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Position 52 in alpha1 glycine receptors is important for the action of ethanol

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Position 52 in alpha1 glycine receptors is important for the action of ethanol

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POSITION 52 IN α1 GLYCINE RECEPTORS IS IMPORTANT FOR
THE ACTION OF ETHANOL

by

Yi Shen

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR PHARMACOLOGY & TOXICOLOGY)

MAY 2008

Copyright 2008 Yi Shen
ii
Acknowledgements

First, I want to thank my advisors, Dr. Ronald Alkana and Dr. Daryl Davies for
their guidance and support. I also want to acknowledge Miriam Fine, Drs. Liana Asatryan
and Kaixun Li for technical assistance. Special thanks to Daniel Crawford, Daya Perkins,
Liya Xu, and Maya Popova, who gave me help in a lot of aspects.
This work was supported in part by research grants NIAAA/NIH AA03972,
AA013890, AA013922, and the USC School of Pharmacy.
Portions of these findings were Presented at the 2005 Scientific Meeting of the
Research Society on Alcoholism, June 24-29, 2005, Santa Barbara, CA. Alcoholism:
Clinical and Experimental Research 29: 142A (No.5), 2005.

iii
Table of Contents

Acknowledgements ii

List of Figures iv

List of Tables v

List of Abbreviations vi

Abstract vii

Chapter 1: Introduction 1
Background 1
Putative Ethanol Targets in the CNS—Ligand Gated Ion Channels 2
Investigating the Molecular Targets of Ethanol 4
A Novel Ethanol Antagonist—Hyperbaric Pressure 6
Position 52 in Receptor Activation 9
Conclusion for the Introduction 10

Chapter 2: Experimental Procedures 13
Materials 13
Mutagenesis and Expression of Human GlyR α1 Subunit cDNA 13
Electrophysiology 14
-- Validation of OpusXpress 6000A 15
-- Glycine Concentration Responses 15
-- Ethanol Potentiation 15
-- Propofol Potentiation 16
Data Analysis 16

Chapter 3: Results 18
Agonist Activation of A52X Mutant α1 GlyR 18
Ethanol Sensitivity of A52X Mutant α1 GlyR 18
Propofol Responsiveness of A52X Mutant α1 GlyRs 20

Chapter 4: Discussion and Conclusions 21

References 39

iv
List of Figures

Figure 1: Glycine concentration--response curves of A52X mutated
GlyRs

26
Figure 2: Correlation analysis between glycine EC
50
and the
physical-chemical properties of amino acid residues at
position 52

27
Figure 3: Correlation analysis between the glycine EC
50
and
physical-chemical properties of the amino acid at position
52 among the mutants who significantly changed EC
50

28
Figure 4: Correlation analysis between Hill Slope and the physical-
chemical properties of amino acid residues at position 52

29
Figure 5: Representative tracing showing 5 successful parallel
recordings on wild type α1 GlyRs

30
Figure 6: Point mutations at position 52 in α1GlyRs significantly
changed the receptor’s sensitivity to 100 mM ethanol

31
Figure 7A: Magnitude of Ethanol potentiation significantly
correlates with glycine EC
50

32
Figure 7B: Magnitude of Ethanol potentiation significantly
correlates with glycine EC
50
among the mutants who
significantly changed both glycine EC
50
and ethanol
potentiation

33
Figure 8: Correlation analysis between the ethanol potentiation
magnitude and the physical-chemical properties among
the mutants who significantly changed ethanol
potentiation

34
Figure 9: Propofol concentration--response curves of WT and
A52X mutated GlyRs

35
Figure 10: Correlation analysis between the glycine EC
50
and the
physical-chemical properties among the mutants who
significantly changed both EC
50
and ethanol potentiation

36

v
List of Tables

Table 1: Concentration-response curves constructed from data
obtained using conventional two-electrode voltage clamp
system and OpusXpress provided similar EC
50
data in
wild type, A52E and A52D mutant α1GlyRs

37
Table 2: Glycine EC
50
, Hill slope and I
max
of WT and mutated α1
GlyRs.
38

vi
List of Abbreviations

AChBP, acetylcholine binding protein

ANOVA, analysis of variance

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

nAChR, nicotinic acetylcholine receptor

TM, transmembrane

WT, wild-type;

vii
Abstract

Glycine receptors (GlyRs) have received considerable attention as putative sites
of action causing the behavioral effects of ethanol. Studies over the last decade have
identified several positions in the transmembrane domain of GlyRs that are critical for
ethanol modulation.
Studies to date in the extracellular domain of α1GlyRs found that mutations at
position 52 in Loop 2 alter receptor sensitivity to ethanol, abolish receptor 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.
The present study uses systematic substitution of the amino acid residue at
position 52 of α1 GlyRs to study possible structure-function relationship between the
physical-chemical properties of the residue at position 52 and receptor sensitivity to
glycine or ethanol. The current investigation also begins to test for possible relationships
between changes in agonist activation produced by these mutations and ethanol
sensitivity. To investigate the specificity of the findings for ethanol, we also tested
propofol, another general anesthetic, which is thought to act at different sites than ethanol
in α1GlyR and GABA
A
Rs.
To accomplish this, we produced mutant α1GlyRs in which the alanine at position
52 in wild-type (WT) α1GlyRs was replaced with each of the remaining nineteen
naturally occurring amino acids and measured their responses to glycine and modulation
by ethanol. Xenopus oocytes expressing WT or mutant α1 GlyRs were voltage-clamped
viii
and tested for the effects of ethanol using an OpusXpress 8 channel two electrode voltage
clamp system (-70mV).
We found that: (1) all mutations produced functional α1 GlyRs, with several of
the mutations significantly reduced the glycine EC
50
; (2) most of the mutations
significantly reduced receptor sensitivity to ethanol; (3) molecular volume and molecular
weight of the amino acid residue at position 52 play roles in determining the
responsiveness of α1GlyRs to both glycine and ethanol; (4) changes in glycine EC
50
were
correlated with changes in magnitude of ethanol potentiation; (5) position 52 of α1 GlyRs
is a target for ethanol action, but not for propofol action.
Together, the present study adds to the evidence that position 52 in the
extracellular domain, in addition to the transmembrane domain, plays an important role in
ethanol modulation in α1 GlyRs. The findings provide insight into structure-function
relationships for GlyRs. This work also provides new evidence that ethanol modulates α1
GlyR function, at least partly, by affecting agonist activation through position 52.
1
Chapter 1
Introduction

Background

Alcohol (ethanol) abuse represents a significant problem in the United States with
approximately 14 million people being affected (NIAAA 2000; Volpicelli 2001). The
economic costs of alcohol-related disorders was estimated to 185 billion dollars per year
in the United States alone (2000), compared to 730 million dollars spent on alcohol-
related research worldwide (Rajendram et al. 2006). To address this issue, considerable
effort has been focused on the development of medications to prevent and treat
alcoholism (Heilig and Egli 2006; Johnson et al. 2007; Steensland et al. 2007). The
development of such medications would be aided by a clear understanding of the
mechanisms and sites of ethanol action.
However, the physical-chemical nature and the low affinities of ethanol limits the
utility of traditional pharmacological receptor ligands as tools for investigating the
mechanism of action for ethanol (Eckenhoff and Johansson 1997; Davies and Alkana
1998; Davies et al. 2003). Nonetheless, considerable evidence implicates ligand-gated
ion channels (LGICs) as initial targets for ethanol action. This master’s thesis focuses on
investigating glycine receptors, one of the primary inhibitory neurotransmitter receptors
in the nervous system, as targets for ethanol action.

2
Putative Ethanol Targets in the CNS—Ligand Gated Ion Channels
Over 100 years ago, ethanol was believed to act by perturbing the lipid membrane
because it has such a simple structure and it requires relatively high millimolar
concentrations to produce behaviorally intoxicating effects (Franks and Lieb 1981;
Suzdak et al. 1986; Sutherland et al. 1988). Recent studies demonstrated that the lipid
bilayer cannot account for all of the alcohol and anesthetic effects in the CNS and that
more specific sites of action, for example, membrane proteins, also play an important role.
In 1986, an important step toward defining the target of ethanol action in the brain was
taken when three groups demonstrated that intoxicating concentration of ethanol enhance
the function of γ-aminobutyric acid type A receptors (GABA
A
Rs), the major inhibitory
neurotransmitter receptors in the brain (Allan and Harris 1986; Suzdak et al. 1986; Ticku
et al. 1986). Since then, the ligand-gated ion channels (LGICs) have received
considerable attention as putative targets of action of ethanol (Deitrich et al. 1989; Zhou
and Lovinger 1996; Mihic et al. 1997; Ye et al. 1998; Cardoso et al. 1999; Harris 1999;
Davies and Alkana 2001a). Research in this area has focused on investigating the effects
of ethanol on three 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 (Lovinger et al.
1989; Dildy-Mayfield and Harris 1995; Ronald et al. 2001); (2) the purinergic (P2X)
superfamily (Li et al. 1998; Li et al. 2000; Davies et al. 2002; Davies et al. 2005); and (3)
the cys-loop receptor superfamily, whose members include nicotinic acetylcholine
receptors (nAChRs), 5-hydroxytryptamine3 receptors (5-HT
3
Rs), γ-aminobutyric acid
3
type-A receptors (GABA
A
Rs) and glycine receptors (GlyRs) (Machu and Harris 1994;
Zhou and Lovinger 1996; Mihic et al. 1997; Ye et al. 1998; Aistrup et al. 1999; Cardoso
et al. 1999; Godden et al. 2001; Davies and Alkana 2001a; Davies et al. 2003; Davies et
al. 2004).
Cys-loop receptor proteins are heteromeric or homomeric pentamers arranged
around a central ion conducting pore. Individual subunits share a similar membrane
topology, with a large extracellular ligand-binding domain made up of β sheets, four
transmembrane α-helical segments (TM1-4), and an intracellular domain. The integral
ion channel is open following ligand binding (Langosch et al. 1988; Ortells et al. 1997;
Brejc et al. 2001; Tang et al. 2002; Unwin 2005). These 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. 2006).
Previous in vivo studies suggest that cys-loop receptors play a role in the
behavioral effects of ethanol. The role of GlyRs has been well studied because of its
pharmacological properties and its localization in the CNS. Previously, glycine was
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). Electrophysiological studies of GlyRs
4
found that behaviorally relevant concentrations of ethanol positively modulate GlyR
function in freshly dissociated rat neurons (Ye et al. 2001a; Ye et al. 2001b; Tao and Ye
2002; Ye et al. 2002; Jiang and Ye 2003; McCool et al. 2003), synaptoneurosomes of
whole-rat brain (Engblom and Akerman 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. 2003) and brain slice preparations
(Darstein et al. 1997; Eggers et al. 2000; Sebe et al. 2003; Eggers and Berger 2004). 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.
1997; Davies et al. 2004; Crawford et al. 2007). More recent studies also suggest that
GlyRs in the nucleus accumbens are targets for ethanol that are involved in ethanol-
induced mesolimbic dopamine release (Molander and Soderpalm 2005; Molander et al.
2007), thus linking GlyRs to the rewarding effects of ethanol.
Taken together, these findings suggest that GlyRs mediate at least a subset of
ethanol-induced behavioral effects. Despite the advances in understanding the effects of
ethanol in vivo and in vitro, the precise molecular sites of action for ethanol in LGICs
and why these site(s) serves as important target for ethanol action are not yet well
understood.

Investigating the Molecular Targets for Ethanol

In 1997, an initial investigation using chimeras first identified a region of 45
amino acid residues within TM2 and TM3 domain of GlyRs and GABA
A
Rs to be
5
important for the enhancement of both receptor functions (Mihic et al. 1997). Within this
region, two specific amino acid residues are found to be critical for the modulation of
both receptors by ethanol: S267 in TM2, A288 in TM3 of α1GlyRs; corresponding
residues S270 and A291 in GABA
A
Rs, (Mihic et al. 1997). The unique role of S270 of
GABA
A
R in the action of ethanol was also confirmed by another study using tryptophan
scanning mutagenesis (Ueno et al. 2000). Subsequent study found that substitution of
S267 with the remaining nineteen amino acid residues dramatically changed the action of
ethanol from enhancement (wild type, S267G), through no effect (S267I, S267V) to
inhibition (S267L, S267Y). Moreover, this work revealed an inverse correlation between
molecular volume of the amino acid substitution and the effect of ethanol (Ye et al.
1998). Potentiation by alcohols and anesthetics was also altered by mutations at I229 in
TM1, A288 in TM3, and W407 and Y410 in TM4 (Greenblatt 1999; Yamakura et al.
1999; Jenkins et al. 2001; 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 ethanol.
Additional investigations using the n-chain alcohol “cutoff” studied the role of
TM residues in the actions of ethanol. The idea of a cutoff is based on the observation
that alcohol potentiation of GlyRs increases with the length of the side chain of a series of
n-alcohols until a certain point (cutoff) is reached (Dildy-Mayfield et al. 1996). Alcohol
cutoff has been used as an indicator of the molecular size of the “binding” cavities of
alcohol within a given protein. The identification of amino acid residues that determine
the cutoff can therefore provide information about the location of the alcohol “binding”
6
site. Specific amino acids in the TM 2 and 3 of Cys-loop LGICs superfamily (S267 in α1
GlyR, I307 and W328 in ρ1 GABA
A
R) are shown to be able to control the size of the
alcohol “binding” cavity (Wick et al. 1998). This provides the evidence of existence of
alcohol “binding” pockets within the TM domains of GlyRs and GABA
A
Rs.
Subsequent studies provided additional evidence that S267 in TM2 and A288 in
TM3 in the α1 GlyR represent specific “binding” site for ethanol by using cysteine
mutagenesis and anesthetic-like thiol or methanethiosulfonate (MTS) reagents (Mascia et
al. 2000). This approach involves mutating critical amino acid residues to cysteine, and
subsequently testing the ability of propyl methanethiosulfonate (PMTS), to covalently
bind to the thiol group of the cysteine residue introduced in these positions. The authors
suggested if the mutated amino acid is the critical ethanol “binding” site, then PMTS
should irreversibly activate the mutant receptor but reversibly activate wild-type receptor.
Consistent with their hypothesis, PMTS covalently bound to and irreversibly enhanced
receptor function for the cysteine mutant created at 267 in GlyRs as well as homologous
position S270 in GABA
A
Rs (Mascia et al. 2000). Collectively, the findings add further
support for the hypothesis that the TM domains of LGICs contribute to an ethanol
binding pocket and play a role in causing ethanol modulation of receptor function.

A Novel Ethanol Antagonist

Despite the aforementioned advances, the precise molecular sites of ethanol
action on LGICs are not yet clear. Traditionally, the mechanism and site of drug action
can be studied using the appropriate receptor agonists and antagonists. This
7
pharmacological approach is based on the concept that a selective antagonist can
differentiate sites of drug action. However, the physical-chemical nature and the low
affinities of ethanol limits the utility of traditional pharmacological receptor ligands as
tools for investigating the mechanism of action for ethanol (Eckenhoff and Johansson
1997; Davies and Alkana 1998; Davies et al. 2003).
To address these issues, increased atmospheric pressure (hyperbaric exposure)
was developed as an alternative to a traditional pharmacological ethanol antagonist.
Initial investigations have shown that exposure to 12 times normal atmospheric pressure
(12 ATA) of helium-oxygen gas (heliox) directly antagonizes the behavioral and
biochemical actions of ethanol. (Alkana and Malcolm 1981; Alkana et al. 1992; Bejanian
et al. 1993; Davies and Alkana 1998; Davies and Alkana 2001a). More recently, these
hyperbaric investigations were extended to two-electrode voltage clamp studies of LGICs
expressed in Xenopus oocytes. Results from these electrophysiological studies add to the
evidence that pressure is a direct, ethanol antagonist with the selectivity and other
properties necessary for it to be used to help differentiate the sites and mechanisms of
ethanol action in GlyRs (Davies et al. 2003).
Initial results showed that α1 GlyRs are sensitive to pressure antagonism of
ethanol at high concentrations (50-200 mM) (Davies et al. 2003). Moreover, the degree of
ethanol antagonism was inversely correlated to the ethanol concentration. These findings
were consistent with the hyperbaric behavioral and biochemical studies and support the
contention that increased atmospheric pressure exhibits characteristics similar to
conventional pharmacological antagonists.
8
Remarkably, low concentrations of ethanol (10 and 25 mM) were completely
insensitive to pressure antagonism (Davies et al. 2004). These concentration-dependent
differences suggest that ethanol acts at more than one site 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 the amino acid sequence identity between GlyR α1 and α2 subunit is
high (79%), 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. 1996a). 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, yielded a receptor similar to α2 GlyR
in regards to its overall ethanol sensitivity (Mascia et al. 1996a). Hyperbaric experiments
demonstrated that A52S mutation in α1 GlyR converts the pressure antagonism sensitive
α1 GlyR into pressure antagonism insensitive (like the α2 GlyR) (Davies et al. 2004).
This result is consistent with the notion that A52 is a site for both pressure antagonism
and ethanol action.
Follow-up studies at normal atmospheric pressure employed cysteine mutagenesis
at position 52 in α1GlyRs. These works showed that PMTS binding to this site caused
irreversible alcohol-like potentiation. In addition, PMTS binding to cysteines substituted
at position 52 in α1GlyRs (A52C) decreased the alcohol cutoff (Crawford et al. 2007).
9
Taken together, these findings suggest that position 52 in the Loop 2 of the extracellular
domain may play an important role in ethanol potentiation of GlyR function.

Position 52 in Receptor Activation
Homozygotic spasmodic (spd/spd) mice are characterized by exhibiting a motor
disorder that resembles hereditary hyperekplexia or seizures due to poisoning by the
glycine receptor antagonist strychnine (Ryan et al. 1994; Saul et al. 1994). The source of
this phenotype was traced to a point mutation at position 52 in the α1GlyRs that reduced
the receptor’s sensitivity to agonist. Recombinant studies also demonstrated that A52S
mutation in α1GlyRs is characterized by normal glycine binding properties but by an
increased glycine EC
50
when examined in either HEK293 cells or oocytes (Ryan et al.
1994; Saul et al. 1994).
Due to the nature of LGICs proteins, molecular modeling has been used to
analyze and visualize LGICs structures. The crystal structure of AChBP has been used as
homology template to generate a proposed structural model for the extracellular domain
for LGICs (Brejc et al. 2001). Threading the α1GlyR sequence onto the backbone atoms
of the AChBP, Position 52 is found to localized in the loop 2 region which has been
implicated to play an important role in coupling ligand binding to channel activation in
GABA
A
Rs and GlyRs (Absalom et al. 2003; Kash et al. 2003; Kash et al. 2004).
Taken together, these findings suggest that position 52 may be part of a signal
transduction site involved in receptor activation.

10
Conclusions for the Introduction

LGICs, which mediate fast synaptic neurotransmission, have been proposed to be
an important target in the mammalian brain for the behavioral effects of ethanol. For cys-
loop LGICs receptors, the TM domains have received considerable attention as a putative
site of action of ethanol. Chimeric studies suggested that S267 and A288 in TM segments
2 and 3 of the α1GlyR subunit are important for allosteric modulation by ethanol. Further
work found that mutations at position 267 changed the sensitivity of the receptor to
ethanol, altered the qualitative ethanol response from potentiation to inhibition, and
decreased the size of a putative alcohol pocket. Additional studies combined the use of
the anesthetic-like PMTS with the substituted cysteine accessibility method to show that
exposure to PMTS caused the predicted alcohol-like irreversible potentiation in S267C
GlyRs. Collectively, these findings suggest that the TM domains play a role in causing
ethanol modulation of α1 GlyR function.
In contrast to the TM domain, the extracellular domain has received relatively
little attention as a potential site of action for ethanol. The use of hyperberic pressure as
an ethanol antagonist has brought attention to the extracellular domain, and in particular,
position 52 of the α1 GlyR. These studies found that a mutation from alanine to serine at
position 52 (A52S) of α1 GlyRs reduced receptor sensitivity to ethanol, eliminated the
subunit-dependent differences in ethanol sensitivity between α1 and α2 GlyRs and
abolished sensitivity to a mechanistic ethanol antagonist—increased atmosphere pressure.
Subsequent studies found that exposing mutant A52C GlyRs to PMTS produced
irreversible potentiation and reduced the alcohol cut-off. Overall, these results suggest
11
that position 52 in the extracellular domain may be an important target for ethanol action
in α1 GlyRs.
Position 52 has also been shown to play an important role in agonist activation of
the receptor. Recombinant studies demonstrated that A52S mutation in α1GlyR increased
glycine EC
50
without altering glycine binding. Homology modeling based on the
structure of nicotinic acetylcholine binding protein (nAChBP) suggests that position 52 is
located in Loop 2 of the extracellular domain. This Loop has been implicated in the
signal transduction pathway underlying receptor activation in both GABA
A
Rs and GlyRs.
Taken together, position 52 of α1 GlyRs is an important site since it contributes to
both agonist activation and ethanol modulation. Therefore, study of this position in more
details is needed to provide a better understanding of the action of ethanol in the
extracellular domain of α1 GlyRs.
The present study uses systematic substitution of the amino acid residue at
position 52 of α1 GlyRs to study the possible structure-function relationship between the
physical-chemical properties of the residue at this position and receptor sensitivity to
glycine or ethanol. In addition, the current investigation also begins to test for possible
relationships between changes in agonist activation produced by these mutations and
ethanol sensitivity. To accomplish this, we produced mutant α1GlyRs in which the
alanine at position 52 in WT α1GlyRs was replaced with each of the remaining nineteen
naturally occurring amino acids and measured their responses to glycine and modulation
by ethanol. To investigate the specificity of the findings for ethanol, we also tested
propofol, another general anesthetic, which is thought to act at different sites than ethanol
12
in α1GlyR and GABA
A
Rs. We found that mutations at this position significantly altered
glycine activation and receptor sensitivity to ethanol. Moreover, our result demonstrated
a significant correlation between the change in glycine sensitivity and in the ethanol
sensitivity. These findings suggest a link between ethanol modulation and agonist
activation in α1GlyRs.

13
CHAPTER 2
EXPERIMENTAL PROCEDURES

Materials
Adult female Xenopus laevis frogs were obtained from Nasco (Fort Atkinson, WI).
Glycine, ethanol, propofol and other reagents for making buffers were purchased from
Sigma (St. Louis, MO).

Mutagenesis and Expression of Human GlyR α α α α1 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 of the vector strands (Microchemical Core Facility, University of
Southrn California). Xenopus laevis oocytes were isolated and injected with 1 ng of wild
type or mutant A52X α1 GlyR cDNA using procedures previously described (Davies et
al. 2003; Davies et al. 2004). Injected oocytes were incubated at 18°C 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, with 1% horse serum and 0.05 mg/mL
gentamycin).

14
Electrophysiology
Electrophysiology measurements were made 1 to 10 days after injection. Two
electrode voltage-clamp technique is routinely used for investigation of ion channel
function. However, using conventional two electrode voltage-clamp technique to
investigate large numbers of mutant receptor function is laborious and time-consuming.
To address this issue, our laboratory has incorporated an automated medium throughput
two-electrode voltage clamp system--OpusXpress 6000A (Axon Instruments, Union City,
CA), to investigate ion channel function. This system increases throughput by
incorporating two-electrode voltage clamp technique to record currents from eight
oocytes in parallel. It was designed to increase the efficiency of drug discovery for ion
channels and transporters by increasing throughput of direct electrophysiological
recordings. Recently, the OpusXpress has been used in several academic laboratories for
studying nAChRs functions.(Marrero et al. 2004; Papke et al. 2004; Xiu et al. 2005).
Oocytes expressing wild type and mutant α1 GlyRs were perfused in a 200 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 [1.2mm ID
thick-walled filamented glass capillaries (WPI, Sarasota, FL)] backfilled with 3 M KCl with
resistances of 0.5 -5 MΩ and voltage clamped (–70 mV). Glycine (Sigma, Louis, MO),
ethanol (Sigma, Sheboygan falls, WI) or propofol (Sigma, Milwaukee, WI) were
delivered from a 96-well compound plate via disposable tips (Rainin, Oakland, CA)
which could eliminate any possibility of cross-contamination.
15
Validation of OpusXpress 6000A -- We first conducted a series of experiments to
compare the data obtained from OpusXpress and conventional two electrode voltage
clamp system in wild-type GlyRs. We found that glycine EC
50
s generated from the
concentration-response curves from a conventional two-electrode voltage clamp system
and from the OpusXpress did not differ significantly from each other. As shown in Table-
1, using OpusXpress, substitution of alanine with an acidic residue glutamic acid (A52E)
did not change glycine EC
50
significantly. However, substitution with another acidic
residue, aspartic acid (A52D) resulted in 35 fold increase of glycine EC
50
. The same
pattern of changes was also found by using conventional two-electrode voltage clamp set
up. These findings illustrate and support the use of the OpusXpress in lieu of
conventional TEVC for the study of agonist activation on GlyRs.
Glycine Concentration Responses -- Oocytes expressing wild type or mutant α1 GlyRs
were exposed to concentrations of glycine ranging from 10µM to 10mM for 12-30s based
on pilot studies which determined the time necessary to achieve maximum response.
Washout periods (6-15 minutes) were allowed between drug applications. Receptor
function was evaluated by determining the glycine EC
50
and Hill Slope obtained from the
glycine concentration-response curve and I
max
.
Ethanol potentiation -- We used a set functional glycine concentration EC10 (an
effective concentration of glycine that produce 10% of the maximal current) to test the
potentiation by 100 mM ethanol. The EC
10
is the concentration of agonist that produces
10% of the maximal current (obtained by a 12 second application of 1-10 mM glycine).
Utilizing a set effective concentration of agonist with each oocytes allowed comparison
16
of drug effects across oocytes and across receptor subtypes while minimizing influence
by differences in levels of receptor expression (Davies et al. 2003; Davies et al. 2004).
Oocytes were pre-incubated with ethanol for 30 seconds before co-application of glycine
and ethanol for 30 seconds. Results are presented as the percent potentiation of EC
10
responses obtained with agonist alone and are expressed as mean ± SEM.
Propofol Potentiation -- The action of propofol was determined using procedures
similar to those described for ethanol potentiation with the following modifications:
multiple concentrations of propofol (1, 2, 5 and 10 μM) were applied sequentially on the
same oocytes in order to construct the propofol concentration response curves.

Data Analysis
Prism (GraphPAD Software, San Diego, CA) was used to perform statistical
analyses, curve fitting and correlation analyses. Concentration response data were plotted on
semi-logarithmic axes and fitted using non-linear regression analysis: [I = I
max
([A] n
H
/
((Absalom et al. 2003) n
H
+ EC
50
n
H
))] where I is the peak current recorded following
application of a range of agonist concentrations, (Absalom et al. 2003); 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 obtained from oocytes from at least two different frogs.
The n refers to the number of different oocytes tested. Data are reported as mean ± SEM.
Polarity values were assigned to amino acid residues using the Zimmerman polarity scale
(Zimmerman et al. 1968). Hydrophobicity values were assigned using the Kyte scale
(Kyte and Doolittle 1982). Molecular volumes were calculated with the Spartan
17
Molecular Modeling Program (Wave Function, San Diego, CA). Statistical significance
was defined as p < 0.05.

18
CHAPTER 3
RESULTS

Agonist activation of A52X mutant α1 GlyR
Our first goal was to determine whether mutation at position 52 altered receptor
response to the agonist-glycine. We found that all nineteen mutations yielded functional
glycine receptors in Xenopus oocytes (Table-2 and Figure-1) with some of the mutations
significantly altered receptor function. Ten mutations significantly changed the glycine
EC
50
. Ten mutations significantly altered the Hill Slope. Seven mutations significantly
altered the I
max
.
Linear regression analyses were performed between glycine EC
50
, Hill Slope, I
max

and the physical-chemical properties of the amino acid residues substituted at position 52,
such as: molecular volume, molecular weight, polarity, charge, and hydropathy. We did
not find a significant correlation between the glycine EC
50
and the physical chemical
properties tested (Figure-2). But there was a non-significant trend between the EC
50
and
the molecular weight among the mutants which significantly changed EC
50
(r
2
=0.33,
p=0.06) (Figure-3). We also found a significant inverse correlation between the
molecular volume and changes in Hill slope (r
2
=0.27, p=0.02) (Figure-4).

Ethanol sensitivity of A52X mutant α1 GlyR
Previously it was shown that amino acid substitution at position 267 in α1 GlyR
altered receptor sensitivity to 200 mM ethanol. Moreover, our and other laboratories have
found that A52S and A52C mutations reduced receptor sensitivity to ethanol (Ye et al.
19
1998; Crawford et al. 2007). We extended their investigations by studying the
modulatory effect of ethanol on all nineteen mutant GlyRs expressed in the Xenopus
oocyte preparation. Each mutant was tested

by applying 100 mM ethanol and the results
were expressed in terms

of potentiation of the response to a test concentration of
glycine—EC
10
.

Representative

tracings were shown in Figure-5. We found, with the
exception of A52F, A52Q and A52E, all mutations significantly reduced receptor
sensitivity to ethanol. The greatest reductions in ethanol sensitivity were found in A52P
(62%), A52D (65%), A52L (66%), A52W (72%) and A52N (84%) mutations (Figure-6).
Interestingly, it seems that mutations which significantly changed receptor
sensitivity to ethanol are the ones which changed receptor function. Correlation analysis
revealed that ethanol potentiation of A52X mutant α1GlyRs inversely correlate with
glycine EC
50
(r
2
=0.26, p=0.02) (Figure-7A), which suggests that ethanol enhance α1
GlyRs function, at least in part, by affecting receptor activation mechanism.
To study further the interaction between ethanol and the amino acid residue at
position 52

in α1 GlyRs, we next investigated the relationship between the specific
physical-chemical properties of the amino acid side chains (i.e. molecular volume,
molecular weight, polarity, charge, and hydropathy) at position 52 and the magnitude of
ethanol modulation. We did not find a significant correlation between the ethanol
potentiation and the physical chemical characteristics tested. However, there are non-
significant trends between molecular volume and molecular weight and the magnitude of
ethanol potentiation (r
2
=0.19, p=0.08) among the mutants which significantly changed
ethanol potentiation (Figure-8).
20
Propofol responsiveness of A52X mutant α1 GlyRs
Previous studies demonstrated that the widely used general anesthetic propofol
potentiated GABA
A
Rs and GlyRs functions (Mascia et al. 1996b), and a point mutation
A52S in α1 GlyRs significantly altered receptor sensitivity to ethanol but did change
receptor sensitivity to propofol (Mascia et al. 1996a). We next used propofol to test the
specificity of position 52 for changing ethanol modulation. This was accomplished by
testing whether mutations which significantly altered ethanol sensitivity also changed the
modulation effect of propofol. We tested the propofol concentration response (1-10 μM)
on wild type, A52S and A52W mutant α1 GlyRs. A52S and A52W mutants were chosen
because: 1) the A52S mutation decreased ethanol sensitivity but did not change propofol
sensitivity in a previous study (Mascia et al. 1996a); 2) the A52W mutation greatly
changed receptor sensitivity to ethanol in the present study (Figure-6). We found that
propofol potentiated glycine-induced currents in a concentration-dependent manner in
wild type, A52S and A52W α1 GlyRs and these mutations did not change receptors
sensitivity to propofol (Figure-9). The lack of change in propofol sensitivity by
mutations which altered ethanol sensitivity indicates that position 52 only plays an
important role as a target for ethanol, but not for propofol in α1 GlyRs.

21
CHAPTER 4
DISCUSSION AND CONCLUSIONS

The present work represents the first systematic investigation of the structure-
activity relationship of position 52 in α1 GlyRs in agonist activation and ethanol
modulation. We investigated whether and how changes in the physical-chemical
properties of the amino acid residue at position 52 alter receptor function and the
sensitivity to ethanol.
The amino acid residue at position 52 in wild-type α1 GlyRs is alanine. The
present work found that mutating this small, neutral, hydrophobic amino acid residue to
the other nineteen amino acid residues significantly changed glycine EC
50
and reduced
the magnitude of ethanol modulation. These findings add to the evidence that the position
52 in the extracellular domain is a site of action for ethanol and also suggest that the
physical-chemical properties of the amino acid residue at position 52 are important for
both agonist activation and ethanol modulation in α1GlyRs.
The present study also tested for possible relationships between changes in
agonist activation and ethanol sensitivity produced by these mutations. We found that
changes in glycine EC
50
were linked to changes in ethanol potentiation magnitude.
Previous in vivo and in vitro studies suggested that Loop 2 residues, including position
52 are involved in agonist-induced receptor activation (Ryan et al. 1994; Saul et al. 1994).
Other work has implicated position 52 as an important target for ethanol modulation
(Mascia et al. 1996a; Crawford et al. 2007). Taken together, previous findings suggest
that position 52 is important for both receptor activation and ethanol modulation. Thus, it
22
is hypothesized that ethanol may modulate GlyR function through position 52. As
predicted, results from the present study support this hypothesis by demonstrating a
relationship between the changes in glycine EC
50
and the changes in the magnitude of
ethanol potentiation (p = 0.02, r
2
= 0.26) (Figure-7A). We found that approximately 50%
of the mutations caused significant changes in both glycine EC
50
and the magnitude of
ethanol potentiation. However, there were about 40% of the mutations did not alter
glycine EC
50
, but significantly reduced the magnitude of ethanol potentiation. Overall,
these findings suggest that changes in glycine EC
50
are sufficient but not necessary to
cause change in ethanol sensitivity.
When we limited our analysis to a subpopulation of mutations which significantly
changed both glycine EC
50
and ethanol potentiation magnitude, the correlation became
stronger (r
2
increased to 0.43) (Figure-7B). Therefore, there appears to be two
populations of substitutions. In one population, ethanol sensitivity always changed when
there was a change in glycine EC
50
and the variance in ethanol potentiation could be
explained to a large extend by the variance in glycine EC
50
. In the other population,
ethanol modulation can be altered independently of changes in glycine sensitivity factors;
other than changes in glycine sensitivity are likely to be important in determining the
sensitivity to ethanol. Although there is no common physical-chemical property shared
among the residues within each population, there is a non-significant trend between EC
50

and molecular weight (r
2
=0.36, p=0.09) observed in the subgroup in which both glycine
EC
50
and ethanol potentiation were significantly changed (Figure-10), suggesting a
relationship between amino acid molecular weight, agonist activation and ethanol
23
modulation. Taken together, these findings support our hypothesis that ethanol modulates
α1 GlyR function, at least in part, by affecting receptor activation mechanism.
Linear regression analysis did not reveal any statistically significant relationship
between the physical-chemical properties (molecular volume, molecular weight, charge,
polarity, and hydropathy) of the amino acid substituted at position 52 and the magnitude
of ethanol potentiation. However, non-significant trends were observed between the
molecular volume and molecular weight of the substituted amino acid and the magnitude
of ethanol potentiation when we limited our analysis for the mutants which significantly
changed the magnitude of ethanol potentiation, suggesting that the physical size of the
residue at position 52 might play a role in dictating the ability of ethanol to potentiate α1
GlyR function. These findings are consistent with previous studies showing that the
molecular volume of the key residue is often correlated with the modulation by
anesthetics (Koltchine et al. 1999; Yamakura et al. 1999) and alcohols (Ye et al. 1998).
Additional analyses revealed no significant relationship between the physical-
chemical properties and glycine EC
50
. However, the absence of a significant relationship
between the physical-chemical properties and glycine EC
50
does not necessarily mean
that structure-function relationship does not exist. One amino acid residue can have
multiple characteristics and thus there may be more complex relationships between
physical chemical parameters and receptor response to glycine.
The current study also provides evidence for the specificity of position 52 as
target for ethanol action. Previous studies suggest that propofol does not act at the same
site as ethanol on GABA
A
R and GlyR (Mascia et al. 1996b). Our results demonstrated
24
that two substitutions (A52S and A52W) which significantly reduced ethanol potentiation
did not significantly affect the magnitude of propofol potentiation. The lack of change in
propofol potentiation is consistent with the previous report suggesting that propofol and
ethanol do not act on the same site on GlyRs.
As stated above, there is a correlation between the ethanol potentiation magnitude
and molecular volume of the residue at position 52. The bigger the molecular volume of
the residue substituted, the less ethanol potentiation was observed. Therefore, it could be
argued that the decreased ethanol sensitivity is not due to a reduced contribution of
position 52 to the modulation of ethanol and that it is the consequences of non-specific
structure alteration resulting from the introduction of bulky residues, such as tryptophan.
The specificity of the A52W mutation affecting modulation by ethanol, but not propofol,
suggests otherwise and thus supports the hypothesis that position 52 is a target specific
for ethanol action.
To summarize, the current investigation suggests that: (1) changes in the physical-
chemical properties of the amino acid residue at position 52 of α1 GlyRs significantly
altered receptor sensitivity to glycine and ethanol; (2) the molecular volume and
molecular weight of the amino acid residue at position 52 play roles in determining the
responsiveness of α1GlyRs to both glycine and ethanol; (3) changes in glycine EC
50
are
correlated to changes in ethanol potentiation magnitude. This result suggests that ethanol
modulates α1 GlyR function, at least in part, by affecting receptor activation mechanism;
(4) position 52 of α1 GlyRs is a target for ethanol action, but not for propofol action.
25
In conclusion, the present study adds to the evidence that position 52 in the
extracellular domain, in addition to the transmembrane domain, plays an important role in
ethanol modulation in α1 GlyRs. The findings provide insight into structure-function
relationships for GlyRs. This work also provides new evidence that ethanol modulates α1
GlyR function, at least partly, by affecting agonist activation through position 52. Given
the high sequence homology within the cys-loop LGIC superfamily, the current findings
provide a new direction for future studies to investigate the relationship between agonist
activation and ethanol modulation in other members of this Cys-loop LGIC superfamily.

26
-5.0 -4.5 -4.0 -3.5 -3.0 -2.5 -2.0
-10
0
10
20
30
40
50
60
70
80
90
100
110
WT
A52C
A52D
A52E
A52F
A52G
A52H
A52I
A52K
A52L
A52M
A52N
A52P
A52R
A52S
A52Q
A52T
A52V
A52W
A52Y
LOG Glycine ConC (M)
Glycine induced Cl- currrent
(normalized)

Figure-1. Glycine concentration--response curves of A52X mutated GlyRs. 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 red curve is
used to illustrate the glycine concentration response in WT GlyRs. The other colorful
curves represent the glycine concentration response in mutant GlyRs by which the
glycine EC
50
s were significantly altered.

27
100 150 200
0
1000
2000
3000
4000
5000
6000
7000
r
2
=0.08
p=0.24
Molecular Volume (A
3
)
Glycine EC50 (μ μ μ μM)
50 75 100 125 150
0
1000
2000
3000
4000
5000
6000
7000
r
2
=0.07
p=0.25
Molecular Volume (ml/mole)
Glycine EC50 (μ μ μ μM)
100 125 150 175 200
-1000
0
1000
2000
3000
4000
5000
6000
7000
r
2
=0.12
p=0.13
Molecular Weight
Glycine EC50 (μ μ μ μM)
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5
1000
2000
3000
4000
5000
6000
7000
r
2
=0.02
p=0.58
Charge
Glycine EC50 (μ μ μ μM)
3 4 5 6 7
0
1000
2000
3000
4000
5000
6000
7000
r
2
=0.01
p=0.64
Polarity
Glycine EC50 (μ μ μ μM)
-5.0 -2.5 0.0 2.5 5.0
500
1500
2500
3500
4500
5500
r
2
=0.05
p=0.35
Hydropathy
Glycine EC50 (μ μ μ μM)

Figure-2. Correlation analysis between glycine EC
50
and the physical-chemical
properties of amino acid residues at position 52. Linear regression analyses were
performed between glycine EC
50
and molecular volume, molecular weight, polarity,
charge, and hydropathy of the amino acid residues at position 52. There was no
significant relationship between glycine EC
50
and these properties.

28
100 150 200
0
1000
2000
3000
4000
5000
6000
7000
r
2
=0.22
p=0.14
A52N
A52D
A52W
A52I
A52L
A52Q
A52R
A52G
WT
A52S
A52P
Molecular Volume (A
3
)
Glycine EC50 (μ μ μ μM)
50 75 100 125
0
1000
2000
3000
4000
5000
6000
7000
r
2
=0.22
p=0.14
A52P
A52N
A52D
A52W
A52I
A52L
A52Q
A52R
A52S
WT
A52G
Molecular Volume (ml/mole)
Glycine EC50 (μ μ μ μM)
75 125 175
-1000
0
1000
2000
3000
4000
5000
6000
7000
r
2
=0.33
p=0.06
A52P
A52N
A52D
A52W
A52I
A52L
A52Q
A52R
A52G
A52S
WT
Molecular Weight
Glycine EC50 (μ μ μ μM)
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5
1000
2000
3000
4000
5000
6000
7000
r
2
=0.03
p=0.62
A52P
A52N
A52D
A52W
A52I
A52L
A52Q
A52R
Charge
Glycine EC50 (μ μ μ μM)
3 4 5 6 7
0
1000
2000
3000
4000
5000
6000
7000
r
2
=0.04
p=0.57
A52P
A52N
A52D
A52W
A52I
A52L
A52Q
A52R
Polarity
Glycine EC50 (μ μ μ μM)
-5.0 -2.5 0.0 2.5 5.0
500
1500
2500
3500
4500
5500
r
2
=0.07
p=0.44
A52P
A52N
A52D
A52W
A52I
A52L
A52Q
A52R
A52S
A52G
WT
Hydropathy
Glycine EC50 (μ μ μ μM)

Figure-3. Correlation analysis between the glycine EC
50
and physical-chemical
properties of the amino acid at position 52 among the mutants who significantly
changed EC
50
. There is a non-significant trend between the EC
50
and the molecular
weight.

29
50 75 100 125
0
1
2
3
4
5
6
r
2
=0.27
p=0.02 Molecular Volume (ml/mole)
Hill Slope
75 100 125 150 175
0
1
2
3
4
5
6
r
2
=0.12
p=0.14 Molecular Weight
Hill Slope
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5
1
2
3
4
5
6
r
2
=0.005
p=0.77
Charge
Hill Slope
3 4 5 6 7
0
1
2
3
4
5
6
r
2
=0.04
p=0.42
Polarity
Hill Slope
-5.0 -2.5 0.0 2.5 5.0
0
1
2
3
4
5
6
r
2
=0.02
p=0.54
Hydropathy
Hill Slope
100 125 150 175 200
0
1
2
3
4
5
6
r
2
=0.19
p=0.05
Molecular Volume (A
3
)
Hill Slope

Figure-4. Correlation analysis between Hill Slope and the physical-chemical
properties of amino acid residues at position 52. Linear regression analyses were
performed between the Hill Slope and molecular volume, molecular weight, polarity,
charge, and hydropathy of the amino acid residues at position 52. There is a significant
correlation between Hill Slope

and molecular volume.

30

Figure-5. Representative tracing showing 5 successful parallel recordings on wild
type α1 GlyRs. From left to right, responses represent effects of EC
10
glycine, EC
10

glycine, EC
10
glycine + 100 mM ethanol, EC
10
glycine and EC
10
glycine. Glycine was
applied for 30 s in duration, followed by 6 min washout; ethanol was applied for 30 s,
and then coapplied with EC
10
glycine for 30 s. Washout time between ethanol and
glycine application was 10 min. EC
10
glycine induced currents completely recovered after
ethanol washout. X-axis presents time in minutes. Y-axis represents current response in
µAs.

31
A F Q E C G T S H Y K R M V I P D L W N
0
25
50
75
100
125
*
*
*
*
**
**
**
**
**
**
**
**
**
**
**
**
A52X mutants
Potentiation of glycine induced Cl
-
current by 100 mM ethanol

Figure-6. Point mutations at position 52 in α1GlyRs significantly changed the
receptor’s sensitivity to 100 mM ethanol. Results are presented as normalized
percentage potentiation of control glycine current responses. Glycine EC
8-12
was
determined for each oocyte and used for measuring the modulation by 100 mM ethanol.
Glycine induced control responses were measured before and after ethanol application to
take into account possible shifts in the control currents as the recording preceded. Each
experiment was carried out with 8-10 oocytes from at least two frogs. (* p<0.05, **
p<0.005)

32
0 1000 2000 3000 4000 5000 6000
0
25
50
75
100
125
WT
C
D
E
F
G
H
I
K
L
M
N
P
Q
R
S
T
W
Y
V
r
2
=0.26
p=0.02
EC50 (uM)
Potentiation (%)

Figure-7A. Magnitude of Ethanol potentiation significantly correlates with glycine
EC
50
. Linear regression was performed between the glycine EC
50
and the ethanol
potentiation (%) among the nineteen A52X mutant and WT α1 GlyRs.

33
0 1000 2000 3000 4000 5000 6000
0
25
50
75
100
125
D
G
L
N
P
R
S
W
r
2
=0.43
p=0.05 I
EC50 (uM)
Potentiation (%)

Figure-7B. Magnitude of Ethanol potentiation significantly correlates with glycine
EC
50
among the mutants who significantly changed both glycine EC
50
and ethanol
potentiation.

34
WT
0 50 100 150 200
0
25
50
75
100
125
WT
polar
nonpolar
acidic
basic
r
2
=0.19
p=0.08
Molecular Volume (ml/mole)
% Potentiation
WT
0 100 200 300
0
25
50
75
100
125
WT
polar
nonpolar
acidic
basic
r
2
=0.19
p=0.08
Molecular Volume (A
3
)
% Potentiation
WT
0 50 100 150 200 250
0
25
50
75
100
125
WT
nonpolar
acidic
basic
polar
r
2
=0.19
p=0.08
Molecular Weight
% Potentiation
WT
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5
25
50
75
100
125
WT
r
2
=0.01
p=0.65
Charge
% Potentiation
WT
-5.0 -2.5 0.0 2.5 5.0
25
50
75
100
125
WT
nonpolar
acidic
basic
polar
r
2
=0.01
p=0.71
Hydropathy
% Potentiation
WT
0.0 2.5 5.0 7.5 10.0 12.5
0
50
100
150
WT
nonpolar
acidic
basic
polar
r
2
=0.007
p=0.76
Polarity
% Potentiation

Figure-8. Correlation analysis between the ethanol potentiation magnitude and the
physical-chemical properties among the mutants who significantly changed ethanol
potentiation. A non-significant trend between the magnitude of ethanol potentiation and
the molecular volume/weight was found.

35
0.0 2.5 5.0 7.5 10.0 12.5
0
25
50
75
100
WT
A52S
A52W
Propofol (μ μ μ μM)
Glycine-Induced Cl
-
Currents
Potentiation (%)

Figure-9. Propofol concentration--response curves of WT and A52X mutated GlyRs.
Propofol potentiated, in a concentration manner, currents induced by glycine in WT,
A52S and A52W α1 GlyRs. No differences in propofol sensitivity were found among the
WT and the two mutant receptors. Values are mean ± standard error of 12-13 oocytes.
Statistical analyses: Two-way ANOVA revealed significant effects of propofol
concentrations [F
3
,
160
=44.45, p<0.0001] but no significant effects between mutations
[F
2
,
160
=0.13, p=0.8814].

36
100 150 200
0
1000
2000
3000
4000
5000
6000
7000
r
2
=0.23
p=0.20
A52N
A52D
A52W
A52I
A52L
A52R
A52G
A52S
A52P
Molecular Volume (A
3
)
Glycine EC50 (μ μ μ μM)
50 75 100 125
0
1000
2000
3000
4000
5000
6000
7000
r
2
=0.22
p=0.20
A52P
A52N
A52D
A52W
A52I
A52L
A52R
A52S
A52G
Molecular Volume (ml/mole)
Glycine EC50 (μ μ μ μM)
75 125 175
0
1000
2000
3000
4000
5000
6000
7000
r
2
=0.36
p=0.09
A52P
A52N
A52D
A52W
A52I
A52L
A52R
A52G
A52S
Molecular Weight
Glycine EC50 (μ μ μ μM)
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5
1000
2000
3000
4000
5000
6000
7000
r
2
=0.03
p=0.64
A52P
A52N
A52D
A52W
A52I
A52L
A52R
Charge
Glycine EC50 (μ μ μ μM)
3 4 5 6 7
0
1000
2000
3000
4000
5000
6000
7000
r
2
=0.05
p=0.55
A52P
A52N
A52D
A52W
A52I
A52L
A52R
Polarity
Glycine EC50 (μ μ μ μM)
-5.0 -2.5 0.0 2.5 5.0
500
1500
2500
3500
4500
5500
r
2
=0.1
p=0.41
A52P
A52N
A52D
A52W
A52I
A52L
A52R
A52S
A52G
Hydropathy
Glycine EC50 (μ μ μ μM)

Figure-10. Correlation analysis between the glycine EC
50
and the physical-chemical
properties among the mutants who significantly changed both EC
50
and ethanol
potentiation. A non-significant trend between the glycine EC
50
and the molecular weight
was found.

37
GlyRs
Glycine EC
50
data collected from
conventional TEVC recording (μM)
Glycine EC
50
obtained from
OpusXpress 6000A (μM)
WT 82±9 85±4
A52E 108±54 116±41
A52D 2891±274* 2427±108*

Table-1. Concentration-response curves constructed from data obtained using
conventional two-electrode voltage clamp system and OpusXpress provided similar
EC
50
data in wild type, A52E and A52D mutant α1GlyRs. To establish concentration-
response curves, oocytes expressing wild type or mutant α1 GlyRs were exposed to
concentrations of glycine ranging from 10µM to 10mM for 12-30s based on initial
studies. Washout periods (6-15 minutes) were allowed between drug applications. Each
data set represents the means ± SEM from 4 different oocytes for conventional TEVC
recording and 8-30 oocytes for OpusXpress recording. (* = p<0.001)

38
A52X Mutant EC
50
(µM) Hill slope Imax (µA) N
I 39±10** 1.0 ± 0.4** 34.2±7.0 4
Q 130±19* 1.9 ± 0.3 31.7±5.7 9
E 181±28 1.9 ± 0.3 35.3±4.9* 9
WT 239±34 2.3 ± 0.1 23.1±2.3 15
H 240±29 2.7 ± 0.2 32.4±7.0 10
F 247±68 1.9 ± 0.4 41.6±7.3** 7
C 250±45 2.8 ± 0.2* 26.6±3.9 10
V 291±126 1.2 ± 0.3** 32.2±4.9 5
T 302±55 3.6±1.4 40.6±4.8** 9
Y 318±139 1.4± 0.2** 26.4±1.6 6
M 343±79 1.4 ± 0.2*** 34.2±8.2 10
K 368±65 1.3± 0.4* 25.1±5.2 9
R 500±70** 1.6 ± 0.3* 33.0±6.2 6
G 520±88** 1.7 ± 0.3 13.7±1.6* 8
S 541±144* 2.2 ± 0.3 40.7±4.6** 10
L 830±278** 1.1 ± 0.5* 12.1±2.8** 5
D 1717±108*** 2.4 ±0.2 17.2±2.2 15
P 2129±403*** 1.6 ± 0.5 15.4±7.1 7
N 3082±785*** 1.1 ± 0.1*** 4.0±0.6*** 7
W 5315±1303*** 0.6 ± 0.1*** 30.5±4.9 6

Table-2. Glycine EC
50
, Hill slope and I
max
of WT and mutated α1 GlyRs. All nineteen
mutations yielded functional glycine receptors. Ten mutations significantly shifted the
glycine EC
50
. Ten mutations significantly altered the Hill Slope. Seven mutations
significantly altered the I
max
. EC
50
, Hill Slope and I
max
were generated from glycine
concentration-response curves of Xenopus oocytes expressing WT or A52X mutant α1
GlyRs. Glycine induced currents were normalized to the maximal currents activated by a
saturating concentration of glycine (ranging from 300 µM to 10 mM depending on the
mutant). Means ± SEM for 4-30 oocytes from at least two frogs are presented for each
measurement. Statistical analyses were done by using one-way ANOVA with Dunnett’s
post-test (*= p< 0.05, **= p< 0.005, ***= p< 0.001).

39
REFERENCES

Absalom, N. L., Lewis, T. M., Kaplan, W., Pierce, K. D. and Schofield, P. R. (2003).
"Role of charged residues in coupling ligand binding and channel activation in the
extracellular domain of the glycine receptor." J Biol Chem 278(50): 50151-7.

Aguayo, L. G. and Pancetti, F. C. (1994). "Ethanol modulation of the gamma-
aminobutyric acidA- and glycine-activated Cl- current in cultured mouse
neurons." J Pharmacol Exp Ther 270(1): 61-9.

Aguayo, L. G., Tapia, J. C. and Pancetti, F. C. (1996). "Potentiation of the glycine-
activated Cl- current by ethanol in cultured mouse spinal neurons." J Pharmacol
Exp Ther 279(3): 1116-22.

Aistrup, G. L., Marszalec, W. and Narahashi, T. (1999). "Ethanol modulation of nicotinic
acetylcholine receptor currents in cultured cortical neurons." Mol Pharmacol
55(1): 39-49.

Alkana, R. L., Finn, D. A., Jones, B. L., Kobayashi, L. S., Babbini, M., Bejanian, M. and
Syapin, P. J. (1992). "Genetically determined differences in the antagonistic effect
of pressure on ethanol-induced loss of righting reflex in mice." Alcohol Clin Exp
Res 16(1): 17-22.

Alkana, R. L. and Malcolm, R. D. (1981). "Low-level hyperbaric ethanol antagonism in
mice. Dose and pressure response." Pharmacology 22(3): 199-208.

Allan, A. M. and Harris, R. A. (1986). "Gamma-aminobutyric acid and alcohol actions:
neurochemical studies of long sleep and short sleep mice." Life Sci 39(21): 2005-
15.

Baur, R., Simmen, U., Senn, M., Sequin, U. and Sigel, E. (2005). "Novel plant substances
acting as beta subunit isoform-selective positive allosteric modulators of GABAA
receptors." Mol Pharmacol 68(3): 787-92.

Beckstead, M. J., Weiner, J. L., Eger, E. I., 2nd, Gong, D. H. and Mihic, S. J. (2000).
"Glycine and gamma-aminobutyric acid(A) receptor function is enhanced by
inhaled drugs of abuse." Mol Pharmacol 57(6): 1199-205.

Bejanian, M., Jones, B. L. and Alkana, R. L. (1993). "Low-level hyperbaric antagonism
of ethanol-induced locomotor depression in C57BL/6J mice: dose response."
Alcohol Clin Exp Res 17(5): 935-9.

40
Brejc, K., van Dijk, W. J., Klaassen, R. V., Schuurmans, M., van Der Oost, J., Smit, A. B.
and Sixma, T. K. (2001). "Crystal structure of an ACh-binding protein reveals the
ligand-binding domain of nicotinic receptors." Nature 411(6835): 269-76.

Cardoso, R. A., Brozowski, S. J., Chavez-Noriega, L. E., Harpold, M., Valenzuela, C. F.
and Harris, R. A. (1999). "Effects of ethanol on recombinant human neuronal
nicotinic acetylcholine receptors expressed in Xenopus oocytes." J Pharmacol
Exp Ther 289(2): 774-80.

Celentano, J. J., Gibbs, T. T. and Farb, D. H. (1988). "Ethanol potentiates GABA- and
glycine-induced chloride currents in chick spinal cord neurons." Brain Res 455(2):
377-80.

Cherubini, E. and Conti, F. (2001). "Generating diversity at GABAergic synapses."
Trends Neurosci 24(3): 155-62.

Crawford, D. K., Trudell, J. R., Bertaccini, E. J., Li, K., Davies, D. L. and Alkana, R. L.
(2007). "Evidence that ethanol acts on a target in Loop 2 of the extracellular
domain of alpha1 glycine receptors." J Neurochem 102(6): 2097-109.

Darstein, M., Loschmann, P. A., Knorle, R. and Feuerstein, T. J. (1997). "Strychnine-
sensitive glycine receptors inducing [3H]-acetylcholine release in rat
caudatoputamen: a new site of action of ethanol?" Naunyn Schmiedebergs Arch
Pharmacol 356(6): 738-45.

Davies, D. L. and Alkana, R. L. (1998). "Direct antagonism of ethanol's effects on
GABA(A) receptors by increased atmospheric pressure." Alcohol Clin Exp Res
22(8): 1689-97.

Davies, D. L. and Alkana, R. L. (2001a). "Direct evidence for a cause-effect link between
ethanol potentiation of GABA(A) receptor function and intoxication from
hyperbaric studies in C57, LS, and SS mice." Alcohol Clin Exp Res 25(8): 1098-
106.

Davies, D. L., Crawford, D. K., Trudell, J. R., Mihic, S. J. and Alkana, R. L. (2004).
"Multiple sites of ethanol action in alpha1 and alpha2 glycine receptors suggested
by sensitivity to pressure antagonism." J Neurochem 89(5): 1175-85.

Davies, D. L., Kochegarov, A. A., Kuo, S. T., Kulkarni, A. A., Woodward, J. J., King, B.
F. and Alkana, R. L. (2005). "Ethanol differentially affects ATP-gated P2X(3)
and P2X(4) receptor subtypes expressed in Xenopus oocytes."
Neuropharmacology 49(2): 243-53.

41
Davies, D. L., Machu, T. K., Guo, Y. and Alkana, R. L. (2002). "Ethanol sensitivity in
ATP-gated P2X receptors is subunit dependent." Alcohol Clin Exp Res 26(6):
773-8.

Davies, D. L., Trudell, J. R., Mihic, S. J., Crawford, D. K. and Alkana, R. L. (2003).
"Ethanol potentiation of glycine receptors expressed in Xenopus oocytes
antagonized by increased atmospheric pressure." Alcohol Clin Exp Res 27(5):
743-55.

Deitrich, R. A., Dunwiddie, T. V., Harris, R. A. and Erwin, V. G. (1989). "Mechanism of
action of ethanol: initial central nervous system actions." Pharmacol Rev 41(4):
489-537.

Dildy-Mayfield, J. E. and Harris, R. A. (1995). "Ethanol inhibits kainate responses of
glutamate receptors expressed in Xenopus oocytes: role of calcium and protein
kinase C." J Neurosci 15(4): 3162-71.

Dildy-Mayfield, J. E., Mihic, S. J., Liu, Y., Deitrich, R. A. and Harris, R. A. (1996).
"Actions of long chain alcohols on GABAA and glutamate receptors: relation to
in vivo effects." Br J Pharmacol 118(2): 378-84.

Eckenhoff, R. G. and Johansson, J. S. (1997). "Molecular interactions between inhaled
anesthetics and proteins." Pharmacol Rev 49(4): 343-67.

Eggers, E. D. and Berger, A. J. (2004). "Mechanisms for the modulation of native glycine
receptor channels by ethanol." J Neurophysiol 91(6): 2685-95.

Eggers, E. D., O'Brien, J. A. and Berger, A. J. (2000). "Developmental changes in the
modulation of synaptic glycine receptors by ethanol." J Neurophysiol 84(5):
2409-16.

Engblom, A. C. and Akerman, K. E. (1991). "Effect of ethanol on gamma-aminobutyric
acid and glycine receptor-coupled Cl- fluxes in rat brain synaptoneurosomes." J
Neurochem 57(2): 384-90.

Findlay, G. S., Wick, M. J., Mascia, M. P., Wallace, D., Miller, G. W., Harris, R. A. and
Blednov, Y. A. (2002). "Transgenic expression of a mutant glycine receptor
decreases alcohol sensitivity of mice." J Pharmacol Exp Ther 300(2): 526-34.

Franks, N. P. and Lieb, W. R. (1981). "Is membrane expansion relevant to anaesthesia?"
Nature 292(5820): 248-51.

42
Godden, E. L., Harris, R. A. and Dunwiddie, T. V. (2001). "Correlation between
molecular volume and effects of n-alcohols on human neuronal nicotinic
acetylcholine receptors expressed in Xenopus oocytes." J Pharmacol Exp Ther
296(3): 716-22.

Greenblatt, E. P. a. M., X. (1999). "A critical amino acid for halothane action."
Anesthesiology 91,(A807).
Harris, R. A. (1999). "Ethanol actions on multiple ion channels: which are important?"
Alcohol Clin Exp Res 23(10): 1563-70.

Harris, R. A., Mihic, S. J., Brozowski, S., Hadingham, K. and Whiting, P. J. (1997).
"Ethanol, flunitrazepam, and pentobarbital modulation of GABAA receptors
expressed in mammalian cells and Xenopus oocytes." Alcohol Clin Exp Res 21(3):
444-51.

Heilig, M. and Egli, M. (2006). "Pharmacological treatment of alcohol dependence:
target symptoms and target mechanisms." Pharmacol Ther 111(3): 855-76.

Hejazi, N., Zhou, C., Oz, M., Sun, H., Ye, J. H. and Zhang, L. (2006). "Delta9-
tetrahydrocannabinol and endogenous cannabinoid anandamide directly potentiate
the function of glycine receptors." Mol Pharmacol 69(3): 991-7.

Jenkins, A., Greenblatt, E. P., Faulkner, H. J., Bertaccini, E., Light, A., Lin, A.,
Andreasen, A., Viner, A., Trudell, J. R. and Harrison, N. L. (2001). "Evidence for
a common binding cavity for three general anesthetics within the GABAA
receptor." J Neurosci 21(6): RC136.

Jiang, Z. L. and Ye, J. H. (2003). "Protein kinase C epsilon is involved in ethanol
potentiation of glycine-gated Cl(-) current in rat neurons of ventral tegmental
area." Neuropharmacology 44(4): 493-502.

Johnson, B. A., Rosenthal, N., Capece, J. A., Wiegand, F., Mao, L., Beyers, K., McKay,
A., Ait-Daoud, N., Anton, R. F., Ciraulo, D. A., Kranzler, H. R., Mann, K.,
O'Malley, S. S. and Swift, R. M. (2007). "Topiramate for treating alcohol
dependence: a randomized controlled trial." Jama 298(14): 1641-51.

Kash, T. L., Jenkins, A., Kelley, J. C., Trudell, J. R. and Harrison, N. L. (2003).
"Coupling of agonist binding to channel gating in the GABA(A) receptor." Nature
421(6920): 272-5.

Kash, T. L., Kim, T., Trudell, J. R. and Harrison, N. L. (2004). "Evaluation of a proposed
mechanism of ligand-gated ion channel activation in the GABAA and glycine
receptors." Neurosci Lett 371(2-3): 230-4.

43
Koltchine, V. V., Finn, S. E., Jenkins, A., Nikolaeva, N., Lin, A. and Harrison, N. L.
(1999). "Agonist gating and isoflurane potentiation in the human gamma-
aminobutyric acid type A receptor determined by the volume of a second
transmembrane domain residue." Mol Pharmacol 56(5): 1087-93.

Kyte, J. and Doolittle, R. F. (1982). "A simple method for displaying the hydropathic
character of a protein." J Mol Biol 157(1): 105-32.

Langosch, D., Thomas, L. and Betz, H. (1988). "Conserved quaternary structure of
ligand-gated ion channels: the postsynaptic glycine receptor is a pentamer." Proc
Natl Acad Sci U S A 85(19): 7394-8.

Laube, B., Kuhse, J., Rundstrom, N., Kirsch, J., Schmieden, V. and Betz, H. (1995).
"Modulation by zinc ions of native rat and recombinant human inhibitory glycine
receptors." J Physiol 483 ( Pt 3): 613-9.

Laube, B., Maksay, G., Schemm, R. and Betz, H. (2002). "Modulation of glycine
receptor function: a novel approach for therapeutic intervention at inhibitory
synapses?" Trends Pharmacol Sci 23(11): 519-27.

Li, C., Peoples, R. W. and Weight, F. F. (1998). "Ethanol-induced inhibition of a
neuronal P2X purinoceptor by an allosteric mechanism." Br J Pharmacol 123(1):
1-3.

Li, C., Xiong, K. and Weight, F. F. (2000). "Ethanol inhibition of adenosine 5'-
triphosphate-activated current in freshly isolated adult rat hippocampal CA1
neurons." Neurosci Lett 295(3): 77-80.

Lobo, I. A., Mascia, M. P., Trudell, J. R. and Harris, R. A. (2004). "Channel gating of the
glycine receptor changes accessibility to residues implicated in receptor
potentiation by alcohols and anesthetics." J Biol Chem 279(32): 33919-27.

Lobo, I. A., Trudell, J. R. and Harris, R. A. (2006). "Accessibility to residues in
transmembrane segment four of the glycine receptor." Neuropharmacology 50(2):
174-81.

Lovinger, D. M., White, G. and Weight, F. F. (1989). "Ethanol inhibits NMDA-activated
ion current in hippocampal neurons." Science 243(4899): 1721-4.

Lynch, J. W. and Callister, R. J. (2006). "Glycine receptors: a new therapeutic target in
pain pathways." Curr Opin Investig Drugs 7(1): 48-53.

44
Machu, T. K. and Harris, R. A. (1994). "Alcohols and anesthetics enhance the function of
5-hydroxytryptamine3 receptors expressed in Xenopus laevis oocytes." J
Pharmacol Exp Ther 271(2): 898-905.

Marrero, M. B., Papke, R. L., Bhatti, B. S., Shaw, S. and Bencherif, M. (2004). "The
neuroprotective effect of 2-(3-pyridyl)-1-azabicyclo[3.2.2]nonane (TC-1698), a
novel alpha7 ligand, is prevented through angiotensin II activation of a tyrosine
phosphatase." J Pharmacol Exp Ther 309(1): 16-27.

Mascia, M. P., Machu, T. K. and Harris, R. A. (1996b). "Enhancement of homomeric
glycine receptor function by long-chain alcohols and anaesthetics." Br J
Pharmacol 119(7): 1331-6.
Mascia, M. P., Mihic, S. J., Valenzuela, C. F., Schofield, P. R. and Harris, R. A. (1996a).
"A single amino acid determines differences in ethanol actions on strychnine-
sensitive glycine receptors." Mol Pharmacol 50(2): 402-6.

Mascia, M. P., Trudell, J. R. and Harris, R. A. (2000). "Specific binding sites for alcohols
and anesthetics on ligand-gated ion channels." Proc Natl Acad Sci U S A 97(16):
9305-10.

McCool, B. A., Frye, G. D., Pulido, M. D. and Botting, S. K. (2003). "Effects of chronic
ethanol consumption on rat GABA(A) and strychnine-sensitive glycine receptors
expressed by lateral/basolateral amygdala neurons." Brain Res 963(1-2): 165-77.

Mihic, S. J., Ye, Q., Wick, M. J., Koltchine, V. V., Krasowski, M. D., Finn, S. E., Mascia,
M. P., Valenzuela, C. F., Hanson, K. K., Greenblatt, E. P., Harris, R. A. and
Harrison, N. L. (1997). "Sites of alcohol and volatile anaesthetic action on
GABA(A) and glycine receptors." Nature 389(6649): 385-9.

Molander, A., Lido, H. H., Lof, E., Ericson, M. and Soderpalm, B. (2007). "The glycine
reuptake inhibitor Org 25935 decreases ethanol intake and preference in male
wistar rats." Alcohol Alcohol 42(1): 11-8.

Molander, A. and Soderpalm, B. (2005). "Accumbal strychnine-sensitive glycine
receptors: an access point for ethanol to the brain reward system." Alcohol Clin
Exp Res 29(1): 27-37.

Monaghan, D. T., Bridges, R. J. and Cotman, C. W. (1989). "The excitatory amino acid
receptors: their classes, pharmacology, and distinct properties in the function of
the central nervous system." Annu Rev Pharmacol Toxicol 29: 365-402.

45
Moore, E. L., Haspel, G., Libersat, F. and Adams, M. E. (2006). "Parasitoid wasp sting: a
cocktail of GABA, taurine, and beta-alanine opens chloride channels for central
synaptic block and transient paralysis of a cockroach host." J Neurobiol 66(8):
811-20.

NIAAA (2000). National Institute on Alcohol Abuse and Alcoholism: Tenth Special
Report to the U.S. Congress on Alcohol and Health.

Olsen, R. W., Chang, C. S., Li, G., Hanchar, H. J. and Wallner, M. (2004). "Fishing for
allosteric sites on GABA(A) receptors." Biochem Pharmacol 68(8): 1675-84.

Ortells, M. O., Barrantes, G. E., Wood, C., Lunt, G. G. and Barrantes, F. J. (1997).
"Molecular modelling of the nicotinic acetylcholine receptor transmembrane
region in the open state." Protein Eng 10(5): 511-7.

Papke, R. L., Meyer, E. M., Lavieri, S., Bollampally, S. R., Papke, T. A., Horenstein, N.
A., Itoh, Y. and Porter Papke, J. K. (2004). "Effects at a distance in alpha 7
nAChR selective agonists: benzylidene substitutions that regulate potency and
efficacy." Neuropharmacology 46(7): 1023-38.

Rajendram, R., Lewison, G. and Preedy, V. R. (2006). "Worldwide alcohol-related
research and the disease burden." Alcohol Alcohol 41(1): 99-106.

Ronald, K. M., Mirshahi, T. and Woodward, J. J. (2001). "Ethanol inhibition of N-
methyl-D-aspartate receptors is reduced by site-directed mutagenesis of a
transmembrane domain phenylalanine residue." J Biol Chem 276(48): 44729-35.

Ryan, S. G., Buckwalter, M. S., Lynch, J. W., Handford, C. A., Segura, L., Shiang, R.,
Wasmuth, J. J., Camper, S. A., Schofield, P. and O'Connell, P. (1994). "A
missense mutation in the gene encoding the alpha 1 subunit of the inhibitory
glycine receptor in the spasmodic mouse." Nat Genet 7(2): 131-5.

Saul, B., Schmieden, V., Kling, C., Mulhardt, C., Gass, P., Kuhse, J. and Becker, C. M.
(1994). "Point mutation of glycine receptor alpha 1 subunit in the spasmodic
mouse affects agonist responses." FEBS Lett 350(1): 71-6.

Sebe, J. Y., Eggers, E. D. and Berger, A. J. (2003). "Differential effects of ethanol on
GABA(A) and glycine receptor-mediated synaptic currents in brain stem
motoneurons." J Neurophysiol 90(2): 870-5.

Sommer, B. and Seeburg, P. H. (1992). "Glutamate receptor channels: novel properties
and new clones." Trends Pharmacol Sci 13(7): 291-6.

46
Steensland, P., Simms, J. A., Holgate, J., Richards, J. K. and Bartlett, S. E. (2007).
"Varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist,
selectively decreases ethanol consumption and seeking." Proc Natl Acad Sci U S
A 104(30): 12518-23.

Sutherland, E., Dixon, B. S., Leffert, H. L., Skally, H., Zaccaro, L. and Simon, F. R.
(1988). "Biochemical localization of hepatic surface-membrane Na+,K+-ATPase
activity depends on membrane lipid fluidity." Proc Natl Acad Sci U S A 85(22):
8673-7.

Suzdak, P. D., Glowa, J. R., Crawley, J. N., Schwartz, R. D., Skolnick, P. and Paul, S. M.
(1986). "A selective imidazobenzodiazepine antagonist of ethanol in the rat."
Science 234(4781): 1243-7.

Suzdak, P. D., Schwartz, R. D., Skolnick, P. and Paul, S. M. (1986). "Ethanol stimulates
gamma-aminobutyric acid receptor-mediated chloride transport in rat brain
synaptoneurosomes." Proc Natl Acad Sci U S A 83(11): 4071-5.

Tang, P., Mandal, P. K. and Xu, Y. (2002). "NMR structures of the second
transmembrane domain of the human glycine receptor alpha(1) subunit: model of
pore architecture and channel gating." Biophys J 83(1): 252-62.

Tao, L. and Ye, J. H. (2002). "Protein kinase C modulation of ethanol inhibition of
glycine-activated current in dissociated neurons of rat ventral tegmental area." J
Pharmacol Exp Ther 300(3): 967-75.

Tapia, J. C., Aguilar, L. F., Sotomayor, C. P. and Aguayo, L. G. (1998). "Ethanol affects
the function of a neurotransmitter receptor protein without altering the membrane
lipid phase." Eur J Pharmacol 354(2-3): 239-44.

Ticku, M. K., Lowrimore, P. and Lehoullier, P. (1986). "Ethanol enhances GABA-
induced 36Cl-influx in primary spinal cord cultured neurons." Brain Res Bull
17(1): 123-6.

Ueno, S., Lin, A., Nikolaeva, N., Trudell, J. R., Mihic, S. J., Harris, R. A. and Harrison,
N. L. (2000). "Tryptophan scanning mutagenesis in TM2 of the GABA(A)
receptor alpha subunit: effects on channel gating and regulation by ethanol." Br J
Pharmacol 131(2): 296-302.

Unwin, N. (2005). "Refined structure of the nicotinic acetylcholine receptor at 4A
resolution." J Mol Biol 346(4): 967-89.

47
van Zundert, B., Albarran, F. A. and Aguayo, L. G. (2000). "Effects of chronic ethanol
treatment on gamma-aminobutyric acid(A) and glycine receptors in mouse
glycinergic spinal neurons." J Pharmacol Exp Ther 295(1): 423-9.

Volpicelli, J. R. (2001). "Alcohol abuse and alcoholism: an overview." J Clin Psychiatry
62 Suppl 20: 4-10.

Wick, M. J., Mihic, S. J., Ueno, S., Mascia, M. P., Trudell, J. R., Brozowski, S. J., Ye, Q.,
Harrison, N. L. and Harris, R. A. (1998). "Mutations of gamma-aminobutyric acid
and glycine receptors change alcohol cutoff: evidence for an alcohol receptor?"
Proc Natl Acad Sci U S A 95(11): 6504-9.

Williams, K. L., Ferko, A. P., Barbieri, E. J. and DiGregorio, G. J. (1995). "Glycine
enhances the central depressant properties of ethanol in mice." Pharmacol
Biochem Behav 50(2): 199-205.

Xiu, X., Hanek, A. P., Wang, J., Lester, H. A. and Dougherty, D. A. (2005). "A unified
view of the role of electrostatic interactions in modulating the gating of Cys loop
receptors." J Biol Chem 280(50): 41655-66.

Yamakura, T., Mihic, S. J. and Harris, R. A. (1999). "Amino acid volume and hydropathy
of a transmembrane site determine glycine and anesthetic sensitivity of glycine
receptors." J Biol Chem 274(33): 23006-12.

Ye, J. H., Tao, L., Ren, J., Schaefer, R., Krnjevic, K., Liu, P. L., Schiller, D. A. and
McArdle, J. J. (2001b). "Ethanol potentiation of glycine-induced responses in
dissociated neurons of rat ventral tegmental area." J Pharmacol Exp Ther 296(1):
77-83.

Ye, J. H., Tao, L., Zhu, L., Krnjevic, K. and McArdle, J. J. (2001a). "Ethanol inhibition
of glycine-activated responses in neurons of ventral tegmental area of neonatal
rats." J Neurophysiol 86(5): 2426-34.

Ye, J. H., Tao, L., Zhu, L., Krnjevic, K. and McArdle, J. J. (2002). "Decay of ethanol-
induced suppression of glycine-activated current of ventral tegmental area
neurons." Neuropharmacology 43(4): 788-98.

Ye, Q., Koltchine, V. V., Mihic, S. J., Mascia, M. P., Wick, M. J., Finn, S. E., Harrison,
N. L. and Harris, R. A. (1998). "Enhancement of glycine receptor function by
ethanol is inversely correlated with molecular volume at position alpha267." J
Biol Chem 273(6): 3314-9.

48
Zhou, Q. and Lovinger, D. M. (1996). "Pharmacologic characteristics of potentiation of
5-HT3 receptors by alcohols and diethyl ether in NCB-20 neuroblastoma cells." J
Pharmacol Exp Ther 278(2): 732-40.

Zimmerman, J. M., Eliezer, N. and Simha, R. (1968). "The characterization of amino acid
sequences in proteins by statistical methods." J Theor Biol 21(2): 170-201.

Ziskind-Conhaim, L., Gao, B. X. and Hinckley, C. (2003). "Ethanol dual modulatory
actions on spontaneous postsynaptic currents in spinal motoneurons." J
Neurophysiol 89(2): 806-13.

Abstract (if available)

Abstract Glycine receptors (GlyRs) have received considerable attention as putative sites of action causing the behavioral effects of ethanol. Studies over the last decade have identified several positions in the transmembrane domain of GlyRs that are critical for ethanol modulation.

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Creator Shen, Yi (author)

Core Title Position 52 in alpha1 glycine receptors is important for the action of ethanol

School School of Pharmacy

Degree Master of Science

Degree Program Molecular Pharmacology

Publication Date 04/19/2008

Defense Date 04/01/2008

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

Tag ethanol,glycine receptor,OAI-PMH Harvest

Language English

Advisor Alkana, Ronald L. (committee chair), Cadenas, Enrique (committee member), Davies, Daryl L. (committee member)

Creator Email yishen@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1163

Unique identifier UC1255491

Identifier etd-Shen-20080419 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-59523 (legacy record id),usctheses-m1163 (legacy record id)

Legacy Identifier etd-Shen-20080419.pdf

Dmrecord 59523

Document Type Thesis

Rights Shen, Yi

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