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Developing ultrasensitive ethanol receptors (USERS) as novel tools for alcohol research: optimizing Loop 2 mutations in α1 GlyRs

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Developing ultrasensitive ethanol receptors (USERS) as novel tools for alcohol research: optimizing Loop 2 mutations in α1 GlyRs

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i

Developing Ultrasensitive Ethanol Receptors (USERS) As Novel Tools for
Alcohol Research: Optimizing Loop 2 Mutations in α1 GlyRs

by

Karan Muchhala

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree,
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)

December 2013

ii

Dedication

To my family for their unwavering support through thick and thin.

To my friends-old and new, for being a constant source of encouragement and relentless
support.

Deepest gratitude

To Drs. Ronald Alkana, Daryl Davies, Kathleen Rodgers and Liana Asatryan for taking
time out of their busy schedules to guide me and assist me to transform my thesis into
what it is today.

To my lab members for making my two years in the lab a wonderful experience.

iii

Acknowledgements

Parts of the research presented in this thesis draws from earlier work published by Daya
Perkins and Daniel Crawford from the Alkana and Davies laboratory. Figures 1.1 and 1.2
are modified from Crawford et al., 2007 and Perkins et al., 2009 respectively.

Material presented in Chapters 2, 3 and 4, Figures 2.1 and 2.2, and Tables 2.1, 2.2, and
2.3 of this thesis will also be incorporated into “Developing Ultrasensitive Ethanol
Receptors (USERs) As Novel Tools for Alcohol Research” by A. Naito, K. Muchhala,
L. Asatryan, J.R. Trudell, G.E. Homanics, D.I. Perkins, K.E. Rodgers, D.L. Davies and
R.L. Alkana. The manuscript is still in preparation.

Data presented in this thesis has been/will be presented at the following scientific
meetings:
• SfN’s (Society for Neuroscience) 43
rd
annual meeting, San Diego, CA,
November 9-13, 2013: “Developing Ultrasensitive Ethanol Receptors
(USERS) As Novel Tools for Alcohol Research. Neuroscience 2013” by A.
Naito, K. Muchhala, L. Asatryan, J.R. Trudell, G.E.Homanics, D.L. Davies,
R.L. Alkana, D.I. Perkins.

• 12
th
Annual Symposium, Moving Targets 2013 “Neurodegenerative Disease:
Therapeutic Challenges and Opportunities” by AAPS-USC student chapter,
Los Angeles, CA, August 23, 2013: “Mutations in GABA
A
Receptor Loop 2
Create Ultrasensitive Ethanol Receptors (USERS) sensitive to micromolar
ethanol concentrations” by K. Muchhala, A. Naito, L. Asatryan, J.R. Trudell,
G.E.Homanics, D.I. Perkins, D.L. Davies, R.L. Alkana.

• RSA 2013, 36th Annual RSA (Research Society for Alcoholism) Scientific
Meeting, Orlando (Grand Cypress), Fl, June 22-26, 2013: “Mutations in
GABA
A
Receptor Loop 2 Create Ultrasensitive Ethanol Receptors (USERS)
sensitive to micromolar ethanol concentrations” by A. Naito, K. Muchhala,

iv

L. Asatryan, J.R. Trudell, G.E.Homanics, D.L. Davies, R.L. Alkana, D.I.
Perkins.

• USC 5
th
Annual Graduate Research Symposium, USC, Los Angeles, CA,
April 2, 2013: “Flare in the Night Receptors: Development of Ultra-Sensitive
Ethanol Receptors (USERs) in Glycine Receptors to Elucidate the Molecular
Mechanism of Alcohol Addiction” by A. Naito, K. Muchhala,
G.E.Homanics, J.R. Trudell, D.L. Davies, D.I. Perkins and R.L. Alkana.

• Undergraduate Research Symposium, USC, Los Angeles, CA, April 2013:
“Developing Ultra-sensitive Ethanol Receptors to Define the Molecular
Mechanism of Ethanol Action” by P. Krishnamani, A. Naito, K. Muchhala,
D.L. Davies and R.L. Alkana.

• “Translational Science Day” by Southern California Clinical and Translational
Science Institute, USC, Los Angeles, CA, November 2012: “Loop 2 sequence
profoundly affects the sensitivity of Alpha 1 Glycine receptors (GlyRs) to
Ethanol” by A. Naito, K. Muchhala, G.E.Homanics, J.R. Trudell, D.I.
Perkins, D.L. Davies, R.L. Alkana.

• Abstract for Poster Presentation. Neuroscience 2012, SfN's (Society for
Neuroscience) 42nd annual meeting, New Orleans, LA, October 2012: “Loop
2 sequence profoundly affects the sensitivity of Alpha 1 Glycine receptors
(GlyRs) to Ethanol” by R.L. Alkana, A. Naito, K. Muchhala, L. Asatryan, K.
Li, G.E. Homanics, J.R. Trudell, D.L. Davies, D.I. Perkins.

v

Table of Contents

Dedication ii
Acknowledgements iii
List of Tables vii
List of Figures viii
Abbreviations ix
Abstract x
Chapter 1 Introduction 1
1.1) Alcohol: A leading global risk factor for disease, death 1
and economic burden
1.2) Mechanisms and sites of alcohol and anesthetic action 4
A) Membrane theories of alcohol and general anesthetic action 4
B) Protein theories of alcohol and general anesthetic action 6
1.3) Protein targets of ethanol action in the CNS 9
A) Ionotropic Receptors 9
B) Metabotropic Receptors 11
1.4) Glycine and GABA
A
Receptors: Key targets in ethanol’s CNS-mediated 13
behavioral effects
A) Glycine Receptors 13
B) GABA
A
Receptors 15
1.5) Sites of ethanol action on GlyRs and GABA
A
Rs 18
A) Intracellular Domain 18
B) Transmembrane Domain 19
C) Extracellular Domain 20
1.6) Ultrasensitive ethanol receptors (USERs) for α1 GlyRs and GABA
A
Rs 26
1.7) Thesis proposal: Optimization of USERs to develop as novel tools 28
1.8) Specific Aims 30

vi

Chapter 2 Optimization Phase 1 – Maximize ethanol sensitivity in 31
α1 (δL2) GlyRs
1. Rationale 31
2. Materials and Methods 33
3. Results 38
Agonist Concentration Response 38
Ethanol Concentration Response 41
4. Conclusion 44

Chapter 3 Optimization Phase 2 – Optimize practicality of USERs for 45
development of transgenic animals
1. Rationale 45
2. Results 46
Agonist Concentration Response 46
Ethanol Concentration Response 47
3. Conclusion 48

Chapter 4 Overall Discussion 49

Bibliography 52

vii

List of Tables
Table 2.1 Loop 2 sequence alignments for human δ GABA
A
R, 34
α1 GlyR WT and δL2 mutant subunits
Table 2.2 Summary of results of non-linear regression analysis for 39
glycine concentration responses in WT and mutant α1 GlyRs.
Table 2.3 Threshold for ethanol sensitivity in GlyR δL2 USERs is 43
bimodal and lower than WT.

viii

List of Figures
Figure 1.1 Molecular model of an α1 GlyR subunit depicting its alcohol 25
action pocket across EC and TM domains
Figure 1.2 Molecular models of WT (A.) and δL2 (B.) α1 GlyRs depicting 27
structural differences in their respective Loop 2 regions
Figure 2.1 Concentration-response curves for glycine (1-3,000 µM) 40
activated chloride currents in Xenopus oocytes expressing
WT and all δL2 mutant α1 GlyR subunits.
Figure 2.2 USER GlyRs have increased sensitivity and bimodal response 42
to ethanol.

ix

Abbreviations
AUD, Alcohol Use Disorder
USERs, Ultrasensitive ethanol receptors
KO, knock-out
KI, knock-in
L2, Loop 2
LGIC, ligand-gated ion channel
GABA
A
R, γ-aminobutyric acid type-A receptor
GlyR, glycine receptor
nAChR nicotinic acetylcholine receptor
NMDA, N-methyl D-aspartate
5HT
3
R, 5-hydroxytryptamine
3
receptor
AMPA, α -amino-3-hydroxyisoxazolepropionic acid
GIRK, G protein-activated inwardly rectifying potassium channel
TM, transmembrane
EC, extracellular
IC, intracellular
PMTS, propyl methanethiosulfonate

x

Abstract

A critical barrier to developing effective drugs to prevent and/or treat Alcohol Use
Disorders (AUDs) has been a lack of specific knowledge about where ethanol acts in the
brain and the resultant cascades leading to behavioral change. This search is complicated
by the multiple receptors and subunit combinations affected by ethanol. Current
strategies that knock-out (KO) specific receptor subunits or knock-in (KI) mutant ethanol
insensitive receptors have provided important insights. However, interpretation of these
studies requires testing with high ethanol concentrations (10 to 40 mM) that affect other
native receptor systems making it difficult to identify the changes in ethanol-induced
behaviors produced by its action on the missing or ethanol-insensitive receptor subtype.
To circumvent these problems, our laboratory is developing novel tools called
ultrasensitive ethanol receptors (USERs) by building on prior findings in our laboratory
that manipulating Loop 2 (L2) structure of glycine receptors (GlyRs) and γ-amino butyric
acid subtype-A (GABA
A
Rs) can significantly increase ethanol sensitivity of mutant
receptors. These USERs would respond to extremely low ethanol concentrations (≤ 1
mM), concentrations too low to affect native receptors. This differential in ethanol
sensitivity between USERs and native receptors provides the basis for exploiting these
mutant receptors to identify ethanol’s neurochemical signaling cascades from its initial
site(s) of action to behavioral outcomes and dissect the role specific receptor subunit
combinations play in each of ethanol’s behavioral effects. The primary objective of my
thesis was to identify and optimize Loop 2 mutations in GlyRs that would maximize
ethanol sensitivity and enhance the potential utility of these receptors when expressed in
vivo to create optimized USER GlyRs. In order to accomplish this, we adopted a two-
pronged approach focused on strategically manipulating specific Loop 2 amino acid
residues of the δL2 USER GlyR developed previously. This two-pronged approach is [1]
to test the hypothesis that reversion of position 52 in δL2 α1 GlyRs from Ser back to Ala
(S52A) will increase the sensitivity of the receptor to ethanol compared to α1 (δL2)
GlyRs [2] to test the hypothesis that limiting δL2 α1 GlyR mutations to Exon 3 can
create USERs. In order to test our hypotheses, we used site-directed mutagenesis to
create USERs-δL2 (S52A) GlyRs and δL2 (E57D, T59R) GlyRs, and tested their

xi

sensitivities to ethanol and to agonist using two-electrode voltage clamp
electrophysiology in a Xenopus oocyte expression system. As a result, we developed
USER GlyRs with [1] ethanol sensitivities ≤ 0.1 mM [2] enhanced ethanol induced
potentiation at extremely low ethanol concentrations versus wild-type receptors [3]
mutations limited to a single Exon [4] normal receptor function. These findings show that
USER GlyRs can be developed as novel tools to trace ethanol’s signaling cascades,
investigate the role that specific receptor subunit combinations play in causing behavioral
effects of ethanol and identify potential targets for medications development not only for
the treatment of AUDs but a myriad of other neurologic disorders. The mutations also
reveal important structure-function relationships of α1 GlyRs. This approach may extend
to other ethanol-sensitive ligand-gated ion channels (LGICs).

1

Chapter 1
Introduction

1.1) Alcohol: A leading global risk factor for disease, death and economic burden

Alcohol and alcoholic beverages have been an integral part of human culture since
ancient times (Hanson, 1995; Mody et al., 2007). Excavated beer jugs have been linked to
the Late Stone Age (~10,000 B.C.), while wine has been depicted in ancient Egyptian
pictographs dated as early as 4000 B.C. It has even been suggested that beer consumption
may have preceded that of bread. Moderate, life-long consumption of alcoholic beverages
is a frequently encountered practice considered safe and acceptable in many social
circles. In fact, several studies underscore health benefits associated with moderate
alcohol consumption (Pinder and Sandler, 2004). However, there is a fine line (often
vague) between moderate, safe alcohol consumption and alcohol misuse and addiction
(Pinder and Sandler, 2004). As a result of the unpredictable risk of progressing to a state
of alcohol dependence and abuse, almost all communities today face the harmful effects
of excessive alcohol consumption and resultant negative effects on global health and
well-being (Pinder and Sandler, 2004; Rehm et al., 2009).

Alcohol consumption is one of the top causal factors for injury, disease, disability and
mortality in the world (Rehm, 2011). Alcohol Use Disorders (AUDs) is defined in the 5
th

edition of the American Psychiatric Association’s Diagnostic and Statistical Manual of
Mental Disorders (DSM-5), as a combination of the disorders of “alcohol abuse” and
“alcohol dependence” (American Psychiatric Association., 2013). In the United States
alone, AUDs affect over 18 million people (Grant et al., 2004; Li et al., 2004). Forty % of
all hospitalized patients are diagnosed with AUDs, and 25% of the population below the
age of 18 is exposed to familial alcohol abuse or dependence (de Wit et al., 2010; Grant
et al., 2004; Li et al., 2004). The total cost attributed to excessive alcohol consumption
has jumped from $184.6 billion in 1998 to $223.5 billion in 2006 (Bouchery et al., 2011;
Grant et al., 2004). The U.S. reportedly spends almost 3% of its GDP on alcohol-related
expenses (Rehm et al., 2009). Alcohol-related causes have accounted for about 80,000

2

deaths and 2.3 million years of life lost each year in the country (Bouchery et al., 2011).
In the United States, AUDs are ranked second only to unipolar depressive disorders in
causing “disease burden” (Li et al., 2004). Moreover, in the U.S., alcoholic beverages are
more affordable today than they have ever been in the last 60 years owing to declining
prices, and reduced federal and state tax rates (Kerr et al., 2013).

At present, 3 FDA approved oral formulations and 1 injectable preparation are available
for treating alcohol dependence. These include: disulfiram (Antabuse or Antabus), oral
naltrexone (Revia or Depade) acamprosate (Campral) and injectable naltrexone (Vivitrol
or Naltrel) (Johnson et al., 2007). These therapies attempt to target alcohol craving and
dependence by either blocking alcohol’s downstream cascade or by inhibiting its
metabolism (Colombo et al., 2007; Gewiss et al., 1991; Johnson et al., 2007; Steensland
et al., 2007). Disulfiram is an acetyl dehydrogenase inhibitor that prevents oxidation of
acetaldehyde to acetic acid, a key step in alcohol metabolism (Heilig and Egli, 2006).
This drug, in presence of alcohol causes numerous undesired events such as headaches,
flushing, nausea and vomiting that is assumed to discourage patients from consuming
more alcohol (Franck and Jayaram-Lindstrom, 2013). Naltrexone, an antagonist of the
mu opioid receptor, is thought to target alcohol craving by blocking alcohol’s rewarding
effects (Bouza et al., 2004). Acamprosate is a drug that is suggested to reduce alcohol’s
withdrawal symptoms by antagonizing the excitatory glutamate receptors (Johnson et al.,
2007). Despite the addition of these relatively new drugs, problems remain over their
sub-optimal efficacy, patient non-compliance and adverse events. In addition to the
aforementioned caveats, all of these compounds have been largely unsuccessful in
treating alcohol-related disorders, with 70% of patients relapsing within a year of
treatment (Johnson, 2008). Therefore, several other compounds such as ivermectin,
dihydromyricetin, allopregnanolone, cabergoline, ondansetron, baclofen, varenicline,
nalmefene, topiramate, and sodium oxybate are being investigated to treat alcohol abuse
and dependence (Addolorato et al., 2013; Davies et al., 2013).

The magnitude of the problem led the National Institute on Alcoholism and Alcohol
Abuse to make the development of new medications to treat AUDs a top priority. Part of

3

the difficulty in developing more effective drugs reflects, at least in part, our poor
understanding of the mechanism of ethanol action causing its misuse. Our current
understanding and gaps are described in the next section.

4

1.2) Mechanisms and sites of alcohol and anesthetic action

A) Membrane theories of alcohol and general anesthetic action

The turn of the 19
th
century saw an emergence of multiple theories about functional
mechanisms and sites of action of anesthetics and alcohols. The Meyer-Overton
correlation was the most celebrated and important. In independent experiments, Charles
Ernest Overton, a botanist, and Hans Horst Meyer, a pharmacologist, observed a strong
correlation between anesthetic potency and its olive oil-water partition coefficient
(Heimburg and Jackson, 2007; Kopp Lugli et al., 2009; Meyer, 1899; Overton, 1899;
Overton, 1901). The Meyer-Overton rule was the first to identify the lipid bilayer as a site
of anesthetic action and highlight the importance of lipid solubility in the mechanism of
anesthesia (Kopp Lugli et al., 2009). K.H. Meyer further refined this unitary theory by
proposing that, “anesthesia occurs when a chemical compound attains a certain
concentration within the lipid bilayer of the cellular membrane. This concentration,
independent of the characteristics of an anesthetic, depends on the animal or cell” (Roth,
1979).

Subsequent refinements of the Meyer-Overton hypothesis centered on physical
mechanisms of anesthetic action emerged over time including: the critical volume
hypothesis, mean excess volume hypothesis, increased membrane fluidity and increased
lateral surface pressure (Cantor, 1997; Galla and Trudell, 1980; Halsey, 1982; Kopp
Lugli et al., 2009; Mori et al., 1984; Perkins et al., 2010; Ueda et al., 1986).

Mullins proposed the ‘critical volume hypothesis’ in 1954. In his hypothesis, Mullins
extended the Meyer-Overton rule by factoring in the “volume” of an anesthetic in the
bilayer (Mullins and Gaffey, 1954). Mullins’s hypothesis was used as a basis to formulate
the ‘membrane expansion theory’. It was suggested that anesthetics get adsorbed into the
membrane and causes it to expand, resulting in disruption of protein function and
anesthesia (Halsey, 1982; Lever et al., 1971; Miller et al., 1973). The critical volume

5

hypothesis also suggested that anesthesia could be reversed by decreasing temperature
and/or increasing atmospheric pressure (Mullins and Gaffey, 1954).

Several studies in isolated systems and animals tested the critical volume hypothesis by
exposing them to augmented hydrostatic and barometric pressures (>100 atmospheres
absolute--ATA). The findings supported Mullins’s critical volume hypothesis.
Specifically, exposure to high pressures antagonizes general anesthetics (‘pressure
reversal’ of anesthesia) (Chin et al., 1976; Galla and Trudell, 1980; Halsey and Wardley-
Smith, 1975; Halsey et al., 1978; Johnson et al., 1942; Johnson and Flagler, 1950; Lever
et al., 1971; Miller and Wilson, 1978; Trudell et al., 1973). However, such high pressures
altered baseline receptor function and caused changes in animal behavior (Brauer et al.,
1979; Halsey et al., 1970; Hunter and Bennett, 1974; Miller, 1977; Wann and
MacDonald, 1988). The latter included an increase in general CNS excitation (HPNS),
which complicated interpretation of the mechanism of the antagonism and, therefore
precluded the use of high pressure as an antagonist to help identify the mechanisms of
ethanol and anesthetic action.

Subsequent studies by our laboratory demonstrated that smaller increases in atmospheric
pressures in the range of 4-12 ATA were sufficient to reverse the acute and chronic
behavioral effects of ethanol (Alkana et al., 1992; Alkana and Malcolm, 1981; Alkana
and Malcolm, 1982a; Alkana and Malcolm, 1982b; Bejanian et al., 1993; Davies and
Alkana, 2001a; Davies et al., 1999; Davies et al., 1994; Syapin et al., 1988). These
studies found that 4-12 ATA pressures directly antagonized the behavioral and
biochemical actions of ethanol without causing the changes in baseline behavior or CNS
excitation that called into question the mechanism of ethanol antagonism through high
pressure reversal (Franks and Lieb, 1994; Little, 1996; Wann and MacDonald, 1988).
Furthermore, the mechanism of hyperbaric ethanol antagonism cannot be explained by
changes in helium partial pressure or pressure-induced changes in body temperature,
ethanol metabolism, rate of blood ethanol decline and brain ethanol distribution
(Malcolm and Alkana, 1982). These studies also demonstrated that pressure was selective
for allosteric modulators such as benzodiazepines, n-alcohols, but did not antagonize

6

direct acting drugs like morphine sulfate (Alkana et al., 1995; Davies and Alkana, 2003;
Davies et al., 1996). The authors concluded that this body of evidence indicates that low-
level hyperbaric exposure is a selective and direct ethanol antagonist that can be used in
place of the classical pharmacological antagonist to identify ethanol’s molecular sites and
mechanisms of action.

B) Protein theories of alcohol and general anesthetic action

In the last two decades, growing evidence indicates that the lipid bilayer may not be the
sole target of general anesthetic and alcohol action (Kopp Lugli et al., 2009). Membrane
perturbation was considered an important mechanism of alcohol and anesthetic action.
However, the lipid bilayer was disrupted only in the presence of extremely high
concentrations of anesthetics and alcohols. Moreover, these high concentrations were
well above the lethal concentration 50 (LC
50
) in animals (Goldstein, 1984). Furthermore,
slight elevations in temperature in vivo (e.g., 101
0
F), which mimicked intoxicating effects
of alcohol on membrane order, did not induce behavioral effects of alcohol intoxication
(Franks and Lieb, 1982; Pang et al., 1980). These findings raised questions about the
nature and target site of alcohol and anesthetic action.

Franks and Lieb, in a landmark experiment, demonstrated that, under lipid-free
conditions, firefly luciferase exposed to clinical concentrations of anesthetics lost its
bioluminescent function (Franks and Lieb, 1984). Evidence from this classical
experiment suggested that proteins are direct targets of general anesthetics.

Further evidence for a protein site of ethanol action came when researchers observed that
anesthetic potency of n-chain alkanes and alcohols progressively increased with
increasing molecular volume and that, beyond a certain point, higher members of these
two series lose their anesthetic potency. This indicated that up to a certain chain length
the Meyer-Overton correlation holds, but at longer chain lengths the anesthetics lose
potency. This phenomenon was described as ‘cut-off’ (Franks and Lieb, 1984; Raines and
Miller, 1994). Moreover, each aliphatic hydrocarbon series had a unique ‘cut-off’ point

7

(Franks and Lieb, 1984). Existing lipid-based theories could not explain the sudden loss
of anesthetic potency beyond the ‘cut-off’ point (Franks and Lieb, 1984; Pringle et al.,
1981). These findings, therefore, suggested that alcohol and other general anesthetics
might not exert their anesthetic effects via membrane perturbation. Moreover, there may
be other sites of action. Furthermore, results from Franks and Lieb’s experiments to
further examine the ‘cut-off’ phenomenon using firefly luciferase strongly suggested the
primary site of alcohol and anesthetic action may be a cavity or binding pocket of finite
size and amphiphilic nature (Franks and Lieb, 1985). Taken together, these data strongly
supported claims that alcohols and other general anesthetics interacted with molecular
target sites of circumscribed size and specific chemistry, such as proteins, to produce
anesthesia.

Biochemical and behavioral studies using the anesthetic isoflurane displayed
stereoselectivity. The (S+) enantiomer was far more potent than the (R-) enantiomer
(Franks and Lieb, 1991; Jones and Harrison, 1993; Lysko et al., 1994). This correlation
between stereoselectivity and anesthetic potency suggested that anesthetic compounds
interacted with optically active sites on a protein (Kopp Lugli et al., 2009).

Eger et al. synthesized a library of chemical compounds with physicochemical properties
similar to general anesthetics. When tested in vivo, these compounds produced an
anesthetic-like effect. However, when tested at concentrations conforming to the Meyer-
Overton rule, these compounds had limited or no efficacy (Fang et al., 1996; Koblin et
al., 1994; Kopp Lugli et al., 2009). These ‘non-anesthetics’ or ‘non-immobilizers’
induced amnesia-like effects in animals by depressing learning and memory, but caused
no effect on movement in response to noxious stimuli. Despite their ability to easily
solubilize in lipids, penetrate the blood brain barrier, and exert a partial pressure
sufficient to induce anesthesia, these compounds in fact defied the Meyer-Overton rule.
(Kandel et al., 1996; Kopp Lugli et al., 2009; Sonner et al., 1998) This indicated that
anesthetics and non-anesthetics act at different target sites. These findings provided
further evidence that the Meyer-Overton correlation may be insufficient to explain the
mechanism of anesthesia, and that proteins are targets of ethanol and anesthetic action.

8

A multitude of targets such as gap junctions of astrocytes, head and tail groups of lipids,
protein kinase C, mitochondrial proteins, membrane proteins and annular lipids of
proteins have been identified in recent years as potential sites of general anesthetic action
(Kopp Lugli et al., 2009; Perkins et al., 2010). These discoveries, coupled with an in-
depth understanding of the inter-play between proteins and lipids in the bilayer of cell
membranes, have enabled researchers to better understand the mechanisms and sites of
action of anesthetics and alcohols. These have shifted focus from a unitary hypothesis to
a combination of physico-chemical, lipid-based theories and protein targets to describe
mechanisms of action of general anesthetics and alcohols.

9

1.3) Protein targets of ethanol action in the CNS

The central nervous system (CNS) is ethanol’s main target responsible for its behavioral
effects (Howard et al., 2011; Sauguet et al., 2013). In the CNS, ethanol primarily
modulates synaptic neurotransmission by interacting with post-synaptic ion channel
receptors (Murail et al., 2012). Additionally, ethanol can modulate release of
neurotransmitters such as GABA and glutamate from pre-synaptic nerve terminals
(Hendricson et al., 2004; Jia et al., 2008; Kelm et al., 2007) Therefore, due to its unique
physical and chemical characteristics, ethanol interacts with a myriad of targets that
initiate multiple cascades leading to a wide range of neuropharmacological and
behavioral effects.

A) Ionotropic Receptors

Ligand-gated ion channels (LGICs) have been extensively explored as putative target
sites of ethanol action both in vivo and in vitro (Asatryan et al., 2010; Asatryan et al.,
2008; Crawford et al., 2007; Davies et al., 2004; Davies et al., 2003; Deitrich et al., 1989;
Harris, 1999; Mihic et al., 1997; Perkins et al., 2012; Perkins et al., 2008; Perkins et al.,
2009; Sauguet et al., 2013). LGICs can be classified into three superfamilies: 1) Cys-loop
superfamily consisting of anion channels such as glycine, γ-amino butyric acid subtype-A
or GABA
A
, GABA
C
and

glutamate-gated anion channels,

cation channels such as
nicotinic acetylcholine (nACh) and 5-hydroxytryptamine
3
(5-HT
3
); 2) Glutamate
superfamily of cation channels such as N-methyl D-aspartate (NMDA), α-amino-3-
hydroxyisoxazolepropionic acid (AMPA) and kainate; 3) Purinergic superfamily of ATP-
gated receptors (P2XRs).

Glycine and GABA
A
receptors, belonging to the Cys-loop receptor family, have been
extensively explored as target sites of alcohol’s actions (See section 1.5). A number of
behavioral effects of ethanol such as anxiolysis, sedation, heightened aggression,
diminished behavioral inhibitions, motor incoordination, withdrawal, dependence, etc.
have been linked to these receptors and are described in detail in section 1.4 (Badanich et

10

al., 2013; Chau et al., 2010; Deitrich et al., 1989; Ericson et al., 2010; Findlay et al.,
2002; Grobin et al., 1998; Kumar et al., 2009; Li et al., 2012; Williams et al., 1995).

Neuronal nAChRs have also been linked to a number of ethanol’s acute behavioral
effects, namely, the ataxic and sedative-hypnotic effects (Kamens et al., 2010). 5-HT
3

receptors are also modulated by ethanol. Evidence indicates that these receptors may be
involved in modulating alcohol consumption and other behavioral effects linked to
alcohol consumption such as nausea, anxiety and seizure (Davies, 2011; Hodge et al.,
2004; Johnson et al., 1993; Knapp and Pohorecky, 1992).

NMDA receptors belonging to the glutamate family also have been substantially explored
as an ethanol target. Molecular modeling strategies have been used to identify putative
sites of alcohol action on these receptors (Chandrasekar, 2013). Moreover, behavioral
symptoms of ethanol such as reward, tolerance, craving, dependence, withdrawal and
relapse have been linked to NMDA receptors (Chandrasekar, 2013; Krystal et al., 2003a;
Krystal et al., 2003b; Krystal et al., 2003c).

The P2X
4
subtype of purinergic receptors has also been recently investigated as a target
site of ethanol’s actions and possible medications development (Asatryan et al., 2011;
Asatryan et al., 2010; Davies et al., 2006; Popova et al., 2010; Popova et al., 2013;
Weight et al., 1999; Yardley et al., 2012). Evidence suggests that this receptor isoform
may be linked to alcohol consumption through modulation of the mesolimbic dopamine
system (Bortolato et al., 2013; Yardley et al., 2012).

Other ion channels have also been explored as putative sites for ethanol’s molecular
actions. Ethanol regulates three potassium ion channels: G protein-activated inwardly
rectifying channels (GIRK, KIR3x), large-conductance calcium-activated channels (BK,
slo-1, KCNMA-1) and Shaw2 (Harris et al., 2008). Sites critical for alcohol’s actions
have been recently identified in the intracellular domain of Shaw2 (Bhattacharji et al.,
2006; Shahidullah et al., 2003). In vivo studies have implicated GIRK in ethanol’s
anxiolytic, sedative-hypnotic, convulsant and anti-nociceptive effects (Blednov et al.,

11

2003b; Blednov et al., 2001; Kozell et al., 2009). Transient receptor potential vanilloid
receptor type 1or TRPV1 belonging to the TRP superfamily of non-selective cation
channels has also been recently implicated in ethanol’s CNS intoxicating effects (Howard
et al., 2011).

B) Metabotropic Receptors

Growing evidence suggests that metabotropic G-protein coupled receptors (GPCRs) play
an important role in ethanol’s pharmacological effects (Kelm et al., 2011; Weiner and
Valenzuela, 2006). Inhibition of Gα
s
- and Gα
q
- coupled GPCRs such as corticotropin-
releasing factor-1 (CRF1) receptor and 5-hydroxytryptamine-2C (5-HT
2C
) respectively
blocked ethanol’s ability to increase GABA release in specific brain regions (Nie et al.,
2004; Roberto et al., 2010a; Roberto et al., 2010b; Theile et al., 2009). Conversely,
stimulation of Gα
i
- coupled GPCRs such as GABA
B
receptor, cannabinoid-1 (CB1)
receptor, nociceptin/orphanin FQ peptide receptor, and δ-Opioid receptor induced a
similar pharmacological effect (Kang-Park et al., 2007; Kelm et al., 2007; Kelm et al.,
2008; Roberto and Siggins, 2006; Silberman et al., 2009; Zhu and Lovinger, 2006). Each
of these GPCRs are linked to an intracellular second messenger-signaling pathway. Gα
s
-
and Gα
i
- coupled GPCRs are associated with the adenylyl cyclase (AC)/protein kinase A
(PKA) pathway, whereas Gα
q
- coupled GPCRs are linked to the phospholipase C (PLC)/
protein kinase C (PKC) pathway (Kelm et al., 2011). Maldve et al. observed that ethanol
regulated PKA-mediated serine/threonine phosphorylation events (Maldve et al., 2002).
Furthermore, knockout, local knockdown and conditional rescue experiments along with
in vitro studies have demonstrated that the signaling protein PKCε modulates ethanol-
mediated GABA
A
R function and is linked to alcohol’s GABA-mediated behavioral
effects such as anxiety and alcohol self-administration (Choi et al., 2002; Hodge et al.,
1999; Kumar et al., 2009; Lesscher et al., 2009; Proctor et al., 2003). Moreover, studies
by Das et al. support the presence of an “alcohol-binding” site in PKC (Das et al., 2004;
Das et al., 2009; Das et al., 2007).

As mentioned earlier, certain GPCRs when activated, trigger the adenylyl cyclase/PKA

12

pathway and thus stimulate synthesis of 3', 5'-monophosphate (Johnson et al., 1993) and
other second messengers (Harris et al., 2008). Adenylyl cyclase can be differentiated into
multiple isozymes. Of all isozymes, AC7 is most sensitive to ethanol enhancement of
cAMP production (Yoshimura and Tabakoff, 1995). Additionally, Yoshimura et al. have
identified “ethanol-responsive domains” in adenylyl cyclase (Yoshimura et al., 2006).
Some groups also demonstrated that acute and chronic ethanol exposure altered Ca
+2

signaling in neurons (Gruol et al., 1997; Mah et al., 2011). Thus ethanol acts on a number
of upstream and downstream targets in the second messenger signaling pathway.

In conclusion, ethanol acts directly and indirectly on a broad spectrum of CNS protein
targets pre-and post-synaptically, making it challenging to trace behavioral effects to
distinct ethanol actions. The subsequent section provides a brief overview of studies
implicating glycine and GABA
A
receptors as key ethanol targets.

13

1.4) Glycine and GABA
A
Receptors: Key targets in ethanol’s CNS-mediated
behavioral effects

A) Glycine Receptors

Glycine receptors (GlyRs) are one of the major inhibitory neurotransmitters in adult
mammalian CNS (Dutertre et al., 2012). They are heteropentameric proteins that bind the
neurotransmitter, glycine (Dutertre et al., 2012; Lynch, 2004; Perkins et al., 2010). These
receptors primarily mediate neurotransmitter inhibition in the spinal cord and are also
distributed across higher brain regions of the cortex, hippocampus, ventral tegmental area
(VTA), striatum, amygdala, nucleus accumbens, cerebellum, and brain stem (Dutertre et
al., 2012; McCracken et al., 2013; Perkins et al., 2010). Furthermore, these receptors are
involved in regulating sensorimotor functions of vision and audition (Dutertre et al.,
2012). Several agents including ethanol modulate the functions of GlyRs (Dutertre et al.,
2012; Perkins et al., 2010).

Since the early 1990s, numerous studies investigating ethanol’s pharmacological actions
have implicated GlyR involvement in causing and/or modulating ethanol’s behavioral
effects. Sensorimotor activities regulated by GlyRs are significantly altered during
ethanol intoxication [reviewed in (Perkins et al., 2010)]. Ethanol-induced loss of righting
reflex was enhanced in mice upon administration of glycine and d-serine, a precursor of
glycine (Williams et al., 1995). On the other hand, Li et al. demonstrated that glycine
microinjections in the VTA of rats reduced ethanol self-administration (Li et al., 2012).
In both of these aforementioned studies, strychnine, a competitive antagonist of GlyRs,
inhibted the effects of glycine, clearly demonstrating the involvement of GlyRs in ethanol
consumption and its central depressant effects (Li et al., 2012; Williams et al., 1995).
Other studies involving microdialysis of glycine or strychnine into the nucleus
accumbens also demonstrated the importance of these receptors in the brain’s reward
pathway (Ericson et al., 2010; Molander et al., 2007; Molander and Soderpalm, 2005).
Transgenic expression of S267Q mutant α1 GlyR subunits displayed decreased
sensitivity in ethanol inhibition of strychnine-induced seizure, loss of righting reflex and

14

motor incoordination (rotarod) in knock-in (KI) mice, further implicating the role of
GlyRs in ethanol’s depressant effects on motor functions and co-ordination (Findlay et
al., 2002). Two lines of α1 GlyR male KI mice- M287L and Q267I displayed a reduction
in ethanol preference in a two-bottle free choice paradigm. Preference to quinine and
sucrose were not affected. In the same study, the Q267I but not the M287L KI mice
showed significantly faster recovery times in rotarod experiments compared to WT mice
after ethanol administration (Blednov et al., 2012). Taken together, results from these
studies implicate α1 GlyRs in ethanol’s rewarding effects and depressant effects on
motor co-ordination. Reports indicated that behavioral effects observed in human
alcoholics such as deficits in behavioral inhibitions, impulsivity and perseveration (Hill et
al., 2009; Tanabe et al., 2009; Verdejo-Garcia et al., 2006), and in mice under chronic
intermittent ethanol exposure such as disruption of reversal learning (Badanich et al.,
2011) reflect symptoms of patients with orbitofrontal cortex (OFC) damage, thus
indicating that OFC neurons may be susceptible to alcohol action (Badanich et al., 2013).
A recent investigation into the actions of acute ethanol exposure on OFC neuron activity
implicated a glycine receptor-dependent mechanism in reducing OFC neuron excitability
by relatively low ethanol concentrations (Badanich et al., 2013). Taken together, these
suggest that glycine receptors in OFC neurons may be involved in modulating cognitive
impairing functions of ethanol (Badanich et al., 2011; Badanich et al., 2013).
Furthermore, acamprosate, an FDA-approved oral drug for AUDs, was shown to reduce
dopamine release and alcohol consumption via a glycine receptor-dependent mechanism
in the nucleus accumbens (Chau et al., 2010).

In addition, a multitude of in vitro studies support ethanol-induced modulation of GlyR
function (Aguayo and Pancetti, 1994; Celentano et al., 1988; Crawford et al., 2007;
Davies et al., 2004; Davies et al., 2003; Engblom and Akerman, 1991; Findlay et al.,
2002; Mascia et al., 1996a; Mascia et al., 1996b; Mihic et al., 1997; Perkins et al., 2008;
Perkins et al., 2009; Yamakura and Harris, 2000; Ye et al., 2001a; Ye et al., 2001b).
Electrophysiological and biochemical studies on Xenopus laevis oocytes expressing
human recombinant α1 and α2 GlyRs, whole-rat brain synaptoneurosomes, embryonic
spinal neurons of chick and mouse, freshly dissociated rat neurons and brain slice

15

preparations demonstrated that pharmacologically relevant concentrations of ethanol
positively modulate receptor function. This work is extensively reviewed in Perkins et al,
2010.

B) GABA
A
Receptors

GABA
A
receptors (GABA
A
Rs) are pentameric protein receptors that mediate fast
inhibitory neurotransmission and are activated by its ligand, GABA and muscimol, a
selective agonist, and inhibited by bicuculline and picrotoxin (Olsen and Sieghart, 2008;
Sigel and Steinmann, 2012). These receptors are the major inhibitory receptors in the
brain and are abundantly distributed throughout the CNS (Mody et al., 2007; Olsen and
Sieghart, 2008). In adults, the most predominant isoform of this receptor constitutes two
α1, two β2, and one γ2 subunit (Sigel and Steinmann, 2012). GABA
A
Rs play a critical
role in almost all physiological functions of the brain and are targeted by a host of
molecules including ethanol (Follesa et al., 2006; Kumar et al., 2009; Olsen and Sieghart,
2008; Perkins et al., 2009).

First examined in the late 1970s as a putative site of ethanol action, GABA
A
Rs have been
the subject of numerous behavioral, neurochemical and electrophysiological experiments
aimed at examining acute and chronic effects of alcohol (Allan and Harris, 1986; Allan
and Harris, 1987; Chandra et al., 2008; Davies and Alkana, 1998; Davies and Alkana,
2001a; Davies and Alkana, 2001b; Deitrich et al., 1989; Follesa et al., 2006; Hanchar et
al., 2006; Harris, 1990; Harris, 1999; Kumar et al., 2009; Liljequist and Engel, 1982;
Liljequist and Engel, 1984; Lobo and Harris, 2008; Martz et al., 1983; Mihic et al., 1997;
Nutt and Lister, 1988; Perkins et al., 2009; Santhakumar et al., 2007; Suzdak et al., 1986;
Wallner et al., 2003; Wallner et al., 2006). Anxiolysis, motor ataxia, impaired cognition,
and sedative-hypnotic, anticonvulsant and pro-aggressive action are behavioral symptoms
seen during consumption of ethanol and mirrored by GABA
A
R agonists such as
benzodiazepines (Deitrich et al., 1989; Follesa et al., 2006; Grobin et al., 1998; Kumar et
al., 2009; Lobo and Harris, 2008; Olsen et al., 2007). Moreover, several GABA
A
R
agonists and antagonists can modulate these ethanol-induced behavioral effects. Drugs

16

enhancing GABA
A
R function such as benzodiazepines and muscimol potentiate ethanol
responses whereas the inverse agonist, Ro15-4513 and antagonists, picrotoxin and
bicuculline diminish ethanol-induced GABA effects (Grobin et al., 1998; Kumar et al.,
2009; Lobo and Harris, 2008). Therefore, a number of ethanol’s behavioral effects have
been linked to GABAergic neurotransmission.

Recent investigations into specific GABA
A
R subtypes have attempted to elucidate the
role distinct subunits play in the behavioral outcomes of ethanol (Hanchar et al., 2006 ;
Kumar et al., 2009; Olsen et al., 2007; Perkins et al., 2009; Santhakumar et al., 2007 ;
Wallner et al., 2003; Wei et al., 2004 ). A multitude of studies using genetically modified
knock-out (KO) mice have implicated α1-GABA
A
Rs in acute effects of ethanol such as
its locomotor-stimulant (Blednov et al., 2003c; June et al., 2007; Kralic et al., 2003) and
sedative-hypnotic effects (Blednov et al., 2003a). In a two bottle-choice drinking
paradigm, α1-GABA
A
R KO mice consumed less alcohol than wild-type mice suggesting
a role for these receptors in ethanol preference (Blednov et al., 2003c; June et al., 2007).
α1-GABA
A
Rs have also been linked to ethanol reinforcement (Harvey et al., 2002) and
ethanol-related pro-aggressive behavior (de Almeida et al., 2004). α1-GABA
A
R selective
antagonist, β-carboline-3-carboxylate-t-butyl ester reduced heightened aggressive
behavior due to ethanol in mice. Furthermore, KO and KI experiments in mice suggest
that ethanol’s sedative-hypnotic effects may involve α2-GABA
A
Rs (Boehm et al., 2004;
Tauber et al., 2003) as well as β2-GABA
A
Rs (Blednov et al., 2003a). KO mice, when
exposed to ethanol, demonstrated reduced loss of righting reflex durations, thus
indicating diminished sedative-hypnotic effects of ethanol (Blednov et al., 2003a; Boehm
et al., 2004). α5- and δ-GABA
A
R KO mice showed reduced preference to ethanol
without altering their preference to saccharin, thus indicating that these receptors may be
involved in ethanol self-administration (Boehm et al., 2004; Mihalek et al., 2001;
Stephens et al., 2005). A study in rats using the α5-GABA
A
R subunit-selective inverse
agonist, RY023 was used to show that these receptors might be linked to ethanol’s
rewarding, motor-impairing and sedative effects (Cook et al., 2005). Furthermore,
studies have found that interaction of ethanol with benzodiazepines increases spatial
memory deficits in animals (Takiguchi et al., 2006) and humans (Simpson and Rush,

17

2002), thus suggesting a role for γ2-GABA
A
Rs in spatial memory impairment during
alcohol exposure.

GABA receptors have also been strongly implicated in chronic effects of ethanol. These
include CNS hyperexcitability during and following alcohol withdrawal (Grobin et al.,
1998; Kumar et al., 2009). Elevated risks for seizures, anxiety, hyperalgesia and
disruptions in sleep states are some effects of chronic ethanol exposure linked to the
GABA
A
R system (Deitrich et al., 1989; Follesa et al., 2006; Grobin et al., 1998; Kumar
et al., 2009). Several experiments using genetically modified mouse models have
demonstrated that α1-GABA
A
Rs might be linked to CNS excitability (Werner et al.,
2009) and β2- and γ2-GABA
A
Rs might be linked to ethanol withdrawal seizure severity
(Blednov et al., 2003c; Hood and Buck, 2000). Additionally, animals that were
pentylenetetrazole (PTZ)-kindled displayed behavioral characteristics similar to those
preconditioned with repeated cycles of ethanol dependency and withdrawal (Davidson et
al., 1999; Ripley et al., 2002). Furthermore, studies showed that the benzodiazepines,
Diazepam and Flumazenil reversed ethanol’s withdrawal hyperalgesia (Gatch, 1999) and
anxiogenic-like effects (Moy et al., 1997) and reduced the intensity of ethanol-induced
seizure(Buck et al., 1991), further linking GABA
A
Rs to alcohol’s behavioral effects.

In addition, biochemical assays for GABA
A
R activity conducted in synaptoneurosomes
and cultured neurons demonstrated an increase in chloride ion uptake, further reinforcing
their involvement in ethanol’s actions (Allan and Harris, 1986; Allan and Harris, 1987;
Suzdak et al., 1986; Ticku and Burch, 1980). Another set of chloride flux assays using 12
times normal atmospheric pressure of a helium-oxygen mixture (heliox) on mouse brain
microsacs linked ethanol’s behavioral effects to ethanol potentiation of GABA
A
Rs
(Davies and Alkana, 1998; Davies and Alkana, 2001a; Davies and Alkana, 2001b).

In summary, there is substantial evidence indicating a role for GlyR and GABA
A
Rs in a
broad spectrum of ethanol’s behavioral effects. Therefore, these receptors have been the
primary focus of investigations in our laboratory aimed at understanding sites and
mechanisms of ethanol action.

18

1.5) Sites of ethanol action on GlyRs and GABA
A
Rs

Our understanding of the sites and mechanisms of ethanol action on GlyRs and
GABA
A
Rs is still evolving. Mutagenesis, transgenesis, chimeric, hyperbaric and
molecular modeling-based strategies have contributed to filling the void in this field
(Kumar et al., 2009; Perkins et al., 2010). Through extensive research conducted over the
last few years, researchers, with the help of these new strategies have been able to
identify specific regions on these receptors targeted by ethanol to exert its actions.

A) Intracellular Domain

Recent studies indicate that the intracellular (IC) domain of α1 GlyRs may play an
important role in ethanol’s molecular cascade (Guzman et al., 2009; Harvey et al., 2004;
Legendre, 2001 ; Lynch, 2004 ; Morrow et al., 2004; Ron and Jurd, 2005; San Martin et
al., 2012; Yevenes et al., 2006; Yevenes et al., 2008). Studies demonstrated that G
proteins through Gβγ heterodimers modulate their function and that interplay between
them is important for ethanol-induced potentiation of α1 GlyRs (Guzman et al., 2009;
San Martin et al., 2012; Yevenes et al., 2006; Yevenes et al., 2008). Intracellular domain
of δ subunit-containing GABA
A
Rs also plays an important role in receptor internalization
following ethanol exposure (Gonzalez et al., 2012). Gonzalez et al. identified two motifs
on the intracellular region of δ subunits responsible for ethanol-induced clathrin-mediated
endocytosis (Gonzalez et al., 2012). This trafficking mechanism may play a role in
ethanol-dependent down-regulation of neurotransmission and tolerance to some effects
observed after acute and chronic ethanol exposure (Gonzalez et al., 2012). From these
observations, it is evident that intracellular signaling molecules, along with the
intracellular loop of GlyRs and GABA
A
Rs likely are involved in mediating and/or
modulating ethanol’s effects on GABA
A
R and GlyR function.

19

B) Transmembrane Domain

The transmembrane (TM) domain of GlyRs and GABA
A
Rs has been extensively
explored as a putative site of ethanol’s actions. Mihic et al. used a chimeric construct to
identify a key 45 amino acid sequence spanning TM 2 and TM 3 domains responsible for
modulating alcohol and volatile anesthetic sensitivity in GlyRs. Replacing amino acid
residues in this ethanol sensitive region with homologous residues from ρ1 GABA
A
Rs
led to the identification of S267 in TM2 and A288 in TM3 as key target sites of ethanol
and volatile anesthetic action in α1 GlyRs. Similar experiment with GABA
A
Rs
implicated positions S270 in TM2 and A291 in TM3 as putative sites of ethanol and
volatile anesthetic action (Mihic et al., 1997).

Subsequently, recombinant α1 GlyRs, created by replacing serine with other amino acid
residues at position 267 in the TM2 region of α1 GlyRs were tested for changes in
agonist sensitivity and allosteric modulation by ethanol. Through these experiments, Ye
et al. concluded that the molecular volume, but not charge or polarity played a critical
role in modulating ethanol sensitivity at position 267 of α1 GlyRs (Ye et al., 1998). ,

As mentioned earlier, the point at which n-alcohols lose their potency is termed ‘n-chain
alcohol cutoff’ (Franks and Lieb, 1985). Harris and colleagues used this phenomenon to
determine if TM domain of GlyRs is a putative ethanol target site. Substituting larger
amino acids at this position significantly reduced n-chain alcohol cutoff (Mascia et al.,
1996a), which indicates that this position in the TM domain is part of a finite alcohol
action pocket that plays a key role in causing ethanol modulation of the receptor

Subsequently, Harris et al. used cysteine substitutions in combination with propyl
methanethiosulphonate (PMTS) reagent at positions 267 and 288 in α1 GlyRs and their
homologous positions, 270 and 291 in α2β1 GABA
A
Rs. Since, PMTS forms a strong
disulfide linkage with cysteine residues, they suggested that creating this irreversible
covalent bond would make the reversible potentiation of GlyRs and GABA
A
Rs
irreversible if these are critical “alcohol-binding” sites. Therefore, they hypothesized that

20

positions 267 and/or 288 in α1 GlyRs and 270 and/or 291 in α2β1 GABA
A
Rs represent
initial alcohol action sites. Consistent with their hypothesis, cysteine mutations at
position 267 in α1 GlyRs and its homologous position, 270 in α2β1 GABA
A
Rs caused an
irreversible potentiation of Cl
-
currents when exposed to PMTS demonstrating the
importance of these TM positions as initial ethanol target sites (Mascia et al., 2000).
Subsequent studies using structural and functional analyses of TM residues added further
evidence implicating the TM region as a putative alcohol target site (Jung et al., 2005;
Jung and Harris, 2006).

Ensuing experiments by several groups identified additional target sites in the TM
domain that modulated alcohol and anesthetic activity (Jenkins et al., 2001; Lobo et al.,
2008; Lobo et al., 2004a; Lobo et al., 2004b; Lobo et al., 2006; Yamakura et al., 1999).
Taken together, these results indicate that the TM domain of GlyRs and GABA
A
Rs
contains one or more alcohol “binding/action” pockets.

C) Extracellular Domain

The extracellular (EC) domain of GlyRs and GABA
A
Rs contains numerous sites for
agonist binding, receptor activation, and allosteric modulation by agents such as
benzodiazepines and Zn
+2
(Barberis et al., 2002; Bloomenthal et al., 1994; Chakrapani et
al., 2004; Kash et al., 2004a; Kash et al., 2004b; Kucken et al., 2000; Mhatre and Ticku,
1989; Perkins et al., 2010; Sigel and Buhr, 1997; Wieland et al., 1992). Furthermore, the
imidazobenzodiazepine Ro15-4513 is a benzodiazepine inverse agonist that antagonizes
ethanol’s behavioral effects. It was suggested that Ro15-4513 occupies a non-
benzodiazepine site on the extracellular region of GABA
A
Rs to inhibit ethanol (Mehta
and Ticku, 1989; Wallner et al., 2006). Moreover, hyperbaric studies suggested that
“pressure antagonized ethanol-induced Cl- uptake by uncoupling a benzodiazepine-like
allosteric modulation” of the receptor (Davies and Alkana, 2003). Taken together, these
results provide indirect evidence implicating the extracellular region of GlyRs and
GABA
A
Rs as a potential site of alcohol action.

21

The EC domain of these receptors also consists of amino acid residues called ‘loops’ that
link extracellular β-strands. These loops are important for LGIC pentamer formation,
functional coupling of the ligand binding domain and the channel pore and receptor
activation (Brejc et al., 2001; Cederholm et al., 2010; Unwin, 2003; Unwin et al., 2002).
Loop 2 (L2) is one of ten such loops and links β1 and β2 strands in the inner β sheet
(Brejc et al., 2001; Cederholm et al., 2010). It is a 10 amino acid region described as a
“tight beta turn” by Perkins and colleagues (Perkins et al., 2009). This region has been
the subject of a number of experiments investigating EC domain as a putative alcohol site
of action.

Initial signs of the involvement of extracellular Loop 2 in ethanol’s actions came from
observations made by Mascia and colleagues using recombinant GlyRs (Mascia et al.,
1996b). They tested α1 GlyRs, α2 GlyRs and α1 (A52S) GlyRs for ethanol sensitivity
and found that α2 GlyRs were less ethanol sensitive than α1 GlyRs. Moreover, an
alanine to serine exchange at position 52 in Loop 2 of α1 (A52S) GlyRs caused a
reduction in ethanol sensitivity to levels comparable to α2 GlyR ethanol sensitivity. This
mutation on position 52 located at the start of Loop 2 (Brejc et al., 2001) occurs naturally
in α1 GlyRs as a spasmodic phenotype in mice (Ryan et al., 1994; Saul et al., 1994).
Sequence alignment also revealed that α2 GlyRs have a threonine (T59) at the
homologous position to 52 in α1 GlyRs (A52) (Mascia et al., 1996b). Thus, Mascia et al.
suggested that this single amino acid in the extracellular Loop 2 region was responsible
for altering ethanol sensitivity of GlyRs.

Subsequently, our laboratory used a novel ethanol antagonist –increased atmospheric
pressure -- to investigate if multiple sites of ethanol action were present in α1 GlyRs
(Davies et al., 2004). In their experiments, pressure antagonized the effects of ethanol at
higher concentrations (>40mM) in a pressure and ethanol concentration dependent
manner, but did not antagonize lower ethanol concentrations. Alkana and Davies
suggested that this bimodal response to pressure could be attributed to the activation
and/or presence of a pressure-antagonism insensitive site at low ethanol concentrations

22

(<10mM) and a pressure-antagonism sensitive site at higher ethanol concentrations
(>40mM).

Daryl Davies (Davies et al., 2004), in our laboratory, then used pressure antagonism to
explore position 52 in the EC domain as a site of ethanol action. Based on their prior
studies indicating that pressure antagonism of ethanol behaved like a direct mechanistic
antagonist, these authors hypothesized that if a mutation altered the sensitivity of the
receptor to both ethanol and antagonism by a mechanistic antagonist, pressure, then that
site is a site of ethanol action. To this end, they substituted serine for alanine at position
52 in α1 GlyRs (A52S). As predicted, this switch changed the sensitivity of the receptor
to ethanol and altered its sensitivity to pressure antagonism. In fact, this point mutation
eliminated the sensitivity of α1 GlyRs to pressure antagonism. This experiment provided
key evidence that position 52 is a site of ethanol action. These authors continued the
investigation of position 52 by testing α2 GlyRs, where a threonine (T59) is present at
position homologous to 52 in α1 GlyRs (A52). They found that α2 GlyRs were
insensitive to pressure at high (>40mM) and low (<10mM) ethanol concentrations
leading the authors to suggest that α2 GlyRs may have a pressure-antagonism insensitive
site but may lack the pressure-antagonism sensitive site present in α1 GlyRs. Subsequent
studies in the Alkana laboratory by Perkins et al. involved replacement of threonine at
position 59 in α2 GlyRs to alanine (α2 T59A GlyRs) (Perkins et al., 2008). This
substitution in α2 GlyR restored sensitivity to pressure antagonism and increased the α2
GlyRs’ sensitivity to ethanol to that seen in α1 GlyRs. Taken together, results from these
hyperbaric studies strongly supported the notion that position 52 in α1 GlyRs is an
important site of ethanol action.

To further investigate the role of position 52 as ethanol’s “action site”, Crawford et al.
examined the effect of cysteine mutations at positions 52 in Loop 2 of the extracellular
domain and position 267 in the TM domain of α1 GlyRs in presence of ethanol and/or
PMTS (Crawford et al., 2007). PMTS produces ethanol-like potentiation of Cl
-
currents
in GlyRs and GABA
A
Rs forming a strong propyl disulfide bond with cysteine residues at

23

the protein site (Borghese et al., 2003; Mascia et al., 2000). Based on prior studies with
PMTS and cysteine mutations of amino acids in the TM region of α1 GlyRs (Mascia et
al., 2000), Crawford et al. predicted that if position 52 were an initial site of ethanol
action, PMTS would produce an irreversible potentiation when cysteine was substituted
at position 52 (A52C) in mutant α1 GlyRs. As predicted, PMTS reagent covalently
bound to cysteine at position 52 to induce an irreversible alcohol-like potentiation
(Crawford et al., 2007). This indicated that in addition to position 267, position 52 in α1
GlyR Loop 2 was an initial site of alcohol action.

In follow-up studies, Crawford et al. introduced a cysteine substitution at positions 52
(A52C) and/or 267 (S267C) with a view to isolate the effects of ethanol on putative target
sites in the EC and TM domains. For this purpose, PMTS and ethanol were sequentially
applied. They hypothesized that PMTS would covalently bind to the site with the cysteine
mutation, thereby blocking ethanol’s access to the site/sites of interest and thus
eliminating resultant effects from ethanol acting on this site. Ethanol induced positive
modulation of glycine currents when position 52 (A52C) was blocked by PMTS bound to
the cysteine mutation, whereas ethanol negatively modulated glycine currents when
position 267 (S267C) was blocked by PMTS. These results indicated that ethanol
produced contrasting effects on the two positions: negative modulation when acting on
position 52 and positive modulation when acting on position 267 in α1 GlyRs.
Furthermore, ethanol produced a small but significant negative modulation of glycine
currents when both these aforementioned sites were blocked by PMTS, indicating that
positions 52 and 267 do not account for all of ethanol’s effects (Crawford et al., 2007).
This indicated that the effect of ethanol on GlyRs might be an aggregate of ethanol’s
action on several sites spanning the EC and TM domains. Overall, these experiments by
Crawford et al. added substantial evidence implicating position 52 in the extracellular
Loop 2 region of GlyRs as a putative site of ethanol action.

As previously mentioned, ‘n-chain alcohol cut-off’ is a term used to describe the
disappearance of potency when n-alcohol chain length exceeds a certain number of
carbon atoms or when the molecular dimensions of alcohol exceed the finite volume of a

24

putative alcohol pocket (Crawford et al., 2007; Pringle et al., 1981; Wick et al., 1998). As
described earlier, Harris et al. used this principle to test if position 267 in the TM region
of α1 GlyRs represented part of an alcohol-binding pocket. By blocking position 267
with a combination of PMTS and cysteine substitution, they were able to reduce n-chain
alcohol cutoff in mutant but not wild-type α1 GlyRs to below octanol from between
decanol and dodecanol (Mascia et al., 1996a; Mascia et al., 2000). This reduction in n-
chain alcohol cut-off indicated that position 267 in the TM region represented a putative
alcohol target site. Crawford et al. wanted to know if position 52 in EC domain of α1
GlyRs also formed part of an alcohol pocket and hence used a similar strategy (Crawford
et al., 2007). They hypothesized that introduction of a cysteine mutation at position 52
would reduce n-chain alcohol cut-off if it represented part of an alcohol pocket. The
cysteine mutation at position 52 in extracellular Loop 2 of α1 GlyRs blocked this site to
n-alcohols by covalently binding to PMTS. As a result, the volume of the alcohol pocket
available to n-alcohols decreased and alcohol cutoff reduced to between octanol and
decanol. When both sites (52 and 267) were blocked by PMTS, the cutoff reduced to
below hexanol. Therefore, these findings suggested that the alcohol pocket extends from
position 52 in the EC domain to position 267 in the TM domain of α1 GlyRs (Crawford
et al., 2007). These results in conjunction with the findings from sequential PMTS
applications on positions 52 and 267 indicate that these two positions constitute the same
ethanol action site in α1 GlyRs (See Figure 1.1).

In ensuing experiments, our laboratory investigated the structure-function relationship of
Loop 2 residues on ethanol sensitivity. Perkins et al. systematically replaced alanine at
position 52 in α1 GlyR Loop 2 with residues having different molecular weight, volume,
polarity and hydrophobicity and observed resultant changes in sensitivity to glycine and
ethanol. In their observations, they noted that the polarity of amino acid residues at
position 52 plays a key role in modulating receptor sensitivity to agonist and ethanol
(Perkins et al., 2008). Perkins et al. extended their investigation to charged residues and
observed that electrostatic charge coupled with specific geometry of amino acids govern
agonist and ethanol receptor sensitivity (Perkins et al., 2012). Collectively, these findings
suggest that both the physical and chemical properties of amino acid residues at position

25

52 in the extracellular Loop 2 can modulate ethanol sensitivity of α1 GlyRs.

In totality, these results demonstrate that amino acid residues at position 52 in the
extracellular Loop 2 region of GlyRs are putative sites of ethanol action and can affect
the sensitivity of the receptor to ethanol.

Figure 1.1

Figure 1.1. Molecular model of an α1 GlyR subunit depicting its alcohol action pocket
across EC and TM domains. The red cavity found by Binding Site Analysis module of Insight
2005L shows putative alcohol action pocket bound by Cα atoms of positive (TM S267) and
negative (EC A52) modulatory sites and separated by a distance of 28 Å (Modified from
(Crawford et al., 2007)).

26

1.6) Ultrasensitive ethanol receptors (USERs) for α1 GlyRs and GABA
A
Rs

It is well established that glycine and GABA
A
receptors significantly differ in their
sensitivities to ethanol. α1 GlyRs are typically unresponsive to ethanol concentrations
below 10 mM (Davies et al., 2003; Mascia et al., 1996b), whereas α1β2γ2 GABA
A
Rs
usually do not respond to ethanol below 50 mM (Weiner et al., 1997; White et al., 1990).
Olsen and others identified extrasynaptic native δ-containing GABA
A
Rs (α4β2/3δ and
α6β2/3δ) that were sensitive to low millimolar concentrations (1–3 mM) of ethanol. Prior
studies have demonstrated that mutating physical and chemical properties of Loop 2
amino acid residues (e.g. alanine at position 52 in α1 GlyRs and threonine at position 59
in α2 GlyRs) altered ethanol sensitivity of GlyRs (Crawford et al., 2007; Davies et al.,
2004; Perkins et al., 2008). Based on this information, Perkins et al. (Perkins et al., 2009)
hypothesized that differences in the structure of Loop 2 between γ2- and δ subunits of
GABA
A
Rs might play a role in their differences in ethanol sensitivity. Consistent with
their hypothesis, sequence alignment showed that the α1 GlyR subunit, and γ2- and δ
subunits of GABA
A
Rs had a number of non-conserved residues in homologous positions
across their extracellular Loop 2 regions. To directly test their hypothesis, Perkins et al.
replaced all non-conserved Loop 2 residues of α1 and γ2 subunits of GlyRs and
GABA
A
Rs respectively with those of the GABA
A
R-δ subunit and tested ethanol
sensitivity of resultant mutant receptors. As hypothesized, the new mutant receptors- α1
(δL2) GlyRs and α1β2γ2 (δL2) GABA
A
Rs were extremely sensitive to ethanol compared
to their respective wild-type receptors. These ultrasensitive ethanol receptors (USERS)
not only had a lower ethanol sensitivity threshold than their respective WT receptors, but
also had increased magnitudes of responses at all ethanol concentrations (1-30 mM EtOH
in GlyRs and 0.5-50 mM EtOH in GABA
A
Rs). Furthermore, when non-conserved Loop
2 residues of α1 GlyRs were replaced with homologous Loop 2 residues from γ2 subunit
of α1β2γ2 GABA
A
Rs, no changes in ethanol sensitivity and magnitude of ethanol
potentiation were observed. Therefore, these results provided key evidence indicating that
Loop 2 structure may play an important role in modulating ethanol sensitivity of GlyRs
and GABA
A
Rs.

27

Figure 1.2

Figure 1.2. Molecular models of WT (A.) and δL2 (B.) α1 GlyRs depicting structural
differences in their respective Loop 2 regions. Surrounding residues interacting with both Loop
2 regions- Lys104 (blue), Leu136 (yellow), Arg218 (pink) and Lys276 (green) are depicted as
colored stick models (Modified from (Perkins et al., 2009)).

A
B

28

1.7) Thesis proposal: Optimization of USERs to develop as novel tools

There is substantial evidence implicating glycine and GABA
A
receptors to several of
ethanol’s behavioral effects (See section 1.4). As described earlier in section 1.5, multiple
sites in the TM and EC domains of these receptors have also been identified as initial
targets of ethanol’s action. However, little is known about ethanol’s actions on specific
receptor-subunit combinations and the resultant cascades leading to alcohol’s behavioral
effects.

Ethanol has a unique mechanism of action due to its distinct physical-chemical
properties. It has low selectivity and low affinity to its targets and hence acts on an array
of molecular targets in the CNS. Its mechanism, though not fully understood, is not based
on structure-activity relationships and high affinity receptor binding. Therefore, these
qualities of ethanol mechanism preclude the use of classical pharmacological approaches
such as the use of selective agonists or antagonists to map ethanol’s receptor-behavior
cascades.

Current strategies that knock-out specific receptor subunits or knock-in mutant ethanol
insensitive receptors have provided some important insights into the link between
specific receptors and receptor subtypes and the behavioral effects of ethanol. Section 1.4
provides a brief overview of the various KI and KO studies that investigate the link
between specific glycine and GABA
A
receptor subtypes and ethanol’s behavioral effects.

An important shortcoming of using null mutant or KO strategies in this regard involves
genetic deletion of the protein, thereby eliminating all associated receptor functions. This
puts various compensatory mechanisms into motion, which may eventually influence the
interpretation of final results (Blednov et al., 2012; Crabbe et al., 2006; Crawley, 1996;
Kumar et al., 2009). Moreover, studies involving functional deletion or reduction in
receptor sensitivity to ethanol require testing with higher ethanol concentrations (10 to 40
mM) to observe behavioral phenotypes (Chandra et al., 2008; Werner et al., 2006). At
these concentrations, ethanol tends to simultaneously activate a number of receptor

29

systems across the CNS, making it difficult to trace molecular cascades and behavioral
outcomes associated with specific receptor subtypes. In addition, the development of KO
and KI animals is time-consuming, expensive and, even with modern
conditional/temporal modifications, is still challenging (Chandra et al., 2008; Werner et
al., 2006).

To circumvent or minimize these problems with traditional KI and KO studies, we are
building on earlier findings by Perkins et al. that structural manipulation of Loop 2 of
GlyRs and GABA
A
Rs can significantly increase ethanol sensitivity by developing a novel
tool--ultrasensitive ethanol receptors (USERs) that are sensitive to extremely low
concentrations of ethanol. These USERs, when expressed in animals would respond to
ethanol concentrations too low to affect other native receptor systems or processes. Thus,
unlike current KI and KO studies, we could initiate responses in the target receptor
system without causing changes in other systems by giving extremely low doses of
ethanol. This should enable us to identify specific receptor-subunit combinations
responsible for causing specific ethanol-induced behavioral effects, map molecular
cascades leading to them and thus define the neurochemical pathways of ethanol from its
initial sites of action to behavioral outcomes.

My research project focused on developing USERs for placement in transgenic animals
as tools to interrogate the cascade of events leading from the initial actions of ethanol on
specific receptors to its behavioral effects underlying reward, addiction and other
behaviors underlying AUDs.

30

1.8) Specific Aims

The main objective of my thesis was to optimize the δL2 GlyR USER mutations in order
to maximize ethanol sensitivity and normalize, as much as possible, USER receptor
function. In order to achieve this, we developed two optimization strategies centered on
strategically manipulating the structure of the δL2 USER GlyRs developed previously
and testing their sensitivities to ethanol and agonist using two-electrode voltage clamp
electrophysiology in Xenopus oocytes. The specific aims of these two strategies were:

[1] To test the hypothesis that reverting Ser52 to Ala52 in α1 (δL2) GlyRs will further
increase ethanol sensitivity of the receptor compared to α1 (δL2) GlyRs.

[2] To test the hypothesis that USERs can be created by limiting δL2 mutations to a
single Exon for improving the practicality of these mutant receptors for transgenic animal
development.

These are addressed in the following Chapters.

31

Chapter 2
Optimization Phase 1 – Maximize ethanol sensitivity in α1 (δL2) GlyRs

Aim 1 – To test the hypothesis that reversion of position 52 in δL2 α1 GlyRs from
Ser back to Ala (S52A) will increase the sensitivity of the receptor to ethanol
compared to α1 (δL2) GlyRs

The overall goal of Phase 1 experiments was to optimize USER GlyRs by maximizing
ethanol sensitivity in α1 (δL2) GlyRs through structural manipulation of Loop 2. Prior
studies in our laboratory (described in Chapter 1, section 1.6) demonstrated that structural
manipulation of Loop 2 could alter ethanol sensitivity of α1 GlyRs (Perkins et al., 2009).
One of the new mutant receptors obtained as a result of these structural manipulations-α1
(δL2) GlyRs decreased the threshold for ethanol sensitivity and increased magnitude of
ethanol potentiation compared to WT GlyRs. As mentioned above, we wanted to build on
these studies to maximize ethanol sensitivity across a wide range of ethanol
concentrations, with the objective of developing receptors that can be sensitized to
ethanol at extremely low concentrations (≤ 1 mM) and normal/higher concentrations of
ethanol (30-50 mM). Optimization of δL2 GlyRs would involve further decreasing the
threshold for ethanol sensitivity (increase ethanol sensitivity at extremely low
concentrations), and increasing the magnitude of ethanol-induced potentiation at higher
concentrations of ethanol (10-50 mM) versus WT receptor responses. In order to achieve
this, our strategy involved altering the amino acid structure of position 52 as a basis for
increasing ethanol sensitivity in δL2 GlyRs.

As described in Chapter 1, Section 1.5, a large body of evidence implicates the
importance of position 52 in modulating ethanol sensitivity of α1 GlyRs. Mascia et al.
demonstrated that an alanine to serine exchange at position 52 (found naturally in
spasmodic mice) was responsible for reducing ethanol sensitivity in α1 GlyRs (Mascia et
al., 1996b). Increased atmospheric pressure- a mechanistic antagonist of ethanol's action
was unable to antagonize ethanol modulation of α1 (A52S) GlyRs (Davies et al., 2004).

32

Conversely, a reverse switch to alanine at a position homologous to 52 in pressure
antagonism insensitive α2 GlyRs made the mutant α2 (T59A) GlyRs sensitive to
pressure antagonism of ethanol’s actions. Moreover, the relatively less ethanol sensitive
α2 GlyRs displayed increased ethanol sensitivity as a result of this mutation (Perkins et
al., 2008). Furthermore, Perkins et al. demonstrated that physical-chemical properties and
geometry of amino acid residues at position 52 play a key role in modulating ethanol
sensitivity of α1 GlyRs (Perkins et al., 2012; Perkins et al., 2008). Taken together, these
demonstrated that physical, chemical, and structural properties associated with an alanine
to serine switch at position 52 drastically reduced α1 GlyRs’ sensitivity to ethanol.

Consequently, we hypothesized that reverting serine at position 52 (S52) in δL2 of
mutant GlyRs to its WT residue alanine would increase ethanol sensitivity of the new
mutant receptor when compared with α1 (δL2) GlyR. Aim 1 experiments, tested this
hypothesis.

33

Materials and Methods

In order to test our hypothesis, we performed site-directed mutagenesis to restore WT
residue, alanine at position 52 in δL2 of α1 GlyRs to create the USER- α1 (δL2 S52A)
GlyR. We then expressed these new mutant receptors in Xenopus oocytes, exposed this
system to a wide range of agonist (0.001-3 mM) and ethanol (0.025-50 mM)
concentrations and measured agonist and ethanol sensitivities using two-electrode voltage
clamp electrophysiology. The materials, methods and results are described in detail
below.

Materials

Adult female Xenopus laevis frogs were purchased from Nasco (Fort Atkinson, WI).
Gentamycin, 3-aminobenzoic acid ethyl ester, glycine, ethanol, and collagenase were
purchased from Sigma (St. Louis, MO). All other chemicals used were of reagent grade.
Glycine stock solutions were prepared from powder and diluted with Modified Barth’s
Solution (MBS) containing (in mM) 88 NaCl, 1 KCl, 10 HEPES, 0.82 MgSO4, 2.4
NaHCO3, 0.91 CaCl2, and 0.33 Ca(NO3)2, adjusted to pH 7.5 .

Site-directed mutagenesis

For the purpose of this study, extracellular Loop 2 region is defined as positions 50-59 in
α1 GlyR subunit and positions 43-52 in δ GABA
A
R subunit. Homologous amino acid
sequences from extracellular Loop 2 regions of α1 GlyR, and δ GABA
A
R subunits were
identified and aligned (Table 2.1). As indicated in Table 2.1, site-directed mutagenesis
was performed in the α1 GlyR subunit cDNA in order to make the receptor’s Loop 2
region identical to δ GABA
A
R Loop 2 (δL2). Subsequently, amino acids at strategic
positions in α1 (δL2) GlyR were reverted back to their wild-type (α1 GlyR) residues to
create new mutant receptors. This site-directed mutagenesis was performed by
subcloning human GlyR cDNA into mammalian vector pCIS2 or pBK-CMV using the
Quick Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) and verified by

34

partial sequencing (DNA Core Facility, University of Southern California).

Table 2.1

SUBUNIT POSITION SEQUENCE
GABA
A
R δ 43 H I S E A N M E Y T
GlyR α1 (WT) 50 S I A E T T M D Y R
GlyR α1 (δL2) 50 H I S E A N M E Y T
GlyR α1
(δL2 S52A)
50 H I A E A N M E Y T
GlyR α1
(δL2 E57D,
T59R)
50 H I S E A N M D Y R

Table 2.1. Loop 2 sequence alignments for human δ GABA
A
R, α1 GlyR WT and δL2
mutant subunits. Non-conserved residues are under-lined while reversions are in red.

35

Expression in Oocytes

Stage V or VI Xenopus oocytes were isolated from adult Xenopus laevis frogs. Davies et
al. have described the isolation and subsequent defolliculation process in detail (Davies et
al., 2004). Once isolated, oocytes were enzymatically treated for 40-60 minutes with two
changes of 0.2-0.3 %w/v collagenase type 1A in 1x OR2 solution containing (in mM) 82
NaCl, 2 KCl, 2 MgCl2, and 5 HEPES (pH 7.5) in order to digest the encircling follicular
layer. Subsequently, collagenase was discarded and oocytes were rinsed six times with 1x
OR2 buffer to arrest digestion and wash off surplus collagenase. Finally, these oocytes
were stored overnight at 18
o
C in incubation medium [88 mM NaCl, 1.8 mM KCl, 5 mM
HEPES, 0.82 mM MgSO4, 2.4 mM NaHCO3, 0.41 mM CaCl2, 0.33 mM Ca(NO3)2, 140
mg pyruvic acid, 500 µl 50 mg/ml of gentamycin supplemented with 10 ml heat
inactivated HyClone® horse serum (VWR, San Dimas, CA) and adjusted to pH 7.5] in
Corning (Corning, NY) petri dishes. All solutions were sterilized by passage through
0.22-µM filters. Nucleus located on the animal pole of each oocyte was then injected (10-
to 15-µm tip size) with human GlyR cDNAs (1 ng/32 nl). Injected oocytes were stored at
18
o
C and used in electrophysiological recordings 24 hours after injection for a period of 1
week.

36

Whole Cell Two-Electrode Voltage Clamp Recordings

Two-electrode voltage clamp recordings were performed using techniques similar to
those previously reported (Davies et al., 2004; Perkins et al., 2009). Briefly, electrodes
pulled (P-30, Sutter Instruments, Novato, CA) from borosilicate glass [1.2mm thick-
walled filamented glass capillaries (WPI, Sarasota, FL)] were filled with 3 M KCl to
yield resistances of 0.5 -3 MΩ. Electrophysiological recordings were conducted in a 100-
µL oocyte recording chamber (Type RC 3Z; Warner Instruments, Hamden, CT) that
holds an impaled oocyte and is placed on a vibration resistant magnetic steel base that
also supports two micro-manipulators (Type MM-33; Warner Instruments, Hamden, CT)
and a bath clamp. The oocyte recording chamber was continuously perfused with MBS ±
drugs using a Dynamax peristaltic pump (Rainin Inst Co., Emeryville, CA) at 3 mL/min
using 18-gauge polyethylene tubing (Becton Dickinson, Sparks, MD). Oocytes were
voltage clamped at a membrane potential of -70 mV using oocyte clamp OC-725C
(Warner Instruments; Hamden, CT). Clamped currents were recorded on a
Barnstead/Thermolyne chart recorder (Barnstead/Thermolyne, Dubuque, IA). All
experiments were performed at room temperature (20-23°C).

Application of Agonist

For agonist concentration response experiments, WT and mutant α1 GlyRs were exposed
to 1 µM – 3 mM glycine for 30 sec. 5-15 min washout periods between applications
ensured complete receptor resensitization.

Application of Ethanol

Potentiation of Cl
-
currents by ethanol is difficult to measure when using high agonist
concentrations, e.g., concentrations that produce 50% of the maximal effect (EC
50
). At
such high concentrations, the degree of alcohol potentiation decreases and the probability
of receptor desensitization increases (Mascia et al., 1996b; Mihic et al., 1994a; Mihic et
al., 1994b). Moreover, alcohol potentiation of Cl
-
currents is more robust and reliable

37

when lower agonist concentrations (EC
2-10
) are used (Davies et al., 2004; Mascia et al.,
1996a; Mascia et al., 1996b). Therefore, agonist concentrations producing 2% of the
maximal effect (EC
2
) were used. EC
2
± 5% agonist concentrations were applied until Cl
-

currents were stable i.e. within + 10% of each other. EC
2
was used as a control pre- and
post-ethanol treatment. Once stable, oocytes were tested for ethanol potentiation. Oocytes
were pre-incubated with ethanol for 60 sec followed by co-application of ethanol and
agonist for 30 sec. Washout periods (5-15 min depending on ethanol concentration
tested) were allowed between agonist and drug applications to ensure complete receptor
resensitization. WT and mutant receptor responses were measured across an ethanol
concentration range of 0.025 mM-50 mM for α1 GlyRs. Holding currents were not
significantly affected during preincubation with ethanol i.e. in the absence of agonist.

Data Analysis

Data for each experiment were obtained from 4-10 oocytes from at least two different
frogs. Sample size ‘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. Prism
(GraphPAD Software, San Diego, CA) was used to perform curve fitting and statistical
analyses. Agonist concentration response data were analyzed using non-linear regression
analysis: [I = I
max
[A]
nH
/ ([A]
nH
+ EC
50

nH
)] where I is the maximal current recorded
following application of a range of agonist concentrations, [A]; I
max
is the estimated
maximum current; EC
50
is the agonist concentration required for a half-maximal response
and nH is the Hill slope. Data were subjected to Student’s t tests, one-way or two-way
Analysis of Variance (ANOVA) with Dunnett’s multiple comparison or Bonferroni post-
tests when warranted. To determine the concentration at which a statistically significant
effect of ethanol was first detected in WT and mutant receptors (threshold concentration),
we compared the absolute values of agonist-induced chloride currents in the presence and
absence of ethanol across ethanol concentrations using two-way ANOVA followed by
Bonferroni post-tests. Statistical significance was defined as * p < 0.05.

38

Results

Agonist Concentration Response

Inward Cl
-
currents were evoked in a concentration dependent manner in WT and mutant
α1 GlyRs upon glycine administration (Figure 2.1). All analyses were conducted using
non-linear regression. Values for glycine EC
50
, Hill slope and I
max
of WT GlyRs (Table
2.3) were consistent with results from previous studies by Crawford et al., 2007 and
Perkins et al., 2009.

T-tests showed no significant differences in Hill slope and I
max
for WT and all mutant α1
GlyRs (Table 2.2). However, δL2 (Consistent with results from Perkins et al., 2009) and
δL2 S52A mutant receptors exhibited a left shift in agonist concentration response curve
with a statistically significant reduction in glycine EC
50
compared to WT α1 GlyRs
(Figure 2.1; Table 2.2). This indicated that δL2 S52A GlyRs were more sensitive to
agonist compared to WT receptors.

39

Table 2.2

Receptor EC
50
(mM) Hill Slope I
max
(nA)
α1 WT 92.3 ± 8.6 2.3 ± 0.3 16,658 ± 923
α1 (δL2) 22.9 ± 4.1
*
1.8 ± 0.1 13,186 ± 761
α1 (δL2 S52A) 47.3 ± 8
*
2.3 ± 0.2 17,710 ± 1290
α1 (δL2 E57D,
T59R)
80.7 ± 9.8 1.6 ± 0.1 13,467 ± 854

Table 2.2. Summary of results of non-linear regression analysis for glycine concentration
responses in WT and mutant α1 GlyRs. Glycine EC
50
, Hill slope (nH) and maximal current
amplitude (I
max
) are presented as mean ± SEM. Statistical significance was defined as p<0.05. T-
tests revealed no significant differences in I
max
and Hill slope between WT and all δL2 mutants.
EC
50
in δL2 and δL2 S52A mutants was significantly reduced compared to WT α1 GlyRs.

40

Figure 2.1

Figure 2.1. Concentration-response curves for glycine (1-3,000 µM) activated chloride
currents in Xenopus oocytes expressing WT and all δL2 mutant α1 GlyR subunits. Glycine-
induced Cl
-
currents were normalized to the maximal current activated by a saturating
concentration of glycine (1000 µM - 3000 µM). The curves represent non-linear regression
analysis of glycine concentration responses in WT and δL2 mutant α1 GlyRs. Details of EC
50
,
I
max
and Hill slope are provided in Table 2.3. Each data point represents mean ± SEM from 4-8
oocytes.

1 10 100 1000 10000
0
25
50
75
100
Gly δL2
Gly δL2 S52A
Gly WT
Gly δL2 E57D, T59R
Glycine, µM
Glycine Response
(% of maximal current)

41

Ethanol Concentration Response

WT GlyRs were insensitive to ethanol concentrations below 30 mM (Figure 2.2). Results
with δL2 GlyRs paralleled previously recorded observations by Perkins and colleagues
(Perkins et al., 2009; Figure 2.2). Substituting Loop 2 of the α1 GlyR subunit with Loop
2 of the δ GABA
A
R subunit significantly increased ethanol sensitivity in δL2 GlyRs
compared to WT GlyRs (Figure 2.2). As hypothesized, a serine to alanine reversion at
position 52 in δL2 of mutant GlyRs markedly increased ethanol sensitivity of δL2
(S52A) GlyRs compared to WT and δL2 GlyRs (Figure 2.2). The decrease in threshold
for ethanol sensitivity was over 100-fold in δL2 (S52A) mutant α1 GlyRs: 30mM EtOH
in WT to 0.075mM EtOH in δL2 S52A (Figure 2.2, Table 2.3). This new mutant receptor
was also more sensitive to ethanol than δL2 GlyRs, signified by its lower ethanol
threshold (Table 2.3). Additionally, the magnitude of ethanol potentiation of glycine
currents was significantly increased across all tested concentrations in δL2 (S52A) USER
GlyRs (Figure 2.2). Furthermore, a bimodal inverted ‘U-shaped’ ethanol concentration
response curve was observed in δL2 (S52A) USER GlyRs but not in WT GlyRs. The first
curve at lower ethanol concentrations started at approximately 0.075 mM extending to 1-
3 mM, and the second curve at higher ethanol concentrations, ranging 10-30 mM ethanol
(Figure 2.2)

42

Figure 2.2

Figure 2.2. USER GlyRs have increased sensitivity and bimodal response to ethanol. This
figure shows ethanol-induced potentiation of glycine activated chloride currents in Xenopus
oocytes expressing WT, δL2, δL2 S52A and δL2 E57D, T59R α1 GlyRs. Each data point
represents the mean ± SEM from 4-9 different oocytes. All δL2 USERs exhibited a bimodal
response to ethanol, decreased threshold for ethanol sensitivity and markedly increased the
magnitude of ethanol response compared to WT GlyRs.

0
15
30
45
60
75
0.025 0.050 0.075 0.100 0.250 0.500 1.000 3.000 10.000 30.000 50.000
0
15
30
45
60
75
α1 GlyR WT
δL2
δL2 S52A
δL2 E57D, T59R
EtOH Concentration, mM
Glycine EC
2
-Induced Cl
-
Currents,
% Potentiation

43

Table 2.3

EtOH
Concentration
(mM)

Receptor
WT δL2 δL2 S52A
δL2 E57D,
T59R
0.025
0.05
0.075 ** *
0.1 ** ** ****
0.25 * ** *
0.5 *** *** ***
1 *** **
3 * *
10 + * *
30 * * ** **
50 ** * ***

Table 2.3. Threshold for ethanol sensitivity in GlyR δL2 USERs is bimodal and lower than
WT. T tests were performed to determine the threshold for ethanol sensitivity in WT and mutant
α1 GlyRs. Threshold for lower ethanol concentrations was observed at 0.075mM EtOH in α1
GlyR- δL2 S52A and - δL2 E57D, T59R, and at 0.1mM EtOH for δL2, compared to 30mM
EtOH in WT. (* denotes statistical significance in absolute values of agonist response in presence
and absence of ethanol. * p < 0.05 and + p<0.06)

44

Conclusion

The results demonstrate that reverting Ser to Ala at position 52 in Loop 2 of δL2 GlyRs
increases ethanol sensitivity (lower ethanol threshold and enhanced ethanol-induced
potentiation) of the mutant receptor compared to δL2 GlyRs, albeit with altered agonist
sensitivity as compared to WT receptors. Overall, the findings support Phase 1 hypothesis
and identified USERs that were more sensitive to ethanol than WT and δL2 GlyR.

45

Chapter 3
Optimization Phase 2 – Optimize practicality of USERs for development of
transgenic animals

Aim 2 – To test the hypothesis that USERs can be created by limiting δL2 α1 GlyR
mutations to Exon 3

In Gly δL2 USERs, the Loop 2 sequence is encoded by base pairs spanning Exons 3
(positions 50-55) and 4 (positions 56-59) of the human GLRA1 gene, making it more
complicated to construct transgenic animals for gene incorporation and functional
expression and increasing the potential for unintentional changes in function of the
resultant mutant receptor. Thus, our goal was to limit all mutations to one Exon to
circumvent possible complications of Gly USER expression and incorporation in vivo.

Consequently, we developed the hypothesis-USERs can be created by limiting α1 (δL2)
GlyR mutations to Exon 3. In order to test this hypothesis, we restored non-conserved
residues (E57 and T59) of Exon 4 in δL2 of mutant GlyRs back to homologous WT
residues (57D and 59R) to create a new USER- α1 (δL2 E57D, T59R) GlyR. We then
expressed new mutant receptors in Xenopus oocytes, exposed this system to a wide range
of agonist (1-3,000 µM) and ethanol (0.025-50 mM) concentrations and measured agonist
and ethanol sensitivities using two-electrode voltage clamp electrophysiology. The
materials and methods used to test this hypothesis were the same as those used in
experiments described in Chapter 2. The results are described in detail below.

46

Results

Agonist Concentration Response

Inward Cl
-
currents were evoked in a concentration dependent manner in WT and mutant
α1 GlyRs upon glycine administration (Figure 2.1). All analyses were conducted using
non-linear regression. Values for glycine EC
50
, Hill slope and I
max
of WT GlyRs (Table
2.3) were consistent with results from previous studies by Crawford et al., 2007 and
Perkins et al., 2009.

T-tests showed no significant differences in Hill slope and I
max
for WT and all mutant α1
GlyRs (Table 2.2). Consistent with results seen in a previous study by Perkins and
colleagues (Perkins et al., 2009), δL2 GlyRs exhibited a left shift in agonist sensitivity
with a statistically significant reduction in glycine EC
50
compared to WT α1 GlyRs
(Figure 2.1; Table 2.2). As expected, amino acid reversions in α1 (δL2 E57D, T59R)
GlyRs caused a significant right shift in agonist sensitivity compared to δL2 GlyRs, as
indicated by a significant increase in Gly EC
50
(Figure 2.1; Table 2.2). As a result, the
agonist concentration response curve overlapped with that of WT receptors. No
statistically significant difference in EC
50
between WT and this mutant receptor was
observed, thus indicating restoration of WT-like agonist sensitivity and normalization of
receptor function. This was not the primary goal when we created this mutation, but it is a
helpful consequence.

47

Ethanol Concentration Response

WT GlyRs were insensitive to ethanol concentrations below 30 mM (Figure 2.2). Results
with δL2 GlyRs paralleled previously recorded observations by Perkins and colleagues
(Perkins et al., 2009, Figure 2.2). Substituting Loop 2 of the α1 GlyR subunit with Loop
2 of the δ GABA
A
R subunit significantly increased ethanol sensitivity in δL2 GlyRs
compared to WT GlyRs (Figure 2.2). Reverting Glu57 and Thr59 to homologous WT
Asp57 and Arg59 residues in Exon 4 of δL2 GlyRs remarkably increased ethanol
sensitivity of δL2 (E57D, T59R) GlyRs compared to WT and δL2 GlyRs (Figure 2.2).
The decrease in threshold for ethanol sensitivity was over 100-fold in δL2 (E57D, T59R)
GlyRs: 30mM EtOH in WT to 0.075mM EtOH in δL2 E57D, T59R (Figure 2.2, Table
2.3). This new mutant receptor was also more sensitive to ethanol than δL2 GlyRs,
signified by its lower ethanol threshold (Table 2.3). Additionally, magnitude of ethanol
potentiation of glycine currents, especially at the lower ethanol concentrations (0.075-
0.5mM), was significantly increased in δL2 (E57D, T59R) USER GlyRs compared to
WT and δL2 GlyRs (Figure 2.2). Furthermore, a bimodal inverted ‘U-shaped’ ethanol
concentration response curve was observed in δL2 (E57D, T59R) USER GlyRs but not in
WT GlyRs. The first curve at lower ethanol concentrations started at approximately 0.075
mM extending to 1-3 mM, and the second curve at higher ethanol concentrations, ranging
10-30 mM ethanol (Figure 2.2).

48

Conclusion

The results demonstrate that USER α1 GlyRs can be created with mutations limited to
Exon 3. In addition, these reversions normalized agonist response in the mutant receptors
suggesting that these positions may be involved in modulating agonist sensitivity of α1
GlyRs. Overall, the findings both support the Phase 2 hypothesis and identified a USER
that meets requirements for use in developing transgenic USER mice.

49

Chapter 4
Overall Discussion

The primary goal of my thesis was to optimize USER GlyRs by maximizing alcohol
sensitivity, and enhancing the potential utility of these receptors when expressed in vivo,
thereby setting the stage for developing transgenic animals with USER GlyRs. Using the
proposed Phase 1 and Phase 2 Optimization strategies, we were able to create two new
optimized USERs: [1] δL2 (S52A) GlyRs that have [i] ultrasensitivity to ethanol [ii]
enhanced ethanol-induced potentiation compared to WT receptors across high and low
ethanol concentrations [2] δL2 (E57D, T59R) GlyRs that have [i] ultrasensitivity to
ethanol [ii] enhanced ethanol- induced potentiation compared to WT receptors at low
ethanol concentrations [iii] mutations confined to only one Exon [iv] normalized WT-like
receptor function . These studies provide evidence that will be helpful in constructing
molecular models that provide insight into structural-functional basis between ethanol
and α1 GlyR Loop 2. The findings also lay a solid foundation for developing transgenic
animals that express USERs as novel tools to characterize neurochemical signaling
cascades stemming from ethanol action and to dissect the role specific receptor subunit
combinations play in ethanol’s behavioral effects.

The molecular interactions behind factors imparting ultrasensitivity to ethanol and
normalizing agonist sensitivity in USER GlyRs are not fully understood. However,
molecular models developed by Perkins and colleagues (Perkins et al., 2012; Perkins et
al., 2009) may provide some insights into possible physical-chemical interactions
between amino acid residues in structure of GlyR Loop 2 responsible for modulating
agonist and ethanol sensitivity. These molecular models show that the δL2 substitution in
α1 GlyRs causes a change in conformation of the loop structure by altering inter- and
intra-subunit salt-bridge interactions. When ethanol is introduced into the equation, the
conformational change and underlying dynamic interactions of δL2 amino acid residues
with surrounding EC and TM residues are likely to contribute to the increased ethanol
sensitivity of δL2 GlyRs (Perkins et al., 2009). When the polar residue- Ser at position 52

50

in the δL2 structure is replaced by the non-polar residue, Ala in the δL2 S52A Gly
USER, the hydrogen bond forces that strongly attract water and block ethanol’s
accessibility to this site may be abolished, thereby, making it easier for ethanol to
displace water and gain access to this site (Perkins et al., 2008). This change in attractive
forces may have been one of the contributing factors of increased ethanol sensitivity of
δL2 S52A GlyRs compared to δL2 GlyRs. Furthermore, in the GlyR Loop 2 molecular
model proposed by Perkins and colleagues (See Figure 1.2), Asp57 in WT Loop 2 is in an
optimal position to participate in salt-bridge interactions with surrounding residues. In
δL2 GlyRs, these interactions are distorted due to different structural properties of the
new glutamate residue (Perkins et al., 2009). The subtle single angstrom change resulting
from the Glu to Asp (E57D) in δL2 (E57D, T59R) GlyRs could be sufficient to restore
and improve the interactions sufficiently to cause a conformational change in the loop
that could subsequently alter receptor properties such as gating and agonist binding
and/or improve accessibility and affinity to ethanol. Finally, restoring the neutral, polar
Thr59 in δL2 to a charged Arg residue (T59R) in δL2 (E57D, T59R) GlyRs may have
restored ionic and hydrogen bond interactions seen previously in WT Loop 2. Taken
together, restoring physical, chemical and structural properties of residues at positions 57
and 59 in δL2 (E57D, T59R) GlyRs may have resulted in increased ethanol sensitivity
and normalized receptor function.

The next step in the development of USERs as tools is to produce transgenic animals that
express USERs in total brain (KI approach) and in specific brain regions (e.g. lenti-viral
vectors) as a brain-mapping tool to investigate the role of specific receptor populations in
causing certain behavioral effects. Furthermore, initial findings from similar studies in
α1β2γ2 GABA
A
Rs attempting to develop and optimize USER GABA
A
Rs are
encouraging and parallel results from our studies with Gly USERs, thereby suggesting
that factors modulating ethanol and agonist sensitivity in GlyRs could extend to
GABA
A
Rs. Therefore, this work and future studies could extend to other ethanol-
sensitive LGICs such as nAChRs, 5-HT
3
Rs and others and may help in identifying and
understanding structural features that influence ethanol’s action sites on these receptors.
Thus, it can provide key evidence that may help in designing molecular models of

51

ethanol’s action sites on specific receptor subtypes, thereby allowing for improved in
silico screening, pharmacophore modeling and exploring/designing new ligands that are
highly specific for specific subunits and receptors. Consequently, this can contribute to
development of novel drugs that can target a discrete set of behaviors and minimize off
target effects for the treatment of AUDs.

52

Bibliography

Addolorato, G., A. Mirijello, and L. Leggio. 2013. Alcohol addiction: toward a patient-
oriented pharmacological treatment. Expert Opin Pharmacother.
Aguayo, L.G., and F.C. Pancetti. 1994. Ethanol modulation of the gamma-aminobutyric
acidA- and glycine-activated Cl- current in cultured mouse neurons. J Pharmacol
Exp Ther. 270:61-69.
Alkana, R.L., D.L. Davies, J. Morland, E.S. Parker, and M. Bejanian. 1995. Low-level
hyperbaric exposure antagonizes locomotor effects of ethanol and n-propanol but
not morphine in C57BL mice. Alcohol Clin Exp Res. 19:693-700.
Alkana, R.L., D.A. Finn, B.L. Jones, L.S. Kobayashi, M. Babbini, M. Bejanian, and P.J.
Syapin. 1992. Genetically determined differences in the antagonistic effect of
pressure on ethanol-induced loss of righting reflex in mice. Alcohol Clin Exp Res.
16:17-22.
Alkana, R.L., and R.D. Malcolm. 1981. Low-level hyperbaric ethanol antagonism in
mice. Dose and pressure response. Pharmacology. 22:199-208.
Alkana, R.L., and R.D. Malcolm. 1982a. Hyperbaric ethanol antagonism in mice: studies
on oxygen, nitrogen, strain and sex. Psychopharmacology. 77:11-16.
Alkana, R.L., and R.D. Malcolm. 1982b. Hyperbaric ethanol antagonism in mice: time
course. Subst Alcohol Actions Misuse. 3:41-46.
Allan, A.M., and R.A. Harris. 1986. Anesthetic and convulsant barbiturates alter gamma-
aminobutyric acid-stimulated chloride flux across brain membranes. J Pharmacol
Exp Ther. 238:763-768.
Allan, A.M., and R.A. Harris. 1987. Acute and chronic ethanol treatments alter GABA
receptor-operated chloride channels. Pharmacol Biochem Behav. 27:665-670.
American Psychiatric Association. 2013. Diagnostic and Statistical Manual of Mental
Disorders, 5th edition. American Psychiatric Association.
Asatryan, L., H.W. Nam, M.R. Lee, M.M. Thakkar, M. Saeed Dar, D.L. Davies, and D.S.
Choi. 2011. Implication of the purinergic system in alcohol use disorders. Alcohol
Clin Exp Res. 35:584-594.
Asatryan, L., M. Popova, D. Perkins, J.R. Trudell, R.L. Alkana, and D.L. Davies. 2010.
Ivermectin antagonizes ethanol inhibition in purinergic P2X4 receptors. J
Pharmacol Exp Ther. 334:720-728.
Asatryan, L., M. Popova, J.J. Woodward, B.F. King, R.L. Alkana, and D.L. Davies.
2008. Roles of ectodomain and transmembrane regions in ethanol and agonist
action in purinergic P2X2 and P2X3 receptors. Neuropharmacology. 55:835-843.
Badanich, K.A., H.C. Becker, and J.J. Woodward. 2011. Effects of chronic intermittent
ethanol exposure on orbitofrontal and medial prefrontal cortex-dependent
behaviors in mice. Behav Neurosci. 125:879-891.
Badanich, K.A., P.J. Mulholland, J.T. Beckley, H. Trantham-Davidson, and J.J.
Woodward. 2013. Ethanol reduces neuronal excitability of lateral orbitofrontal
cortex neurons via a glycine receptor dependent mechanism.
Neuropsychopharmacology. 38:1176-1188.
Barberis, A., E.M. Petrini, E. Cherubini, and J.W. Mozrzymas. 2002. Allosteric
interaction of zinc with recombinant alpha(1)beta(2)gamma(2) and
alpha(1)beta(2) GABA(A) receptors. Neuropharmacology. 43:607-618.

53

Bejanian, M., B.L. Jones, and R.L. Alkana. 1993. Low-level hyperbaric antagonism of
ethanol-induced locomotor depression in C57BL/6J mice: dose response. Alcohol
Clin Exp Res. 17:935-939.
Bhattacharji, A., B. Kaplan, T. Harris, X. Qu, M.W. Germann, and M. Covarrubias. 2006.
The concerted contribution of the S4-S5 linker and the S6 segment to the
modulation of a Kv channel by 1-alkanols. Mol Pharmacol. 70:1542-1554.
Blednov, Y.A., J.M. Benavidez, G.E. Homanics, and R.A. Harris. 2012. Behavioral
characterization of knockin mice with mutations M287L and Q266I in the glycine
receptor alpha1 subunit. J Pharmacol Exp Ther. 340:317-329.
Blednov, Y.A., S. Jung, H. Alva, D. Wallace, T. Rosahl, P.J. Whiting, and R.A. Harris.
2003a. Deletion of the alpha1 or beta2 subunit of GABAA receptors reduces
actions of alcohol and other drugs. J Pharmacol Exp Ther. 304:30-36.
Blednov, Y.A., M. Stoffel, H. Alva, and R.A. Harris. 2003b. A pervasive mechanism for
analgesia: activation of GIRK2 channels. Proc Natl Acad Sci U S A. 100:277-282.
Blednov, Y.A., M. Stoffel, S.R. Chang, and R.A. Harris. 2001. Potassium channels as
targets for ethanol: studies of G-protein-coupled inwardly rectifying potassium
channel 2 (GIRK2) null mutant mice. J Pharmacol Exp Ther. 298:521-530.
Blednov, Y.A., D. Walker, H. Alva, K. Creech, G. Findlay, and R.A. Harris. 2003c.
GABAA receptor alpha 1 and beta 2 subunit null mutant mice: behavioral
responses to ethanol. J Pharmacol Exp Ther. 305:854-863.
Bloomenthal, A.B., E. Goldwater, D.B. Pritchett, and N.L. Harrison. 1994. Biphasic
modulation of the strychnine-sensitive glycine receptor by Zn2+. Mol Pharmacol.
46:1156-1159.
Boehm, S.L., 2nd, I. Ponomarev, A.W. Jennings, P.J. Whiting, T.W. Rosahl, E.M.
Garrett, Y.A. Blednov, and R.A. Harris. 2004. gamma-Aminobutyric acid A
receptor subunit mutant mice: new perspectives on alcohol actions. Biochem
Pharmacol. 68:1581-1602.
Borghese, C.M., L.A. Henderson, V. Bleck, J.R. Trudell, and R.A. Harris. 2003. Sites of
excitatory and inhibitory actions of alcohols on neuronal alpha2beta4 nicotinic
acetylcholine receptors. J Pharmacol Exp Ther. 307:42-52.
Bortolato, M., M.M. Yardley, S. Khoja, S.C. Godar, L. Asatryan, D.A. Finn, R.L.
Alkana, S.G. Louie, and D.L. Davies. 2013. Pharmacological insights into the role
of P2X4 receptors in behavioural regulation: lessons from ivermectin. Int J
Neuropsychopharmacol. 16:1059-1070.
Bouchery, E.E., H.J. Harwood, J.J. Sacks, C.J. Simon, and R.D. Brewer. 2011. Economic
costs of excessive alcohol consumption in the U.S., 2006. Am J Prev Med.
41:516-524.
Bouza, C., M. Angeles, A. Munoz, and J.M. Amate. 2004. Efficacy and safety of
naltrexone and acamprosate in the treatment of alcohol dependence: a systematic
review. Addiction. 99:811-828.
Brauer, R.W., W.M. Mansfield, Jr., R.W. Beaver, and H.W. Gillen. 1979. Stages in
development of high-pressure neurological syndrome in the mouse. J Appl
Physiol. 46:756-765.
Brejc, K., W.J. van Dijk, R.V. Klaassen, M. Schuurmans, J. van Der Oost, A.B. Smit, and
T.K. Sixma. 2001. Crystal structure of an ACh-binding protein reveals the ligand-
binding domain of nicotinic receptors. Nature. 411:269-276.

54

Buck, K.J., H. Heim, and R.A. Harris. 1991. Reversal of alcohol dependence and
tolerance by a single administration of flumazenil. J Pharmacol Exp Ther.
257:984-989.
Cantor, R.S. 1997. The lateral pressure profile in membranes: a physical mechanism of
general anesthesia. Biochemistry. 36:2339-2344.
Cederholm, J.M., N.L. Absalom, S. Sugiharto, R. Griffith, P.R. Schofield, and T.M.
Lewis. 2010. Conformational changes in extracellular loop 2 associated with
signal transduction in the glycine receptor. J Neurochem. 115:1245-1255.
Celentano, J.J., T.T. Gibbs, and D.H. Farb. 1988. Ethanol potentiates GABA- and
glycine-induced chloride currents in chick spinal cord neurons. Brain Res.
455:377-380.
Chakrapani, S., T.D. Bailey, and A. Auerbach. 2004. Gating dynamics of the
acetylcholine receptor extracellular domain. J Gen Physiol. 123:341-356.
Chandra, D., D.F. Werner, J. Liang, A. Suryanarayanan, N.L. Harrison, I. Spigelman,
R.W. Olsen, and G.E. Homanics. 2008. Normal acute behavioral responses to
moderate/high dose ethanol in GABAA receptor alpha 4 subunit knockout mice.
Alcohol Clin Exp Res. 32:10-18.
Chandrasekar, R. 2013. Alcohol and NMDA receptor: current research and future
direction. Front Mol Neurosci. 6:14.
Chau, P., H. Hoifodt-Lido, E. Lof, B. Soderpalm, and M. Ericson. 2010. Glycine
receptors in the nucleus accumbens involved in the ethanol intake-reducing effect
of acamprosate. Alcohol Clin Exp Res. 34:39-45.
Chin, J.H., J.R. Trudell, and E.N. Cohen. 1976. The compression-ordering and solubility-
disordering effects of high pressure gases on phospholipid bilayers. Life Sci.
18:489-497.
Choi, D.S., D. Wang, J. Dadgar, W.S. Chang, and R.O. Messing. 2002. Conditional
rescue of protein kinase C epsilon regulates ethanol preference and hypnotic
sensitivity in adult mice. J Neurosci. 22:9905-9911.
Colombo, G., A. Orru, P. Lai, C. Cabras, P. Maccioni, M. Rubio, G.L. Gessa, and M.A.
Carai. 2007. The cannabinoid CB1 receptor antagonist, rimonabant, as a
promising pharmacotherapy for alcohol dependence: preclinical evidence. Mol
Neurobiol. 36:102-112.
Cook, J.B., K.L. Foster, W.J. Eiler, 2nd, P.F. McKay, J. Woods, 2nd, S.C. Harvey, M.
Garcia, C. Grey, S. McCane, D. Mason, R. Cummings, X. Li, J.M. Cook, and
H.L. June. 2005. Selective GABAA alpha5 benzodiazepine inverse agonist
antagonizes the neurobehavioral actions of alcohol. Alcohol Clin Exp Res.
29:1390-1401.
Crabbe, J.C., T.J. Phillips, R.A. Harris, M.A. Arends, and G.F. Koob. 2006. Alcohol-
related genes: contributions from studies with genetically engineered mice. Addict
Biol. 11:195-269.
Crawford, D.K., J.R. Trudell, E.J. Bertaccini, K. Li, D.L. Davies, and R.L. Alkana. 2007.
Evidence that ethanol acts on a target in Loop 2 of the extracellular domain of
alpha1 glycine receptors. J Neurochem. 102:2097-2109.
Crawley, J.N. 1996. Unusual behavioral phenotypes of inbred mouse strains. Trends
Neurosci. 19:181-182; discussion 188-189.

55

Das, J., G.H. Addona, W.S. Sandberg, S.S. Husain, T. Stehle, and K.W. Miller. 2004.
Identification of a general anesthetic binding site in the diacylglycerol-binding
domain of protein kinase Cdelta. J Biol Chem. 279:37964-37972.
Das, J., S. Pany, G.M. Rahman, and S.J. Slater. 2009. PKC epsilon has an alcohol-
binding site in its second cysteine-rich regulatory domain. Biochem J. 421:405-
413.
Das, J., S. Shunmugasundararaj, T. Stehle, and K.W. Miller. 2007. Crystal structure of an
alcohol complexed with the cysteine-rich domain of protein kinase C δ. Alcohol
Clin Exp Res. 31:202A.
Davidson, M., W. Chen, and P.A. Wilce. 1999. Behavioral analysis of PTZ-kindled rats
after acute and chronic ethanol treatments. Pharmacol Biochem Behav. 64:7-13.
Davies, D.L., and R.L. Alkana. 1998. Direct antagonism of ethanol's effects on
GABA(A) receptors by increased atmospheric pressure. Alcohol Clin Exp Res.
22:1689-1697.
Davies, D.L., and R.L. Alkana. 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. 25:1098-1106.
Davies, D.L., and R.L. Alkana. 2001b. Ethanol enhances GABAA receptor function in
short sleep and long sleep mouse brain membranes. Alcohol Clin Exp Res.
25:478-483.
Davies, D.L., and R.L. Alkana. 2003. Benzodiazepine agonist and inverse agonist
coupling in GABAA receptors antagonized by increased atmospheric pressure.
Eur J Pharmacol. 469:37-45.
Davies, D.L., L. Asatryan, S.T. Kuo, J.J. Woodward, B.F. King, R.L. Alkana, C. Xiao,
J.H. Ye, H. Sun, L. Zhang, X.Q. Hu, V. Hayrapetyan, D.M. Lovinger, and T.K.
Machu. 2006. Effects of ethanol on adenosine 5'-triphosphate-gated purinergic
and 5-hydroxytryptamine receptors. Alcohol Clin Exp Res. 30:349-358.
Davies, D.L., M. Bejanian, E.S. Parker, J. Morland, M.B. Bolger, R.D. Brinton, and R.L.
Alkana. 1996. Low level hyperbaric antagonism of diazepam's locomotor
depressant and anticonvulsant properties in mice. J Pharmacol Exp Ther.
276:667-675.
Davies, D.L., M.B. Bolger, R.D. Brinton, D.A. Finn, and R.L. Alkana. 1999. In vivo and
in vitro hyperbaric studies in mice suggest novel sites of action for ethanol.
Psychopharmacology. 141:339-350.
Davies, D.L., M. Bortolato, D.A. Finn, M.J. Ramaker, S. Barak, D. Ron, J. Liang, and
R.W. Olsen. 2013. Recent advances in the discovery and preclinical testing of
novel compounds for the prevention and/or treatment of alcohol use disorders.
Alcohol Clin Exp Res h. 37:8-15.
Davies, D.L., D.K. Crawford, J.R. Trudell, S.J. Mihic, and R.L. Alkana. 2004. Multiple
sites of ethanol action in alpha1 and alpha2 glycine receptors suggested by
sensitivity to pressure antagonism. J Neurochem. 89:1175-1185.
Davies, D.L., J. Morland, B.L. Jones, and R.L. Alkana. 1994. Low-level hyperbaric
antagonism of ethanol's anticonvulsant property in C57BL/6J mice. Alcohol Clin
Exp Res. 18:1190-1195.

56

Davies, D.L., J.R. Trudell, S.J. Mihic, D.K. Crawford, and R.L. Alkana. 2003. Ethanol
potentiation of glycine receptors expressed in Xenopus oocytes antagonized by
increased atmospheric pressure. Alcohol Clin Exp Res. 27:743-755.
Davies, P.A. 2011. Allosteric modulation of the 5-HT(3) receptor. Curr Opin Pharmacol.
11:75-80.
de Almeida, R.M., J.K. Rowlett, J.M. Cook, W. Yin, and K.A. Miczek. 2004.
GABAA/alpha1 receptor agonists and antagonists: effects on species-typical and
heightened aggressive behavior after alcohol self-administration in mice.
Psychopharmacology. 172:255-263.
de Wit, M., G.K. Wiaterek, N.D. Gray, K.E. Goulet, A.M. Best, J.N. Clore, and L.B.
Sweeney. 2010. Relationship between alcohol use disorders, cortisol
concentrations, and cytokine levels in patients with sepsis. Crit Care. 14:R230.
Deitrich, R.A., T.V. Dunwiddie, R.A. Harris, and V.G. Erwin. 1989. Mechanism of
action of ethanol: initial central nervous system actions. Pharmacol Rev. 41:489-
537.
Dutertre, S., C.M. Becker, and H. Betz. 2012. Inhibitory glycine receptors: an update. J
Biol Chem. 287:40216-40223.
Engblom, A.C., and K.E. Akerman. 1991. Effect of ethanol on gamma-aminobutyric acid
and glycine receptor-coupled Cl- fluxes in rat brain synaptoneurosomes. J
Neurochem. 57:384-390.
Ericson, M., R.B. Clarke, P. Chau, L. Adermark, and B. Soderpalm. 2010. beta-Alanine
elevates dopamine levels in the rat nucleus accumbens: antagonism by strychnine.
Amino acids. 38:1051-1055.
Fang, Z., J. Sonner, M.J. Laster, P. Ionescu, L. Kandel, D.D. Koblin, E.I. Eger, 2nd, and
M.J. Halsey. 1996. Anesthetic and convulsant properties of aromatic compounds
and cycloalkanes: implications for mechanisms of narcosis. Anesth Analg.
83:1097-1104.
Findlay, G.S., M.J. Wick, M.P. Mascia, D. Wallace, G.W. Miller, R.A. Harris, and Y.A.
Blednov. 2002. Transgenic expression of a mutant glycine receptor decreases
alcohol sensitivity of mice. J Pharmacol Exp Ther. 300:526-534.
Follesa, P., F. Biggio, G. Talani, L. Murru, M. Serra, E. Sanna, and G. Biggio. 2006.
Neurosteroids, GABAA receptors, and ethanol dependence.
Psychopharmacology. 186:267-280.
Franck, J., and N. Jayaram-Lindstrom. 2013. Pharmacotherapy for alcohol dependence:
status of current treatments. Curr Opin Neurobiol. 23:692-699.
Franks, N.P., and W.R. Lieb. 1982. Molecular mechanisms of general anaesthesia.
Nature. 300:487-493.
Franks, N.P., and W.R. Lieb. 1984. Do general anaesthetics act by competitive binding to
specific receptors? Nature. 310:599-601.
Franks, N.P., and W.R. Lieb. 1985. Mapping of general anaesthetic target sites provides a
molecular basis for cutoff effects. Nature. 316:349-351.
Franks, N.P., and W.R. Lieb. 1991. Stereospecific effects of inhalational general
anesthetic optical isomers on nerve ion channels. Science. 254:427-430.
Franks, N.P., and W.R. Lieb. 1994. Molecular and cellular mechanisms of general
anaesthesia. Nature. 367:607-614.

57

Galla, H.J., and J.R. Trudell. 1980. Asymmetric antagonistic effects of an inhalation
anesthetic and high pressure on the phase transition temperature of dipalmitoyl
phosphatidic acid bilayers. Biochim Biophys Acta. 599:336-340.
Gatch, M.B. 1999. Effects of benzodiazepines on acute and chronic ethanol-induced
nociception in rats. Alcohol Clin Exp Res. 23:1736-1743.
Gewiss, M., C. Heidbreder, L. Opsomer, P. Durbin, and P. De Witte. 1991. Acamprosate
and diazepam differentially modulate alcohol-induced behavioural and cortical
alterations in rats following chronic inhalation of ethanol vapour. Alcohol
Alcohol. 26:129-137.
Goldstein, D.B. 1984. The effects of drugs on membrane fluidity. Annu Rev Pharmacol
Toxicol. 24:43-64.
Gonzalez, C., S.J. Moss, and R.W. Olsen. 2012. Ethanol promotes clathrin adaptor-
mediated endocytosis via the intracellular domain of delta-containing GABAA
receptors. J Neurosci. 32:17874-17881.
Grant, B.F., D.A. Dawson, F.S. Stinson, S.P. Chou, M.C. Dufour, and R.P. Pickering.
2004. The 12-month prevalence and trends in DSM-IV alcohol abuse and
dependence: United States, 1991-1992 and 2001-2002. Drug Alcohol Depend.
74:223-234.
Grobin, A.C., D.B. Matthews, L.L. Devaud, and A.L. Morrow. 1998. The role of
GABA(A) receptors in the acute and chronic effects of ethanol.
Psychopharmacology. 139:2-19.
Gruol, D.L., K.L. Parsons, and N. DiJulio. 1997. Acute ethanol alters calcium signals
elicited by glutamate receptor agonists and K+ depolarization in cultured
cerebellar Purkinje neurons. Brain Res. 773:82-89.
Guzman, L., G. Moraga-Cid, A. Avila, M. Figueroa, G.E. Yevenes, J. Fuentealba, and
L.G. Aguayo. 2009. Blockade of ethanol-induced potentiation of glycine
receptors by a peptide that interferes with Gbetagamma binding. J Pharmacol Exp
Ther. 331:933-939.
Halsey, M.J. 1982. Effects of high pressure on the central nervous system. Physiol Rev.
62:1341-1377.
Halsey, M.J., E.I. Eger, 2nd, D.W. Kent, and P.J. Warne. 1970. High pressure studies of
anesthesia. in Progress in Anesthesiology (Fink BR ed).
Halsey, M.J., and B. Wardley-Smith. 1975. Pressure reversal of narocsis produced by
anaesthetics, narcotics and tranquillisers. Nature. 257:811-813.
Halsey, M.J., B. Wardley-Smith, and C.J. Green. 1978. Pressure reversal of general
anaesthesia--a multi-site expansion hypothesis. Br J Anaesth. 50:1091-1097.
Hanchar, H.J., P. Chutsrinopkun, P. Meera, P. Supavilai, W. Sieghart, M. Wallner, and
R.W. Olsen. 2006. Ethanol potently and competitively inhibits binding of the
alcohol antagonist Ro15-4513 to alpha4/6beta3delta GABAA receptors. Proc
Natl Acad Sci U S A. 103:8546-8551.
Hanson, D.J. 1995. Preventing alcohol abuse : alcohol, culture, and control. Praeger,
Westport, Conn. xiv, 140 p. pp.
Harris, R.A. 1990. Alcohol sensitivity. Nature. 348:589.
Harris, R.A. 1999. Ethanol actions on multiple ion channels: which are important?
Alcohol Clin Exp Res. 23:1563-1570.

58

Harris, R.A., J.R. Trudell, and S.J. Mihic. 2008. Ethanol's molecular targets. Sci Signal.
1:re7.
Harvey, R.J., U.B. Depner, H. Wassle, S. Ahmadi, C. Heindl, H. Reinold, T.G. Smart, K.
Harvey, B. Schutz, O.M. Abo-Salem, A. Zimmer, P. Poisbeau, H. Welzl, D.P.
Wolfer, H. Betz, H.U. Zeilhofer, and U. Muller. 2004. GlyR alpha3: an essential
target for spinal PGE2-mediated inflammatory pain sensitization. Science.
304:884-887.
Harvey, S.C., K.L. Foster, P.F. McKay, M.R. Carroll, R. Seyoum, J.E. Woods, 2nd, C.
Grey, C.M. Jones, S. McCane, R. Cummings, D. Mason, C. Ma, J.M. Cook, and
H.L. June. 2002. The GABA(A) receptor alpha1 subtype in the ventral pallidum
regulates alcohol-seeking behaviors. J Neurosci. 22:3765-3775.
Heilig, M., and M. Egli. 2006. Pharmacological treatment of alcohol dependence: target
symptoms and target mechanisms. Pharmacol Ther. 111:855-876.
Heimburg, T., and A.D. Jackson. 2007. The thermodynamics of general anesthesia.
Biophys J. 92:3159-3165.
Hendricson, A.W., J.R. Sibbald, and R.A. Morrisett. 2004. Ethanol alters the frequency,
amplitude, and decay kinetics of Sr2+-supported, asynchronous NMDAR
mEPSCs in rat hippocampal slices. J Neurophysiol. 91:2568-2577.
Hill, S.Y., S. Wang, B. Kostelnik, H. Carter, B. Holmes, M. McDermott, N. Zezza, S.
Stiffler, and M.S. Keshavan. 2009. Disruption of orbitofrontal cortex laterality in
offspring from multiplex alcohol dependence families. Biol Psychiatry. 65:129-
136.
Hodge, C.W., S.P. Kelley, A.M. Bratt, K. Iller, J.P. Schroeder, and J. Besheer. 2004. 5-
HT(3A) receptor subunit is required for 5-HT3 antagonist-induced reductions in
alcohol drinking. Neuropsychopharmacology. 29:1807-1813.
Hodge, C.W., K.K. Mehmert, S.P. Kelley, T. McMahon, A. Haywood, M.F. Olive, D.
Wang, A.M. Sanchez-Perez, and R.O. Messing. 1999. Supersensitivity to
allosteric GABA(A) receptor modulators and alcohol in mice lacking
PKCepsilon. Nat Neurosci. 2:997-1002.
Hood, H.M., and K.J. Buck. 2000. Allelic variation in the GABA A receptor gamma2
subunit is associated with genetic susceptibility to ethanol-induced motor
incoordination and hypothermia, conditioned taste aversion, and withdrawal in
BXD/Ty recombinant inbred mice. Alcohol Clin Exp Res. 24:1327-1334.
Howard, R.J., P.A. Slesinger, D.L. Davies, J. Das, J.R. Trudell, and R.A. Harris. 2011.
Alcohol-binding sites in distinct brain proteins: the quest for atomic level
resolution. Alcohol Clin Exp Res. 35:1561-1573.
Hunter, W.L., and P.B. Bennett. 1974. The Causes, Mechanisms and Prevention of the
High Pressure Nervous Syndrome. Undersea Biomed Res. 1:1-28.
Jenkins, A., E.P. Greenblatt, H.J. Faulkner, E. Bertaccini, A. Light, A. Lin, A. Andreasen,
A. Viner, J.R. Trudell, and N.L. Harrison. 2001. Evidence for a common binding
cavity for three general anesthetics within the GABAA receptor. J Neurosci.
21:RC136.
Jia, F., D. Chandra, G.E. Homanics, and N.L. Harrison. 2008. Ethanol modulates synaptic
and extrasynaptic GABAA receptors in the thalamus. J Pharmacol Exp Ther.
326:475-482.

59

Johnson, B.A. 2008. Update on neuropharmacological treatments for alcoholism:
scientific basis and clinical findings. Biochem Pharmacol. 75:34-56.
Johnson, B.A., G.M. Campling, P. Griffiths, and P.J. Cowen. 1993. Attenuation of some
alcohol-induced mood changes and the desire to drink by 5-HT3 receptor
blockade: a preliminary study in healthy male volunteers. Psychopharmacology.
112:142-144.
Johnson, B.A., N. Rosenthal, J.A. Capece, F. Wiegand, L. Mao, K. Beyers, A. McKay, N.
Ait-Daoud, R.F. Anton, D.A. Ciraulo, H.R. Kranzler, K. Mann, S.S. O'Malley,
and R.M. Swift. 2007. Topiramate for treating alcohol dependence: a randomized
controlled trial. JAMA. 298:1641-1651.
Johnson, F.H., D. Brown, and D. Marsland. 1942. A Basic Mechanism in the Biological
Effects of Temperature, Pressure and Narcotics. Science. 95:200-203.
Johnson, F.H., and E.A. Flagler. 1950. Hydrostatic pressure reversal of narcosis in
tadpoles. Science. 112:91-92.
Jones, M.V., and N.L. Harrison. 1993. Effects of volatile anesthetics on the kinetics of
inhibitory postsynaptic currents in cultured rat hippocampal neurons. J
Neurophysiol. 70:1339-1349.
June, H.L., Sr., K.L. Foster, W.J. Eiler, 2nd, J. Goergen, J.B. Cook, N. Johnson, B.
Mensah-Zoe, J.O. Simmons, H.L. June, Jr., W. Yin, J.M. Cook, and G.E.
Homanics. 2007. Dopamine and benzodiazepine-dependent mechanisms regulate
the EtOH-enhanced locomotor stimulation in the GABAA alpha1 subunit null
mutant mice. Neuropsychopharmacology. 32:137-152.
Jung, S., M.H. Akabas, and R.A. Harris. 2005. Functional and structural analysis of the
GABAA receptor alpha 1 subunit during channel gating and alcohol modulation.
J Biol Chem. 280:308-316.
Jung, S., and R.A. Harris. 2006. Sites in TM2 and 3 are critical for alcohol-induced
conformational changes in GABA receptors. J Neurochem. 96:885-892.
Kamens, H.M., J. Andersen, and M.R. Picciotto. 2010. The nicotinic acetylcholine
receptor partial agonist varenicline increases the ataxic and sedative-hypnotic
effects of acute ethanol administration in C57BL/6J mice. Alcohol Clin Exp Res.
34:2053-2060.
Kandel, L., B.S. Chortkoff, J. Sonner, M.J. Laster, and E.I. Eger, 2nd. 1996.
Nonanesthetics can suppress learning. Anesth Analg. 82:321-326.
Kang-Park, M.H., B.L. Kieffer, A.J. Roberts, G.R. Siggins, and S.D. Moore. 2007.
Presynaptic delta opioid receptors regulate ethanol actions in central amygdala. J
Pharmacol Exp Ther. 320:917-925.
Kash, T.L., M.J. Dizon, J.R. Trudell, and N.L. Harrison. 2004a. Charged residues in the
beta2 subunit involved in GABAA receptor activation. J Biol Chem. 279:4887-
4893.
Kash, T.L., J.R. Trudell, and N.L. Harrison. 2004b. Structural elements involved in
activation of the gamma-aminobutyric acid type A (GABAA) receptor. Biochem
Soc Trans. 32:540-546.
Kelm, M.K., H.E. Criswell, and G.R. Breese. 2007. Calcium release from presynaptic
internal stores is required for ethanol to increase spontaneous gamma-
aminobutyric acid release onto cerebellum Purkinje neurons. J Pharmacol Exp
Ther. 323:356-364.

60

Kelm, M.K., H.E. Criswell, and G.R. Breese. 2008. The role of protein kinase A in the
ethanol-induced increase in spontaneous GABA release onto cerebellar Purkinje
neurons. J Neurophysiol. 100:3417-3428.
Kelm, M.K., H.E. Criswell, and G.R. Breese. 2011. Ethanol-enhanced GABA release: a
focus on G protein-coupled receptors. Brain Res Rev. 65:113-123.
Kerr, W.C., D. Patterson, T.K. Greenfield, A.S. Jones, K.A. McGeary, J.V. Terza, and
C.J. Ruhm. 2013. U.S. alcohol affordability and real tax rates, 1950-2011. Am J
Prev Med. 44:459-464.
Knapp, D.J., and L.A. Pohorecky. 1992. Zacopride, a 5-HT3 receptor antagonist, reduces
voluntary ethanol consumption in rats. Pharmacol Biochem Behav. 41:847-850.
Koblin, D.D., B.S. Chortkoff, M.J. Laster, E.I. Eger, 2nd, M.J. Halsey, and P. Ionescu.
1994. Polyhalogenated and perfluorinated compounds that disobey the Meyer-
Overton hypothesis. Anesth Analg. 79:1043-1048.
Kopp Lugli, A., C.S. Yost, and C.H. Kindler. 2009. Anaesthetic mechanisms: update on
the challenge of unravelling the mystery of anaesthesia. Euro J Anaesthesiol.
26:807-820.
Kozell, L.B., N.A. Walter, L.C. Milner, K. Wickman, and K.J. Buck. 2009. Mapping a
barbiturate withdrawal locus to a 0.44 Mb interval and analysis of a novel null
mutant identify a role for Kcnj9 (GIRK3) in withdrawal from pentobarbital,
zolpidem, and ethanol. J Neurosci. 29:11662-11673.
Kralic, J.E., M. Wheeler, K. Renzi, C. Ferguson, T.K. O'Buckley, A.C. Grobin, A.L.
Morrow, and G.E. Homanics. 2003. Deletion of GABAA receptor alpha 1
subunit-containing receptors alters responses to ethanol and other anesthetics. J
Pharmacol Exp Ther. 305:600-607.
Krystal, J.H., I.L. Petrakis, E. Krupitsky, C. Schutz, L. Trevisan, and D.C. D'Souza.
2003a. NMDA receptor antagonism and the ethanol intoxication signal: from
alcoholism risk to pharmacotherapy. Ann N Y Acad Sci. 1003:176-184.
Krystal, J.H., I.L. Petrakis, D. Limoncelli, E. Webb, R. Gueorgueva, D.C. D'Souza, N.N.
Boutros, L. Trevisan, and D.S. Charney. 2003b. Altered NMDA glutamate
receptor antagonist response in recovering ethanol-dependent patients.
Neuropsychopharmacology. 28:2020-2028.
Krystal, J.H., I.L. Petrakis, G. Mason, L. Trevisan, and D.C. D'Souza. 2003c. N-methyl-
D-aspartate glutamate receptors and alcoholism: reward, dependence, treatment,
and vulnerability. Pharmacol Ther. 99:79-94.
Kucken, A.M., D.A. Wagner, P.R. Ward, J.A. Teissere, A.J. Boileau, and C. Czajkowski.
2000. Identification of benzodiazepine binding site residues in the gamma2
subunit of the gamma-aminobutyric acid(A) receptor. Mol Pharmacol. 57:932-
939.
Kumar, S., P. Porcu, D.F. Werner, D.B. Matthews, J.L. Diaz-Granados, R.S. Helfand,
and A.L. Morrow. 2009. The role of GABA(A) receptors in the acute and chronic
effects of ethanol: a decade of progress. Psychopharmacology. 205:529-564.
Legendre, P. 2001. The glycinergic inhibitory synapse. Cellular and molecular life
sciences : CMLS. 58:760-793.
Lesscher, H.M., M.J. Wallace, L. Zeng, V. Wang, J.K. Deitchman, T. McMahon, R.O.
Messing, and P.M. Newton. 2009. Amygdala protein kinase C epsilon controls
alcohol consumption. Genes Brain Behav. 8:493-499.

61

Lever, M.J., K.W. Miller, W.D. Paton, and E.B. Smith. 1971. Pressure reversal of
anaesthesia. Nature. 231:368-371.
Li, J., H. Nie, W. Bian, V. Dave, P.H. Janak, and J.H. Ye. 2012. Microinjection of
glycine into the ventral tegmental area selectively decreases ethanol consumption.
J Pharmacol Exp Ther. 341:196-204.
Li, T.K., B.G. Hewitt, and B.F. Grant. 2004. Alcohol use disorders and mood disorders: a
National Institute on Alcohol Abuse and Alcoholism perspective. Biol Psychiatry.
56:718-720.
Liljequist, S., and J. Engel. 1982. Effects of GABAergic agonists and antagonists on
various ethanol-induced behavioral changes. Psychopharmacology. 78:71-75.
Liljequist, S., and J.A. Engel. 1984. The effects of GABA and benzodiazepine receptor
antagonists on the anti-conflict actions of diazepam or ethanol. Pharmacol
Biochem Behav. 21:521-525.
Little, H.J. 1996. How has molecular pharmacology contributed to our understanding of
the mechanism(s) of general anesthesia? Pharmacol Ther. 69:37-58.
Lobo, I.A., and R.A. Harris. 2008. GABA(A) receptors and alcohol. Pharmacol Biochem
Behav. 90:90-94.
Lobo, I.A., R.A. Harris, and J.R. Trudell. 2008. Cross-linking of sites involved with
alcohol action between transmembrane segments 1 and 3 of the glycine receptor
following activation. J Neurochem. 104:1649-1662.
Lobo, I.A., M.P. Mascia, J.R. Trudell, and R.A. Harris. 2004a. Channel gating of the
glycine receptor changes accessibility to residues implicated in receptor
potentiation by alcohols and anesthetics. J Biol Chem. 279:33919-33927.
Lobo, I.A., J.R. Trudell, and R.A. Harris. 2004b. Cross-linking of glycine receptor
transmembrane segments two and three alters coupling of ligand binding with
channel opening. J Neurochem. 90:962-969.
Lobo, I.A., J.R. Trudell, and R.A. Harris. 2006. Accessibility to residues in
transmembrane segment four of the glycine receptor. Neuropharmacology.
50:174-181.
Lynch, J.W. 2004. Molecular structure and function of the glycine receptor chloride
channel. Physiol Rev. 84:1051-1095.
Lysko, G.S., J.L. Robinson, R. Casto, and R.A. Ferrone. 1994. The stereospecific effects
of isoflurane isomers in vivo. Eur J Pharmacol. 263:25-29.
Mah, S.J., M.W. Fleck, and T.A. Lindsley. 2011. Ethanol alters calcium signaling in
axonal growth cones. Neuroscience. 189:384-396.
Malcolm, R.D., and R.L. Alkana. 1982. Hyperbaric ethanol antagonism: role of
temperature, blood and brain ethanol concentrations. Pharmacol Biochem Behav.
16:341-346.
Maldve, R.E., T.A. Zhang, K. Ferrani-Kile, S.S. Schreiber, M.J. Lippmann, G.L. Snyder,
A.A. Fienberg, S.W. Leslie, R.A. Gonzales, and R.A. Morrisett. 2002. DARPP-32
and regulation of the ethanol sensitivity of NMDA receptors in the nucleus
accumbens. Nat Neurosci. 5:641-648.
Martz, A., R.A. Deitrich, and R.A. Harris. 1983. Behavioral evidence for the involvement
of gamma-aminobutyric acid in the actions of ethanol. Eur J Pharmacol. 89:53-
62.

62

Mascia, M.P., T.K. Machu, and R.A. Harris. 1996a. Enhancement of homomeric glycine
receptor function by long-chain alcohols and anaesthetics. Br J Pharmacol.
119:1331-1336.
Mascia, M.P., S.J. Mihic, C.F. Valenzuela, P.R. Schofield, and R.A. Harris. 1996b. A
single amino acid determines differences in ethanol actions on strychnine-
sensitive glycine receptors. Mol Pharmacol. 50:402-406.
Mascia, M.P., J.R. Trudell, and R.A. Harris. 2000. Specific binding sites for alcohols and
anesthetics on ligand-gated ion channels. Proc Natl Acad Sci U S A. 97:9305-
9310.
McCracken, L.M., Y.A. Blednov, J.R. Trudell, J.M. Benavidez, H. Betz, and R.A. Harris.
2013. Mutation of a zinc-binding residue in the glycine receptor alpha1 subunit
changes ethanol sensitivity in vitro and alcohol consumption in vivo. J Pharmacol
Exp Ther. 344:489-500.
Mehta, A.K., and M.K. Ticku. 1989. Chronic ethanol treatment alters the behavioral
effects of Ro 15-4513, a partially negative ligand for benzodiazepine binding
sites. Brain Res. 489:93-100.
Meyer, H. 1899. Welche Eigenschaft der Anästhetika bedingt ihre narkotische Wirkung?
Arch Exp Pathol Pharmakol (Naunyn-Schmiedebergs). 42:109–118.
Mhatre, M., and M.K. Ticku. 1989. Chronic ethanol treatment selectively increases the
binding of inverse agonists for benzodiazepine binding sites in cultured spinal
cord neurons. J Pharmacol Exp Ther. 251:164-168.
Mihalek, R.M., B.J. Bowers, J.M. Wehner, J.E. Kralic, M.J. VanDoren, A.L. Morrow,
and G.E. Homanics. 2001. GABA(A)-receptor delta subunit knockout mice have
multiple defects in behavioral responses to ethanol. Alcohol Clin Exp Res.
25:1708-1718.
Mihic, S.J., P.J. Whiting, and R.A. Harris. 1994a. Anaesthetic concentrations of alcohols
potentiate GABAA receptor-mediated currents: lack of subunit specificity. Eur J
Pharmacol. 268:209-214.
Mihic, S.J., P.J. Whiting, R.L. Klein, K.A. Wafford, and R.A. Harris. 1994b. A single
amino acid of the human gamma-aminobutyric acid type A receptor gamma 2
subunit determines benzodiazepine efficacy. J Biol Chem. 269:32768-32773.
Mihic, S.J., Q. Ye, M.J. Wick, V.V. Koltchine, M.D. Krasowski, S.E. Finn, M.P. Mascia,
C.F. Valenzuela, K.K. Hanson, E.P. Greenblatt, R.A. Harris, and N.L. Harrison.
1997. Sites of alcohol and volatile anaesthetic action on GABA(A) and glycine
receptors. Nature. 389:385-389.
Miller, K.W. 1977. The opposing physiological effects of high pressures and inert gases.
Fed Proc. 36:1663-1667.
Miller, K.W., W.D. Paton, R.A. Smith, and E.B. Smith. 1973. The pressure reversal of
general anesthesia and the critical volume hypothesis. Mol Pharmacol. 9:131-143.
Miller, K.W., and M.W. Wilson. 1978. The pressure reversal of a variety of anesthetic
agents in mice. Anesthesiology. 48:104-110.
Mody, I., J. Glykys, and W. Wei. 2007. A new meaning for "Gin & Tonic": tonic
inhibition as the target for ethanol action in the brain. Alcohol. 41:145-153.
Molander, A., H.H. Lido, E. Lof, M. Ericson, and B. Soderpalm. 2007. The glycine
reuptake inhibitor Org 25935 decreases ethanol intake and preference in male
wistar rats. Alcohol Alcohol. 42:11-18.

63

Molander, A., and B. Soderpalm. 2005. Accumbal strychnine-sensitive glycine receptors:
an access point for ethanol to the brain reward system. Alcohol Clin Exp Res.
29:27-37.
Mori, T., N. Matubayasi, and I. Ueda. 1984. Membrane expansion and inhalation
anesthetics. Mean excess volume hypothesis. Mol Pharmacol. 25:123-130.
Morrow, A.L., K. Ferrani-Kile, M.I. Davis, J.A. Shumilla, S. Kumar, R. Maldve, and
S.C. Pandey. 2004. Ethanol effects on cell signaling mechanisms. Alcohol Clin
Exp Res. 28:217-227.
Moy, S.S., D.J. Knapp, H.E. Criswell, and G.R. Breese. 1997. Flumazenil blockade of
anxiety following ethanol withdrawal in rats. Psychopharmacology. 131:354-360.
Mullins, L.J., and C.T. Gaffey. 1954. The depolarization of nerve by organic compounds.
Proc Soc Exp Biol Med. 85:144-149.
Murail, S., R.J. Howard, T. Broemstrup, E.J. Bertaccini, R.A. Harris, J.R. Trudell, and E.
Lindahl. 2012. Molecular mechanism for the dual alcohol modulation of Cys-loop
receptors. PLoS Comput Biol. 8:e1002710.
Nie, Z., P. Schweitzer, A.J. Roberts, S.G. Madamba, S.D. Moore, and G.R. Siggins.
2004. Ethanol augments GABAergic transmission in the central amygdala via
CRF1 receptors. Science. 303:1512-1514.
Nutt, D.J., and R.G. Lister. 1988. Antagonizing the anticonvulsant effect of ethanol using
drugs acting at the benzodiazepine/GABA receptor complex. Pharmacol Biochem
Behav. 31:751-755.
Olsen, R.W., H.J. Hanchar, P. Meera, and M. Wallner. 2007. GABAA receptor subtypes:
the "one glass of wine" receptors. Alcohol. 41:201-209.
Olsen, R.W., and W. Sieghart. 2008. International Union of Pharmacology. LXX.
Subtypes of gamma-aminobutyric acid(A) receptors: classification on the basis of
subunit composition, pharmacology, and function. Update. Pharmacol Rev.
60:243-260.
Overton, E. 1899. Ueber die allgemeinen osmotischen Eigenschaften der Zelle, ihre
vermutlichen Ursachen und ihre Bedeutung für die Physiologie. Vierteljahres schr
Naturforsch Ges Zürich. 44:88-135.
Overton, E. 1901. Studien über die Narkose. Zugleich ein Beitrag zur allgemeinen
Pharmakologie. Jena, Germany: Gustav Fischer.
Pang, Y.C., P.E. Reid, D.E. Brooks, K.M. Leighton, and C. Bruce. 1980. Uptake and
distribution of halothane in dog blood. Can J Physiol Pharmacol. 58:1078-1085.
Perkins, D.I., J.R. Trudell, L. Asatryan, D.L. Davies, and R.L. Alkana. 2012. Charge and
geometry of residues in the loop 2 beta hairpin differentially affect agonist and
ethanol sensitivity in glycine receptors. J Pharmacol Exp Ther. 341:543-551.
Perkins, D.I., J.R. Trudell, D.K. Crawford, R.L. Alkana, and D.L. Davies. 2008. Targets
for ethanol action and antagonism in loop 2 of the extracellular domain of glycine
receptors. J Neurochem. 106:1337-1349.
Perkins, D.I., J.R. Trudell, D.K. Crawford, R.L. Alkana, and D.L. Davies. 2010.
Molecular targets and mechanisms for ethanol action in glycine receptors.
Pharmacol Ther. 127:53-65.
Perkins, D.I., J.R. Trudell, D.K. Crawford, L. Asatryan, R.L. Alkana, and D.L. Davies.
2009. Loop 2 structure in glycine and GABA(A) receptors plays a key role in
determining ethanol sensitivity. J Biol Chem. 284:27304-27314.

64

Pinder, R.M., and M. Sandler. 2004. Alcohol, wine and mental health: focus on dementia
and stroke. J Psychopharmacol. 18:449-456.
Popova, M., L. Asatryan, O. Ostrovskaya, L.R. Wyatt, K. Li, R.L. Alkana, and D.L.
Davies. 2010. A point mutation in the ectodomain-transmembrane 2 interface
eliminates the inhibitory effects of ethanol in P2X4 receptors. J Neurochem.
112:307-317.
Popova, M., J. Trudell, K. Li, R. Alkana, D. Davies, and L. Asatryan. 2013. Tryptophan
46 is a site for ethanol and ivermectin action in P2X4 receptors. Purinergic
Signal.
Pringle, M.J., K.B. Brown, and K.W. Miller. 1981. Can the lipid theories of anesthesia
account for the cutoff in anesthetic potency in homologous series of alcohols?
Mol Pharmacol. 19:49-55.
Proctor, W.R., W. Poelchen, B.J. Bowers, J.M. Wehner, R.O. Messing, and T.V.
Dunwiddie. 2003. Ethanol differentially enhances hippocampal GABA A
receptor-mediated responses in protein kinase C gamma (PKC gamma) and PKC
epsilon null mice. J Pharmacol Exp Ther. 305:264-270.
Raines, D.E., and K.W. Miller. 1994. On the importance of volatile agents devoid of
anesthetic action. Anesth Analg. 79:1031-1033.
Rehm, J. 2011. The risks associated with alcohol use and alcoholism. Alcohol Res Health.
34:135-143.
Rehm, J., C. Mathers, S. Popova, M. Thavorncharoensap, Y. Teerawattananon, and J.
Patra. 2009. Global burden of disease and injury and economic cost attributable to
alcohol use and alcohol-use disorders. Lancet. 373:2223-2233.
Ripley, T.L., S.J. Dunworth, and D.N. Stephens. 2002. Consequences of amygdala
kindling and repeated withdrawal from ethanol on amphetamine-induced
behaviours. Eur J Neurosci. 16:1129-1138.
Roberto, M., M. Cruz, M. Bajo, G.R. Siggins, L.H. Parsons, and P. Schweitzer. 2010a.
The endocannabinoid system tonically regulates inhibitory transmission and
depresses the effect of ethanol in central amygdala. Neuropsychopharmacology.
35:1962-1972.
Roberto, M., M.T. Cruz, N.W. Gilpin, V. Sabino, P. Schweitzer, M. Bajo, P. Cottone,
S.G. Madamba, D.G. Stouffer, E.P. Zorrilla, G.F. Koob, G.R. Siggins, and L.H.
Parsons. 2010b. Corticotropin releasing factor-induced amygdala gamma-
aminobutyric Acid release plays a key role in alcohol dependence. Biol
Psychiatry. 67:831-839.
Roberto, M., and G.R. Siggins. 2006. Nociceptin/orphanin FQ presynaptically decreases
GABAergic transmission and blocks the ethanol-induced increase of GABA
release in central amygdala. Proc Natl Acad Sci U S A. 103:9715-9720.
Ron, D., and R. Jurd. 2005. The "ups and downs" of signaling cascades in addiction. Sci
STKE. 2005:re14.
Roth, S.H. 1979. Physical mechanisms of anesthesia. Annu Rev Pharmacol Toxicol.
19:159-178.
Ryan, S.G., M.S. Buckwalter, J.W. Lynch, C.A. Handford, L. Segura, R. Shiang, J.J.
Wasmuth, S.A. Camper, P. Schofield, and P. O'Connell. 1994. A missense
mutation in the gene encoding the alpha 1 subunit of the inhibitory glycine
receptor in the spasmodic mouse. Nat Genet. 7:131-135.

65

San Martin, L., F. Cerda, V. Jimenez, J. Fuentealba, B. Munoz, L.G. Aguayo, and L.
Guzman. 2012. Inhibition of the ethanol-induced potentiation of alpha1 glycine
receptor by a small peptide that interferes with Gbetagamma binding. J Biol
Chem. 287:40713-40721.
Santhakumar, V., M. Wallner, and T.S. Otis. 2007. Ethanol acts directly on extrasynaptic
subtypes of GABAA receptors to increase tonic inhibition. Alcohol. 41:211-221.
Sauguet, L., R.J. Howard, L. Malherbe, U.S. Lee, P.J. Corringer, R.A. Harris, and M.
Delarue. 2013. Structural basis for potentiation by alcohols and anaesthetics in a
ligand-gated ion channel. Nat Commun. 4:1697.
Saul, B., V. Schmieden, C. Kling, C. Mulhardt, P. Gass, J. Kuhse, and C.M. Becker.
1994. Point mutation of glycine receptor alpha 1 subunit in the spasmodic mouse
affects agonist responses. FEBS Lett. 350:71-76.
Shahidullah, M., T. Harris, M.W. Germann, and M. Covarrubias. 2003. Molecular
features of an alcohol binding site in a neuronal potassium channel. Biochemistry.
42:11243-11252.
Sigel, E., and A. Buhr. 1997. The benzodiazepine binding site of GABAA receptors.
Trends Pharmacol Sci. 18:425-429.
Sigel, E., and M.E. Steinmann. 2012. Structure, function, and modulation of GABA(A)
receptors. J Biol Chem. 287:40224-40231.
Silberman, Y., O.J. Ariwodola, and J.L. Weiner. 2009. Differential effects of GABAB
autoreceptor activation on ethanol potentiation of local and lateral paracapsular
GABAergic synapses in the rat basolateral amygdala. Neuropharmacology.
56:886-895.
Simpson, C.A., and C.R. Rush. 2002. Acute performance-impairing and subject-rated
effects of triazolam and temazepam, alone and in combination with ethanol, in
humans. J Psychopharmacol. 16:23-34.
Sonner, J.M., J. Li, and E.I. Eger, 2nd. 1998. Desflurane and the nonimmobilizer 1,2-
dichlorohexafluorocyclobutane suppress learning by a mechanism independent of
the level of unconditioned stimulation. Anesth Analg. 87:200-205.
Steensland, P., J.A. Simms, J. Holgate, J.K. Richards, and S.E. Bartlett. 2007.
Varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist,
selectively decreases ethanol consumption and seeking. Proc Natl Acad Sci U S A.
104:12518-12523.
Stephens, D.N., J. Pistovcakova, L. Worthing, J.R. Atack, and G.R. Dawson. 2005. Role
of GABAA alpha5-containing receptors in ethanol reward: the effects of targeted
gene deletion, and a selective inverse agonist. Eur J Pharmacol. 526:240-250.
Suzdak, P.D., R.D. Schwartz, P. Skolnick, and S.M. Paul. 1986. Ethanol stimulates
gamma-aminobutyric acid receptor-mediated chloride transport in rat brain
synaptoneurosomes. Proc Natl Acad Sci U S A. 83:4071-4075.
Syapin, P.J., J. Chen, D.A. Finn, and R.L. Alkana. 1988. Antagonism of ethanol-induced
depression of mouse locomotor activity by hyperbaric exposure. Life Sci.
43:2221-2229.
Takiguchi, A., T. Masuoka, Y. Yamamoto, A. Mikami, and C. Kamei. 2006. Potentiation
of ethanol in spatial memory deficits induced by some benzodiazepines. J
Pharmacol Sci. 101:325-328.

66

Tanabe, J., J.R. Tregellas, M. Dalwani, L. Thompson, E. Owens, T. Crowley, and M.
Banich. 2009. Medial orbitofrontal cortex gray matter is reduced in abstinent
substance-dependent individuals. Biol Psychiatry. 65:160-164.
Tauber, M., E. Calame-Droz, L. Prut, U. Rudolph, and F. Crestani. 2003. alpha2-gamma-
Aminobutyric acid (GABA)A receptors are the molecular substrates mediating
precipitation of narcosis but not of sedation by the combined use of diazepam and
alcohol in vivo. Eur J Neurosci. 18:2599-2604.
Theile, J.W., H. Morikawa, R.A. Gonzales, and R.A. Morrisett. 2009. Role of 5-
hydroxytryptamine2C receptors in Ca2+-dependent ethanol potentiation of
GABA release onto ventral tegmental area dopamine neurons. J Pharmacol Exp
Ther. 329:625-633.
Ticku, M.K., and T. Burch. 1980. Purine inhibition of [3H]-gamma-aminobutyric acid
receptor binding to rat brain membranes. Biochem Pharmacol. 29:1217-1220.
Trudell, J.R., W.L. Hubbell, and E.N. Cohen. 1973. Pressure reversal of inhalation
anesthetic-induced disorder in spin-labeled phospholipid vesicles. Biochim
Biophys Acta. 291:328-334.
Ueda, I., M. Hirakawa, K. Arakawa, and H. Kamaya. 1986. Do anesthetics fluidize
membranes? Anesthesiology. 64:67-71.
Unwin, N. 2003. Structure and action of the nicotinic acetylcholine receptor explored by
electron microscopy. FEBS Lett. 555:91-95.
Unwin, N., A. Miyazawa, J. Li, and Y. Fujiyoshi. 2002. Activation of the nicotinic
acetylcholine receptor involves a switch in conformation of the alpha subunits. J
Mol Biol. 319:1165-1176.
Verdejo-Garcia, A., A. Bechara, E.C. Recknor, and M. Perez-Garcia. 2006. Executive
dysfunction in substance dependent individuals during drug use and abstinence:
an examination of the behavioral, cognitive and emotional correlates of addiction.
J Int Neuropsychol Soc. 12:405-415.
Wallner, M., H.J. Hanchar, and R.W. Olsen. 2003. Ethanol enhances alpha 4 beta 3 delta
and alpha 6 beta 3 delta gamma-aminobutyric acid type A receptors at low
concentrations known to affect humans. Proc Natl Acad Sci U S A. 100:15218-
15223.
Wallner, M., H.J. Hanchar, and R.W. Olsen. 2006. Low-dose alcohol actions on
alpha4beta3delta GABAA receptors are reversed by the behavioral alcohol
antagonist Ro15-4513. Proc Natl Acad Sci U S A. 103:8540-8545.
Wann, K.T., and A.G. MacDonald. 1988. Actions and Interactions of High Pressure and
General Anesthetics. Prog Neurobiol. 30:271-307.
Wei, W., L.C. Faria, and I. Mody. 2004. Low ethanol concentrations selectively augment
the tonic inhibition mediated by delta subunit-containing GABAA receptors in
hippocampal neurons. J Neurosci. 24:8379-8382.
Weight, F.F., C. Li, and R.W. Peoples. 1999. Alcohol action on membrane ion channels
gated by extracellular ATP (P2X receptors). Neurochem Int. 35:143-152.
Weiner, J.L., C. Gu, and T.V. Dunwiddie. 1997. Differential ethanol sensitivity of
subpopulations of GABAA synapses onto rat hippocampal CA1 pyramidal
neurons. J Neurophysiol. 77:1306-1312.
Weiner, J.L., and C.F. Valenzuela. 2006. Ethanol modulation of GABAergic
transmission: the view from the slice. Pharmacol Ther. 111:533-554.

67

Werner, D.F., Y.A. Blednov, O.J. Ariwodola, Y. Silberman, E. Logan, R.B. Berry, C.M.
Borghese, D.B. Matthews, J.L. Weiner, N.L. Harrison, R.A. Harris, and G.E.
Homanics. 2006. Knockin mice with ethanol-insensitive alpha1-containing
gamma-aminobutyric acid type A receptors display selective alterations in
behavioral responses to ethanol. J Pharmacol Exp Ther. 319:219-227.
Werner, D.F., A.R. Swihart, C. Ferguson, W.R. Lariviere, N.L. Harrison, and G.E.
Homanics. 2009. Alcohol-induced tolerance and physical dependence in mice
with ethanol insensitive alpha1 GABA A receptors. Alcohol Clin Exp Res.
33:289-299.
White, G., D.M. Lovinger, and F.F. Weight. 1990. Ethanol inhibits NMDA-activated
current but does not alter GABA-activated current in an isolated adult mammalian
neuron. Brain Res. 507:332-336.
Wick, M.J., S.J. Mihic, S. Ueno, M.P. Mascia, J.R. Trudell, S.J. Brozowski, Q. Ye, N.L.
Harrison, and R.A. Harris. 1998. Mutations of gamma-aminobutyric acid and
glycine receptors change alcohol cutoff: evidence for an alcohol receptor? Proc
Natl Acad Sci U S A. 95:6504-6509.
Wieland, H.A., H. Luddens, and P.H. Seeburg. 1992. A single histidine in GABAA
receptors is essential for benzodiazepine agonist binding. J Biol Chem. 267:1426-
1429.
Williams, K.L., A.P. Ferko, E.J. Barbieri, and G.J. DiGregorio. 1995. Glycine enhances
the central depressant properties of ethanol in mice. Pharmacol Biochem Behav.
50:199-205.
Yamakura, T., and R.A. Harris. 2000. Effects of gaseous anesthetics nitrous oxide and
xenon on ligand-gated ion channels. Comparison with isoflurane and ethanol.
Anesthesiology. 93:1095-1101.
Yamakura, T., S.J. Mihic, and R.A. Harris. 1999. Amino acid volume and hydropathy of
a transmembrane site determine glycine and anesthetic sensitivity of glycine
receptors. J Biol Chem. 274:23006-23012.
Yardley, M.M., L. Wyatt, S. Khoja, L. Asatryan, M.J. Ramaker, D.A. Finn, R.L. Alkana,
N. Huynh, S.G. Louie, N.A. Petasis, M. Bortolato, and D.L. Davies. 2012.
Ivermectin reduces alcohol intake and preference in mice. Neuropharmacology.
63:190-201.
Ye, J.H., L. Tao, J. Ren, R. Schaefer, K. Krnjevic, P.L. Liu, D.A. Schiller, and J.J.
McArdle. 2001a. Ethanol potentiation of glycine-induced responses in dissociated
neurons of rat ventral tegmental area. J Pharmacol Exp Ther. 296:77-83.
Ye, J.H., L. Tao, L. Zhu, K. Krnjevic, and J.J. McArdle. 2001b. Ethanol inhibition of
glycine-activated responses in neurons of ventral tegmental area of neonatal rats.
J Neurophysiol. 86:2426-2434.
Ye, Q., V.V. Koltchine, S.J. Mihic, M.P. Mascia, M.J. Wick, S.E. Finn, N.L. Harrison,
and R.A. Harris. 1998. Enhancement of glycine receptor function by ethanol is
inversely correlated with molecular volume at position alpha267. J Biol Chem.
273:3314-3319.
Yevenes, G.E., G. Moraga-Cid, L. Guzman, S. Haeger, L. Oliveira, J. Olate, G.
Schmalzing, and L.G. Aguayo. 2006. Molecular determinants for G protein
betagamma modulation of ionotropic glycine receptors. J Biol Chem. 281:39300-
39307.

68

Yevenes, G.E., G. Moraga-Cid, R.W. Peoples, G. Schmalzing, and L.G. Aguayo. 2008. A
selective G betagamma-linked intracellular mechanism for modulation of a
ligand-gated ion channel by ethanol. Proc Natl Acad Sci U S A. 105:20523-20528.
Yoshimura, H., M. Honjo, N. Segami, K. Kaneyama, T. Sugai, Y. Mashiyama, and N.
Onoda. 2006. Cyclic AMP-dependent attenuation of oscillatory-activity-induced
intercortical strengthening of horizontal pathways between insular and parietal
cortices. Brain Res. 1069:86-95.
Yoshimura, M., and B. Tabakoff. 1995. Selective effects of ethanol on the generation of
cAMP by particular members of the adenylyl cyclase family. Alcohol Clin Exp
Res. 19:1435-1440.
Zhu, P.J., and D.M. Lovinger. 2006. Ethanol potentiates GABAergic synaptic
transmission in a postsynaptic neuron/synaptic bouton preparation from
basolateral amygdala. J Neurophysiol. 96:433-441.

Abstract (if available)

Abstract A critical barrier to developing effective drugs to prevent and/or treat Alcohol Use Disorders (AUDs) has been a lack of specific knowledge about where ethanol acts in the brain and the resultant cascades leading to behavioral change. This search is complicated by the multiple receptors and subunit combinations affected by ethanol. Current strategies that knock-out (KO) specific receptor subunits or knock-in (KI) mutant ethanol insensitive receptors have provided important insights. However, interpretation of these studies requires testing with high ethanol concentrations (10 to 40 mM) that affect other native receptor systems making it difficult to identify the changes in ethanol-induced behaviors produced by its action on the missing or ethanol-insensitive receptor subtype. To circumvent these problems, our laboratory is developing novel tools called ultrasensitive ethanol receptors (USERs) by building on prior findings in our laboratory that manipulating Loop 2 (L2) structure of glycine receptors (GlyRs) and γ-amino butyric acid subtype-A (GABAARs) can significantly increase ethanol sensitivity of mutant receptors. These USERs would respond to extremely low ethanol concentrations (≤ 1 mM), concentrations too low to affect native receptors. This differential in ethanol sensitivity between USERs and native receptors provides the basis for exploiting these mutant receptors to identify ethanol’s neurochemical signaling cascades from its initial site(s) of action to behavioral outcomes and dissect the role specific receptor subunit combinations play in each of ethanol’s behavioral effects. The primary objective of my thesis was to identify and optimize Loop 2 mutations in GlyRs that would maximize ethanol sensitivity and enhance the potential utility of these receptors when expressed in vivo to create optimized USER GlyRs. In order to accomplish this, we adopted a two-pronged approach focused on strategically manipulating specific Loop 2 amino acid residues of the δL2 USER GlyR developed previously. This two-pronged approach is [1] to test the hypothesis that reversion of position 52 in δL2 α1 GlyRs from Ser back to Ala (S52A) will increase the sensitivity of the receptor to ethanol compared to α1 (δL2) GlyRs [2] to test the hypothesis that limiting δL2 α1 GlyR mutations to Exon 3 can create USERs. In order to test our hypotheses, we used site-directed mutagenesis to create USERs-δL2 (S52A) GlyRs and δL2 (E57D, T59R) GlyRs, and tested their sensitivities to ethanol and to agonist using two-electrode voltage clamp electrophysiology in a Xenopus oocyte expression system. As a result, we developed USER GlyRs with [1] ethanol sensitivities ≤ 0.1 mM [2] enhanced ethanol induced potentiation at extremely low ethanol concentrations versus wild-type receptors [3] mutations limited to a single Exon [4] normal receptor function. These findings show that USER GlyRs can be developed as novel tools to trace ethanol’s signaling cascades, investigate the role that specific receptor subunit combinations play in causing behavioral effects of ethanol and identify potential targets for medications development not only for the treatment of AUDs but a myriad of other neurologic disorders. The mutations also reveal important structure-function relationships of α1 GlyRs. This approach may extend to other ethanol-sensitive ligand-gated ion channels (LGICs).

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Asset Metadata

Creator Muchhala, Karan H. (author)

Core Title Developing ultrasensitive ethanol receptors (USERS) as novel tools for alcohol research: optimizing Loop 2 mutations in α1 GlyRs

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 10/08/2013

Defense Date 10/08/2013

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

Tag Alcohol,ethanol,GlyRs,Loop 2,OAI-PMH Harvest,USERS

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Alkana, Ronald L. (committee chair), Davies, Daryl L. (committee chair), Rodgers, Kathleen E. (committee member)

Creator Email karanmuchhala@gmail.com,kmuchhal@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-334919

Unique identifier UC11295309

Identifier etd-MuchhalaKa-2087.pdf (filename),usctheses-c3-334919 (legacy record id)

Legacy Identifier etd-MuchhalaKa-2087.pdf

Dmrecord 334919

Document Type Thesis

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Rights Muchhala, Karan H.

Type texts

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

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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