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Ultra-sensitive ethanol receptors as novel tools for alcohol and brain research: optimizing loop 2 mutations in α2 glycine receptors, γ2 and α1 γ-aminobutyric acid type A receptors

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Ultra-sensitive ethanol receptors as novel tools for alcohol and brain research: optimizing loop 2 mutations in α2 glycine receptors, γ2 and α1 γ-aminobutyric acid type A receptors

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Ultra-Sensitive Ethanol Receptors as Novel Tools for Alcohol and Brain
Research: Optimizing Loop 2 Mutations in α2 Glycine Receptors, γ2 and α1
γ-Aminobutyric Acid Type A Receptors

by

Yihui Wang

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
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)

May 2015

i

Dedication

To
my parents and friends
for their constant encouragement and support.

ii

Acknowledgements

To the late Dr. Ronald Alkana, who guided me in my research here and enlightened my
interests in this area.

To Drs. Curtis T. Okamoto, Enrique Cadenas and Bangyan L. Stiles for guiding and
assisting me in the writing of my thesis.

To Dr. Daryl L. Davies, Dr. Liana Asatryan, Anna Naito, Karan H. Muchhala and all lab
members for teaching, guiding and helping me in my experiments.

Some of the material and data presented in this thesis were incorporated in “Glycine and
GABA
A
Ultra-Sensitive Ethanol Receptors as Novel Tools for Alcohol and Brain
Research” by Anna Naito, Karan H. Muchhala, Liana Asatryan, James R. Trudell, Gregg
E. Homanics, Daya I. Perkins, Daryl L. Davies, and Ronald L. Alkana.

iii

Table of Contents

Dedication i

Acknowledgements ii
List of Tables vii

List of Figures vii

Abstract viii

Chapter 1 Introduction 1

1.1)
Alcohol: A double-edged sword for public health 1

1.2)
Mechanisms and sites of alcohol action 4

A) Membrane theories of alcohol action 4

B) Protein theories of alcohol action 5
1.3) Protein targets of ethanol action in CNS 6
1.4) Glycine and GABA
A
Receptors: key targets in ethanol’s CNS-mediated behavioral
effects 9
A) Glycine Receptors 9
B) GABA
A
Receptors 10
1.5) Sites of ethanol action on GlyRs and GABA
A
Rs 12
A) Intracellular Domain 13
B) Transmembrane Domain 13
C) Extracellular Domain 14
1.6) Tools for ethanol study 16
A) Knock out and knock in rodent models 17
B) Ultrasensitive ethanol receptors (USERs) for α1 GlyRs 18
1.7) Thesis proposal: Application of USERs in different subunits of Cys Loop
receptors-α2 GlyR, γ2 and α1 GABA
A
R USERs 19
1.8) Specific Aims 20

iv

Chapter 2 Experimental Procedures 22

2.1) Materials 22
2.2) Site-directed Mutagenesis 22
2.3) Expression in Oocytes 23
2.4) Whole Cell Two-electrode Voltage Clamp Recordings 24
A) Application of Agonist 24
B) Application of Ethanol 24
2.5) Data Analysis 25
Chapter 3 Loop 2 Manipulations in Multiple Subunits of GlyRs – α2 GlyR USER 27

3.1) Rationale 27
3.2) Results 28
A) Agonist Concentration Response 28
B) Ethanol Concentration Response 29
3.3) Conclusion 29
Chapter 4 Loop 2 Manipulations in Multiple Receptors of the Cys-loop superfamily
– γ2 GABA
A
R USERs 30

4.1) Rationale 30
4.2) Results 31
A) Agonist Concentration Response 31
B) Ethanol Concentration Response 31
4.3) Conclusion 32
Chapter 5 Loop 2 Manipulations in γ2 GABA
A
R USERs with Normalized Receptor
Characteristics 33

5.1) Rationale 33
5.2) Results 34
A) Agonist Concentration Response 34
B) Ethanol Concentration Response 34

v

5.3) Conclusion 35
Chapter 6 Loop 2 Manipulations in Multiple Subunits of GABA
A
Rs – α1 GABA
A
R
USER 36

6.1) Rationale 36
6.2) Results 37
A) Agonist Concentration Response 37
B) Ethanol Concentration Response 37
6.3) Conclusion 37
Chapter 7 Overall Discussion 39

Tables 42

Figures 45

Bibliography 52

vi

List of Tables

Table 1 Loop 2 sequence alignment and receptor characteristics for the human WT
and α1 and α2 GlyR USERs, and α1 and γ2 GABA
A
R USERs.
Table 2 Threshold for ethanol sensitivity in α1 and α2 GlyR USERs is bimodal and
lower than WT.
Table 3 Threshold for ethanol sensitivity of γ2 and α1 GABA
A
R USERs is bimodal
and lower than α1β2γ2 GABA
A
R WT.

vii

List of Figures

Figure 1 α1 GlyR USERs ethanol and agonist concentration response.
Figure 2 α2 GlyR USER ethanol and agonist concentration response.
Figure 3 γ2 GABA
A
R USERs ethanol and agonist concentration response.
Figure 4 α1 GABA
A
R USER ethanol and agonist concentration response.
Figure 5a Schematic of neurochemical pathways activated in response to high ethanol
concentrations (10-50 mM)
Figure 5b Schematic of USER receptor subunit activated by ultra-low ethanol
concentrations.
Figure 6 Molecular model of α1 GlyR.
Figure 7 Molecular model of the α1β2γ2 GABA
A
R with mutations in loop 2 of the γ2
subunit.

viii

Abstract

The lack of specific knowledge on where and how ethanol acts in the CNS and the
resultant neurochemical cascades leading to behavioral change limited the investigation
of ethanol action and the development of effective medications to prevent or/and treat
alcohol use disorders. To identify the role of specific individual receptor subunits in
ethanol induced behaviors, ultrasensitive ethanol receptors (USERs) were developed by
manipulating by mutagenesis the physical-chemical characteristics of extracellular
domain loop 2 region in glycine receptors (GlyRs) and γ-aminobutyric acid type A
receptors (GABA
A
Rs), which are implicated in causing many behavioral effects linked to
ethanol abuse. These mutant receptors would then theoretically allow activation of a
single receptor subunit population sensitized to extremely low ethanol concentrations
while keeping other receptor systems silenced. Based on previous studies of USERs in α1
GlyRs, in the current study we observed increased ethanol sensitivity in different subunits
of GlyRs and GABA
A
Rs. Expression in Xenopus oocytes and testing by two-electrode
voltage clamp, the last generation of USERs indicated an increase in ethanol sensitivity
of 100-fold over wild-type receptors by significantly decreasing the threshold and
increasing the magnitude of ethanol response, without altering general receptor properties.
Further application of USERs may lead to the elucidation ethanol action and mechanisms
behind complicated behavioral effects.

1

Chapter 1
Introduction
1.1) Alcohol: A double-edged sword for public health
Alcoholic beverages have been produced and consumed by human beings since the
Neolithic Era (around 10,000 B.C.) (McGovern, 2009). Global consumption of alcoholic
beverages in 2010 was equivalent to 6.2 litres of pure alcohol consumed per person aged
15 years or older, which translates into 13.5 grams of pure alcohol per day. 50.1% of the
total recorded alcohol consumption is in the form of spirits, beer accounts for 34.8%,
while only 8.0% is consumed in the form of wine (World Health Organization, 2014).
There is growing evidence that moderate consumption of alcoholic beverages may have
beneficial implications on public health (Pinder and Sandler, 2004). It has been proven
that wine has antimicrobial and antifungal properties, and may protect against many
diseases such as coronary heart disease, atherosclerosis and some forms of cancer
(Leikert et al., 2002; Vinson et al., 2001). These potential health benefits of wine may be
attributed to the high content of polyphenol antioxidants such as phenolic acids, stilbenes
and flavonoids (Kammerer et al., 2004). Similarly, recent in vitro studies have suggested
that certain compounds found in beer may possess antioxidant, anti-carcinogenic,
anti-inflammatory, estrogenic and/or antiviral properties (Arranz et al., 2012). Additional
benefits associated with moderate consumption of alcoholic beverages are still under
study (Barron et al., 2014; Mazue et al., 2014).

However, consumption of alcohol in excess has severe implications on human health and
has been identified as one of five major global risk factors for chronic disease and injury
(Lim et al., 2013; World Health Organization, 2013). Moreover, excessive alcohol

2

consumption is associated with abundant social problems including violence, psychiatric
illness, drunk driving, drug use, unsafe sex, suicide and premature death (Hill et al., 2009;
Lam and Chim, 2010)). 40% of individuals in the United States experience an
alcohol-related adverse event at some time in their lives, with alcohol accounting for up
to 55% of fatal driving events (American Psychiatric Association, 2013), thus
contributing to considerable morbidity and mortality in the United States. There is no
doubt that uncontrollable consumption of alcohol constitutes a heavy burden to health,
society and economy globally, with approximately 3.3 million deaths annually (or 5.9%
of all deaths) and 4.6% of global disability-adjusted life-years (World Health
Organization, 2014; Rehm et al., 2009). In the U.S., excessive alcohol consumption has
been associated with costs of $223.5 billion in 2006 including lost productivity,
healthcare costs, criminal justice costs and other effects and is responsible for 87,800
deaths on average from 2006 to 2010 (Bouchery et al., 2011; Prevention, 2014; Sacks et
al., 2013).

One of the most severe manifestations of excessive alcohol consumption is Alcohol Use
Disorder (AUD). AUDs affect 18.3 million Americans, 7.3% of the population, while an
estimated 21% to 42% of patients admitted to general hospital wards have AUDs (de Wit
et al., 2010). AUDs are defined as “a cluster of behavioral and physical symptoms, which
can include withdrawal, tolerance, and craving” in the 5
th
edition of the American
Psychiatric Association’s Diagnostic and Statistical Manual of Mental Disorders (DSM-5)
and can be diagnosed as “a problematic pattern of alcohol use leading to clinically
significant impairment or distress” (American Psychiatric Association, 2013). Individuals
with severe AUDs demonstrate heightened cue sensitivity, compulsive seeking, craving,
and continued alcohol use in the face of negative consequences (O'Tousa and Grahame,
2014). Repeated intake of high doses of alcohol due to AUDs can affect nearly every

3

organ system, especially the gastrointestinal tract, cardiovascular system, and the central
and peripheral nervous systems. In 2012, 33.4% of all alcohol-attributable deaths caused
by cardiovascular diseases and diabetes, 17.1% caused by unintentional injuries and 16.2%
caused by gastrointestinal diseases followed by cancers, intentional injuries, infectious
diseases and neuropsychiatric disorders (World Health Organization, 2014).

Current treatments for alcoholic patients aim at reducing or stopping alcohol
consumption and preventing relapse. There are 3 oral formulations (disulfiram,
acamprosate and oral naltrexone) and 1 injectable preparation (injectable naltrexone) that
were approved by FDA to treat AUDs (Addolorato et al., 2013; Clapp, 2012; Davies et
al., 2013). Some of them target symptoms of alcohol withdrawal working as aversion
therapy, some of them work on alcohol action receptors or metabolism pathways.
Disulfiram involves the disruption of normal alcohol metabolism. It prevents oxidation of
acetaldehyde to acetic acid by inhibiting aldehyde dehydrogenase (ALDH) enzyme
activity. Further, the accumulation of acetaldehyde in blood causes unpleasant
physiologic reactions that result in avoidance of continued drinking (Clapp, 2012). As a
functional N-methyl-D-aspartate receptor (NMDA) modulator, acamprosate works as an
antagonist of NMDA glutamate receptors, which restores the balance between excitatory
and inhibitory neurotransmission (Bouza et al., 2004; Clapp, 2012; Davies et al., 2013).
Naltrexone is an opioid antagonist, which blocks the release of alcohol-induced dopamine,
thereby reducing the stimulus and reinforcing effects of ethanol (Bouza et al., 2004).
Further it avoids the ensuing craving to drink and loss of control. However, these
medications remain problematic in their suboptimal effects and only work for a specific
subgroup of patients, because of the heterogeneity and complicated causes of alcoholism
(Addolorato et al., 2013; Franck and Jayaram-Lindstrom, 2013). Furthermore, while
several other compounds like topiramate, balclofen, ondansetron, sodium oxybate,

4

varenicline and nalmefene have been tested, only nalmefene has recently received
approval in Europe (Addolorato et al., 2013). Thus, more understanding of the
mechanism of ethanol action is of significance in developing effective drugs to treat
alcoholism.
1.2) Mechanisms and sites of alcohol action
Although alcohol affects widely the human body not only physiologically but also
behaviorally, the exact mechanisms of how it acts to produce these effects are still poorly
understood (Harris et al., 2008; Peoples et al., 1996). For over a century, ethanol was
long believed to act nonspecifically on cell membranes through the perturbation of lipid
bilayers; however, growing evidence indicates that lipid bilayers may not be the only
target. In the last two decades proteins are considered as direct targets of alcohol and is
the core of most current theories.
A) Membrane theories of alcohol action
The original membrane theories of alcohol and general anesthetic action, as formulated
by H. Meyer (Meyer, 1899, 1901) and Overton (Overton, 1901), stated that anesthetic
potency is correlated with the lipid solubility of various alcohols and anesthetics without
mentioning a proposed molecular mechanism of action. This Meyer-Overton correlation
was subsequently refined by K.H. Meyer (Meyer, 1937) and Ferguson (Ferguson, 1939),
who proposed that alcohols and anesthetics act by achieving a certain molar
concentration or thermodynamic activity (equivalent to the concentration of the agent
divided by its saturating concentration) in cellular lipids. The following refinements of
the Meyer-Overton theory include anesthetic induced volume expansion of the cell
membrane (Halsey, 1982; Mullins, 1954; Mullins and Gaffey, 1954; Seeman, 1972),
increased fluidity of the cell membrane (Ueda et al., 1986), and increased lateral surface

5

pressure (Cantor, 1997; Galla and Trudell, 1980; Halsey, 1982; Lugli et al., 2009; Perkins
et al., 2010). Further supports for these hypotheses came from the finding that animals
under conditions of increased hydrostatic or barometric pressure were resistant to the
effects of volatile anesthetics (Bailey et al., 1977). These observations, termed pressure
reversal of anesthesia, led to further development of theories focused on the disruption of
membrane structure by increased membrane fluidity (Bailey et al., 1977; Lugli et al.,
2009; Ueda et al., 1986). However, these theories conflicted with other whole organism
data showing the elevated body temperature, which disrupts membrane fluidity as much
as anesthetics, actually reduced the effects of anesthetics (Eger 2nd et al., 1965).

However, growing evidence in the last two decades indicates that the membrane lipid
bilayers may not be the sole target of alcohol and general anesthetic action (Franks and
Lieb, 1994; Koblin et al., 1994; Lugli et al., 2009; Lysko et al., 1994; Raines and Miller,
1994)
B) Protein theories of alcohol action
The idea that besides lipid bilayers there are other potential targets for alcohol action led
researchers to study protein targets as proposed mechanisms of action. The hypotheses
includes proposing alcohol acts on both lipids and proteins (Seeman, 1972), by binding
directly to proteins, producing conformational changes that diminish or abolish their
activity (Eyring et al., 1973). It has been proposed that a cavity or binding pocket of finite
size and amphiphilic nature as a primary site of alcohol action on proteins (Franks and
Lieb, 1994). To support protein theories of alcohol action, Franks and Lieb revealed that
proteins also contain lipophilic domains consistent with the continuing importance of the
Meyer-Overton correlation (Franks and Lieb, 1994).

6

A variety of protein targets have been identified in recent years range from gap junctions
of astrocytes, protein kinase C, mitochondrial proteins, membrane proteins and annular
lipids of proteins (Lugli et al., 2009; Perkins et al., 2010). These discoveries enabled
researchers to better understand the mechanisms and sites of action of alcohol behind its
physiological and behavioral effects. However, it also shifts the problem from
discovering the key physicochemical effect of alcohol on a homogenous lipid membrane
to the new burden of too many possible protein targets (Lugli et al., 2009).
1.3) Protein targets of ethanol action in CNS
Although the primary site of action differs in the two theories, both theories postulate that
the central nervous system (CNS) effects of ethanol ultimately result from alterations in
protein function (Peoples et al., 1996). As ethanol’s main target responsible for its
behavioral effects, the CNS contains multiple post-synaptic ion channels, which may be
primarily responsible for the modulation of synaptic neurotransmission of ethanol
(Howard et al., 2011; Murail et al., 2012; Sauguet et al., 2013). In addition, evidence
showed that ethanol could 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., 2011). Ethanol interacting with a myriad of targets may explain the wide
range of neuropharmacological and behavioral effects of ethanol’s interaction with
multiple signaling cascades.

Ligand-gated ion channel (LGIC) membrane proteins have recently been recognized as
important molecular targets for anesthetics and ethanol in the brain, supported by
physiological, genetic, and biochemical studies (Asatryan et al., 2010; Crawford et al.,
2007; Davies et al., 2004; Deitrich et al., 1989; Harris, 1999; Mihic et al., 1997; Olsen et
al., 2014; Perkins et al., 2012; Perkins et al., 2008; Perkins et al., 2009; Sauguet et al.,

7

2013). LGICs can be classified into three superfamilies: 1) Cys-loop superfamily
consisting of anion channel receptors for glycine, γ-amino butyric acid subtype-A
(GABA
A
) and glutamate-gated anion channels receptors, and cation channel receptors
such as nicotinic acetylcholine (nAch) and 5-hydroxytryptamine
3
(5-HT
3
) receptors; 2)
glutamate superfamily of cation channel receptors such as N-methyl D-aspartate (NMDA)
and α-amino-3-hydroxyisoxazolepropionic acid (AMPA) receptors; 3) purinergic
superfamily of ATP-gated receptors (P2XRs).

GABA
A
receptors (GABA
A
Rs) and glycine receptors (GlyRs) are inhibitory ion channels
belonging to the Cys-loop receptor family and constitute the major inhibitory
neurotransmitter systems in the brain and brainstem/spinal cord, respectively (Ortells and
Lunt, 1995). Their roles in ethanol action have been extensively studied for over decades.
As primary mediators of inhibitory neurotransmission in the mammalian CNS,
potentiation or inhibition of these receptor systems should alter the balance of neuronal
excitatory and inhibitory influences, and thus be expected to alter behavior (Mihic, 1999).
A number of behavioral effects have been attributed to ethanol actions at the GABA
A
Rs
and GlyRs including anxiolysis, sedation, dependence, reward, tolerance and impaired
cognition (Badanich et al., 2013; Chau et al., 2010; Deitrich et al., 1989; Hunt and
Majchrowicz, 1983; Li et al., 2012). Details will be described in the following section.

Cation channel receptors in Cys-loop superfamily have also been linked to a number of
ethanol’s acute behavioral effects. Kamens et al. demonstrated that nAchRs might be
involved in the ataxic and sedative effects of ethanol (Kamens et al., 2010). 5-HT
3

receptors, which may be involved in modulating alcohol consumption and other
behavioral effects such as nausea, anxiety and seizure, have been paid more and more
attention by researchers (Hauser et al., 2015; Parker et al., 1996; Schuckit et al., 1999).

8

Besides the Cys-loop superfamily, which has long been considered as the primary targets
of ethanol, protein receptors in the glutamate and purinergic families have also been
explored recently. Molecular modeling strategies designed by Chandrasekar have been
used to identify putative sites of ethanol action on NMDA receptors (Chandrasekar,
2013). Moreover, behavioral symptoms of ethanol including reward, tolerance, craving,
dependence, withdrawal and relapse have also been linked to NMDA receptors
(Chandrasekar, 2013; Krystal et al., 2003a; Krystal et al., 2003b). The P2X receptors of
purinergic receptors have also been recently investigated as a target site of ethanol
actions and possible medication development supported by evidence that these receptors
may be linked to alcohol consumption through modulation of the mesolimbic dopamine
system (Asatryan et al., 2010; Bortolato et al., 2013; Popova et al., 2013; Yardley et al.,
2012).

However, for most effects of alcohol characterized thus far, it is still unknown whether
the protein whose function is being studied actually binds alcohol, or if alcohol is instead
binding to another protein that then indirectly affects the functioning of the protein being
studied (Harris et al., 2008). Undoubtedly, ethanol acts directly and indirectly on the
plethora of CNS protein targets pre-and post-synaptically. The lack of specific knowledge
about where and how ethanol acts in the brain and the resultant neurochemical cascades
that lead to behavioral changes make it difficult to study distinct ethanol action
mechanisms and possible medication treatment. Here in our current study, we aim at
focusing on the role of individual receptor subunits in ethanol-induced behaviors
specifically in GlyRs and GABA
A
Rs.

9

1.4) Glycine and GABA
A
Receptors: key targets in ethanol’s CNS-mediated
behavioral effects
A) Glycine Receptors
As mentioned before, glycine receptors constitute the major inhibitory neurotransmitter
receptor system in the brainstem and spinal cord, however they have also been found in
higher brain regions such as the olfactory bulb, midbrain, cerebellum and cerebral cortex,
which are linked to a number of behavioral effects such as motor, sensory and other
functions (Betz, 1991; Mihic, 1999). Evidence to support the relationship between
glycine receptors and some of the behavioral effects include intracerebroventricular
administration of glycine and its precursor serine enhanced ethanol-induced loss of
righting reflex in mice in a manner that was blocked by the GlyR antagonist strychnine
(Williams et al., 1995), and a lack of glycine receptors in C. elegans may be responsible
for the failure to observe pressure antagonism of ethanol-induced immobility in these
animals (Eckenhoff and Yang, 1994). To further evaluate the effects of ethanol on
glycine receptors, electrophysiological studies showed ethanol enhanced GlyR function
in mouse and chick embryonic spinal neurons in a concentration-dependent manner
(Aguayo and Pancetti, 1994; Celentano et al., 1988); the glycine EC
50
was decreased in
the presence of 100 mM ethanol with no effect on the maximal glycinergic currents
(Aguayo et al., 1996).

GlyRs are heteropentameric proteins that consist of five subunits, which are assembled to
form a single chloride ion channel (Fig. 6). GlyR subunits cloned to date include four α
subunits (1 to 4) and one β subunit, which provide for significant potential diversity
(Lynch, 2004). Researchers have already found that adult GlyRs are constituted by a 2:3
αβ stoichiometry (Webb and Lynch, 2007); however, most of the information obtained

10

regarding the molecular structure and function of GlyRs, including ethanol studies, have
used homomeric GlyRs both in vitro and in vivo.

Previous studies have shown that GlyRs mediate part of the immobility produced by
anesthetics (Quinlan et al., 2002; Zhang et al., 2003). This conclusion is further supported
by studies in which the transgenic expression of a S276Q mutant α1 GlyR subunits
decreased sensitivity to ethanol-induced loss of righting reflex, motor incoordination
(rotarod) and inhibition of strychnine-induced seizures (Findlay et al., 2002). Recent
studies also found that GlyRs are important for regulating voluntary ethanol intake
and
may act as an enhancing point into the brain reward system, and demonstrated a strong
positive correlation between α1 subunit expression and ethanol intake (Ericson et al.,
2010; Jonsson et al., 2009; Molander et al., 2007; Molander and Söderpalm, 2005;
Perkins et al., 2010). Although the function of α1 GlyR subunits have been widely
explored, the in vivo role of α2 GlyR subunits in ethanol responses have not been
characterized despite high expression levels in the nucleus accumbens and amygdala,
areas that are important for the reward behaviors. Increasingly to date, studies have
shown the importance of α2 GlyR subunits in ethanol-related behaviors including its
regulation of ethanol consumption and the aversive response to ethanol (Blednov et al.,
2015; Davies et al., 2004; Jonsson et al., 2009; McCracken et al., 2013). Our studies here
focus on further exploration of α1 and α2 GlyR subunits.
B) GABA
A
Receptors
GABA
A
Rs are chloride channels and the major inhibitory receptors abundantly
distributed throughout the CNS (Mihic, 1999). Because of their widespread localization
throughout the CNS, they play a major role in virtually all brain physiological functions
and serve as targets of numerous classes of drugs including ethanol. GABA
A
Rs are gated

11

by GABA and mediate rapid phasic inhibitory synaptic transmission and also tonic
inhibition by producing current in extrasynaptic and presynaptic locations (Farrant and
Nusser, 2005; Mody and Pearce, 2004). They were first identified pharmacologically as
being activated by GABA and its selective agonist muscimol, blocked by bicuculline and
picrotoxin, and modulated by benzodiazepines, barbiturates, and certain other CNS
depressants (Macdonald and Olsen, 1994; Sieghart et al., 1999). GABA
A
Rs have been
explored over decades.

A number of behavioral effects have been attributed to ethanol actions at the GABA
A
Rs,
including reward, anxiolysis, sedation, motor ataxia, impaired cognition, aggression and
other behaviors related to alcohol consumption. Specifically, the duration of loss of
righting reflex induced by ethanol was increased by the GABA
A
R agonist muscimol and
decreased by the antagonist bicuculline (Liljequist and Engel, 1982; Martz et al., 1983).
Injection of bicuculline into the ventral tegmental area of the brain decreased alcohol
consumption by rats, suggesting that tonic GABA
A
R activity regulates alcohol drinking
(Nowak et al., 1998). Inverse agonists and antagonists at the benzodiazepine receptor site
on the GABA
A
R/chloride channel complex also decreased alcohol consumption by rats
(June et al., 1998a; June et al., 1998b)

Currently, the number of GABA
A
R isoforms existing in CNS is unknown. Up to date,
GABA
A
Rs are assembled from a family of 19 homologous subunit gene products and
form numerous, mostly hetero-oligomeric, pentamers (Olsen and Sieghart, 2008). Most
native GABA
A
Rs are thought to contain α, β and γ subunits (McKernan and Whiting,
1996). The vast majority of GABA
A
Rs in the CNS contain the γ2 subunit, and this is the
most abundant subunit in rat brain and in most regions based on in situ hybridization of
mRNA and immunostaining (Laurie et al., 1992; Persohn et al., 1991, 1992; Wisden et al.,

12

1992). Approximately 75 to 80% of GABA
A
Rs contain the γ2 subunit (Sieghart and Ernst,
2005; Whiting et al., 2000). Moreover, a polymorphism of the γ2 subunit of the
GABA
A
R has recently been associated with the severity of alcohol withdrawal signs
(Buck and Hood, 1998). These evidences, taken together, thus implicate the γ2 GABA
A
R
subtype as a major site for the in vivo actions of ethanol. Although less widespread than
γ2 subunits, the α1 subtype is the most abundant among all α subunits (Sieghart and
Sperk, 2002). Knockout of the α1 subunit causes total GABA
A
R content in mouse brain
to decrease by 50% (Sur et al., 2001). Besides the distribution of single subunit, in the
possible arrangements of a pentamer consisted of subunits comprised of the three
different subunit types, α1β2γ2 GABA
A
Rs are the major isoform of GABA
A
Rs (Fig. 7)
(Sigel and Steinmann, 2012). In our current study, α1β2γ2 GABA
A
R are used as
GABA
A
R WT to compare with other GABA
A
receptors with γ2 and α1 mutant subunits.
1.5) Sites of ethanol action on GlyRs and GABA
A
Rs
As mentioned above, GlyRs and GABA
A
Rs are the members of the Cys-loop pentameric
ligand-gated ion channel superfamily and share structural and functional homology on
the order of 30% identity but even greater similarity at the level of secondary and tertiary
structure with other members of that family. Such receptors are all organized as
pentameric membrane spanning proteins surrounding a central pore that forms the ion
channel through the membrane (Olsen and Sieghart, 2008). The specific structural
features shared by GlyRs and GABA
A
Rs includes a large intracellular (IC) loop between
the third and fourth TM domains, containing consensus sequences for phosphorylation by
protein kinases; four transmembrane (TM) domains; and an extracellular (EC) N-terminal
ligand binding domain (Béchade et al., 1994; DeLorey and Olsen, 1992; Kuhse et al.,
1995).

13

A) Intracellular Domain
Previous studies indicated the importance of the IC domain of α1 GlyRs in ethanol’s
molecular cascade, which may be initiated by PKA, PKC and Gβγ heterodimers (Burgos
et al., 2015; Harvey et al., 2004; Legendre, 2001; Lynch, 2004). Yevenes and colleagues
revealed that α1 GlyRs are modulated by G proteins through Gβγ heterodimers (Yevenes
et al., 2006). This work suggests the possibility of intracellular signaling elements, such
as G proteins, interact with GlyRs through the IC domain to control ethanol’s signals. As
expected, the IC domain of δ subunit-containing GABA
A
Rs also plays a significant role
in receptor internalization following ethanol exposure (Gonzalez et al., 2012). Gonzalez
and colleges identified two motifs on the intracellular region of δ subunits responsible for
ethanol-induced clathrin-mediated endocytosis, which 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). Taken together,
this evidence indicated that the intracellular loop of GlyRs and GABA
A
Rs as well as
intracellular signaling molecules are likely involved in mediating and/or modulating
ethanol’s effects on GlyR and GABA
A
R function.
B) Transmembrane Domain
Numerous experimental techniques show that the TM domain is composed of four
membrane spanning α-helices (TM1-4) (Thompson et al., 2010). The TM domains of
GlyRs and GABA
A
Rs have long been extensively explored as a putative site of ethanol’s
actions. Initial work used α1 GlyRs and ρ1 GABA
A
Rs as molecular targets for ethanol
since both receptors can be formed as functional homomers and have qualitatively
different responses to ethanol: α1 GlyRs are potentiated by ethanol while ρ1 GABA
A
Rs
are inhibited by ethanol (Mihic et al., 1997; Perkins et al., 2010). To search for the

14

potential important regions of the TM domain for ethanol’s actions, Mihic and colleges
developed chimeras of these receptors as tool to identify a key 45 amino acid sequence
spanning TM2 and TM3 domains (Mihic et al., 1997). 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. Similarly, positions S270 in TM2 and A291 in TM3 were
recognized as putative sites of ethanol action within GABA
A
Rs.

Subsequent experiments confirmed the TM domain of GlyR and GABA
A
R as an alcohol
target site, containing one or more alcohol “binding/action” pockets (Jenkins et al., 2001;
Jung et al., 2005; Jung and Harris, 2006; Lobo et al., 2008; Mascia et al., 1996a; Mascia
et al., 2000; Yamakura et al., 1999). Ye et al. concluded that molecular volume, but not
charge or polarity of the amino acids played a critical role in modulating ethanol
sensitivity at position 267 of α1 GlyRs (Ye et al., 1998).
C) Extracellular Domain
The extracellular (EC) domains of GlyRs and GABA
A
R are recognized as the sites for
agonist binding, receptor activation and allosteric modulation by agents such as
benzodiazepines and Zn
2+
(Barberis et al., 2002; Chakrapani et al., 2004; Crawford et al.,
2008; Kash et al., 2003; Kash et al., 2004). Based on this knowledge, researchers found
that the imidazobenzodiazepine Ro15-4513, a benzodiazepine inverse agonist,
antagonized 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 action
(Mehta and Ticku, 1989; Wallner et al., 2006). These studies suggested that the
extracellular region of GlyRs and GABA
A
Rs may paly a role as another potential site of
ethanol action.

15

Studies using recombinant expression systems provided more direct evidence to confirm
the role of EC domain in ethanol action. The initial recombinant studies demonstrated
that α1 GlyRs were more sensitive to ethanol than α2 GlyRs, which was further explained
by the one residue difference between α1 and α2 GlyRs (Mascia et al., 1996b); (Perkins
et al., 2010). In these studies, the researchers found that an alanine to serine exchange at
position 52 (A52S), located at the beginning of EC domain loop 2 in α1 GlyR, decreased
the ethanol sensitivity of the α1 GlyR to the point where the ethanol responses resembled
that of the α2 GlyR (Perkins et al., 2010). This finding implicated residues in the EC
domain, particularly position 52 in loop 2, as a possible site of ethanol action and/or
modulation (Fig. 6-7).

As described, 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 the mutant α1 (A52S) GlyRs (Davies et al., 2004). Conversely, a reverse
switch to alanine at 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 the 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). Perkins et al. systematically replaced alanine at
position 52 in α1 GlyR loop 2 with residues having different molecular weight, volume,

16

polarity and hydrophobicity and observed resultant changes in sensitivity to ethanol
(Perkins et al., 2008). They also studied the concentration-response for glycine activated
chloride currents in Xenopus oocytes expressing WT and mutant α1 GlyR subunits.
However, these mutants also showed changes in their receptor properties, which perform
as the changes in sensitivity to its agonist. There is a commonly seen left shift in their
concentration-response curve for glycine, implicating the abnormal receptor functions.
Furthermore, they extended the investigation to charged residues and observed the
electrostatic charge coupled with specific geometry of amino acids govern agonist and
ethanol receptor sensitivity (Perkins et al., 2012). Taken together, these data
demonstrated that physical, chemical, and structural properties associated with an alanine
to serine switch at position 52 drastically reduced α1 GlyR’s sensitivity to ethanol.

The importance of the amino acid in position 52 in loop 2 further lead to our study in
ultrasensitive ethanol receptors for α1 GlyRs.
1.6) Tools for ethanol study
The lack of a high-affinity structure-activity relationship between ethanol and its
potential targets precludes the classic approach of using specific agonist and antagonists
to identify the sites and mechanisms of ethanol action (Deitrich et al., 1989; Little, 1991).
This problem is further complicated by the study of Trudell et al. that multiple receptor
subunit combinations will be affected by ethanol, moreover, acute and chronic actions of
ethanol may occur by different mechanisms (Trudell et al., 2014). To this end, current
strategies are to develop knock out (KO) receptor subunits or knock in (KI) mutant
ethanol-insensitive receptors (Blednov et al., 2003; Chandra et al., 2008; Liang et al.,
2008; Moore et al., 2010; Trudell et al., 2014).

17

A) Knock out and knock in rodent models
As promising approaches to study the regulation of alcohol consumption and, perhaps,
the development of alcoholism, transgenic and knock out (null mutant) mice have been
extensively developed to study how receptor subunit composition influences the
pharmacological and behavioral effects of ethanol (Lobo and Harris, 2008; Trudell et al.,
2014). Up to date, mice have been developed that have individually knocked out the α1,
α2, α5, α6, β2, β3, γ2S+L, γ2L and δ GABA
A
R subunits and have been examined to
identify subunit-specific changes in the behavioral effects of alcohol (Boehm Ii et al.,
2004; Crabbe et al., 2006). Moreover, 93 ethanol-related genes in mice have been
genetically modified, including gene overexpression, gene knock out and gene knock in
to understand the role of specific proteins in ethanol action (Crabbe et al., 2006). Notably,
the knock out model of GABA receptor subunits showed pervasive evidence in the role
of this receptor in alcohol consumption when conducted the mice in the continuous
two-bottle choice test (Crabbe et al., 2006). On the other hand, even though genetic
modification of rodent models provides useful tools for ethanol study, it is important to
note that genetic deletion of key neuronal proteins, such as GABA receptors, usually
leads to compensatory changes in gene expression and brain function (Ponomarev et al.,
2006).

Knock in mice models can be used to examine the specific point mutations that alter only
a single aspect of receptor function. Using mutagenesis and a recombinant expression
system, Wieland and colleges investigated the action of benzodiazepine on GABA
A
Rs in
the CNS by a knock in mouse model (Wieland et al., 1992). Unlike the knock out mouse
model, the knock in receptor usually functions normally in all other aspects, and
compensation effects of other receptors and subunits are less likely to occur (Lobo and
Harris, 2008). However, either knock out or even knock in mice, involving functional

18

deletion or reduction in receptor sensitivity to ethanol, respectively, required the use of
relatively high ethanol concentrations to activate their receptors (Blednov et al., 2011;
Blednov et al., 2010; Borghese et al., 2006)
B) Ultrasensitive ethanol receptors (USERs) for α1 GlyRs
Decades of studies with GlyRs and GABA
A
Rs showed their differences in ethanol
sensitivity. α1 GlyRs are insensitive to ethanol concentration below 10 mM (Davies and
Alkana, 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). Moreover, 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. Based on this
knowledge together with mutating physical and chemical properties of position 52 of
loop 2 in α1 GlyRs, Perkins and colleagues in our laboratory hypothesized that by
replacing all non-conserved loop 2 residues of α1 and γ2 subunits of GlyRs and
GABA
A
Rs respectively with those of the δ GABA
A
R subunit (α1 GlyR USER 1 and γ2
GABA
A
R USER 1) their sensitivity to ethanol will increase compared to their respective
wide type (WT) receptors (Crawford et al., 2008; Perkins et al., 2008; Perkins et al., 2009;
Perkins et al., 2012). These first generation ultrasensitive ethanol receptors (USERs) not
only had a lower ethanol sensitivity threshold, but also had increased magnitudes of
responses at all ethanol concentrations (1-30 mM in GlyRs and 0.5-50 mM in GABA
A
Rs).
Therefore, these results provided a basis for further modification in other receptor
subunits to build ultrasensitive tools for ethanol study by activating single mutant
subunits of receptors possessing these subunits, while keeping others silent (Fig. 5).

To maximize ethanol sensitivity and normalize USER receptor function, Anna Naito and
Karan Muchala in our laboratory developed two optimization strategies centered on

19

manipulating the structure of the α1 GlyR USER 1 developed previously and testing their
sensitivities to ethanol and agonist using two-electrode voltage clamp electrophysiology
in Xenopus oocytes. Further experiments showed that α1 GlyR USER 2 created by
reverting Ser52 to Ala52 in α1 GlyR USER 1 has increased ethanol sensitivity compared
to α1 GlyR USER 1. They also successfully created α1 USER 3 by limiting mutations to
a single exon for improving the practicality of these mutant receptors for transgenic
animal development.

The subsequently created α1 GlyR USER 2 and 3 both can be sensitized to ethanol at
extremely low concentrations (≤ 1 mM) and normal/higher concentrations of ethanol
(30-50 mM). They both have a decreased threshold for ethanol sensitivity (i.e., an
increased ethanol sensitivity at extremely low concentrations of ethanol), as well as
increased magnitude of ethanol-induced potentiation at higher concentrations of ethanol
(10-50 mM) versus WT receptor responses. However, α1 GlyR USER 1 (consistent with
results from Perkins et al., 2009) and α1 GlyR USER 2 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 (Fig. 1; Table 1), indicating that these USERs
were more sensitive to agonist compared to WT receptors.
1.7) Thesis proposal: Application of USERs in different subunits of Cys Loop
receptors-α2 GlyR, γ2 and α1 GABA
A
R USERs
To further develop USERs in γ2 GABA
A
Rs, we used the same strategy when creating α1
GlyR USER 2 from α1 GlyR USER 1 by reverting the amino acid at the homologous
position in γ2 GABA
A
R USER 1 back to WT. The success in α1 GlyR USER 2 will
guide the application of USER strategy in γ2 GABA
A
Rs.

20

As stated in previous sections, α2 GlyRs and α1 GABA
A
Rs have been found previously
as key targets also widespread in CNS, which highlights the importance of applying the
USER strategy to these subunits to study the mechanisms of ethanol action and
circumvent or minimize the problems caused by traditional knock out studies. Thus, we
could initiate responses in the target receptor system without causing changes in other
systems by giving extremely low doses of ethanol.

In current project, agonist EC
50
, Hill slope and I
max
will be measured to indicate the
function of mutant and WT receptor subunits. Since GlyRs and GABA
A
Rs are chloride
ion channels, EC
2
-induced Cl
–
currents potentiation will be measured to indicate their
ethanol sensitivity; percentage of maximal Cl
–
currents will be measured to indicate their
agonist sensitivity. Concentration response curve of both ethanol and agonist will be
described as shown in Fig. 1-4.
1.8) Specific Aims
The main objective of my thesis was to develop α2 GlyR USER mutations based on α1
GlyR USERs developed previously by colleagues in order to not only improve their
ethanol sensitivity but also normalize their response to agonist the same as the WT.
Furthermore, the success of GlyR USER development leads us to focus on other ethanol
related receptors like GABA
A
R. Another goal of my project is developing GABA
A
R
USER mutations on different subunits including γ2 and α1 GABA
A
R subunits. In order
to achieve this, we developed several optimization strategies centered on strategically
manipulating the structure of α2 GlyR subunit and α1β2γ2 GABA
A
R subunits,
respectively. We tested their sensitivities to ethanol and agonist using two-electrode
voltage clamp electrophysiology in Xenopus oocytes. The specific aims of these steps
were:

21

[1] To demonstrate the applicability of USERs across different subunits of GlyRs, our
hypothesis is to develop USER in a new subunit, α2 GlyR, and to further increase its
ethanol sensitivity and normalize USER receptor response to agonist the same as α2
GlyR WT.

[2] To test the hypothesis that reverting serine at the homologous position in γ2 GABA
A
R
USER 1 back to WT asparagine (S66N) will increase the sensitivity of the receptor to
ethanol compared to γ2 GABA
A
R USER 1.

[3] To test the hypothesis that restoring additional loop 2 residues in γ2 GABA
A
R USER
1 back to WT will normalize EC
50
and concentration-response to its agonist GABA the
same as α1β2γ2 GABA
A
R WT with the increased ethanol sensitivity.

[4] To test the hypothesis that the USER strategy can be applied to different subunits of
GABA
A
Rs with the increased ethanol sensitivity and normal receptor response to agonist
as their WT.

These are addressed in the following Chapters.

22

Chapter 2
Experimental Procedures

In order to test our hypothesis, we performed site-directed mutagenesis to create the α2
GlyR USER 1 based on α1 GlyR USER 3 as well as the γ2 and α1 GABA
A
R USERs. We
then expressed these new mutant receptors in Xenopus oocytes, exposed this system to a
wide range of concentrations of agonist (1 µM to 10mM for glycine and GABA) and
ethanol (0.025-50 mM for GlyRs, 0.1-50 mM for GABA
A
Rs and measured agonist and
ethanol sensitivities using two-electrode voltage clamp electrophysiology. The materials,
methods and results are described in detail below.
2.1) 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 MgSO
4
, 2.4
NaHCO
3
, 0.91 CaCl
2
, and 0.33 Ca(NO
3
)
2
, adjusted to pH 7.5.
2.2) Site-directed Mutagenesis
For the purpose of this study, the extracellular loop 2 region is defined as positions 50-59
in α1 GlyR subunit, positions 57-66 in α2 GlyR subunit, positions 64-73 in γ2 GABA
A
R
subunit, positions 43-52 in α2 GABA
A
R subunit and positions 43-52 in δ GABA
A
R
subunit. Homologous amino acid sequences from extracellular Loop 2 regions of α1

23

GlyR, α2 GlyR, γ2 GABA
A
R and α2 GABA
A
R subunits were identified and aligned
(Table 2.1). As indicated in Table 1, site-directed mutagenesis was performed in the α2
GlyR, γ2 GABA
A
R and α2 GABA
A
R subunits cDNA respectively based on the
development of α1 GlyR USERs in order to make the receptor’s loop 2 region identical to
δ GABA
A
R loop 2. This site-directed mutagenesis was performed by subcloning human
GlyR and GABA
A
R cDNA into mammalian vector pCIS2 or pBK-CMV using the Quick
Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) and verified by partial
sequencing (DNA Core Facility, University of Southern California).
2.3) 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 MgCl
2
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℃ in incubation medium [88 mM NaCl, 1.8 mM KCl, 5 mM
HEPES, 0.82 mM MgSO
4
, 2.4 mM NaHCO
3
, 0.41 mM CaCl
2
, 0.33 mM Ca(NO
3
)
2
, 2
mM sodium pyruvate, 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 or GABA
A
R cDNAs (1 ng/32 nl). Injected oocytes
were stored at 18℃ and used in electrophysiological recordings 24 hours after injection
for a period of 1 week.

24

2.4) 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℃).
A) Application of Agonist
GlyR or GABA
A
R WT and USERs were exposed to 1 µM-10 µM glycine or GABA,
respectively, for 30 seconds at a rate of 3 ml/min, with 5- to 15-minute washout periods
between applications to ensure complete receptor resensitization.
B) 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

25

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
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 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 0.1-50 mM for α2 GlyRs, γ2 and α1 GABA
A
Rs. Holding currents
were not significantly affected during preincubation with ethanol, i.e., in the absence of
agonist.
2.5) Data Analysis
Data for each experiment were obtained from 4-23 oocytes from at least three 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 responses 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 concentration, [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

26

Analysis of Variance (ANOVA) with Dunnett’s multiple comparison or Bonferroni
post-test 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.

27

Chapter 3
Loop 2 Manipulations in Multiple Subunits of GlyRs – α2 GlyR USER

Aim 1 – To demonstrate the applicability of USERs across different subunits of
GlyRs, our hypothesis is to develop USER in a new subunit, α2 GlyR, and to further
increase its ethanol sensitivity and normalize USER receptor response to agonist the
same as α2 GlyR WT.
3.1) Rationale
α2 GlyRs are the predominant receptor subtype found in the adult brain, and are believed
to play an important role in ethanol-induced reward (Delaney et al., 2010; Jonsson et al.,
2012). In addition, emerging evidence suggests that, unlike α1 GlyRs, which are
predominantly expressed as heteromers (α1β) in the spinal cord, α2 GlyRs are expressed
as homomers in the brain (Adermark et al., 2011; Chen et al., 2011; Eichler et al., 2009;
Weltzien et al., 2012).

Prior studies demonstrated that structural manipulation of loop 2 could alter ethanol
sensitivity of α1 GlyRs (Perkins et al., 2009). One of the mutant receptors obtained as a
result of these structural manipulations, α1 GlyR USER 1, exhibited decreased threshold
for ethanol sensitivity and increased magnitude of ethanol potentiation compared to WT
GlyRs, but there was a commonly seen left shift in their concentration-response curve for
glycine, implicating the abnormal receptor functions.. Furthermore, colleagues in our
laboratory altered the amino acid structure and sequence based on position 52 in exon 3
and 4 of the α1 GlyR USER1 to create α1 GlyR USER 2 and 3 for increasing ethanol
sensitivity and normalizing receptor response to agonist as its WT (Table 1; Fig. 1).

28

Notably, there were no statistically significant differences in the EC
50
, Hill slope and I
max

value of α1 GlyR USER 3 compared with its WT receptors (Table 1; Fig. 1). The overall
goal of this part of experiments was to develop α2 GlyR USER by maximizing ethanol
sensitivity and normalizing receptor function in α2 GlyRs through structural
manipulation of loop 2 based on the amino acid sequence of α1 GlyR USER 3.

We hypothesized that by applying the identical homologous amino acid sequences of α1
GlyR USER 3 at extracellular loop 2 regions to create α2 GlyR USER 1 (S57H, V58I,
T59S, T61A, T62N) (Table 1) we would increase ethanol sensitivity without altering
agonist response of the new mutant receptor when compared with α2 GlyR WT. This part
of the experiments was conducted on my own as my thesis project.
3.2) Results
A) Agonist Concentration Response
Inward Cl
–
currents were evoked in a concentration dependent manner in WT and mutant
α2 GlyR USER 1 upon glycine administration (Fig. 2). All analyses were conducted
using non-linear regression. Values for glycine EC
50
, Hill slope and I
max
of α2 GlyR WT
and α2 GlyR USER 1 are shown in Table 1.

Remarkably, t-tests showed no significant changes in agonist sensitivity relative to α2
GlyR WT, as indicated by similar EC
50
values (Fig. 2; Table 1). The other receptor
characteristics of α2 GlyR USER 1, including Hill slope and I
max
, did not differ
significantly from α2 GlyR WT (Table 1).

29

B) Ethanol Concentration Response
In α2 GlyR WT ethanol produced concentration-dependent potentiation of
glycine-induced Cl
-
currents with significant effects starting at 50 mM ethanol (Fig. 2;
Table 2). There were no significant effects of ethanol on these WT receptors at
concentrations below 50 mM.

As predicted, α2 GlyR USER 1 demonstrated an increase in ethanol sensitivity by
decreasing the ethanol threshold from 50 mM in α2 GlyR WT to 0.25 mM (Fig. 2; Table
2). At higher concentrations, ethanol produced a concentration-dependent effect with
significant potentiation in α2 GlyR USER 1 beginning at 10 mM (Fig. 2; Table 2). As
with other GlyR USERs, the ethanol concentration responses followed a bimodal pattern
(Fig. 2).
3.3) Conclusion
The results demonstrate that by applying the same rationale of α1 GlyR USER 3 that
limiting loop 2 mutations to exon 3 in α2 GlyR USER 1, the new mutant USERs showed
increasing ethanol sensitivity (lower ethanol threshold and enhanced ethanol-induced
potentiation) with no changes in agonist sensitivity compared to α2 GlyR WT, which
share the similar characteristics with α1 GlyR USER 3 that exhibited increased ethanol
sensitivity with the same response to agonist as WT. Overall, the findings support the
Aim 1 hypothesis and confirm that the strategy to generate USERs can be applied across
different subunits of GlyRs.

30

Chapter 4
Loop 2 Manipulations in Multiple Receptors of the Cys-loop superfamily – γ2
GABAAR USERs

Aim 2 – To test the hypothesis that reverting serine at the homologous position in γ2
GABA
A
R USER 1 back to WT asparagine (S66N) will increase the sensitivity of the
receptor to ethanol compared to γ2 GABA
A
R USER 1.
4.1) Rationale
Earlier studies reported that loop 2 mutant γ2 GABA
A
R USER 1 had increased ethanol
sensitivity compared with GABA
A
R WT in response to 0.25-50 mM ethanol (Perkins et
al., 2009). In the current study, we identified a lower ethanol threshold in γ2 GABA
A
R
USER 1 at 0.25 mM, compared with the previously reported 0.5 mM (Table 3) (Perkins
et al., 2009). At ultra-low concentrations, ethanol produced a concentration-dependent
effect with significant potentiation starting at 0.25 mM. Thus, the threshold for ethanol
sensitivity in γ2 GABA
A
R USER 1 was reduced from 50 mM to 0.25 mM compared with
GABA
A
R WT (Table 3). As seen with all GlyR USERs, the ethanol concentration
response demonstrated a bimodal trend, with the first curve following an inverse U
pattern ranging 0.1-1 mM, and a second curve beginning at 3 mM with a
concentration-dependent increase in ethanol response (Fig. 3).

As showed in α1 GlyR USERs, reverting position 52 from serine to the WT alanine in α1
GlyR USER 2 increased ethanol sensitivity relative to α1 GlyR WT. We hypothesized
that by using the similar approach that reverting serine at the homologous position in γ2
GABA
A
R USER 1 back to WT asparagine (S66N) to create γ2 GABA
A
R USER 2 (P64H,

31

V65I, A67E, I68A) (Table 1) would increase ethanol sensitivity. Anna Naito and I
conducted this part of the experiments.
4.2) Results
A) Agonist Concentration Response
Inward Cl
–
currents were evoked in concentration dependent manner in WT and mutant
γ2 GABA
A
R USER 1 and 2 upon GABA administration (Fig. 3). All analyses were
conducted using non-linear regression. Values for glycine EC
50
, Hill slope and I
max
of
WT GABA
A
R (Table 1) were consistent with results form previous studies by Perkins et
al., 2009.

T-tests showed no significant differences in Hill slope and I
max
between γ2 GABA
A
R
USER 1 and 2 and GABA
A
R WT (Table 1). However, in both γ2 GABA
A
R USER 1 and
2, loop 2 mutations produced a left shift in the agonist concentration-response curve
relative to GABA
A
R WT with a significant decrease in EC
50
(Fig. 3; Table 1).
B) Ethanol Concentration Response
In α1β2γ2 GABA
A
R WT ethanol produced concentration-dependent potentiation of
GABA-induced Cl
-
currents with significant effects starting at 50 mM ethanol (Fig. 3;
Table 3). There were no significant effects of ethanol on these WT receptors at
concentrations below 50 mM.

Similar to results for γ2 GABA
A
R USER 1, the resultant γ2 GABA
A
R USER 2
demonstrated an increase in ethanol sensitivity with decreased ethanol threshold from 50
mM in GABA
A
R WT to 0.25 mM (Fig. 3; Table 3) and increased magnitude of ethanol

32

potentiation (Fig. 3; Table 3). At higher concentrations, ethanol produced a
concentration-dependent effect with significant potentiation in γ2 GABA
A
R USER 2
beginning at 10 mM (Fig. 3; Table 3). As with other USERs, the ethanol concentration
responses followed a bimodal pattern (Fig. 3). However, the ethanol threshold and
magnitude of potentiation between the two mutant γ2 GABA
A
R USERs remained similar
(Fig. 3).
4.3) Conclusion
The results demonstrate that by reverting serine at the homologous position in γ2
GABA
A
R USER 1 back to WT asparagine (S66N), the new mutant USERs showed
increasing ethanol sensitivity (lower ethanol threshold and enhanced ethanol-induced
potentiation). However, both γ2 GABA
A
R USER 1 and 2 produced a left shift in the
agonist concentration-response curve relative to GABA
A
R WT. Moreover, there is no
significant increase in ethanol sensitivity between γ2 GABA
A
R USER 1 and 2.

33

Chapter 5
Loop 2 Manipulations in γ2 GABAAR USERs with Normalized Receptor
Characteristics

Aim 3 – To test the hypothesis that restoring additional loop 2 residues in γ2
GABA
A
R USER 1 back to those in WT will normalize EC
50
and
concentration-response to its agonist GABA the same as α1β2γ2 GABA
A
R WT with
the increased ethanol sensitivity.
5.1) Rationale
Crawford et al. (2008) demonstrated that removal of the negative charge associated with
the glutamate residue at position 53 in α1 GlyRs caused a right shift in agonist sensitivity
with respect to α1 GlyR WT, indicating that the physical-chemical properties at this
position may influence agonist sensitivity.

Therefore, we hypothesized that substituting the charged glutamate at position 67 with
the neutral WT alanine in γ2 GABA
A
R USER 1 to created γ2 GABA
A
R USER 3 (P64H,
V65I, N66S, I68A) would right shift and thus normalize agonist sensitivity compared
with GABA
A
R WT.

On the other hand, since α1 GlyR USER 3 was ultra-sensitive to ethanol with WT-like
agonist response, we hypothesized that substitution of the terminal loop 2 residues of α1
GlyR USER 3 in γ2 GABA
A
R USERs would also increase ethanol sensitivity while
normalizing agonist response. Following this strategy, we created γ2 GABA
A
R USER 4
(P64H, V65I, N66S, A67E, I68A, E71D, T73R) (Table 1).

34

Both of the hypotheses will be tested and showed in the following results. Anna Naito
and I conducted this part of the experiments.
5.2) Results
A) Agonist Concentration Response
Inward Cl
–
currents were evoked in concentration dependent manner in the mutant γ2
GABA
A
R USER 3 and 4 upon GABA administration (Fig. 3). All analyses were
conducted using non-linear regression. Values for glycine EC
50
, Hill slope and I
max
of γ2
GABA
A
R USER 3 and 4 are shown in Table 1.

T-tests showed no significant differences in Hill slope and I
max
between γ2 GABA
A
R
USER 3 and 4 and GABA
A
R WT (Table 1). As predicted, the GABA
concentration-response curve for γ2 GABA
A
R USER 3 was right shifted compared with
γ2 GABA
A
R USER 1 and 2 (Fig. 3). However, γ2 GABA
A
R USER 3 was still
significantly more sensitive to agonist compared with GABA
A
R WT (Fig. 3; Table 1).
Thus, these loop 2 manipulations in γ2 GABA
A
R USER 3 partially restored agonist
sensitivity toward WT values. Moreover, mutations in γ2 GABA
A
R USER 4 kept left
shifting the agonist sensitivity (Fig. 3; Table 1).
B) Ethanol Concentration Response
γ2 GABA
A
R USER 3 demonstrated an increase in ethanol sensitivity with decreased
ethanol threshold from 50 mM in GABA
A
R WT to 0.25 mM (Fig. 3; Table 3). The
magnitude of ethanol response at ultra-low concentrations was higher than with γ2
GABA
A
R USER 1. At higher concentrations, ethanol produced a

35

concentration-dependent effect with significant potentiation in γ2 GABA
A
R USER 3
beginning at 10 mM (Fig. 3; Table 3). The ethanol concentration response for γ2
GABA
A
R USER 3 followed a bimodal pattern similar to that of other γ2 GABA
A
R
USERs (Fig. 3).

In contrast to the expected increase in ethanol sensitivity, γ2 GABA
A
R USER 4 was
insensitive to ethanol across all concentrations tested (Fig. 3; Table 3).
5.3) Conclusion
The results demonstrate that by substituting the charged glutamate at position 67 with the
neutral WT alanine in γ2 GABA
A
R USER 1, the created γ2 GABA
A
R USER 3 partially
restored agonist sensitivity with the increased ethanol sensitivity.

On the other hand, the results show that by substituting the terminal loop 2 residue
sequence of α1 GlyR USER 3 in γ2 GABA
A
R USERs, γ2 GABA
A
R USER 4 was
insensitive to ethanol across all concentrations tested with a left shifted agonist
sensitivity.

36

Chapter 6
Loop 2 Manipulations in Multiple Subunits of GABAARs – α1 GABAAR USER

Aim 4 – To test the hypothesis that USER strategy can be applied in different
subunits of GABA
A
Rs with the increased ethanol sensitivity and normal receptor
response to agonist as their WT.
6.1) Rationale
α1β2γ2 GABA
A
Rs are the predominantly expressed form of GABA
A
Rs in the brain, and
are believed to play a role in producing ethanol-induced behaviors (Borghese et al., 2006;
Kumar et al., 2009; Werner et al., 2006). α1 subtype of GABA
A
R, although less
widespread than γ2 subunits, is the most abundant among α subunits (Sieghart and Sperk,
2002). Knockout of the α1 subunit causes total GABA
A
R content in mouse brain to
decrease by 50% (Sur et al., 2001). Therefore, it is important to know whether they are
also involved in ethanol action. The overall goal of this part of the experiments was to
develop an α1 GABA
A
R USER by maximizing ethanol sensitivity and normalizing
receptor function in α1 GABA
A
R through structural manipulation of loop 2 based on the
amino acid sequence of γ2 GABA
A
R USER 1.

We hypothesized that by applying the identical homologous amino acid sequences of γ2
GABA
A
R USER 1 at extracellular loop 2 regions to create α1 GABA
A
R USER 1 (P43H,
V44I, D46E, H47A, D48N) (Table 1) we would increase ethanol sensitivity without
altering agonist response of the new mutant receptor when compared with α1β2γ2
GABA
A
R WT. The experiments in Aim 4 tested this hypothesis. Anna Naito and I
conducted this part of the experiments.

37

6.2) Results
A) Agonist Concentration Response
Inward Cl
–
currents were evoked in a concentration dependent manner in the mutant α1
GABA
A
R USER 1 upon GABA administration (Figure 4). All analyses were conducted
using non-linear regression. Values for glycine EC
50
, Hill slope and I
max
of α1 GABA
A
R
USER 1 are shown in Table 1.

Remarkably, α1 GABA
A
R USER 1 produced no change in agonist sensitivity relative to
GABA
A
R WT, as indicated by similar EC
50
values (Fig. 4; Table 1). The other receptor
characteristics of α1 GABA
A
R USER 1, including Hill slope and I
max
did not differ
significantly from GABA
A
R WT (Table 1).
B) Ethanol Concentration Response
As expected, α1 GABA
A
R USER 1 had markedly increased ethanol sensitivity compared
with GABA
A
R WT (Fig. 4), with an ethanol sensitivity threshold at 0.25 mM compared
with 50 mM in GABA
A
R WT (Fig. 4; Table 3). At ultra-low ethanol concentrations, α1
GABA
A
R USER 1 demonstrated an increase in the magnitude of ethanol response
compared with WT (Fig. 4). The ethanol concentration response exhibited a bimodal
pattern, consistent with other GlyR and GABA
A
R USERs (Fig. 4; Table 3).
6.3) Conclusion
The results demonstrate that by applying the identical homologous amino acid sequences
of γ2 GABA
A
R USER 1 at extracellular loop 2 regions, the new mutant α1 GABA
A
R
USER 1 showed increased ethanol sensitivity with no changes in agonist sensitivity

38

compared to GABA
A
R WT. Overall, the findings support Aim 4’s hypothesis and
confirm that the strategy of USER can be applied across different subunits of GABA
A
Rs.

39

Chapter 7
Overall Discussion

Based on previous studies of USERs of α1 GlyRs, we applied the USER strategy across
multiple subunits of GlyRs and GABA
A
Rs and observed the increased ethanol sensitivity
(over 100-fold) by significantly decreasing the threshold and increasing the magnitude of
ethanol response, without altering general receptor properties (Fig. 1-4; Table 1-3), such
as agonist binding. The findings also lay a solid foundation for elucidating ethanol action
and mechanisms contributing to complicated behavioral effects.

One important characteristic of USERs is the significant decreased threshold
concentrations for ethanol sensitivity, which is less than 0.25 mM (Blood Ethanol
Concentration ≤ 0.001%). In humans, behavioral effects of ethanol can be detected at
BECs as low as 0.03% (w/v) (7 mM) (Kumar et al., 2009; Ogden and Moskowitz, 2004).
Because of the ethanol concentration threshold of USERs is far below that which
produces known responses in vivo and in vitro, USERs would circumvent or minimize
the probability of activating multiple targets of ethanol and associated behaviors, thus
allowing activation of specific receptor subunits. Furthermore, results of USER
application in Xenopus oocytes suggest the exciting possibility of knock in animals
expressing USERs should respond similarly to ultra-low-dose ethanol as if they received
higher ethanol doses in the 30-50 mM range. This provides an insight of using USERs
conjugated with current transgenic approaches to investigate the pharmacological and
physiologic effects of ethanol action by establishing precise links between specific
receptor subunits and behavioral outcomes.

40

The successful application of the USER strategy across multiple subunits of GlyRs and
GABA
A
Rs implicates the reality of applying USER in other members of the Cys-loop
superfamily of LGICs, including the nAch receptors and 5-HT
3
receptors, since they
share 30% homology sequence with the subunits of Cys-loop receptors (Olsen and
Sieghart, 2008) and have been linked to a number of behavioral effects of ethanol
administration (Hodge et al., 2004; Kamens et al., 2010). Thus, by expressing in oocyte
system or knocking in animal models USERs could serve as an advantageous
brain-mapping tool to investigate different receptor subunits and provide further insight
into specific neural cascades at the resolution of the receptor subunit level. Further α2
GlyR USER 1 knock in mice are currently under study. Animal behavior studies in these
knock in mice will illustrate 1) how these mutant receptors express and work in animal
model; 2) which behavior changes will occur and further link to the specific receptor
subunits; 3) whether these USER knock in animal models are ready for the further study
in investigating the ethanol action in brain.

The ethanol responses of USERs showed a typical bimodal pattern, which combined with
findings from previous studies (Crawford et al., 2007; Davies et al., 2004), supports the
possible existence of multiple potentiating as well as inhibitory sites of ethanol action. A
potential explanation could be that these loop 2 mutations lead to the creation of an
ultrasensitive ethanol site that produces robust potentiation of the receptor at
submillimolar concentrations. While at concentration between 1 and 3 mM, ethanol could
act at both inhibitory and potentiating sites that potentially reduce and/or mask the
contribution of the ultrasensitive site. Further at higher concentrations higher than 3 mM,
ethanol could act at WT potentiating sites. This funding is supported by studies of other
Cys-loop allosteric modulators, such as zinc, propofol, pentobarbital, and neuro-steroids
(Bloomenthal et al., 1994; Evers et al., 2010; Laube et al., 1995; Maksay and Bíró, 2002;

41

Morrow et al., 1990), suggesting the possibility of exploring the actions of such other
allosteric agents on these USERs.

Overall, USER strategy can be used as tools to reveal the specific role of individual
receptor subunits in ethanol action and may provide insights on developing effective
medications to prevent or/and treat alcohol use disorders.

42

Tables

Table 1
Table 1 Loop 2 sequence alignment and receptor characteristics for the human WT
and α1 and α2 GlyR USERs, and α1 and γ2 GABA
A
R USERs.
Loop 2 of α1 and α2 GlyRs spans exon 3 and exon 4. In WT α1 and γ2 GABA
A
R USERs,
the GABA
A
R isoform represented is α1β2γ2. Loop 2 of α1 and γ2 GABA
A
R USER

43

subunits spans exon 3. GlyR and GABA
A
R EC
50
, Hill slope, and I
max
are presented as
mean ± S.E.M. from at least 4-23 oocytes. *P < 0.05 compared to respective WT
receptors.

Table 2

Table 2 Threshold for ethanol sensitivity in α1 and α2 GlyR USERs is bimodal and
lower than WT.
α1 and α2 GlyR USERs exhibited a bimodal pattern in response to ethanol. Two
threshold concentrations are denoted for USERs to represent the bimodal effect at
ultra-low and higer (> 3 mM) ethanol concentrations. Each data point represents the mean
± S.E.M. from at least 4-16 oocytes. All threshold values were statistically significant
with P < 0.05.

44

Table 3
Table 3 Threshold for ethanol sensitivity of γ2

and α1 GABA
A
R USERs is bimodal
and lower than α1β2γ2 GABA
A
R WT.
γ2 GABA
A
R USERs 1, 2 and 3, and α1 GABA
A
R USER 1 exhibited a bimodal pattern in
response to ethanol. Two threshold concentrations are denoted for USERs to represent
the bimodal effect at ultra-low and higher (> 3 mM) ethanol concentrations. All
GABA
A
R USERs represent the α1β2γ2 isoform. Each data point represents the mean ±
S.E.M. from at least 4-13 oocytes. All threshold values were statistically significant with
P < 0.05.

45

Figures

Figure 1

Figure 1 α1 GlyR USERs ethanol and agonist concentration response.
(a) Ethanol-induced potentiation of glycine EC
2
-activated Cl
-
currents in Xenopus oocytes
expressing α1 GlyR USERs exhibited a bimodal response to ethanol. Values for ethanol
potentiation are presented as the percentage of glycine EC
2
control. The glycine EC
2

concentrations used ranged from 5 to 10 µM in USERs and 18 to 25 µM for WT. Each
data point represents the mean ± S.E.M. from at least 4-9 oocytes. (b) Glycine-induced
Cl
-
currents were normalized to the maximal current activated by a saturating
concentration of glycine (1000-10,000 µM). The curves represent nonlinear regression
analysis of the glycine concentration responses in α1 GlyR USERs and WT. Each data
point represents the mean ± S.E.M. from at least 4-23 oocytes.

46

Figure 2

Figure 2 α2 GlyR USER ethanol and agonist concentration response.
(a) Ethanol-induced potentiation of glycine EC
2
-activated Cl
-
currents in Xenopus oocytes
expressing α2 GlyR USER 1 exhibited a bimodal response to ethanol. Values for ethanol
potentiation are presented as the percentage of glycine EC
2
control. The glycine EC
2

concentrations used ranged from 5 to 10 µM in USER and WT. Each data point
represents the mean ± S.E.M. from at least 4-16 oocytes. (b) Glycine-induced Cl
-
currents
were normalized to the maximal current activated by a saturating concentration of glycine
(1000-10,000 µM). The curves represent nonlinear regression analysis of the glycine
concentration responses in α2 GlyR USER 1 and WT. Each data point represents the
mean ± S.E.M. from at least 4-5 oocytes.

47

Figure 3
Figure 3 γ2 GABA
A
R USERs ethanol and agonist concentration response.
(a) Ethanol-induced potentiation of GABA EC
2
-activated Cl
-
currents in Xenopus oocytes
expressing γ2 GABA
A
R USERs 1, 2 and 3 with α1β2γ2 composition exhibited a bimodal
response to ethanol. Values for ethanol potentiation are presented as the percentage of
GABA EC
2
control. The GABA EC
2
concentrations used were 5 µM for USERs and 10
µM for WT. Each data point represents the mean ± S.E.M. from at least 4-9 oocytes. (b)
GABA-induced Cl
-
currents were normalized to the maximal current activated by a
saturating concentration of GABA (1000-10,000 µM). The curves represent nonlinear
regression analysis of the GABA concentration responses in γ2 GABA
A
R USERs and
WT. Each data point represents the mean ± S.E.M. from at least 4-23 oocytes.

48

Figure 4
Figure 4 α1 GABA
A
R USER ethanol and agonist concentration response.
(a) Ethanol-induced potentiation of GABA EC
2
-activated Cl
-
currents in Xenopus oocytes
expressing α1 GABA
A
R USERs 1 with α1β2γ2 composition exhibited a bimodal
response to ethanol. Values for ethanol potentiation are presented as the percentage of
GABA EC
2
control. The GABA EC
2
concentrations used were 8-10 µM for USERs and
WT. Each data point represents the mean ± S.E.M. from at least 4-13 oocytes. (b)
GABA-induced Cl
-
currents were normalized to the maximal current activated by a
saturating concentration of GABA (1000-10,000 µM). The curves represent nonlinear
regression analysis of the GABA concentration responses in α1 GABA
A
R USER 1 and
WT. Each data point represents the mean ± S.E.M. from at least 4-13 oocytes.

49

Figure 5a
Figure 5a Schematic of neurochemical pathways activated in response to high
ethanol concentrations (10 – 50 mM).

Figure 5b
Figure 5b Schematic of USER receptor subunit activated by ultra-low ethanol
concentrations.

50

Figure 6

Figure 6 Molecular model of α1 GlyR.
(a) The structure of a pentameric GlyR based on the template of a prokaryotic
ligand-gated ion channel (LGIC). (b) The structure of a single subunit viewed from the
plane of the membrane. (c) Zoom view of the domain interface. (d) Detailed view of
residues in loop 2 (modified from (Crawford et al., 2008)).

51

Figure 7

Figure 7 Molecular model of the α1β2γ2 GABA
A
R with mutations in loop 2 of the γ2
subunit.
(a) Model of the full receptor α1β2γ2 GABA
A
R. (b) Zoom view of loop 2.

52

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Abstract (if available)

Abstract The lack of specific knowledge on where and how ethanol acts in the CNS and the resultant neurochemical cascades leading to behavioral change limited the investigation of ethanol action and the development of effective medications to prevent or/and treat alcohol use disorders. To identify the role of specific individual receptor subunits in ethanol induced behaviors, ultrasensitive ethanol receptors (USERs) were developed by manipulating by mutagenesis the physical-chemical characteristics of extracellular domain loop 2 region in glycine receptors (GlyRs) and γ-aminobutyric acid type A receptors (GABAARs), which are implicated in causing many behavioral effects linked to ethanol abuse. These mutant receptors would then theoretically allow activation of a single receptor subunit population sensitized to extremely low ethanol concentrations while keeping other receptor systems silenced. Based on previous studies of USERs in α1 GlyRs, in the current study we observed increased ethanol sensitivity in different subunits of GlyRs and GABAARs. Expression in Xenopus oocytes and testing by two-electrode voltage clamp, the last generation of USERs indicated an increase in ethanol sensitivity of 100-fold over wild-type receptors by significantly decreasing the threshold and increasing the magnitude of ethanol response, without altering general receptor properties. Further application of USERs may lead to the elucidation ethanol action and mechanisms behind complicated behavioral effects.

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Asset Metadata

Creator Wang, Yihui (author)

Core Title Ultra-sensitive ethanol receptors as novel tools for alcohol and brain research: optimizing loop 2 mutations in α2 glycine receptors, γ2 and α1 γ-aminobutyric acid type A receptors

School School of Pharmacy

Degree Master of Science

Degree Program Molecular Pharmacology and Toxicology

Publication Date 04/20/2015

Defense Date 03/20/2015

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

Tag AUD,ethanol,GABA receptor,glycine receptor,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Okamoto, Curtis Toshio (committee chair), Cadenas, Enrique (committee member), Stiles, Bangyan L. (committee member)

Creator Email yhw723@gmail.com,yihuiwan@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-553142

Unique identifier UC11297330

Identifier etd-WangYihui-3334.pdf (filename),usctheses-c3-553142 (legacy record id)

Legacy Identifier etd-WangYihui-3334.pdf

Dmrecord 553142

Document Type Thesis

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Rights Wang, Yihui

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...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA