), neutral
(MTSEH) or negatively (MTSES) charged MTS reagents to the substituted cysteine
residue on the magnitude of glycine induced currents in E53C GlyRs.
WT GlyRs: The glycine EC
50
and Hill slope for WT GlyRs exposed to
MTSEA, MTSEH, MTSES, and MTSET did not significantly differ from WT GlyRs
that were not exposed to an MTS reagent (Figure 2 and Table 2). This absence of a
change in baseline function or agonist activation in WT GlyRs indicates that: 1) the
MTS reagents do not bind to cysteines residues in wildtype α1 GlyRs or 2) any MTS
binding that occurs in wildtype GlyRs does not appreciably alter receptor function.
E53C GlyRs: Replacing the negatively charged glutamic acid at position 53
in wildtype GlyRs with the neutral cysteines (E53C) right shifted the glycine
concentration response resulting in a significant increase in the glycine EC
50
(Figure
2 and Table 2). Exposure to the negatively charged MTSES significantly left shifted
the glycine concentration response curve of the E53C GlyR, resulting in an EC
50
and
Hill slope which did not significantly differ from WT GlyRs. These changes
71
induced by cysteine substitution and MTSES binding at position 53 support the
notion that a negative charge at this position is important in promoting chloride ion
flow following agonist activation.
Exposing E53C GlyRs to neutral MTSEH did not significantly change the
glycine sensitivity of E53C GlyRs (Figure 2 and Table 2). This absence of change
reinforces the conclusion that the right shift in glycine response in E53C GlyRs
versus WT GlyRs reflects the change from the negative glutamic acid residue to the
neutral cysteine.
Exposing E53C GlyRs to positively charged MTSEA or MTSET also did not
significantly change the glycine sensitivity of E53C GlyRs (Figure 2 and Table 2).
The absence of change in glycine response suggests that it is not having a charged
residue per se present at position 53 that promotes chloride ion movement in
response to glycine. Together, these MTS findings provides substantive evidence
that a negative charge at position 53 increases response to the agonist and that
neutral and positive substitutions do not.
It should be noted that the lack of change in glycine sensitivity of E53C
GlyRs to neutral and positive MTS reagents could reflect a lack of accessibility due
to their charge or lack thereof. To assess this possibility, we tested the effects of
MTSES on E53C GlyRs that had been exposed to MTSEA, MTSET or MTSEH.
We reasoned that MTSES would bind to position 53 and change glycine responses
only if the first exposure to MTS agents did not bind to the sites. Therefore, if the
follow-up exposure to MTSES changed glycine response, it would indicate that the
72
lack of response in the initial test reflected inaccessibility. However, the findings did
not support this possibility. That is, follow-up exposure to MTSES did not
significantly change the glycine respone in E53C GlyRs (data not shown). These
results indicate that position 53 is accessible to MTS reagents regardless of charge.
Collectively, these results suggest that the negative charge at position 53 is important
for chloride ion placement and movement in the pore.

Table 2. Summary of non-linear regression analysis results for the glycine
concentration responses in WT and mutant α1 GlyRs. EC
50
, Hill slope (n
H
), and
maximal current amplitude (I
max
) are presented as mean ± SEM from 4-5 different
oocytes (as shown in Figure 1). Statistical significance from WT α1 GlyRs was
assessed using one-way ANOVA with Dunnett’s post test. (* p < 0.05, ** p < 0.01)

73
74

Table 3. Summary of non-linear regression analysis results for the glycine
concentration responses in MTS-exposed WT and E53C GlyRs. EC
50
, Hill slope
(n
H
), and maximal current amplitude (I
max
) are presented as mean ± SEM from 4-5
different oocytes (as shown in Figure 2).

75 75

Figure 9. Cysteine point mutations in α1 GlyRs produce position-specific shifts
in the glycine concentration responses. The curves represent non-linear regression
analysis of the glycine concentration responses from 4-5 different oocytes. Each
data point represents the mean ± SEM. The dashed red curve is used to illustrate the
glycine concentration response in WT GlyRs. The grey curves are used to highlight
the glycine responses in cysteine mutant GlyRs with significant changes in glycine
EC
50
as compared to WT.

76

Figure 10. The negative charge at position 53 is important for agonist
activation and chloride ion flux through the channel. The curves represent non-
linear regression analysis of the glycine concentration responses from 4-5 different
oocytes expressing (A.) WT (black) or (B.) E53C GlyRs (grey). Each data point
represents the mean ± SEM. The dashed curve represents the glycine concentration
response in WT GlyRs that were not exposed to MTS reagents.
77

Figure 11. Molecular model of an α1GlyR subunit. (A.) The interface between
the extracellular and TM domains. The amino acid backbone of the GlyR α1
subunit is shown as a ribbon, with the β sheet structure of the extracellular domain
shown in blue and the α-helical structure of the TM domain shown in purple. (B.)
Zoom view. To visualize residues tested in the current study, the area of Loop 2 was
expanded to focus on the interface between the extracellular and TM domains. Here,
the amino acid backbone is shown as a stick structure with the C α atoms at each
apex. The atoms of Loop 2 residues are colored red, black, white, and blue for
oxygen, carbon, hydrogen, and nitrogen, respectively. Alanine 52 is highlighted in
pink in reference to previous work at this position.

78

Figure 12. Comparison of the changes in agonist EC
50
for α1 GlyRs and α7
nAChRs (McLaughlin et al., 2007). Residues 50-58 of the α1GlyR were aligned
with residues 41-48 of the α7 nAChR as suggested by Sixma and colleagues (Brejc
et al., 2001). The pattern of changes in EC
50
is consistent with a β-strand structure
for Loop 2 for both α1GlyRs (black) and α7 nAChRs (grey); however the pattern
reversal suggests that the proposed sequence alignment of these receptors is off by
one residue. Statistical significance from WT α1GlyRs or α7 nAChRs was assessed
using one-way ANOVA with Dunnett’s post test. (* p < 0.05 for WT α1 GlyRs, † p
< 0.05 for WT α7 nAChRs)

79
80
DISCUSSION
The current study represents the first systematic investigation into the role of
Loop 2 residues as sites involved in agonist activation of α1GlyRs. The findings add
evidence that: 1) Each residue within Loop 2 is important for GlyR agonist activation
and 2) The pattern of changes in glycine sensitivity is consistent with β strand-like
structure of the Loop. The findings also suggest that the negative charge at position
53 is important for chloride flux through the GlyR pore. Together, this work led to
the generation of a new molecular model of the α1GlyR based on these findings.
The present study adds substantive new evidence that residues within Loop 2
are important for GlyR agonist activation. We found that cysteine mutations caused
position-specific shifts in glycine sensitivity and reduced the Hill slope. These
findings build upon previous work, which investigated the importance of the Loop 2
region in agonist activation of GlyRs (Saul et al., 1994;Ryan et al., 1994;Mascia et
al., 1996b;Absalom et al., 2003;Miller et al., 2004) and expand our knowledge of the
sites involved in GlyR agonist activation to include other residues in Loop 2.
The pattern of changes in glycine sensitivity provides the first evidence for
the secondary structure of Loop 2 in the extracellular domain of cys-loop receptors.
Cysteine substitutions at positions 51, 53, 55 and 57 increased the glycine EC
50
. In
contrast, cysteine substitution at position 54 decreased the glycine EC
50
. These
position specific changes in glycine sensitivity are consistent with a β strand-like
structure of the Loop (i.e., a β turn) and suggest that the odd numbered positions in
81
this sequence interact with other agonist-activation elements at the interface between
extracellular and TM domains.
Based on this evidence, we developed a new molecular model of the α1GlyR
that depicts Loop 2 with β strand structure (Figure 3). This model suggests that
residues at either end of Loop 2 (positions 51 and 57 in α1GlyRs) could act like a
hinge which provides flexibility to allow movement of Loop 2 with respect to the
more stable β sheet structure that makes up most of the extracellular domain. The
notion of a hinge and the resulting flexibility in relation to other structures at the
interface between the extracellular and TM domains provides a mechanism for Loop
2 movement in agonist activation.
Recent work in the chick α7 nicotinic acetylcholine receptor (nAChR), which
also utilized cysteine scanning mutagenesis of the Loop 2 region, investigated
conformational changes in the inner β -sheet of nAChRs that occur following agonist
binding (McLaughlin et al., 2007). Interestingly, cysteine mutations in the Loop 2
region of nAChRs display a pattern of changes in agonist sensitivity similar to that
found in α1GlyRs, with significant rightward shifts in nAChR agonist sensitivity at
residues that appear to correspond to positions 52, 54, and 56 of the α1GlyR. The
pattern of changes in agonist sensitivity for α1GlyRs and α7 nAChRs appears to be
opposite (Figure 4). This pattern reversal suggests that there are small differences in
the structure of Loop 2 between GlyRs and nAChRs. These structural differences
might underlie discrepancies in the role of certain Loop 2 residues in agonist
activation observed previously in cys-loop receptors (McLaughlin et al., 2007; Sine
82
and Engel, 2006; Xiu et al., 2005; Sala et al., 2005; Reeves et al., 2005; Kash et al.,
2003; Absalom et al., 2003). On the other hand, the pattern reversal might reflect a
difference in sequence alignment between α1GlyRs and α7 nAChRs in which the
alignment is off by a single residue in this region. Preliminary analysis of the
sequence alignment, which varied the gap penalties and the scoring matrices used in
ClustalW, did not support the latter notion. Further research is necessary to
investigate both scenarios. Regardless, these findings are consistent with Loop 2
being a β-turn in both α1GlyRs and α7 nAChRs.
The current study also supports the hypothesis that Loop 2 plays a role in
influencing the presence and movement of chloride ions within the channel. The
cysteine mutation at position 53 removes a negative charge associated with a glutamic
acid residue under physiological conditions and results in a significant right shift in
glycine sensitivity. Binding a negatively charged MTS reagent (MTSES) to the
substituted cysteine residue shifts agonist sensitivity back to resemble that of WT
GlyRs. These findings agree with previous molecular models, which suggested that the
region of the channel pore formed by the Loop 2 region creates an energy well that
hinders chloride ion movement (O’Mara et al., 2003; O’Mara et al., 2005). We also
found that binding positively charged MTS reagents (MTSEA or MTSET) does not
further decrease the glycine EC
50
for E53C GlyRs beyond that of the mutation alone.
Together, these findings suggest that the presence of a negative charge at position 53 is
important for chloride ion permeability and that, once this negative charge is removed,
additional positive charge in this region does not increase the negative effect chloride
83
ion movement. Further studies are necessary at position 57, the other Loop 2 charged
residue which does not face into the channel pore, to determine if these charge results
generalize to other locations. Regardless, the present findings are the first to
demonstrate that the charge associated with a Loop 2 residue influences chloride ion
movement in the channel pore.
Collectively, these findings suggest that the extracellular domain plays an
important role, both structurally and functionally, in GlyR agonist activation. The
findings provide a new model for agonist activation involving the Loop 2 region,
offer insight into the structure-function relationships in GlyRs and suggest that the
present findings may generalize to other ligand-gated ion channels.
84
CHAPTER 4
ETHANOL AND ANESTHETIC SENSITIVITY OF α1 GLYCINE
RECEPTORS SELECTIVELY ALTERED BY CYSTEINE MUTATIONS IN
THE LOOP 2 REGION

ABSTRACT
Recent work suggests that Loop 2 of the α1 glycine receptor (GlyR) plays a
role in agonist activation and that position 52 is a target for ethanol action. The
present study systematically tested the role of Loop 2 residues in agonist activation
and general anesthetic modulation by investigating the effect of cysteine point
mutations at positions 50-60 on the responses to ethanol, isoflurane, propofol and
PMTS in α1GlyRs expressed in Xenopus oocytes. Cysteine mutations at positions
51 and 53 changed the magnitude of ethanol potentiation, whereas cysteines
substitution at position 57 switched the ethanol response to inhibition. Potentiation
by isoflurane and propofol was also altered by cysteine mutations in this region,
however the role of specific residues within the Loop 2 region in ethanol or
anesthetic action appeared to differ. PMTS produced irreversible modulation in all
of the Loop 2 cysteine mutant GlyRs except V60C. These findings suggest that each
Loop 2 residue is capable of producing alcohol- or anesthetic-like potentiation.
Collectively, these findings support the hypothesis that residues in the Loop 2 region
play an important role in allosteric modulation by ethanol and other general
85
anesthetics in α1GlyRs. These findings also provide the basis for a new molecular
model of the Loop 2 region and offer new insight into the structure-function
relationships in GlyRs and possibly other ligand-gated ion channels.

INTRODUCTION
Glycine is a major inhibitory neurotransmitter in the adult mammalian central
nervous system (Rajendra et al., 1997;Lynch, 2004). It reduces central nervous
system excitability via activation of a ligand-gated receptor linked to an integral
chloride channel. Strychnine-sensitive glycine receptors (GlyRs) belong to a
superfamily of ligand-gated ion channels (LGICs) known as cys-loop receptors
(Ortells and Lunt, 1995;Xiu et. al., 2005) whose members also include γ-
aminobutyric acid type-A (GABA
A
), nicotinic acetylcholine and 5-
hydroxytryptamine
3
, all of which assemble to form ion channels with a pentameric
structure. Cys-loop receptor subunits share significant sequence homology and
consist of four transmembrane (TM) α-helical segments, an intracellular component
for cytosolic interactions, and a large, extracellular ligand-binding domain (Ortells
1997;Brejc et al., 2001;Tang et al., 2002,Unwin, 2005).
Cys-loop receptors have received considerable attention as putative sites of
action causing the behavioral effects of alcohol and other general anesthetics
(Dietrich et al., 1999;Harris, 1999;Krasowski and Harrison, 1999;Davies et al.,
2003). Studies over the last decade identified several positions in the TM domain
86
that play a critical role in causing ethanol modulation of the inhibitory cys-loop
receptors - GlyRs and GABA
A
Rs (Greenblatt and Meng, 1999;Jenkins et. al.,
2001;Mihic et. al., 1997;Ueno et. al., 2000;Wick et. al., 1998;Yamakura et. al.,
1999;Ye et. al., 1998;Lobo et. al., 2006;Mascia et al., 2000;Hall et al., 2005).
Growing evidence indicates that Loop 2 in the extracellular domain of
α1GlyRs, as defined by Sixma and colleagues (Brejc et al., 2001), is also a target for
alcohol action (Davies et al., 2004;Crawford et al., in press). The evidence for Loop
2 as a target for ethanol began with work which found that a serine mutation at
position 52 (A52S) altered ethanol sensitivity without altering sensitivity to the
intravenous anesthetic, propofol (Mascia et. al., 1996b), and abolished α1GlyR
sensitivity to a direct ethanol antagonist (Davies et al., 2004).
Subsequent studies tested the hypothesis that position 52 is a target for
ethanol action in α1GlyRs using propyl methanethiosulfonate (PMTS), a thiol agent
held to mimic ethanol action (Mascia et al., 2000). As predicted, PMTS produced
irreversible alcohol-like potentiation in α1GlyRs with cysteine mutations at position
52 (Crawford et al., in press). These findings with PMTS indicate that position 52 is
capable of producing alcohol-like potentiation. Moreover, PMTS binding to cysteine
mutations at position 52 (Crawford et al., in press) and position 267 (Mascia et al.,
2000) each reduced the n-chain alcohol cutoff, suggesting that these sites for ethanol
action form part of a single alcohol pocket.
Further studies used PMTS to isolate the effects of ethanol on putative
ethanol targets in the extracellular and TM domains (Crawford et al., in press). This
87
work found that ethanol acting on the TM domain produced potentiation when
access to sites in Loop 2 were blocked by PMTS binding to cysteine substitutions at
position 52. In contrast, ethanol acting on the extracellular domain produced
negative modulation when access to sites in the TM domain were blocked by PMTS
binding to cysteine substitutions at position 267.
Collectively, these findings in GlyRs suggest that there are sites of ethanol
action in the extracellular and TM domains and that these sites play opposing roles
(negative and positive modulation, respectively) in ethanol modulation of α1GlyRs.
The question remains whether other residues in the Loop 2 region are also targets for
ethanol and what their role is in producing ethanol effects.
The current investigation addressed these questions by systematically
scanning through the Loop 2 region to study the role each residue plays in alcohol
and anesthetic modulation. To accomplish this goal, we tested the effect of cysteine
point mutations in Loop 2 (positions 50-60) on α1GlyR agonist, ethanol and other
general anesthetics, and PMTS sensitivities.

MATERIALS AND METHODS
Materials. Adult female Xenopus laevis frogs were obtained from Nasco (Fort
Atkinson, WI). Glycine, ethanol, and propofol were purchased from Sigma (St. Louis,
MO). Isoflurane was purchased from Abbott Laboratories (North Chicago, IL). PMTS
was purchased from Toronto Research Chemicals Inc. (North York, Toronto, Canada).
88
10 mM stock solutions of isoflurane and propofol were prepared in DMSO and serially
diluted with buffer (DMSO ≤ 0.1%) immediately prior to testing. 100 mM stock
solutions of PMTS in DMSO, stored in 10 µL aliquots at 4°C, were thawed and diluted
with buffer similar to the other anesthetics. Pilot studies found that DMSO at this
concentration, with or without glycine, had no appreciable effect on GlyR currents in
WT or mutant receptors (Crawford et al., in press).

Loop 2 Cysteine Mutagenesis and Expression of α1 GlyR Subunit cDNA. To
investigate the role of Loop 2 residues in ethanol modulation of α1 GlyR function, we
performed cysteine mutagenesis on residues in and adjacent to Loop 2. Loop 2 is
defined as positions 51-57 per alignment with the acetylcholine binding protein
(AChBP) as suggested by Sixma and colleagues (Brejc et al., 2001). The residues
tested in the present study extend beyond this defined range to positions 50-60 to test if
positions near, but outside of Loop 2, play a role in agonist activation or modulation.
Site-directed mutagenesis in the human GlyR α1 subunit was performed on cDNA
subcloned into the pBK-CMV N/B 200 vector using the QuikChange Site-Directed
Mutagenesis kit (Stratagene, La Jolla, CA). Point mutations were verified by partial
sequencing (DNA Core Facility, University of Southern California). Xenopus laevis
oocytes were isolated and injected with 1-10 ng of wildtype (WT) or mutant α1GlyR
cDNA using procedures previously described (Davies et. al., 2003;Davies et. al.,
2004;Crawford et al., in press).

89
Electrophysiology. Electrophysiological measurements were made 2 to 10 days after
oocyte injection as previously described (Davies et. al., 2003;Davies et. al.,
2004;Crawford et al., in press). Briefly, oocytes expressing wildtype and mutant
α1GlyRs were perfused in a 100 μl volume oocyte bath with Modified Barth’s Saline
(MBS) ± drugs at 4.0 ml/min using a peristaltic pump (Rainin Instruments, Oakland,
CA). MBS contains in mM: 83 NaCl, 1 KCl, 10 HEPES, 0.82 MgSO
4
, 2.4 NaHCO
3
,
0.91 CaCl
2
and 0.33 Ca(NO
3
)
2
adjusted to pH 7.5. Oocytes were impaled with two
electrodes (1.2mm ID thick-walled filamented glass capillaries) backfilled with 3 M
KCl with resistances of 0.5 -3 MΩ and voltage clamped (–70 mV) using a Warner
Instruments Model OC-725C (Hamden, CT) oocyte clamp. Currents were
continuously recorded with a strip-chart recorder (Barnstead/Thermolyne, Dubuque,
IA).

Glycine Concentration Response. Oocytes expressing wildtype or mutant α1GlyRs
were exposed to glycine for 30s, using 5-15 min washouts between applications to
ensure complete resensitization (Mascia et. al. 1996a;Mascia et. al. 1996b;Davies et
al 2004). Pilot experiments found that WT and cysteine mutant GlyR responses
using a 1 min glycine application reached maximal response, which did not differ
appreciably from results using 30s applications (data not shown). Therefore, we
used the shorter application time to increase efficiency and to minimize
desensitization at the higher glycine concentrations. Responses were normalized to
90
the maximal glycine response. Concentration response curves were analyzed using
non-linear regression.

Ethanol Concentration Response. The effect of ethanol on WT and mutant GlyRs
was tested using an effective concentration of agonist that produced 10±2% of the
maximal current for the oocyte (EC
10
). We standardized the agonist effect in order
to facilitate comparison of drug effects across oocytes and receptor subtypes, while
minimizing influence by differences in receptor expression levels (Davies et. al.,
2003;Davies et. al., 2004;Crawford et al., in press). We used an EC
10
for these
experiments based on prior studies, which showed that alcohols robustly potentiate
GlyR currents at low (EC
2-10
), but not high (EC
50+
) glycine ECs (Mascia et al.,
1996a, Davies et al., 2004). Glycine was applied for 60s in order to establish an
EC
10
for each oocyte. After a 5 min washout period, oocytes were pre-incubated
with ethanol (25-100 mM) for 60s before co-application of EC
10
glycine plus ethanol
for 60s. Following an 8 min washout, the glycine EC
10
was applied for 60s.
Applications of low and high ethanol concentrations were counter-balanced to
minimize order effects. Results are presented as the percent modulation of the EC
10

glycine response. The ethanol concentrations tested are used commonly in
electrophysiological recordings of GlyRs and GABA
A
Rs and roughly correspond to
ethanol concentrations that cause behavioral intoxication (a blood alcohol
concentration of 0.08% is approximately 17 mM) and anesthesia (the minimum
91
alveolar concentration or MAC for ethanol is 138 mM) (Krasowski and Harrison,
1999).

General Anesthetic Concentration Responses. Oocytes expressing WT and
mutant GlyRs were tested for their responses to isoflurane and propofol using the
procedures and conditions described above for ethanol. We prepared concentrations
of isoflurane (100-400 μM) and propofol (5-10 μM) to functionally match the
potentiation produced by 25-100 mM ethanol in WT GlyRs. The actual bath
concentrations of isoflurane and propofol may have been less than the prepared
concentration due to losses during delivery (Krasowski and Harrison, 2000;Lopreato
et al., 2003). The anesthetic concentrations tested are used commonly in
electrophysiological recordings of GlyRs and GABA
A
Rs and roughly correspond to
concentrations that cause anesthesia (the MAC for isoflurane and propofol are 260
μM and 0.4 μM, respectively) (Krasowski and Harrison, 1999).

Single PMTS Application. The effect of PMTS on WT and mutant GlyRs was
tested using an EC
10
glycine in order to facilitate comparison with prior studies at
position 52 and in the TM domain (Crawford et al., in press;Mascia et al., 2000;Lobo
et al., 2004). Oocytes were perfused with 100 µM PMTS for 60s before a 60s co-
application with EC
10
glycine to test for PMTS binding under conditions in which
channels are open. These conditions are based on previous studies which found that
PMTS accessibility to position 52 was greater in the open state versus the resting
92
state of the receptor (Crawford et al., in press). This prior work in A52C GlyRs also
indicated that 100 μM PMTS was sufficient to saturate the substituted cysteine
residues at position 52. MBS was perfused for at least 10 min after PMTS exposure
to ensure that unbound reagent completely washed out. The responses to EC
10

glycine and/or PMTS were expressed as percent control of the initial glycine EC
10

response.

Sequential PMTS - Ethanol Applications. We extended the Single PMTS
Application protocol described above to include exposure to 100 mM ethanol to test
the notion that residues in Loop 2 and the TM domain can account for all of the
effects of ethanol in α1 GlyRs. Previous experiments have used this protocol to
isolate the effects of ethanol acting on different sites in α1 GlyRs (Crawford et al., in
press). EC
10
glycine, 300 µM PMTS and/or ethanol were applied as follows, with
MBS washout between applications: 1) Glycine; 2) PMTS pre-incubation/PMTS +
Glycine co-application; 3) Glycine; 4) Ethanol pre-incubation/Ethanol + Glycine co-
application; 5) Glycine.

Molecular Modeling. To help visualize Loop 2 characteristics which incorporated
the current findings, we built a model of the α1GlyR by threading the human
α1GlyR subunit primary sequence onto the backbone coordinates of a template,
essentially as previously described (Crawford et al., in press; Trudell and Bertaccini,
2004; Trudell, 2002). In this case, the template was the cryo-electron micrograph of
93
the nicotinic acetylcholine receptors (nAChR) - PDB ID 2BG9 (Unwin, 2005). We
used the Homology module of Insight 2005L (Accelrys, San Diego, CA). The
alignment of the extracellular domain of GlyR with nAChR was as suggested by
Sixma and coworkers (Brejc et al., 2001) and the alignment of the TM domain was
as suggested by Bertaccini (Bertaccini and Trudell, 2002). Loops and gaps in the
threaded structure were generated with the same Homology module. The resulting
α1GlyR structure was refined by optimizations to a gradient of 0.1 kcal/Å in which
the backbone harmonic restraints were successively reduced from 100 to 10, 1, and 0
kcal/Å
2
using the Discover_3 module of Insight 2005L. We considered interactions
among several regions at the interface of the extracellular domain and the TM
domain: Loop 2 (residues 51-57), Loop 7 (cys-loop, residues 138-152), Loop 9
(residues 182-188), the pre-TM1 segment (residues 216-221), and the TM2-TM3
loop (residues 276-283).

Data Analysis. Data for each experiment were obtained from oocytes from at least
two different frogs. The n refers to the number of oocytes tested. Results are
expressed as mean ± SEM. Where no error bars are shown, they are smaller than the
symbols. We used Prism (GraphPAD Software, San Diego, CA) to perform curve
fitting and statistical analyses. Concentration response data were analyzed using non-
linear regression analysis: [I = I
max
[A]
nH
/ ([A]
nH
+ EC
50

nH
)] where I is the peak
current recorded following application of a range of agonist concentrations, [A]; I
max
is
the estimated maximum current; EC
50
is the glycine concentration required for a half-
94
maximal response and n
H
is the Hill slope. Data were subjected to one-way or two-way
Analysis of Variance (ANOVA) with Dunnett’s multiple comparison post test when
warranted. Statistical significance was defined as p < 0.05.

RESULTS
Glycine Concentration Response. We first tested the effect of cysteine point
mutations at positions 50-60 on the glycine sensitivity of α1GlyRs. These findings
were presented in Chapter 3 and reproduced here as Figure 1 and Table 1. The
changes in glycine sensitivity are consistent with the notion that many of the residues
in the Loop 2 region play an important role in agonist activation.

Ethanol Concentration Response. We next tested the effect of cysteine point
mutations on the ethanol sensitivity of α1GlyRs (Figure 2). Exposure to 25 or 100
mM ethanol potentiated WT α1GlyR function in a concentration-dependent manner
that was consistent with previous reports (Figure 2) (Mascia et al., 1996b;Davies et.
al., 2003;Davies et. al., 2004;Crawford et al., in press). Seven mutations (S50C,
A52C, T54C, T55C, Y58C, R59C, and V60C) did not significantly alter ethanol
potentiation as compared to WT α1GlyRs. The I51C mutation significantly
increased potentiation by 25 mM ethanol, but did not significantly alter potentiation
by 100 mM ethanol. The E53C mutation did not significantly affect potentiation by
25 mM ethanol, but significantly decreased potentiation by 100 mM ethanol.
95
Cysteine substitution at position 57 switched the ethanol response from potentiation
to inhibition. The magnitude of inhibition in D57C GlyRs was greater with 100 mM
ethanol than with 25 mM ethanol. These findings with ethanol are consistent with
the notion that several residues in the Loop 2 region play a role in ethanol
modulation of α1GlyRs and that some positions may play different roles in ethanol
modulation than do others.

Isoflurane Concentration Response. We next tested the effect of cysteine point
mutations on the isoflurane sensitivity of α1GlyRs (Figure 3). Previous studies
suggest that the volatile anesthetic, isoflurane, acts at the same TM sites as ethanol
(Mihic et al., 1997;Krasowski et al., 1998;Hall et al., 2005). The current study tests
the hypothesis that isoflurane also acts at the same sites as ethanol in the Loop 2
region of the extracellular domain.
Exposure to 100 or 400 μM isoflurane potentiated WT α1GlyR function in a
concentration-dependent manner that was consistent with previous reports (Harrison
et al., 1993;Downie et al., 1996;Schofield et al., 2004;Yamakura et al., 2000). Three
mutations (S50C, D57C, and Y58C) did not significantly alter isoflurane potentiation
as compared to WT α1GlyRs. Two mutations (I51C, T54C) increased potentiation
by 100 and 400 μM isoflurane as compared to WT GlyRs. Five mutations (A52C,
E53C, T55C, R59C, V60C) increased potentiation by only 400 μM isoflurane.
Interestingly, the D57C mutation, which switched the effect of ethanol from
potentiation to inhibition, did not significantly change receptor response to
96
isoflurane. These findings support the hypothesis that the Loop 2 region plays a role
in isoflurane modulation of α1GlyRs and that the roles of specific residues within
the Loop 2 region may differ from their roles for ethanol.

Propofol Concentration Response. We also tested the effect of cysteine point
mutations on the propofol sensitivity of α1GlyRs (Figure 4). Propofol, an
intravenous anesthetic, is believed to have sites of action in the TM domain of
GABA
A
Rs distinct from those for ethanol and isoflurane (Krasowski et al., 1998;Bali
et al., 2004). By homology, the same has been suggested for α1GlyRs (Beckstead et
al., 2000). Previous work has shown that a point mutation at position 52 in Loop 2
of the extracellular domain, which alters ethanol sensitivity, does not affect propofol
sensitivity (Mascia et al., 1996b). Here, we tested if the same distinction holds true
for other residues in the Loop 2 region.
Exposure to 5 or 10 μM propofol potentiated WT α1GlyR function in a
concentration-dependent manner that was consistent with previous reports (Mascia et
al., 1996b). The cysteine mutation at position 51 significantly increased propofol
potentiation. The significant increase in propofol potentiation in I51C GlyRs
parallels the increased potentiation by ethanol, PMTS, and isoflurane resulting from
the same mutation. No other cysteine mutation significantly altered propofol
potentiation at the concentrations tested. These findings suggest that Loop 2, and
position 51 in particular, plays a role in propofol modulation of α1GlyRs.
Alternatively, the increases in potentiation by ethanol, isoflurane and propofol due to
97
cysteine mutation at position 51 could indicate that the I51C GlyR results reflect a
change in allosteric modulation that is related to agonist activation, and thus, not
specifically due to binding of these agents at this site.

Single PMTS Application. We also tested the hypothesis that multiple residues
within the Loop 2 region of the α1GlyR are targets for ethanol capable of causing
alcohol-like receptor modulation by applying PMTS to cysteine substituted GlyRs.
PMTS is a thiol agent which causes alcohol-like effects on GlyRs and GABA
A
Rs
(Mascia et al., 2000). Previous work supports the notion that PMTS covalently binds
to a substituted cysteine residue and changes the normal effect of PMTS (reversible
potentiation) to irreversible potentiation if the site is capable of causing alcohol-like
potentiation (Mascia et al., 2000;Lobo et al., 2004;Roberts et al., 2005;Crawford et al.,
in press).
Exposure to 100 µM PMTS with EC
10
glycine significantly potentiated
glycine responses in WT α1GlyRs (Figure 5). The WT GlyR response to PMTS was
completely reversible. That is, following washout, the glycine response in WT
GlyRs did not differ from the pre-PMTS response. The absence of irreversible
potentiation in WT GlyRs indicates that PMTS potentiation does not result from it
acting on and/or binding to cysteine residues that are present in WT GlyRs. As with
WT GlyRs, the glycine response post-washout in V60C GlyRs did not differ from
the pre-PMTS response. The lack of irreversible potentiation in V60C GlyRs
suggests that: 1) Position 60 is not accessible to PMTS, or 2) Position 60 is
98
accessible to PMTS and PMTS binds to the substituted cysteine residue, however the
reaction does not produce alcohol-like potentiation as a functional result. The
present experiments are unable to distinguish between these scenarios.
In contrast to WT and V60C GlyRs, the EC
10
glycine responses following
PMTS washout in eight mutants (S50C, I51C, A52C, E53C, T54C, T55C, Y58C,
and R59C) were significantly greater than their glycine responses prior to PMTS
exposure (Figure 5). The irreversible potentiation in these mutant GlyRs indicates
that the Loop 2 region is accessible to PMTS. Moreover, PMTS binding to these
cysteine substituted residues produces alcohol-like potentiation of GlyR function and
suggests that each residue in the Loop 2 region is capable of producing alcohol or
anesthetic modulation of receptor function.
Exposure to 100µM PMTS with EC
10
glycine significantly inhibited glycine
responses in D57C GlyRs (Figure 5). Following washout, the responses to EC
10

glycine remained significantly inhibited when compared to the glycine responses
prior to PMTS exposure. The inhibition of D57C GlyRs by PMTS parallels the
inhibition by ethanol and suggests that ethanol acting on position 57 causes negative
modulation of GlyR function.

Sequential PMTS – Ethanol Applications. Previous studies from our laboratory
used PMTS binding to substituted cysteine residues to block ethanol action at one
site in order to determine its effect at the other site(s) (Crawford et al., in press). We
reasoned that PMTS would bind to and saturate cysteine substituted residues,
99
produce maximal irreversible potentiation, and would effectively block further
modulation through these bound sites. Subsequent ethanol applications would be
active and produce reversible effects only if there were sites of action still available
that were not bound or sterically hindered by PMTS binding to the substituted
cysteine. The nature of the reversible response would reflect the effects of ethanol
on the remaining sites. This approach is analogous to using specific receptor
antagonists to help isolate and characterize the action of ligands at different sites.
Given this reasoning, we tested the effects of sequential applications of
PMTS followed by 100 mM ethanol in WT and mutant α1 GlyRs in order to isolate
the effects of ethanol acting on different sites (Crawford et al., in press). In these
situations, ethanol caused negative modulation when acting at position 52 and
positive modulation when acting at position 267. When positions 52 and 267 were
both blocked, ethanol caused a small, but significant, amount of reversible negative
modulation. This small amount of residual negative modulation in the double mutant
GlyR indicates that positions 52 and 267 do not account for all of the effects of
ethanol. Given the current results with ethanol and PMTS in GlyRs with cysteine
mutations in Loop 2, we hypothesized that occupation of two sites in Loop 2 (I51
and D57) and a site in the TM domain (S267) could account for all of the effects of
ethanol. To test this hypothesis, we created a triple mutant GlyR in which we
substituted cysteine residues into positions 51, 57, and 267.
We first tested the effect of the three mutations on the glycine sensitivity of
α1GlyRs (data not shown). Inward Cl
-
currents were evoked in a concentration-
100
dependent manner by glycine in triple mutant GlyRs. Student’s t-test revealed
significant differences between WT and triple mutant GlyRs in EC
50
(97.4 ± 9 μM
versus 777.7 ± 71 μM, n = 4, p < 0.0001), Hill slope (2.3 ± 0.2 versus 1.3 ± 0.1, n =
4, p < 0.005) and/or maximal current amplitude (11313 ± 2176 nA versus 1763 ±
382 nA, n = 4, p < 0.005). These glycine findings in the triple mutant are similar to
the I51C and D57C GlyR individual cysteine point mutations.
We next tested the PMTS – Ethanol protocol described above. These initial
studies found that PMTS (300 μM) exposure caused irreversible potentiation in the
triple mutant GlyRs (Figure 6). Subsequent ethanol exposure reversibly reduced the
magnitude of the irreversible potentiation from the initial PMTS exposure. The
ability of ethanol to produce an effect when access to positions 51, 57 and 267 is
blocked or sterically hindered suggests that these targets do not account for all of the
effects of ethanol in α1GlyRs. Alternatively, the cysteine substitutions at position 51
and 57 could have spontaneously formed a disulfide bond, which in turn, prevented
PMTS binding. Without PMTS binding to and irreversibly acting at positions 51 and
57, ethanol might be able to act on these or neighboring targets. The PMTS –
Ethanol responses of the triple mutant resemble those for S267C GlyRs (Crawford et
al., in press), thereby suggesting that the latter notion is true. Further research is
necessary to distinguish between these scenarios.

Table 4. Summary of non-linear regression analysis results for the glycine
concentration responses in WT and mutant α1 GlyRs. EC
50
, Hill slope (n
H
), and
maximal current amplitude (I
max
) are presented as mean ± SEM from 4-5 different
oocytes (as shown in Figure 1). Statistical significance from WT α1 GlyRs was
assessed using one-way ANOVA with Dunnett’s post test. (* p < 0.05, ** p < 0.01)
(from Chapter 3)

101

Figure 13. Cysteine point mutations in α1 GlyRs produce position-specific
shifts in the glycine concentration responses. The curves represent non-linear
regression analysis of the glycine concentration responses from 4-5 different oocytes.
Each data point represents the mean ± SEM. The dashed red curve is used to
illustrate the glycine concentration response in WT GlyRs. The grey curves are used
to highlight the glycine responses in cysteine mutant GlyRs with significant changes
in glycine EC
50
as compared to WT. (from Chapter 3)

102

Figure 14. Cysteine substitutions in α1 GlyRs produce position-specific
differences in the ethanol responses. (A.) 25 and (B.) 100 mM ethanol. Mean ±
SEM percent ethanol modulation of the EC
10
glycine response for WT and mutant
α1 GlyRs as measured in 4-6 oocytes. Shaded bars indicate the results from residues
within the defined range for Loop 2 (positions 51-57). Two-way ANOVA revealed
significant main effects of receptor mutation [F
10,76
= 18.4, p < 0.001], ethanol
concentration [F
1,76
= 146, p < 0.001] and an interaction between main effects [F
10,76

= 3.17, p < 0.01].
103

Figure 15. Cysteine substitutions in α1 GlyRs produce position-specific
increases in the isoflurane responses. (A.) 100 and (B.) 400 μM isoflurane.
Mean ± SEM percent isoflurane modulation of the EC
10
glycine response for WT
and mutant α1 GlyRs as measured in 4-5 oocytes. Shaded bars indicate the results
from residues within the defined range for Loop 2 (positions 51-57). Two-way
ANOVA revealed significant main effects of receptor mutation [F
10,70
= 23.6, p <
0.001], isoflurane concentration [F
1,70
= 290, p < 0.001], and an interaction between
main effects [F
10,70
= 2.13, p < 0.05].
104

Figure 16. Propofol sensitivity in Loop 2 cysteine mutant GlyRs is only altered
in I51C GlyRs. (A.) 2.5 and (B.) 5 μM propofol. Mean ± SEM percent propofol
modulation of the EC
10
glycine response for WT and mutant α1 GlyRs as measured
in 4-7 oocytes. Shaded bars indicate the results from residues within the defined
range for Loop 2 (positions 51-57). Two-way ANOVA revealed significant main
effects of receptor mutation [F
10,72
= 69.6, p < 0.0001], propofol concentration [F
1,72

= 172, p < 0.0001] and an interaction between main effects [F
10,72
= 4.49, p <
0.0001].
105

Figure 17. PMTS exposure in the presence of glycine irreversibly potentiates
some, but not all, cysteine mutant GlyRs. Mean ± SEM percent control glycine
response for WT and mutant GlyRs as measured in 4 oocytes. A subset of results in
(A.) are shown on an expanded scale (B.) to facilitate interpretation of the lower
magnitude of PMTS potentiation in several Loop 2 cysteine mutant GlyRs. The
shaded box indicates GlyR responses in the presence of PMTS. Two-way ANOVA
revealed significant main effects of treatment [F
2,66
= 131.5, p < 0.0001], receptor
mutation [F
10,66
= 42.2, p < 0.0001], and an interaction between main effects [F
10,66
=
40.6, p < 0.0001].

106

Figure 18. PMTS binding in the triple mutant GlyR does not block all of the
effects of ethanol. Mean ± SEM percent control glycine response for the I51C-
D57C-S267C α1GlyR (n = 3). The shaded boxes indicate GlyR responses in the
presence of 300 μM PMTS (grey) or 100 mM ethanol (yellow).
107

Figure 19. Molecular model of an α1GlyR subunit. (A.) The interface between
the extracellular and TM domains. The amino acid backbone of the GlyR α1
subunit is shown as a ribbon, with the β sheet structure of the extracellular domain
shown in blue and the α-helical structure of the TM domain shown in purple. (B.)
Zoom view. To visualize residues tested in the current study, the area of Loop 2 was
expanded to focus on the interface between the extracellular and TM domains. Here,
the amino acid backbone is shown as a stick structure with the Cα atoms at each
apex. The atoms of Loop 2 residues are colored red, black, white, and blue for
oxygen, carbon, hydrogen, and nitrogen, respectively. Alanine 52 is highlighted in
pink in reference to previous work at this position. (from Chapter 3)

108
109
DISCUSSION
The current study represents the first systematic investigation into the role of
Loop 2 residues as sites of ethanol and general anesthetic modulation in α1GlyRs.
The findings suggest that multiple residues within Loop 2 are important for
determining ethanol sensitivity in GlyRs, that some positions may play different
roles in ethanol modulation than do others ad that the targets for ethanol may differ
from those for isoflurane and propofol. The current study also offers an
interpretation of the molecular model presented in the previous chapter and provides
a mechanism for modulation by ethanol and other general anesthetics.
The present study expands upon previous work from our laboratory which
suggested that the effect of ethanol in WT GlyRs represents the summation of
opposing ethanol effects in the extracellular and TM domains (Crawford et al., in
press). The current study suggests that ethanol produces positive and negative
modulation by its action on targets within Loop 2. The cysteine mutation at position
51 increased ethanol potentiation, whereas the cysteine mutation at position 57
switched the ethanol response to inhibition. Consistent with these ethanol findings,
PMTS produced irreversible potentiation in I51C GlyRs and irreversible inhibition in
D57C GlyRs. These results suggest that ethanol acting on position 51 produces
positive modulation and ethanol acting on position 57 produces negative modulation.
Alternatively, the switch from ethanol potentiation to inhibition by cysteine mutation
at position 57 could reflect a conformational change induced by the mutation that
110
alters the WT GlyR response to ethanol. Further research is necessary to investigate
this scenario.
Cysteine mutations in the Loop 2 region also significantly altered isoflurane
potentiation of GlyR function, yet yielded a different pattern of responses than the
changes in ethanol potentiation. The ability of Loop 2 cysteine point mutations to
significantly alter isoflurane sensitivity suggests that, in addition to acting at targets
in the TM domain (Mihic et al., 1997;Krasowski et al., 1998;Hall et al., 2005),
isoflurane acts at targets in Loop 2 of the extracellular domain of α1GlyRs. The
current results suggest that residues across Loop 2 are important for isoflurane
modulation, whereas Loop 2 residues proximal to the β sheets (positions 51 and 57)
are important for ethanol modulation. Therefore, based on the ability of cysteine
mutations to alter potentiation by each agent, ethanol appears to have fewer targets in
Loop 2 than isoflurane. However, further studies are needed before conclusions can
be drawn in this regard. Nevertheless, differences in response to ethanol and
isoflurane may be useful in understanding the structure-function relationships that
underlie the action of general anesthetics in the Loop 2 region and in determining the
molecular mechanism of alcohol and anesthetic action.
Interestingly, previous work that employed alanine-scanning mutagenesis
revealed several positions within Loop 7 that are important for isoflurane modulation
(Schofield et al., 2004). In light of the current study, these findings suggest that
Loop 7 deserves additional attention and may potentially function as another target
for ethanol action.
111
Based on this evidence, we modified our interpretation of the glycine
activation model presented in Chapter 3 (presented here as Figure 7) to suggest a
mechanism for ethanol and general anesthetic modulation of α1GlyR function. This
model suggests that residues at either end of Loop 2 (positions 51 and 57) act like a
hinge. This hinge could provide flexibility to allow movement of Loop 2 with
respect to the more stable β sheet structure that makes up most of the extracellular
domain. Ethanol and other general anesthetic could act upon this flexible section to
alter Loop 2 interaction with other agonist activation elements. Together, this model
provides a mechanism for Loop 2 movement in agonist activation and could help to
explain why these residues are so important for ethanol modulation of GlyR
function.
Collectively, these findings suggest that the extracellular domain plays an
important role, both structurally and functionally, in GlyR allosteric modulation by
ethanol and other general anesthetics. The findings provide a new model for the
Loop 2 region and offer new insight into the structure-function relationships in
GlyRs. Further study is necessary to investigate whether the present findings
generalize to other ligand-gated ion channels.
112
CHAPTER 5
SUMMARY AND OVERALL CONCLUSIONS

Our understanding of the molecular targets for alcohols and anesthetics has
increased markedly over the past twenty years. Initially, these agents were believed
to act in a non-specific manner by perturbing the lipid membrane (Meyer, 1899;
Meyer, 1901; Overton, 1901; Meyer, 1937; Mullins, 1954; Lever et al., 1971; Miller
et al., 1973). Starting with the notion that anesthetics “act by competing with
endogenous ligands for binding to specific receptors” proposed by Franks and Lieb
(Franks and Lieb, 1984), substantial effort has been spent on identifying which
receptor targets are important for the behavioral effects of alcohols and anesthetics
and on understanding the molecular sites of action for these receptors. LGICs of the
cys-loop receptor family have received considerable attention as a putative site of
ethanol action. Several lines of evidence suggest that the TM domains of GlyRs and
GABA
A
Rs contribute to an alcohol pocket and play a role in causing alcohol
modulation of α1 GlyR function (Mihic et al., 1997; Ye et al., 1998; Wick et al.,
1998; Yamakura et al., 1999; Mascia et al., 2000). Recent work using increased
atmospheric pressure as an ethanol antagonist brought attention to the extracellular
domain of α1 GlyRs and suggested that position 52 in Loop 2 might be a target for
ethanol (Davies et al., 2004).
The overall goal of this dissertation was to increase our understanding of the
sites of ethanol action in the extracellular and TM domains of α1 GlyRs. The initial
113
work tested whether cysteine substitutions at position 52 alter ethanol sensitivity in
α1GlyRs and if action at this residue directly produces alcohol-like effects (Chapter
2). We found that cysteine mutations at position 52 significantly reduced ethanol
sensitivity when compared to WT GlyRs. Moreover, PMTS binding to cysteine
substitutions at position 52 produced irreversible alcohol-like potentiation. Together,
these findings support the hypothesis that position 52 is a site of alcohol action.
PMTS binding to the cysteine mutations at position 52 and/or position 267 (Mascia
et al., 2000) also reduced the n-chain alcohol cutoff, suggesting that these sites for
ethanol action form part of a single alcohol action pocket. Molecular modeling of
the α1GlyR revealed a cavity at the interface between the TM and extracellular
domain that could function as this putative alcohol action pocket.
We then used PMTS binding to substituted cysteine residues was also used to
block ethanol action at one site in order to determine its effect at other sites within
the α1 GlyR. From this perspective, ethanol caused negative modulation when
acting at position 52 and positive modulation when acting at position 267. However,
positions 52 and 267 cannot account for all of the effects of ethanol in GlyRs and
ethanol acting on the remaining site(s) causes negative modulation. Together, these
findings suggest that the net effect of ethanol on α1 GlyRs represents the summation
of positive and negative modulatory effects on multiple targets in the TM and
extracellular domains. The question remained whether other residues in the Loop 2
region are also targets for ethanol and what their role is in producing ethanol effects.
114
The subsequent investigations addressed these questions by scanning through
the Loop 2 region to study the role each residue plays in agonist activation (Chapter
3) and modulation by alcohols and anesthetics (Chapter 4). This cysteine
substitution approach first yielded information regarding the structure and function
of Loop 2 in GlyR agonist activation. Cysteine mutations within the defined range
for Loop 2 (positions 51-57) caused position-specific shifts in glycine sensitivity
(EC
50
), reduced the Hill slope, and in one case, drastically reduced the maximal
current amplitude. The pattern of changes in glycine sensitivity provided the first
suggestion of secondary structure for Loop 2 in the extracellular domain of cys-loop
receptors. The position specific changes in glycine sensitivity are consistent with β
strand-like structure of the Loop and suggest that the odd numbered positions in this
sequence interact with other agonist-activation elements at the interface between
extracellular and TM domains. The residues at either end of Loop 2 (positions 51
and 57) could act like a hinge and provide flexibility to allow movement of Loop 2,
which may provide a mechanism for Loop 2 movement in agonist activation.
Findings from this GlyR agonist activation study also supported the
hypothesis that Loop 2 plays a role in influencing the presence and movement of
chloride ions within the channel. The cysteine mutation at position 53 removes a
negative charge associated with a glutamic acid residue under physiological conditions
and results in a significant right shift in glycine sensitivity. Binding a negatively
charged MTS reagent (MTSES) to the substituted cysteine residue shifts agonist
sensitivity back to resemble that of WT GlyRs, whereas binding positively charged
115
MTS reagents (MTSEA or MTSET) did not. These findings are the first to
demonstrate that the charge associated with a Loop 2 residue influences chloride ion
movement in the channel pore and agree with previous molecular models which
suggested that the region of the channel pore formed by the Loop 2 region creates an
energy well that hinders chloride ion movement.
Further work began to address the question of whether residues in the Loop 2
region are targets for ethanol and what their role is in producing ethanol effects
(Chapter 4). This study found that several Loop 2 cysteine mutations caused
changes in ethanol sensitivity and that one mutation could switch the qualitative
ethanol response from potentiation to inhibition. In addition, PMTS binding to Loop
2 cysteine substitutions resulted in irreversible alcohol-like modulation that
paralleled changes in ethanol sensitivity. These findings with PMTS and ethanol
provide evidence that several residues in the Loop 2 region play a role in ethanol
modulation of α1 GlyRs.
Loop 2 cysteine mutations also significantly altered isoflurane and propofol
sensitivity, however, the pattern of changes in ethanol sensitivity were different than
the changes in sensitivity for these anesthetics. Residues across the Loop 2 region
appear to be important for isoflurane sensitivity, whereas residues more proximal to
the β sheets appear to be important for ethanol sensitivity. Interestingly, only the
cysteine mutation at position 51 changed propofol sensitivity, but it is noteworthy
that the cysteine mutation at position 51 significantly increased potentiation by
ethanol, PMTS, and isoflurane as well. Collectively, these findings suggest that
116
Loop 2 plays an important role in the allosteric modulation pathway connecting
agonist binding to channel gating. Further study is necessary to understand the
structure-function relationships that underlie the action of general anesthetics in the
Loop 2 region and to determine whether the present findings generalize to other
LGICs.

Conclusions
The current work provides insight into molecular targets for alcohols and
anesthetics within the α1 GlyR and the complex interaction between sites of action
in the extracellular and TM domains. The findings provide strong evidence that: 1.)
several residues in Loop 2 of the α1 GlyR are targets for ethanol and other general
anesthetics, 2.) the extracellular and TM domains are part of a single alcohol action
pocket, 3.) there are positive and negative modulatory sites for ethanol in the
extracellular and TM domains, and 4.) the effect of ethanol on GlyR function reflects
summation of these positive and negative modulatory effects. This work also
suggests that : 1.) Each residue in Loop 2 plays a role in agonist activation, and 2.)
The charge at position 53 in Loop 2 influences chloride ion permeability in the
channel. Together, these studies generated two different molecular models of the
α1GlyR which may help to explain the structural-functional relationships that occur
in this receptor. Further study is necessary to understand the intricacies of agonist
activation and the mechanism underlying allosteric modulation by ethanol and other
general anesthetics in the Loop 2 region.
117
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Abstract (if available)

Abstract Ligand-gated ion channels of the cys-loop receptor family have received considerable attention as putative sites of action causing the behavioral effects of ethanol. Studies over the last decade identified several positions in the transmembrane domain critical for ethanol modulation of glycine receptors (GlyRs). Studies to date in the extracellular domain of alpha1GlyRs found that mutations at position 52 in Loop 2 change ethanol sensitivity, alter sensitivity to an ethanol antagonist and can eliminate subunit-dependent differences in ethanol sensitivity between alpha1 and alpha2GlyRs. These findings suggest that the extracellular domain also represents a target for ethanol in GlyRs. We tested this hypothesis by investigating the effect of cysteine substitutions at positions 52 and 267 on responses to n-alcohols and propyl methanethiosulfonate (PMTS) in alpha1GlyRs. We also tested the role of Loop 2 in agonist activation and ethanol modulation by investigating the effect of cysteine point mutations at positions 50-60. Xenopus oocytes expressing human alpha1 wildtype (WT) or mutant GlyRs were voltage-clamped and tested 3-10 days post-injection. In support of the hypothesis, we found that: 1) The A52C mutation changed ethanol sensitivity compared to WT GlyRs

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Asset Metadata

Creator Crawford, Daniel Kenneth (author)

Core Title Positive and negative modulatory sites for ethanol in alpha1 glycine receptors

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 07/25/2007

Defense Date 03/27/2007

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag ethanol,glycine receptor,molecular model,OAI-PMH Harvest,two-electrode voltage clamp,Xenopus oocyte

Language English

Advisor Alkana, Ronald L. (committee chair), Brinton, Roberta Diaz (committee member), Byerly, Lou (committee member), Davies, Daryl L. (committee member), Haworth, Ian S. (committee member)

Creator Email dkcrawfo@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m670

Unique identifier UC1472070

Identifier etd-Crawford-20070725 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-528607 (legacy record id),usctheses-m670 (legacy record id)

Legacy Identifier etd-Crawford-20070725.pdf

Dmrecord 528607

Document Type Dissertation

Rights Crawford, Daniel Kenneth

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu