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Development of glycine and GABAA ultra-sensitive ethanol receptors (USERs) as novel tools for alcohol and brain research

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Development of glycine and GABAA ultra-sensitive ethanol receptors (USERs) as novel tools for alcohol and brain research

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Content DEVELOPMENT OF GLYCINE AND GABAA ULTRA-SENSITIVE ETHANOL
RECEPTORS (USERs) AS NOVEL TOOLS FOR ALCOHOL AND BRAIN RESEARCH

by

Anna Naito

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

AUGUST 2015
2

Authorship

Published works by the Author incorporated into the Dissertation

Naito A, Muchhala KH, Asatryan L, Trudell JR, Homanics GE, Perkins DI, Davies DL,
and Alkana RL. (2014). Glycine and GABA(A) ultra-sensitive ethanol receptors as novel
tools for alcohol and brain research. Mol Pharm 86:635-646. Incorporated as Chapter 2.
Naito A, Muchhala KH, Trang J, Asatryan L, Trudell JR, Homanics GE, Alkana RL, and
Davies DL. (2015). Manipulations of extracellular Loop 2 in alpha1 GlyR ultra-sensitive
ethanol receptors (USERs) enhance receptor sensitivity to isoflurane, ethanol, and
lidocaine, but not propofol. Neuroscience 297:68-77. Incorporated as Chapter 3.

Research Support
NIH National Institute on Alcohol Abuse and Alcoholism AA10422 (GEH), AA20980
(JRT), AA17243 (LA), AA22448 (DLD)
American Foundation for Pharmaceutical Education (AFPE) Pre-Doctoral Fellowship
USC School of Pharmacy

3

Dedication

To Dr. Ron Alkana,

Who taught me how to see things that never were,
and ask, why not?

With deepest gratitude to:
Drs. Asatryan and Davies
for their mentorship and guidance;
Drs. Trudell and Homanics
for sharing their expertise;
Dr. Okamoto
for shaping me into the scientist that I am today;
My family and friends
for their unconditional support.

4

Statement of Contributions to Works Contained in this Dissertation

This dissertation is composed of the author’s original work and contains no material
previously published or written by any other individual except where due reference is made. All
data contained herein was collected and analyzed by A. Naito. Molecular modeling was carried
out by Dr. James Trudell at Stanford University, genetically engineered mice were provided by
Dr. Gregg Homanics at University of Pittsburgh. Drs. Davies, Asatryan and Okamoto provided
discussion and revisions to the manuscript.

5

TABLE OF CONTENTS
Authorship 2
Dedication 3
Statement of Contributions to Works Contained in this Dissertation 4
List of Figures 7
List of Tables 9
List of Abbreviations 10
Abstract 13
Chapter 1 Introduction 15
A. Background 15
B. Mechanism of ethanol and anesthetic action 18
Lipid membrane theory 18
Protein Theory 20
C. Protein Sites of Ethanol Action in the CNS 22
Ionotropic Receptors 22
Glycine Receptors (GlyRs) 24
GABA
A
Receptors 25
Metabotropic Receptors 27
D. Molecular Targets of Ethanol Action on Cys-Loop Receptors 28
Transmembrane 28
Intracellular 29
Extracellular 29
E. Current Strategies for Identifying the Role of Receptors in Ethanol Action 32
Molecular Approaches 32
DREADD 33
Optogenetics 34
Molecular Modeling 35
Genetically engineered animal models 36
F. USERs as Novel Tools for Alcohol and Brain Research 36
Chapter 2 Glycine and GABA
A
Ultra-Sensitive Ethanol Receptors (USERs)
as Novel Tools for Alcohol and Brain Research 38
CHAPTER 2 ABSTRACT 38
INTRODUCTION 39
MATERIALS AND METHODS 42
RESULTS 47
Manipulation of Loop 2 results in GlyR and GABA
A
R USERs that are
sensitive to ultra-low ethanol concentrations (<1 mM) 49
6

Agonist sensitivity of α1 GlyR and γ2 GABA
A
R USERs 1 and 2 were
modestly altered compared to respective WT 55
Manipulation of the Loop 2 structure results in GlyR and GABA
A
R
USERs with normalized receptor characteristics 56
Loop 2 manipulations in multiple subunits of GlyRs and GABA
A
Rs
result in USERs 58
γ2 and α1 GABA
A
R USERs do not have altered sensitivity to the
neurosteroid, Allopregnanolone (3α,5α-THP) 63
DISCUSSION 65
Chapter 3 Manipulations of Extracellular Loop 2 in α1 GlyR Ultra-Sensitive
Ethanol Receptors (USERs) Enhance Receptor Sensitivity to Isoflurane,
Ethanol, and Lidocaine, but not Propofol 73
CHAPTER 3 ABSTRACT 73
INTRODUCTION 75
MATERIALS AND METHODS 80
RESULTS 86
α1 GlyR USERs 1 and 2 have increased sensitivity to isoflurane 86
α1 GlyR USERs 1 and 2 have increased sensitivity to lidocaine 89
α1 GlyR USERs exhibit no significant changes in propofol sensitivity
compared to WT 91
Loop 2 mutations exclusive to exon 4 of α1 GlyRs are sufficient to
increase ethanol, isoflurane and lidocaine sensitivity 94
DISCUSSION 97
Chapter 4 Development and Initial Behavioral Testing of α2 GlyR USER
Knock-In (KI) in Mice 103
CHAPTER 4 ABSTRACT 103
INTRODUCTION 105
METHODS 108
RESULTS 112
α2 GlyR USER KI mice demonstrated no differences in motor
incoordination compared to WT on accelerating rotarod 112
α2 GlyR USER KI mice demonstrate no change in sensitivity to a
hypnotic dose of ethanol in LORR 115
α2 GlyR USER KI mice exhibit significance increase in anxiolytic
effects at 1% ethanol exposure 116
DISCUSSION 119
Chapter 5 Overall Discussion and Conclusions 124
References 130

7

List of Figures

Figure 2.1 Loop 2 mutations in α1 GlyRs produce ultra-sensitive ethanol
receptors that are sensitive to ≤ 0.5 mM ethanol 47
Figure 2.2 α1 GlyR USERs have increased ethanol sensitivity and bimodal
response 51
Figure 2.3 γ2 GABA
A
R USERs 1, 2 and 3 have increased ethanol
sensitivity and bimodal response 54
Figure 2.4 α2 GlyR USERs have increased ethanol sensitivity and bimodal
response 60
Figure 2.5 α1 GABA
A
R USERs have increased ethanol sensitivity and
bimodal response to ethanol 62
Figure 2.6 Loop 2 mutations do not alter sensitivity to 3α,5α-THP for γ2
GABA
A
R USER 3 and α1 GABA
A
R USER 1 64
Figure 2.7 A molecular model of the α1β2γ2 GABA
A
R with mutations in
Loop 2 of the γ2 subunit 70
Figure 3.1 α1 GlyR USERs 1 and 2 have increased sensitivity to isoflurane
compared to WT 88
Figure 3.2 Representative tracings of α1 GlyR USERs in response to
isoflurane 89
Figure 3.3 α1 GlyR USERs 1 and 2 have increased sensitivity to lidocaine
compared to WT 91
Figure 3.4 α1 GlyR USERs show no changes to propofol sensitivity
compared to WT 92
Figure 3.5 Representative tracings of α1 GlyR USERs in response to
propofol 93
Figure 3.6 α1 GlyR USER 4 demonstrates increased sensitivity to ethanol,
isoflurane, and lidocaine, but not propofol 95
Figure 4.1 WT and α2 GlyR USER KI mice on the accelerating rotarod in
response to 0.1% ethanol 113
Figure 4.2 WT and α2 GlyR USER KI mice on the accelerating rotarod in
response to 1% ethanol 113
Figure 4.3 WT and α2 GlyR USER KI mice on the accelerating rotarod in
response to 10% ethanol 114
Figure 4.4 α2 GlyR USERs exhibit no significant differences to the hypnotic
effect of 3.6 g/kg ethanol compared to WT 115
Figure 4.5 α2 GlyR USERs exhibit no significant differences in locomotor
activity compared to WT 116
Figure 4.6 α2 GlyR USERs exhibit no significant differences in thigmotaxic
behavior compared to WT 117
8

Figure 4.7 α2 GlyR USERs exhibit increased rearing behavior compared to
WT at 1% ethanol 117
Figure 4.8 α2 GlyR USERs exhibit differences in grooming behavior
compared to WT 118

9

List of Tables
Table 2.1 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 48
Table 2.2 Threshold for ethanol sensitivity in α1 and α2 GlyR USERs is
bimodal and lower than WT 52
Table 2.3 Threshold for ethanol sensitivity of γ2

and α1 GABA
A
R USERs is
bimodal and lower than α1β2γ2 GABA
A
R WT 55
Table 3.1 Loop 2 sequence alignment and mutations for the human WT
and α1 GlyR USERs 82
Table 3.2 Approximate EC50 values for isoflurane and propofol in WT and
α1 GlyR USERs 93

10

List of Abbreviations

AUD Alcohol use disorder
USER Ultra-sensitive ethanol receptor
GlyR Glycine receptor
GABA
A
R γ-aminobutyric acid subtype-A receptor
DSM-5 Diagnostic and statistical manual 5
th
edition
NIAAA National institute on alcohol abuse and alcoholism
WHO World health organization
FDA Food and drug administration
CNS Central nervous system
US United States
C Celsius
LGIC Ligand-gated ion channel
nAChR Nicotinic acetylcholine receptor
5-HT
3
5-hydroxytryptamine
3

NMDA N-methyl D-aspartate
AMPA α-amino-3-hydroxyisoxazolepropionic acid
NAc Nucleus accumbens
VTA Ventral tegmental area
LORR Loss of righting reflex
TM Transmembrane
GPCR G-protein coupled receptor
AC Adenylyl cyclase
PKC Protein kinase C
11

CRF-1 Corticotropin-Releasing Factor 1
CB 1 Cannabinoid-1
EC Extracellular
IC Intracellular
MTS Methylthiolsulfate
PMTS Propyl Methylthiolsulfate
DREADD Designer Receptors Exclusively Activated by Designer Drugs
P2X Purinoreceptor
ATA Atmosphere of pressure
OFC Orbitofrontal cortex
PFC Prefrontal cortex
mM Millimolar
EC
50
Half-maximal effective concentration
EC
2
Effective concentration at 2% of the maximal response
I
max
Maximum current
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
MAC Minimum alveolar concentration
SEM Standard error of the mean
WT Wild type
TALEN Transcriptional activator-like effector nucleases
RPM Revolutions per minute
IP Intraperitoneal
ANOVA Analysis of variance
S Seconds
Cm Centimeter
12

F
0
Initial parent generation
F
1
First filial generation
DUI Driving under the influence

13

Abstract

A critical obstacle to developing effective medications to prevent and/or treat an alcohol-
use disorder (AUD) is the lack of specific knowledge regarding the role of individual
receptors in mediating ethanol-induced reward, addiction, and dependence. The studies
presented in this dissertation introduce a transformative tool to investigate the primary
sites of ethanol action on individual receptor subunits that are expressed in the reward
and addiction brain regions. Here, we developed a novel class of Ultra-Sensitive
Ethanol Receptors (USERs) that allow activation of a single receptor subunit population
sensitized to extremely low ethanol concentrations. USERs were created by mutating as
few as two residues in the extracellular Loop 2 region of glycine receptors (GlyRs) or γ-
aminobutyric acid type A receptors (GABA
A
Rs), which are implicated in causing many
behavioral effects linked to ethanol abuse. USERs, expressed in Xenopus oocytes and
tested using two-electrode voltage clamp electrophysiology, demonstrated an increase
in ethanol sensitivity of 100-fold over wild-type receptors by significantly decreasing the
threshold for ethanol sensitivity and increasing the magnitude of ethanol response,
without altering general receptor properties including sensitivity to the neurosteroid,
allopregnanolone. These profound changes in ethanol sensitivity were observed across
multiple subunits of GlyRs and GABA
A
Rs. We also demonstrated the selectivity of
these USERs by reporting that the extracellular Loop 2 region is a common site of
action for anesthetics including ethanol, isoflurane, and lidocaine in α1 GlyRs.
Interestingly, Loop 2 was not a site of action for the general anesthetic, propofol. We
further employed USER technology in genetically engineered knock-in mice to
demonstrate proof-of-concept that USERs expressed in vivo can be activated by
14

extremely low ethanol concentrations. Ultimately, USERs provide a unique tool to
increase understanding of the individual role of receptors in mediating the reward and
addiction pathways of ethanol. Thus, USERs could help identify key sites of ethanol
action and potential drug targets that could treat and/or prevent alcohol addiction.

15

Chapter 1 Introduction
A. Background
The prevalence of alcohol abuse in our society results in a significant burden on
global health and economics. According to the current edition of the Diagnostic and
Statistical Manual (DSM-5), the diagnosis for an Alcohol Use Disorder (AUD)
encompasses characteristics of both alcohol abuse and alcohol dependence (DSM-5,
2013). AUD constitutes a spectrum of disorders that range from mild, moderate to
severe classifications. The National Institute on Alcohol Abuse and Alcoholism (NIAAA)
characterizes an AUD as a disease that involves symptoms of alcohol craving,
dependence, loss of control, and tolerance (NIAAA, 2014). In the United States (U.S.)
alone, approximately 18 million people suffer from an AUD, which results in a financial
burden that exceeds $200 billion, and nearly 100,000 deaths on an annual basis
(Bouchery et al., 2011b; Grant et al., 2004; Harwood, 2000). In fact, alcohol abuse is
considered the third leading preventable cause of death in the U.S. (NIAAA, 2014).
According to the World Health Organization (WHO), AUD was responsible for over 3.3
million annual deaths worldwide in 2013 (WHO, 2014). As of 2012, approximately 5.9%
of global deaths were attributable to AUD, a figure greater than global annual deaths
caused by HIV/AIDS, tuberculosis, and violence (WHO, 2014). Importantly, AUD is
considered a causal factor for at least 60 types of diseases and injuries including, but
not limited to, liver cirrhosis, neurodegeneration, diabetes, cancers, and psychological
disorders. Despite these serious risks, and the global prevalence of AUD, efficacious
therapies are minimal, and largely unsuccessful, as evidenced by the rising deaths and
costs associated with AUD.
16

Currently there are three Food and Drug Administration (FDA)-approved
medications for the treatment of AUD: disulfiram (Antabuse), naltrexone (oral
formulation: Revia, Depade; injectable formulation: Vivitrol, Naltrel), and accamprosate
(Campral) (Johnson, 2008). Despite the availability of these medications, adverse
effects, patient compliance, and limited efficacy have resulted in nearly 70% of patients
relapsing back to heavy drinking within the first year of treatment, inclusive of
pharmacotherapy and psychotherapy interventions (Johnson, 2008). The marginal
success of these medications for the prevention and/or treatment of AUD may be due to
their mechanisms of action; which mainly interfere with the downstream effects of
ethanol action by either blocking metabolism or curbing symptoms such as craving and
dependence (Colombo et al., 2007; Gewiss et al., 1991; Johnson et al., 2007).
Disulfiram, an acetylaldehyde dehydrogenase inhibitor, is thought to act by
preventing the oxidation of acetylaldehyde (a primary metabolite of alcohol) to acetic
acid. As a result, disulfiram causes a buildup of acetylaldehde, which is toxic to humans,
and leads to undesirable side-effects including headache, nausea, tachycardia, and
shortness of breath, upon alcohol consumption. Patients who are unable to abstain from
alcohol while taking disulfiram face serious medical risks of liver failure and toxicity due
to acetylaldehyde accumulation in the bloodstream (Franck and Jayaram-Lindstrom,
2013; Heilig and Egli, 2006). Thus, the strongest predictor of disulfiram efficacy
depends on close supervision during administration of the drug (Krampe et al., 2011).
Naltrexone, a competitive antagonist against the µ, δ, and κ opioid receptors is
thought to inhibit the rewarding effects of alcohol by preventing release of opioid
peptides that ultimately result in mesolimbic dopamine release (Sinclair et al., 2002).
17

Naltrexone has recently been found to be effective in reducing relapse rates in patients
carrying the 118G allele (Oslin et al., 2003). In addition, a 3-month multicenter clinical
trial revealed a 25% decrease in heavy drinking days compared to placebo in men, but
not in women (Garbutt et al., 2005). However, with a black box warning label issued by
the FDA for the risk of liver damage associated with both oral and injectable
formulations, safety issues associated with naltrexone results in limited therapeutic
success.
Acamprosate is thought to act as a glutamate receptor antagonist to regulate
elevated extracellular glutamate levels resulting from chronic alcohol consumption;
however, this mechanistic explanation requires further characterization and remains
questionable (De Witte et al., 2005). Notably, a limiting factor that contributes to patient
compliance with acamprosate is the high dosage and dosing regimen involving two 333-
mg tablets three times a day (Heilig and Egli, 2006). Importantly, the overall lack of
success associated with these medications is due to the lack of knowledge regarding
the precise targets of alcohol (ethanol) action in the central nervous system (CNS), and
the neurocircuitry underlying the behavioral effects of ethanol.
Understanding the initial sites of ethanol action and its downstream cascades
would increase knowledge regarding the pharmacological mechanism of ethanol action
and related anesthetics. More importantly, deciphering the important targets that
mediate ethanol-induced reward and addiction would contribute to the development of
more effective medications to treat and/or prevent AUD. The current theories
surrounding the mechanism of ethanol action are described in the following section.
18

B. Mechanism of ethanol and anesthetic action
Lipid membrane theory
Initially, researchers believed that the pharmacological action of anesthetics
(including alcohols) occurs by perturbation of the lipid membrane. Studies conducted
independently by H.H. Meyer and C.E. Overton in the early 1900s led to the hallmark
discovery of the Meyer-Overton theory, which suggested that the potency of alcohols
and anesthetics are proportional to their partition coefficient between the aqueous and
oil phase (Meyer, 1899, 1901; Overton, 1901). Thus, the correlation between lipid
solubility and potency implicates lipids as an important site for alcohol (ethanol) and
anesthetic action. Further studies by Meyer describe that anesthesia occurs by a
physical mechanism of membrane perturbation, which occurs when accumulation of a
chemical compound reaches a critical concentration in the lipid bilayer of the cell
(Meyer, 1937). The theoretical evolution regarding the physical and chemical
mechanisms of alcohol and anesthetics are described below.
Mullins integrated volume as a factor that causes anesthesia by describing that
the volume occupied by a chemical compound affects membrane expansion, and thus,
the permeability of ions through the cell (Mullins and Gaffey, 1954). Following this work,
Mullins proposed the ‘critical volume hypothesis’, which described that anesthesia
occurs when adsorption of an inert substance causes expansion of a hydrophobic
region, such as the lipid membrane, beyond a critical volume, thereby reducing ion
permeability and cellular excitability (Lever et al., 1971; Mullins and Gaffey, 1954). The
critical volume hypothesis also suggested that anesthesia could be reversed if the
disturbed hydrophobic region was restored by changes in pressure or temperature
19

(Lever et al., 1971; Mullins and Gaffey, 1954). In fact, studies demonstrated that
exposures to high pressure (> 100 atmosphere of pressure (ATA)) reversed the effects
of anesthesia by ethanol and other general anesthetic agents in vivo (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 et al., 1978; Trudell et
al., 1973).
Extension of the critical volume theory led to the development of the ‘membrane
expansion theory’, which suggested that the adsorption of an anesthetic agent could
cause membrane expansion and result in the disruption of membrane protein function
(Halsey, 1982; Miller et al., 1973). Furthermore, studies correlated hypothermia (low
body temperature) with increased ethanol potency and sensitivity in mice, supporting
the membrane expansion-fluidization theories of anesthesia (Malcom and Alkana 1983
(Alkana et al., 1988; Finn et al., 1990; Finn et al., 1991; Malcolm and Alkana, 1983).
Interestingly, the membrane expansion theory explained the protection of erythrocytes
from hemolysis due to anesthetic exposure by isotropically expanding the membrane in
three dimensions (Seeman and Roth, 1972). However, the membrane expansion
caused by anesthetics was approximated to be 10-fold greater in volume than the
molecules themselves (Seeman, 1972), implicating a discrepancy in the expansion
theory. Trudell et al. resolved this discrepancy by proposing that the overall membrane
surface thins when the membrane expands (Trudell, 1977).
Further studies demonstrated that pressure could reverse the effects of
anesthesia, by proposal of the “mean excess volume hypothesis” (Mori et al., 1984;
Ueda and Mashimo, 1982). This concept described a ‘critical volume’ by which the sum
20

of the molar volumes of the anesthetic and the target site of action should be less than
the total volume of the system. When this system is disrupted by exceeding the ‘critical
volume’ caused by conformational changes in the protein-lipid interfaces or creation of
cavities, pressure could act to restore the system and displace molecules back to a
lower-volume state.
While understanding the physical mechanism of anesthetic action is important, it
is also critical to consider the vast structural diversity among the chemical compounds
that are known to cause anesthesia in animals, which range from inert rare gases to
large steroid molecules (Seeman and Roth, 1972). Therefore, the actions of alcohol on
its putative targets involves non-specific and non-selective pharmacological action
(Trudell, 1977). Thus, these findings provide mounting evidence that alcohols and
anesthetics may also act on proteins embedded within the lipid membrane and/or
expressed independently of a lipid membrane environment (Buck et al., 1989; Hunt,
1985). Studies found that the physical mechanism of membrane perturbation alone was
not sufficient to define the mechanism of anesthesia. In fact, changes in temperature
alone of 3/10 °C resulted in membrane perturbation similar to that seen with intoxicating
concentrations of alcohols (Franks and Lieb, 1982; Pang et al., 1980). However, these
changes in membrane fluidity did not cause behavioral effects of alcohol intoxication,
indicating that physical changes in the membrane structure alone cannot account for the
mechanism of anesthesia or intoxication.
Protein Theory
Over half a decade after the Meyer-Overton theory was proposed, a seminal
study conducted by Franks and Lieb reported that general anesthetics act directly on
21

proteins. When general anesthetics were applied to purified, lipid-free firefly luciferin
and luciferase, the presence of anesthetics inhibited the normally light-emitting firefly
luciferase reaction, suggesting that anesthetics directly bind to ‘specific receptors’ and
compete with endogenous ligands (Franks and Lieb, 1984; Ueda and Mashimo, 1982).
Furthermore, optical isomers of the volatile anesthetic, isoflurane that disrupt membrane
fluidity equally as well as isoflurane, were found to stereoselectively exert their effects
on specific ion channels such as neuronal nicotinic acetylcholine receptors and gamma-
aminobutyric acid receptors (GABA
A
Rs) (Franks and Lieb, 1991; Hall et al., 1994; Jones
and Harrison, 1993). These stereoselective effects of isoflurane enantiomers on ion
channels also correlated with effects in vivo, in which the S(+)-isomer of isoflurane
induced a longer isoflurane sleep time than the R(-)-isomer, suggesting a difference in
the structure activity relationship of anesthetics to its target site of action (Harris et al.,
1992; Lysko et al., 1994). Recent investigations in protein targets of alcohols and
anesthetics have culminated in the development of atomic level resolution of crystal
structures of alcohols and anesthetics bound to ion channel proteins that are known
targets of these agents (Nury et al., 2011; Sauguet et al., 2013; Sauguet et al., 2014).
Taken together, these findings demonstrate that both the lipid bilayer and proteins are
targets of ethanol and anesthetic action in the central nervous system (CNS).
The non-selective and non-specific nature of the pharmacological action of
ethanol and other anesthetics implicate tremendous diversity in potential targets and
mechanisms of action for these agents. Current evidence implicates a multitude of
potential sites of alcohol and anesthetic action including: gap junctions in astrocytes,
head and tail groups of lipids, annular lipid proteins, signaling molecules such as protein
22

kinase C, mitochondrial proteins, and membrane proteins (Forman and Miller, 2011;
Howard et al., 2014; Olsen et al., 2014; Perkins et al., 2010). Thus, recent efforts have
investigated the complex interactions among these molecules and their targets to
increase understanding of the neurochemical cascades in the CNS that mediate the
behavioral effects of alcohol and anesthetics.
C. Protein Sites of Ethanol Action in the CNS
Ionotropic Receptors
Ligand-gated ion channels (LGICs) are transmembrane proteins that are activated
by the binding of a ligand to allow passage of specific ions through the cell membrane.
LGICs are implicated as the initial targets of ethanol action in the CNS that mediate a
myriad of ethanol-induced behavioral effects such as anxiolysis, hypnosis, aggression,
sedation, memory impairment, reward, addiction, motor incoordination, withdrawal,
dependence and tolerance (Badanich et al., 2013; Cardoso et al., 1999; Chau et al.,
2010a; Davies and Alkana, 2001b; Deitrich et al., 1989; Ericson et al., 2011; Findlay et
al., 2002; Harris, 1999; Kumar et al., 2009b; Mihic et al., 1997; Molander et al., 2007;
Molander and Söderpalm, 2005; Rewal et al., 2009; Ye et al., 1998; Zhou and Lovinger,
1996). LGICs consist of three major superfamilies: 1) Cys-loop superfamily, which are
pentameric in structure and include γ-amino butyric acid subtype-A (GABA
A
) and
subtype-C (GABA
C
), nicotinic acetylcholine (nACh), 5-hydroxytryptamine
3
(5-HT
3
) and
glycine receptors (GlyRs) (Cardoso et al., 1999; Crawford et al., 2007; Davies et al.,
2004; Davies et al., 2002; Grant, 1995; Mihic and Harris, 1996; Perkins et al., 2008,
2010; Zhou and Lovinger, 1996); 2) Glutamate superfamily, which are tetrameric in
structure and include N-methyl D-aspartate (NMDA), α-amino-3-
23

hydroxyisoxazolepropionic acid (AMPA) and kainate receptors (Monaghan et al., 1989;
Sommer and Seeburg, 1992); and 3) Purinergic superfamily, which are trimeric in
structure and include purinoreceptors (P2X) (Asatryan et al., 2011; Weight et al., 1999;
Xiong et al., 1999).
Pentameric LGICs of the Cys-loop superfamily have been a focus of alcohol and
anesthetic action due to their involvement in both inhibitory and excitatory
neurotransmission in humans. Generally, Cys-loop receptors can be separated into two
main categories of inhibitory and excitatory activity linked to behavioral processes
including: reward, addiction, consciousness, nociception, motor incoordination,
sedation, hypnosis, learning, memory, tolerance and withdrawal (Howard et al., 2014;
Kumar et al., 2009b). Inhibitory channels include GlyRs and GABA
A
Rs, as well as
glutamate-gated chloride channels of lower organisms such as worms (Caenorhabditis
elegans, c. elegans) and insects. Excitatory channels are cation-selective (primarily
sodium and calcium) and include nAChRs, and 5-HT
3
Rs. Traditionally, the potentiation
of excitatory channels has been implicated in stimulating the rewarding effects of
ethanol, while the enhancement of inhibitory channels by ethanol has been linked to the
physiologic effects caused by inhibitory action on the nervous system. However, recent
evidence reveals that GlyRs and GABA
A
Rs are increasingly important in regulating
intricate neurochemical pathways of both the physiological and psychological effects of
ethanol as described below. The following section will focus on the sites and
mechanisms of ethanol action on GlyRs and GABA
A
Rs.
24

Glycine Receptors (GlyRs)
Glycine receptors (GlyRs) are anion-selective LGICs that belong to the Cys-loop
superfamily. GlyRs are considered one of the major inhibitory neurotransmitter receptor
systems in the human central and peripheral nervous system (Dutertre et al., 2012;
Perkins et al., 2008). GlyRs are widely expressed throughout the CNS, and particularly
constitute neurotransmission in the brainstem and spinal cord (Betz, 1991). Three
human α subunits (α1-3) and an additional α subunit in mice (Matzenbach et al., 1994)
and chicks (α4) (Harvey et al., 2000), along with one β subunit can form pentameric
receptors that can be expressed either α-homomerically or heteromerically (αβ)
(Grenningloh et al., 1990; Pless and Lynch, 2009; Xiong et al., 2014). Heteromeric
GlyRs are typically arranged in a ratio of two α subunits: three β subunits (Weltzien et
al., 2012). GlyRs are selectively inhibited by the neurotoxin, strychnine.
Ethanol potentiates, or enhances GlyR activity, which ultimately augments dopamine
release in key brain regions linked to reward and addiction, including the striatum (Yadid
et al., 1993), nucleus accumbens (NAc) (Ericson et al., 2006), and the ventral tegmental
area (VTA) (Ye et al., 2004). Studies found that glycine microdialysis in the NAc of mice
increased release of extracellular dopamine and decreased ethanol consumption in a
strychnine-sensitive manner, suggesting that GlyRs are responsible for the rewarding
properties of ethanol (Molander and Söderpalm, 2005). Microdialysis of the selective
GlyR antagonist, strychnine, in the same brain region reversed these effects. In
addition, accamprosate, a current FDA-approved therapeutic for the treatment of AUD,
is thought to interact with GlyRs to increase dopamine release, and ultimately reduce
alcohol intake (Chau et al., 2010a; Chau et al., 2010b).
25

Broad expression of GlyRs throughout the brain and in the spinal cord indicates
involvement of these receptors in a myriad of physiological manifestations of ethanol.
For example, α1 subunit-containing GlyRs are mainly expressed in the brainstem and
spinal cord, and thus mediate motor control, spinal reflexes, and respiration (Legendre
(Legendre, 2001; Lynch, 2004). On the other hand, α2 and α3 subunit-containing GlyRs
are found mainly in the reward pathways including amygdala and NAc at equal or
greater expression levels compared to α1 GlyRs (Delaney et al., 2010; Jonsson et al.,
2009; Jonsson et al., 2012). Systemic administration of strychnine in mice reduced
ethanol-induced loss of righting reflex (LORR) implicating GlyR activity in the hypnotic
effects of ethanol (Ye et al., 2009). Mutations in the transmembrane (TM) region of the
α1 subunit of GlyRs in mice conferred changes in ethanol-induced motor incoordination
and a reduction in ethanol-induced LORR (Aguayo et al., 2014; Borghese et al., 2012;
McCracken et al., 2013). Mice lacking the α2 GlyR subunit demonstrated reduced
ethanol intake and preference, and increased initial aversive responses to ethanol
(Blednov et al., 2015). Mice lacking the α3 GlyR subunit exhibited increased ethanol
intake and preference, and increased aversive learning to ethanol. These findings
differentiate the contributions of individual subunits of GlyRs in the physical
manifestations of ethanol.
GABA
A
Receptors
GABA
A
Rs are anion-selective LGICs that constitute much of the inhibitory
neurotransmission in the CNS. Historically, GABA
A
Rs have served as pharmacological
targets for sedative-hypnotic and anxiolytic agents to reduce anxiety, motor activity, and
addiction (Garcia et al., 2010; Kumar et al., 2009b). There are at least 20 different
26

subunit isoforms that encode GABA
A
Rs characterized by the following subunit groups:
α, β, δ, ε, γ and ρ (Vithlani et al., 2011). GABA
A
Rs form pentameric structures, in which
the most widely expressed isoform in the adult human brain is α1β2γ2 in a ratio of 2:2:1
(Sieghart and Sperk, 2002). GABA
A
Rs are inhibited by bicuculline and picrotoxin (Olsen
and Sieghart, 2009).
GABA
A
Rs have long been implicated as a target of ethanol action and are
involved in mediating much of the behavioral effects of ethanol. Early biochemical
approaches such as chloride ion flux studies using cultured neurons demonstrated
GABA
A
R-dependent chloride ion uptake upon ethanol exposure (Allan and Harris, 1986,
1987; Suzdak et al., 1986; Ticku and Burch, 1980). Direct involvement of GABA
A
R
potentiation by ethanol was demonstrated by Davies and Alkana, who showed that
exposure to pressurized heliox, a physical antagonist of ethanol, on mouse membrane
vesicles inhibited GABA-activated ethanol potentiation by reducing total chloride uptake
(Davies and Alkana, 1998).
Early behavioral studies in mice implicated GABA
A
Rs as a direct target of n-
alcohols and anesthetics (Davies and Alkana, 1998, 2001a, b; Davies et al., 1997;
Davies et al., 2001a; Davies et al., 1999; Davies et al., 2002; Davies et al., 2001b;
Davies et al., 1998; Hanchar et al., 2006; Wallner et al., 2003, 2006). Ethanol action on
GABA
A
Rs has been found to cause anxiolysis, motor ataxia, impaired cognition,
sedation, hypnosis, tolerance, dependence and anti-convulsion (Deitrich et al., 1989;
Follesa et al., 2006; Grobin et al., 1998b; Kumar et al., 2009b; Lobo et al., 2008; Mody
et al., 2007; Olsen et al., 2007). Pharmacological antagonists, picrotxin and bicuculline,
reduce ethanol-induced behavioral effects in mice, further substantiating the role of
27

GABAergic activity in mediating the physiological effects of ethanol (Grobin et al.,
1998b; Harris et al., 2008; Kumar et al., 2009b).
Therefore, both GlyRs and GABA
A
Rs are responsible for a broad range of the
physiologic effects of ethanol action. Evidence from these earlier studies provides the
rationale for our focus on identifying the molecular targets and mechanisms of ethanol
action on GlyRs and GABA
A
Rs.
Metabotropic Receptors
In addition to LGICs, metabotropic G-protein coupled receptors (GPCRs) have
also been implicated in mediating the pharmacological effects of ethanol action (Kelm et
al., 2011b; Weiner and Valenzuela, 2006). Ethanol action on GPCRs triggers
intracellular second messenger signaling cascades including stimulation of the
PKA/adenylyl cyclase (AC) pathway (Kelm et al., 2011b) and protein kinase C (PKC)
pathway to alter GABA-mediated anxiety and alcohol self-administration (Choi et al.,
2002; Khasar et al., 1999; Kumar et al., 2009b; Lesscher et al., 2009; Proctor et al.,
2003). Recent studies by Yevenes et al. demonstrate that the sedative effects of
ethanol can be linked to a Gβγ-dependent mechanism that modulates channel gating of
α1 GlyRs (Yevenes et al., 2008). Other studies report that ethanol-induced GABAergic
neurotransmission in the CNS is driven by G(s) and G(q/11) coupled corticotropin-
releasing factor 1 (CRF 1) receptors in the central amygdala (the brain’s motivation
center) (Nie et al., 2004). A myriad of other Gα
i
-coupled GPCRs including GABA
B
,
cannabinoid-1 (CB1), nocieptin/orphanin FQ peptide, and δ-opioid receptor also
stimulate GABAergic inhibitory neurotransmission in the CNS (Kang-Park et al., 2007;
28

Kelm et al., 2011b; Roberto and Siggins, 2006; Silberman et al., 2009; Zhu and
Lovinger, 2006).
Thus, both ionotropic and metabotropic receptors serve a broad range of targets
for ethanol action. The sheer diversity of potential ethanol targets contributes to the
challenges of identifying the direct cause and effect of ethanol action resulting in the
behavioral effects that are responsible for developing AUD. The focus of this
dissertation will be on the mechanism of ethanol-induced reward and addiction
pathways of GlyRs and GABA
A
Rs.
D. Molecular Targets of Ethanol Action on Cys-Loop Receptors
While LGICs may differ widely by function, receptor activity, and activating
ligands, they share general common structural features that are conserved across
superfamilies including a TM domain, extracellular (EC) domain, and intracellular (IC)
domain. Studies have explored each of these domains as potential targets of ethanol
action.
Transmembrane
The TM region of GlyRs and GABA
A
Rs has been defined as an important site of
ethanol action. The use of a cysteine mutation at TM 267 and methylthiolsulfate (MTS),
an anesthetic-like agent that covalently binds to cysteine residues, blocked this amino
acid residue from ethanol action, and prevented ethanol modulation of the resultant
GABA
A
R, suggesting the involvement of position 267 in ethanol and anesthetic action
(Jung and Harris, 2006; Mascia et al., 2000). Radiophotolabeling studies further
corroborated TM 267 in the involvement of ethanol and anesthetic action in GABA
A
Rs
(Chiara et al., 2012; Chiara et al., 2014; Li et al., 2006). Mutagenesis and chimera
29

studies revealed additional sites within the TM domain of GlyRs in controlling ethanol
modulation of these receptors (Borghese et al., 2012; Lobo et al., 2008).
Intracellular
Residues in the IC loop of GlyRs interact with cell signaling molecules such as
Gβγ and regulate GlyR modulation by ethanol. Disruption of this IC loop independently
reduces ethanol potentiation of GlyRs without affecting other Gβγ signaling pathways
(San Martin et al., 2012). In addition, Gβγ heterodimer interaction with the IC region of
α1 GlyRs has been implicated in ethanol-induced potentiation (Guzman et al., 2009;
San Martin et al., 2012; Yevenes et al., 2006; Yevenes et al., 2008). The IC domain of δ
GABA
A
Rs is also involved in receptor internalization following ethanol exposure, linking
this domain to ethanol tolerance (Gonzalez et al., 2012). In fact, clathrin-mediated
endocytosis following ethanol exposure of δ GABA
A
Rs is dependent upon trafficking
mediated by the IC region of the receptor (Gonzalez et al., 2012). Thus, the IC domain
is likely important when considering chronic effects of ethanol exposure.
Extracellular
Recent evidence also implicates the EC domain, particularly Loop 2, as a critical
site of ethanol action that alters ethanol sensitivity of GlyRs and GABA
A
Rs (Crawford et
al., 2008; Naito et al., 2014; Olsen et al., 2014; Perkins et al., 2008, 2010; Perkins et al.,
2009; Wallner et al., 2014). Loop 2 spans a 10 amino acid region that is conserved
among all receptors that belong to the Cys-loop superfamily. Early studies found that
substitution of the amino acid residue at position 52 from alanine to a serine (A52S) in
α1 GlyRs significantly reduced ethanol sensitivity in the resultant receptor (Mascia et al.,
1996b), implicating this region as an important site of ethanol action. Further studies
30

from our laboratory using atmospheric pressure, a mechanistic antagonist of ethanol,
demonstrated that the A52S mutation in α1 GlyRs eliminated pressure antagonism of
ethanol (Davies et al., 2004); suggesting that position 52 is a key site of ethanol action.
Interestingly, an alanine substitution at the homologous position of the less ethanol-
sensitive α2 GlyR restored antagonism of ethanol in the presence of pressure (Perkins
et al., 2008). More importantly, in the absence of pressure, these A52S α2 GlyRs
demonstrated a significant increase in ethanol sensitivity, thus indicating that position 52
is a direct site of ethanol action.
Follow-up studies by Crawford et al. confirmed that EC Loop 2, particularly
position 52, forms a common ethanol action pocket with the TM domain spanning TM 2
and TM 3 regions (including TM 267) (Crawford et al., 2008). When position 52 was
blocked using cysteine substitution and PMTS, an agent that forms covalent disulfide
bonds with free cysteine residues, there was a significant increase in ethanol
potentiation of these α1 GlyRs. Interestingly, when TM 267 was blocked using the same
technique, the resultant effect of ethanol was negative modulation of α1 GlyR activity
(Crawford et al., 2007). Together, these findings indicate the existence of multiple sites
of ethanol action that mediate both positive and negative modulation by ethanol on
GlyRs. More importantly, these findings demonstrate that we must consider the
aggregate effects of ethanol on both the positive and negative modulatory sites when
investigating the structural and functional components that underlie ethanol action on
these receptors.
To determine the size of the ethanol action pocket formed between EC 52 and
TM 267, Crawford et al. investigated the n-chain alcohol cutoff by blocking these sites
31

independently and together. In earlier studies, blocking TM 267 using PMTS decreased
the volume necessary for alcohol action in this region and reduced the n-chain alcohol
cut-off from dodecanol to below octanol (Mascia et al., 1996a; Mascia et al., 2000).
Blocking position 52 similarly reduced the n-chain alcohol cut-off from dodecanol to
octanol (Crawford et al., 2007). Interestingly, blocking both TM 267 and EC 52 further
reduced the n-chain alcohol cut-off to below hexanol (Crawford et al., 2007). Hence,
these findings demonstrate that the TM region forms a common alcohol action pocket
with EC Loop 2 at position 52. The common structural homology among Cys-loop
receptors implies that these findings could also apply to GABA
A
Rs.
Further investigations of Loop 2 led by Perkins et al. involved production of
chimeric receptors that replaced the EC Loop 2 regions of α1 GlyRs and γ2 GABA
A
Rs
with Loop 2 of the ethanol-sensitive δ GABA
A
Rs. The rationale for swapping the Loop 2
regions for that of δ GABA
A
Rs is based on the discovery of native δ-subunit containing
GABA
A
Rs (α4β2/3δ and α6β2/3δ) that are sensitive to low ethanol concentrations in the
1-3 mM range (Olsen et al., 2007). In contrast, α1β2γ2 GABA
A
Rs, the most widely
expressed GABA
A
R isoform in the CNS, do not respond to ethanol concentrations
below 50 mM (Weiner et al., 1997). Based on earlier studies involving the alterations in
ethanol sensitivity through the manipulation of the amino acid residue at position 52,
Perkins and colleagues hypothesized that the amino acid sequence of Loop 2 of δ
GABA
A
Rs may be responsible for the increased ethanol sensitivity seen in δ GABA
A
Rs
(Perkins et al., 2009). Thus, the δ Loop 2 substitution significantly increased ethanol
sensitivity in α1 GlyRs, supporting their hypothesis. As a negative control, similar
substitutions of Loop 2 using the less ethanol-sensitive γ2 GABA
A
Rs did not increase
32

ethanol sensitivity of the resultant chimeric receptor. Therefore, EC Loop 2 is an
important target of ethanol action, and its structural elements are important
determinants of ethanol sensitivity for GlyRs.
E. Current Strategies for Identifying the Role of Receptors in Ethanol Action
A wide variety of approaches ranging from biochemical to biotechnological
advancements are utilized to explore the role of specific receptors in ethanol action.
While great strides have been made to produce high-resolution models of receptors and
the sites of ethanol action, much of our understanding of the direct cause and effect
mechanisms of ethanol reward and addiction remains poorly characterized. Much of the
difficulty in understanding the distinct mechanisms of ethanol action in the CNS is
attributable to its pharmacological nature. Ethanol acts with low affinity, and low
selectivity, and lacks structure activity relationship to its targets. As a result, the vast
diversity of ethanol targets precludes the use of classical pharmacological agonists and
antagonists to identify the distinct sites and mechanisms of ethanol action. Novel
approaches including mutagenesis, genetically engineered animals, Designer
Receptors Exclusively Activated by Designer Drugs (DREADD), and optogenetics have
been utilized to address this issue. These approaches are briefly described below.
Molecular Approaches
Biochemical methods have been a classical approach towards gaining insight
into identifying molecular targets and understanding how protein structures influence
drug action. Mutagenesis of receptors that are sensitive to ethanol have been the
predominant method of choice to investigate whether certain amino acid residues
mediate ethanol action. A classic example of this is the cysteine substitution approach
33

using an anesthetic-like agent, PMTS, which covalently binds to cysteine residues, and
ultimately blocks ethanol from interacting with this site. Mascia et al. were the first to
demonstrate that S267 in TM 2 and A288 in TM 3 are key sites of ethanol action of α1
GlyRs. Mutation of the serine at position 267 to cysteine (S267C) and the co-application
of PMTS and ethanol resulted in diminished ethanol-induced potentiation of α1 GlyRs in
the two-electrode voltage clamp electrophysiology model (Mascia et al., 2000). Other
studies have used mutagenesis approaches to identify sites of ethanol action that, when
mutated to a different amino acid residue, results in an ethanol-insensitive receptor.
Early studies in our laboratory confirmed findings by Mascia et al., who found that a
single point mutation at position 52 in α1 GlyRs from an alanine to serine (A52S) results
in an ethanol-insensitive receptor (Davies et al., 2004; Mascia et al., 1996b). These
studies have contributed important information regarding the sites of ethanol action.
Other traditional biochemical approaches have utilized western blots to
determine brain-regional receptor expression levels. For example, Badanich et al. were
the first to characterize the existence of extrasynaptic GlyRs that regulate ethanol-
induced excitatory projections in the lateral orbitofrontal cortex (OFC) neurons
(Badanich et al., 2013). Additional studies have identified tonic inhibition mediated by
extrasynaptic GlyRs that influence neuronal excitability in the hippocampus and
prefrontal cortex (PFC) (Badanich et al., 2013; Chattipakorn and McMahon, 2002; Keck
and White, 2009; Lu and Ye, 2011; Xu and Gong, 2010).
DREADD
Designer Receptors Exclusively Activated by Designer Drugs (DREADD)
involves pharmacological activation of DREADD-expressing neurons using clozapine N-
34

oxide (Khan, 2013). These DREADD-expressing neurons provide the capability of
mapping neurochemical pathways at neuronal resolution. However, this technique
utilizes an artificial agonist and is limited to providing information regarding the general
pathways based on stimulation of neuronal populations that express DREADD, rather
than identifying molecular target sites of ethanol action. Thus, while this approach
provides a much-needed overview of signaling pathways that are up-regulated as a
result of ethanol action, it is incapable of specifying the initial sites of action, and the
subsequent cause-effect cascades that result in neuronal excitation and/or inhibition.
Optogenetics
Optogenetics is the next-generation brain mapping technology that affords both
temporal and brain-regional control of specific neuronal populations of interest. This
strategy involves microbial opsins that allow photo activation or inhibition of defined
neuron populations, axonal pathways, or brain regions (Aston-Jones and Deisseroth,
2013). Thus, optogenetics helps characterize the cause-effect relationships between
stimulation of certain opsin-expressing neurons and the resultant neurochemical
pathway (Fenno et al., 2011). For example, NpHR, derived from the halobacterium
Natronomonas pharaonis, is a chloride pump activated by yellow light (Zhang et al.,
2007). NpHR has been used to enhance inhibitory projections in the NAc to
demonstrate the role of inhibitory striatal medium spiny neurons in regulating cocaine
conditioning in mice (Witten et al., 2010). However, despite these efforts, the exact sites
of drug action on specific receptor subunits in mediating these inhibitory and excitatory
neurochemical cascades in the CNS remain unclear.
35

A current challenge regarding the use of optogenetics is the capability to improve
optical stimulation of single cells. To improve upon this, Deisseroth et al. have explored
two-photon excitation studies to achieve finer spatiotemporal resolution (Prakash et al.,
2012). However, this approach requires the use of high average irradiance levels 2 x
10
6
W/cm
2
(Prakash et al., 2012). Irradiation levels on the order of 20 W/cm
2
are
sufficient to elicit thermal stimulation of temperature-sensitive neurons (Deng et al.,
2014). Thus, achieving precise single cell opsin stimulation is difficult because the
contribution of both light and temperature from opsins cannot be distinguished and
could lead to confounding results for genetically engineered opsin-expressing neuronal
populations that are close in proximity to temperature-sensitive neurons (Deng et al.,
2014). Taken together, while optogenetics is a promising novel brain-mapping tool,
achieving specificity at the receptor level, and precision for stimulation of desired
neuronal populations using this approach requires further refinement.
Molecular Modeling
Recent efforts have utilized radiophotolabeling approaches to visualize and
confirm sites of anesthetic action for pentameric LGICs including GlyRs and GABA
A
Rs.
However, the lack of a crystalized structure of human GlyRs and GABA
A
Rs pose
significant challenges to accurately predict the sites of drug action. To mitigate this,
current strategies involve the use of homologous X-ray crystallographic structures of
Gloeobacterviolaceus Ligand Gated Ion Channel (GLIC) as a template to model the
dynamic structures of alcohol- and anesthetic-bound pentameric LGICs at atomic
resolution (Nury et al., 2011; Sauguet et al., 2013). As a result, strictly
radiophotolabeling alone is not a feasible approach to identify sites of ethanol action.
36

Thus, functional in vitro studies using cellular systems such as electrophysiology are
necessary to help predict and validate the identification of binding sites as well as
develop potential pharmacophores that act to inhibit or mimic these sites of action.
Genetically engineered animal models
A variety of genetically engineered animal models have been developed that
express mutant receptors that are ethanol-insensitive. Current strategies that knock out
(KO) specific receptor subunits, or knock in (KI) mutant ethanol-insensitive receptors
have provided important insights (Blednov et al., 2013; Blednov et al., 2010; Chandra et
al., 2008; Liang et al., 2008; Moore et al., 2010). However, studies involving functional
deletion or reduction in receptor sensitivity to ethanol require testing with high ethanol
concentrations (10 to 40 mM) that affect other native receptor systems (Chandra et al.,
2008; Werner et al., 2006). Thus, although one ethanol-sensitive target is eliminated,
ethanol’s action on other receptors makes it difficult to isolate and identify the changes
in ethanol-induced behavioral responses produced by the mutations. Moreover, KO
studies can be complicated by complex compensatory responses that often arise during
development of genetically modified animals (Chandra et al., 2008; Findlay et al., 2003;
Mihalek et al., 2001; Werner et al., 2006). As a result, interpretation of these studies
must take into consideration confounding variables that could affect the overall results
obtained by these animal models.
F. USERs as Novel Tools for Alcohol and Brain Research
Collectively, molecular strategies, DREADD, optogenetics, molecular modeling,
and genetically engineered animal models have contributed complementary approaches
that have identified important targets and mechanisms of ethanol action. To date, there
37

are no tools available that allow direct activation of single receptor subunits in response
to allosteric modulators such as ethanol and anesthetics that enable us to decipher the
role of individual receptor subunits in causing the behavioral effects of ethanol and
anesthetics. The goal of the present work is to help fill these deficiencies in knowledge
and tools by developing a novel, potentially transformative strategy to identify the
physiological and behavioral effects caused by the actions of ethanol on specific
subunits of GlyRs and GABA
A
Rs.
The present dissertation investigates the hypothesis that Loop 2 manipulations
produce ultra-sensitive ethanol receptors (USERs) that significantly increase ethanol
sensitivity in GlyRs and GABA
A
Rs and provide a novel tool to identify the structural
features and mechanisms that control ethanol and anesthetic sensitivity. In support of
this hypothesis, the findings demonstrate two key concepts: 1) identification of new
extracellular targets for ethanol and anesthetics, and 2) provide proof-of-concept that
USERs represent a novel tool that can be employed to map the cascade of events
leading from ethanol modulation of specific populations of GlyRs expressed in addiction
and reward brain regions to behavioral change in mice.

38

Chapter 2 Glycine and GABA
A
Ultra-Sensitive Ethanol Receptors (USERs) as
Novel Tools for Alcohol and Brain Research

CHAPTER 2 ABSTRACT
A critical obstacle to developing effective medications to prevent and/or treat alcohol-
use disorders (AUDs) is the lack of specific knowledge regarding the plethora of
molecular targets and mechanisms underlying alcohol (ethanol) action in the brain. To
identify the role of individual receptor subunits in ethanol-induced behaviors, we
developed a novel class of Ultra-Sensitive Ethanol Receptors (USERs) that allow
activation of a single receptor subunit population sensitized to exly low ethanol
concentrations. USERs were created by mutating as few as four residues in the
extracellular Loop 2 region of glycine receptors (GlyRs) or γ-aminobutyric acid type A
receptors (GABA
A
Rs), which are implicated in causing many behavioral effects linked to
ethanol abuse. USERs, expressed in Xenopus oocytes and tested using two-electrode
voltage clamp, demonstrated 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 including sensitivity to the
neurosteroid, allopregnanolone. These profound changes in ethanol sensitivity were
observed across multiple subunits of GlyRs and GABA
A
Rs. Collectively, our studies set
the stage for employing USER technology in genetically engineered animals as a
unique tool to increase understanding of the neurobiological basis of the behavioral
effects of ethanol.
39

INTRODUCTION
Alcohol use disorders (AUDs) have a serious impact on global health and
economics. In the United States alone, AUDs affect over 18 million people, cause
approximately 100,000 deaths and cost over $200 billion annually (Bouchery et al.,
2011a; Grant et al., 2004; Harwood, 2000; Litten et al., 2012; Rehm et al., 2009).
Unfortunately, the success rate of available drugs has been limited, with approximately
70% of patients relapsing back to heavy drinking within the first year of treatment
(Johnson, 2008; Litten et al., 2012). Thus, the development of new pharmacotherapies
to treat AUDs is an important endeavor.
A critical barrier to the development of medications to prevent and/or treat AUDs
has been the lack of specific knowledge about where and how alcohol (ethanol) acts in
the brain and the resultant neurochemical cascades leading to behavioral change. This
paucity of knowledge largely reflects the physical-chemical mechanism of ethanol action
and low potency that requires millimolar (mM) concentrations to alter brain function. The
resultant lack of high affinity structure-activity relationship precludes the classical
approach of employing specific agonists and antagonists to identify the sites and
mechanisms of ethanol action (Deitrich et al., 1989; Little, 1991). This problem is further
complicated by the multiple receptor subunit combinations affected by ethanol and the
complex acute and chronic mechanisms of ethanol action (Trudell et al., 2014).
Current strategies that knock-out (KO) receptor subunits or knock-in (KI) mutant
ethanol-insensitive receptors have provided important insights (Blednov et al., 2010;
Blednov et al., 2003; Chandra et al., 2008; Liang et al., 2008; Moore et al., 2010; Trudell
et al., 2014). However, these studies involving functional deletion or reduction in
40

receptor sensitivity to ethanol require the use of relatively high ethanol concentrations
(10 to 50 mM) (Blednov et al., 2011; Blednov et al., 2010; Borghese et al., 2006) that
may affect other native receptor systems and signaling pathways that modulate
additional physiological processes (Chandra et al., 2008; Harris et al., 2008; Howard et
al., 2011; Kelm et al., 2011a; Kumar et al., 2009a; Liang et al., 2008). In comparison, 17
mM ethanol is equivalent to the 0.08% blood ethanol concentration (BEC) legal driving
limit in the United States (Ogden and Moskowitz, 2004; Wallner et al., 2003). Moreover,
KO studies can be complicated by developmental compensatory responses that can
alter the expression levels of different receptor subtypes/families in the genetically
modified animals (Brickley et al., 2001; Homanics et al., 2005; Peng et al., 2002;
Ponomarev et al., 2006). Thus, the observed changes in ethanol-induced behaviors
may be a result of the indirect effects of the interplay among compensatory responses
resulting from the gene replacement, thereby complicating the interpretation of results.
GlyRs and GABA
A
Rs are the primary inhibitory ligand-gated ion channels
(LGICs) in the brain that have been implicated in causing many acute and chronic
behavioral effects of ethanol including: tolerance, dependence, reward, anxiolysis,
motor ataxia, impaired cognition, sedation, and aggression (Deitrich et al., 1989;
Dutertre et al., 2012; Follesa et al., 2006; Grobin et al., 1998a; Kumar et al., 2009a;
Lobo and Harris, 2008; Olsen et al., 2007). Prior studies identified extracellular Loop 2
of GlyRs and GABA
A
Rs as a site of ethanol action, and that structural modifications in
this region profoundly influences receptor sensitivity to ethanol (Crawford et al., 2007;
Davies and Alkana, 2003; Davies et al., 2004; Mascia et al., 1996b; Perkins et al.,
2008). This initial work found that replacing Loop 2 of α1 GlyR and γ2 GABA
A
R subunits
41

with Loop 2 of the more ethanol-sensitive δ GABA
A
Rs significantly increased ethanol
sensitivity of the resultant receptor and identified important physical-chemical properties
of Loop 2 that alter receptor sensitivity to ethanol and agonist (Crawford et al., 2008;
Perkins et al., 2012; Perkins et al., 2009).
The objective of our current study was to further characterize Loop 2 as a novel
tool that would distinguish the contribution of individual receptor subunits in ethanol
action. Thus, we manipulated the physical-chemical characteristics of Loop 2 to develop
Ultra-Sensitive Ethanol Receptors (USERs) in GlyRs and GABA
A
Rs that: 1) are
sensitive to ethanol concentrations lower than those that affect any other receptor
system; 2) have wild-type (WT)-like receptor properties; and 3) can be produced across
multiple receptor subunits of LGICs. The unique characteristics of USERs provide the
rationale for exploiting these receptors in genetically engineered animals to link the role
of specific receptor subunits in behaviors mediated by ethanol action without affecting
other targets. Ultimately, USERs would increase understanding of the neurological
basis of AUDs.

42

MATERIALS AND METHODS
Materials
Adult female Xenopus laevis frogs were purchased from Nasco (Fort Atkinson, WI).
Gentamycin, 3-aminobenzoic acid ethyl ester, glycine, GABA, ethanol, and collagenase
were purchased from Sigma (St. Louis, MO), allopregnanolone (3α,5α-THP) was
purchased from Steraloid (Newport, RI). All chemicals used were of reagent grade.
Glycine and GABA 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 (Davies,
2003). 10 mM 3α,5α-THP stock solution was prepared from powder and diluted with
DMSO and serially diluted with MBS (DMSO ≤ 0.05%) immediately prior to testing. Pilot
studies found that DMSO at this concentration, with or without agonist, had no
appreciable effect on α1β2γ2 GABA
A
R currents in WT or mutant receptors.
Mutagenesis and Expression of GlyRs and GABA
A
Rs in Oocytes
Homologous amino acid sequences of extracellular Loop 2 regions of WT α1 and α2
GlyR, α1 and γ2 GABA
A
R subunits were identified (Table 2.1). Loop 2 for the purpose of
this study is defined as the following amino acid positions: 50-59 in α1 GlyR subunit, 57-
66 in α2 GlyR subunit, 43-52 α1 GABA
A
R subunit and 64-73 in γ2 GABA
A
R subunit
(Mihic et al., 1997; Perkins et al., 2009). α1 GlyR and γ2 GABA
A
R USER 1 were
developed according to methods described previously (Perkins et al., 2009). Briefly,
site-directed mutagenesis was performed in α1 GlyR and γ2 GABA
A
R subunit cDNA so
that the resulting receptors’ Loop 2 regions were identical to the δ GABA
A
R Loop 2 to
produce α1 GlyR and γ2 GABA
A
R USER 1. Subsequently, we used prior structure
43

activity relationships gleaned from mutational studies in Loop 2 to develop second
generation GlyR and GABA
A
R USERs. Site-directed mutagenesis was performed by
subcloning human α1 and α2 GlyR, and α1 and γ2 GABA
A
R cDNA into mammalian
vector pCIS2 or pBK-CMV using Quick Change Site-Directed Mutagenesis kit
(Stratagene, La Jolla, CA) and verified by partial sequencing (DNA Core Facility,
University of Southern California) as described previously (Davies et al., 2003). Stage V
or VI Xenopus oocytes were isolated and injected with homomeric α1

or α2 GlyR cDNA
(1 ng/32 nl) or α1β2γ2 GABA
A
R cDNA (1:1:10 ratio for a total volume of 1 ng of
α1β2γ2).
Oocytes were stored in incubation medium [ND96 supplemented with 2 mM sodium
pyruvate, 50 mg/ml of gentamycin and 10 ml heat inactivated HyClone® horse serum
(VWR, San Dimas, CA), adjusted to pH 7.5] in Petri dishes (Corning, NY). All solutions
were sterilized by passage through 0.22-µM filters. Injected oocytes were stored at 18°C
and used in electrophysiological experiments 24 - 48 hours after injection for a period of
1 week.
Whole Cell Two-Electrode Voltage Clamp Recordings
Two-electrode voltage clamp recording was performed using techniques according
to those previously reported (Davies et al., 2003; Perkins et al., 2009). Briefly, oocytes
were voltage clamped at a membrane potential of -70 mV using oocyte clamp OC-725C
(Warner Instruments; Hamden, CT) and the oocyte recording chamber was
continuously perfused with MBS ± ethanol and/or agonist using a Dynamax peristaltic
pump (Rainin Inst Co., Emeryville, CA) at 3 ml/min using an 18-gauge polyethylene tube
(Becton Dickinson, Sparks, MD) and resultant currents were recorded.
44

Application of Agonist – GlyR or GABA
A
R WT and USERs were exposed to 1 µM - 10
mM glycine or GABA for 30 s at a rate of 3 ml/min, with 5 - 15 min washout periods
between applications to ensure complete receptor re-sensitization.
Application of Ethanol – Potentiation of Cl
−
currents by ethanol is difficult to measure
since the degree of ethanol potentiation is decreased and probability of receptor de-
sensitization is increased when using agonist concentrations close to EC
50
(Mascia et
al., 1996b; Mihic et al., 1994a; Mihic et al., 1994b). In general, most studies involving
mutagenesis of GlyRs and GABA
A
Rs often result in changes in EC
50
for agonists, while
the maximum current remains unchanged (Davies et al., 2004; Mascia et al., 1996a;
Mascia et al., 1996b). However, in these mutant receptors, the agonist concentrations to
achieve EC
2
- EC
10
are not significantly different from that of WT. 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 s
followed by co-application of ethanol and agonist for 30 s at a perfusion rate of 3 ml/min
(Davies et al., 2004). Washout periods (5 - 15 min depending on ethanol concentration
tested) were allowed between agonist and drug applications to ensure complete
receptor re-sensitization. WT and mutant receptor responses were measured across
ethanol concentrations ranging 0.025 mM - 50 mM for α1 GlyRs, and 0.1 mM - 50 mM
for α2 GlyRs. α1 and γ2 GABA
A
R USER responses were measured across ethanol
concentrations ranging 0.1 mM - 50 mM and were compared to α1β2γ2 GABA
A
R WT.
45

Holding currents were not significantly affected during pre-incubation with ethanol, i.e. in
the absence of agonist.
Application of Allopregnanolone (3α,5α-THP) – To investigate the effects of endogenous
neurosteroids on USERs, we tested two physiologically relevant concentrations of
3α,5α-THP (20 nM and 100 nM) on α1β2γ2 GABA
A
Rs and α1 and γ2 GABA
A
R USERs.
The concentrations of 3α,5α-THP were based on findings describing 3α,5α-THP levels
in the temporal cortex of post-mortem brain tissue in cognitively intact patients ranging
5.644 ng/g – 32.11 ng/g (18 nM – 100 nM) (Naylor et al., 2010); and the highest
concentration of serum 3α,5α-THP levels in pregnant women during the third trimester
reported at 50 ng/ml (157 nM) (Luisi et al., 2000). Electrophysiological recordings were
conducted according to the methods described above for ethanol application.
Molecular Modeling
Homology models of α1β2γ2 GABA
A
R with mutations in Loop 2 of the γ2 subunit
were built by concatenating the primary sequence of GABA
A
R in the order γ/α/β/α/β.
This sequence was aligned with the five subunits of the glutamate-gated chloride
channel (GluCl, PDB ID 3RHW) (Hibbs and Gouaux, 2011). As shown in Table 1,
mutations in the sequence of γ2 Loop 2 were performed to correspond to γ2 GABA
A
R
USERs 1 and 4. The models were built with the Modeler Module of Discovery Studio 3.5
(Accelrys, San Diego, CA) and then optimized as described previously (Perkins et al.,
2012).
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
46

expressed as mean ± SEM. Where no error bars are shown, they are smaller than the
symbols. Non-linear regression analysis was used to analyze EC
50
, Hill slope and I
max

for the agonist concentration response curves. The thresholds for ethanol sensitivity for
GlyR and GABA
A
R WT and USERs were determined by comparing the percent change
in agonist EC
2
-induced Cl
-
currents in the presence and absence of ethanol across all
tested ethanol concentrations using Student’s t test. Statistical significance was defined
as * p<0.05.
47

RESULTS
The structure-activity relationships identified by previous mutational studies in Loop 2
(Crawford et al., 2008; Perkins et al., 2009; Perkins, 2012) served as the basis for
selecting mutations in the homologous regions of α1 and α2 GlyRs and α1 and γ2
GABA
A
Rs to develop USERs as described below. Representative tracings for the WT and
mutant α1 GlyRs are shown below in Figure 2.1. Loop 2 sequence alignments of USER
subunits are shown in Table 2.1.
FIGURE 2.1

Figure 2.1
Loop 2 mutations in α1 GlyRs produce ultra-sensitive ethanol receptors that are
sensitive to ≤ 0.5 mM ethanol.
Two electrode voltage clamp electrophysiology tracings of homomeric α1 GlyR WT and α1 GlyR USERs
expressed in Xenopus oocytes. (A) α1 GlyR WT in response to 0.5 mM and 50 mM ethanol; (B) α1 GlyR
USER 1 in response to 0.5 mM ethanol; (C) α1 GlyR USER 2 in response to 0.5 mM ethanol; and (D) α1
GlyR USER 3 in response to 0.1 mM ethanol. Effects of ethanol were tested with EC
2
glycine (5 - 25 µM).

48

TABLE 2.1

Table 2.1 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 (indicated by solid line) and Exon 4 (indicated by dotted line). 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 subunits spans Exon 3. GlyR and GABA
A
R EC
50
, Hill slope and Imax are presented as
mean ± SEM from at least 4 - 23 oocytes. T tests revealed no significant differences in Imax and Hill
slope between GlyR and GABA
A
R WT and USERs. There were no statistically significant differences in
the EC50 values of α1 GlyR USER 3, α2 GlyR USER 1 and α1 GABA
A
R USER 1 compared to that of
respective WT receptors. EC50 values of α1 GlyR USERs 1 and 2, and γ2 GABA
A
R USERs 1 - 4 were
significantly reduced compared to that of respective WT receptors. EC
50
values of α1 GlyR USER 3, α2
GlyR USER 1 and α1 GABA
A
R USER 1 were not significantly different from respective WT receptors.

49

Manipulation of Loop 2 results in GlyR and GABA
A
R USERs that are sensitive to
ultra-low ethanol concentrations (<1 mM)
α1 GlyR WT
Ethanol produced concentration-dependent potentiation of glycine-induced Cl
-

currents in α1 GlyR WT with significant effects starting at 30 mM ethanol (Fig. 2.2A,
Table 2.2). There were no significant effects of ethanol on these WT receptors at
concentrations below 30 mM.
α1 GlyR USER 1 (First generation α1 GlyR USER)
Earlier studies reported that Loop 2 mutant, α1 GlyR USER 1 had increased ethanol
sensitivity compared to WT receptors at 1 mM - 30 mM ethanol (Perkins et al., 2009).
To establish the ethanol threshold in these receptors, we expanded the ethanol
concentration range from 0.025 mM - 50 mM. At ultra-low concentrations (<1 mM)
1
,
ethanol produced a concentration-dependent effect with significant potentiation starting
at 0.075 mM. Thus, the threshold for ethanol sensitivity in α1 GlyR USER 1 was
significantly reduced to 0.075 mM from 30 mM in α1 GlyR WT (Fig. 2.2A, Table 2.2). At
higher concentrations (>3 mM)
2
, ethanol produced a concentration-dependent effect
with significant potentiation in α1 GlyR USER 1 beginning at 3 mM (Fig. 2.2A, Table
2.2).
Notably, the ethanol concentration response demonstrated a bimodal pattern (Fig.
2.2A). The first curve followed an inverse U pattern ranging 0.025 mM - 1 mM, and the
second curve started at 3 mM, with concentration-dependent increases in potentiation
until 30 mM ethanol, where the response appeared to plateau.
α1 GlyR USER 2
50

We and others previously reported that a single point mutation at position 52 in WT
α1 GlyRs from alanine to serine (A52S) significantly reduced the sensitivity of the
receptor to ethanol (Davies et al., 2004; Mascia et al., 1996b). Interestingly, the α1 GlyR
USER 1 includes the same alanine to serine mutation at position 52. We therefore
tested whether reverting serine at position 52 in α1 GlyR USER 1 to the WT alanine
would further increase ethanol sensitivity of this new mutant receptor (α1 GlyR USER 2,
Table 2.1).
The resultant α1 GlyR USER 2 showed an increase in ethanol sensitivity compared
to α1 GlyR WT by decreasing the ethanol threshold and increasing the magnitude of
ethanol response (Fig. 2.2A, Table 2.2). While retaining significant ethanol sensitivity
relative to WT, the mutations in α1 GlyR USER 2 did not cause an incremental increase
in ethanol sensitivity relative to α1 GlyR USER 1 with respect to both ethanol threshold
(0.075 mM and 3 mM, respectively for the ultra-low and high concentration ranges) and
the magnitude of ethanol response. The ethanol concentration response followed a
bimodal pattern for α1 GlyR USER 2 similar to that of α1 GlyR USER 1.

51

FIGURE 2.2

Figure 2.2 α1 GlyR USERs have increased ethanol sensitivity and bimodal 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 (denoted by black curves). The first curve followed
an inverse U pattern that extended from 0.025 mM to 3 mM ethanol, and the second curve extended
across higher concentrations beginning at 3 mM ethanol with concentration-dependent increases in
potentiation until 50 mM. Values for ethanol potentiation are presented as percentage of glycine EC
2

control. The glycine EC
2
concentrations utilized ranged from 5 - 10 µM in USERs and 18 - 25 µM for WT.
There was a significant decrease in the threshold for ethanol sensitivity and increase in the magnitude of
ethanol response in α1 GlyR USERs compared to WT. Each data point represents the mean ± SEM from
at least 4 - 9 oocytes.
Figure 2B. Manipulation of Loop 2 restores the agonist concentration response for
α1 GlyR USER 3.
[B] Glycine-induced Cl− currents were normalized to the maximal current activated by a saturating
concentration of glycine (1,000 µM - 10,000 µM). The curves represent non-linear regression analysis of
the glycine concentration responses in α1 GlyR USERs and WT. There was a significant left shift in the
agonist concentration response curve for α1 GlyR USERs 1 and 2 relative to WT. The agonist
concentration response curve for α1 GlyR USER 3 was fully restored to WT. Each data point represents
the mean ± SEM from at least 4 - 23 oocytes.

52

TABLE 2.2

Lower Ethanol Threshold (mM) Upper Ethanol Threshold (mM)
α1 GlyR WT n/a 30
α1 GlyR USER 1 0.075

3
α1 GlyR USER 2 0.075 3
α1 GlyR USER 3 0.05 10
α2 GlyR WT n/a 50
α2 GlyR USER 1 0.25 10

Table 2.2 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 higher (> 3 mM) ethanol
concentrations. The threshold for ethanol sensitivity was reduced from 30 mM ethanol in α1 GlyR WT to
0.075 mM in α1 GlyR USERs 1 and 2, and 0.05 mM in α1 GlyR USER 3. At higher ethanol
concentrations, α1 GlyR USERs 1 and 2 exhibited a threshold of 3 mM ethanol, while α1 GlyR USER 3
exhibited a threshold of 10 mM ethanol. In α2 GlyR USERs, thresholds were significantly reduced from 50
mM ethanol in α2 GlyR WT to 0.25 mM and 10 mM in α2 GlyR USER 1. Each data point represents the
mean ± SEM from at least 4 - 16 oocytes. All threshold values were statistically significant with p values <
0.05.
α1β2γ2 GABA
A
R WT (GABA
A
R WT)
Ethanol produced concentration-dependent potentiation of GABA-induced Cl
-

currents in GABA
A
R WT with significant effects starting at 50 mM ethanol (Fig. 2.3A,
Table 2.3). There were no significant effects of ethanol on these WT receptors at
concentrations below 50 mM.
γ2 GABA
A
R USER 1 (First generation γ2 GABA
A
R USER)
Earlier studies reported that Loop 2 mutant γ2 GABA
A
R USER 1 had increased
ethanol sensitivity compared to GABA
A
R WT in response to 0.25 mM - 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 to the previously reported 0.5 mM (Table 2.3)
(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 to 0.25 mM from 50 mM in
53

GABA
A
R WT (Table 2.3). 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 mM - 1 mM, and a second curve beginning at 3 mM with a
concentration-dependent increase in ethanol response (Fig. 2.3A).
γ2 GABA
A
R USER 2
The reversion at position 52 from serine to the WT alanine in α1 GlyR USER 2
increased ethanol sensitivity relative to α1 GlyR WT. We used a similar approach in γ2
GABA
A
R USER 2 by reverting serine at the homologous position in γ2 GABA
A
R USER
1 back to WT asparagine (S66N) to test whether this would increase ethanol sensitivity.
Similar to results for γ2 GABA
A
R USER 1, the resultant γ2 GABA
A
R USER 2
showed an increase in ethanol sensitivity compared to WT with decreased ethanol
threshold and increased magnitude of ethanol potentiation (Fig. 2.3A, Table 2.3).
However, the ethanol threshold and magnitude of potentiation between the two mutant
γ2 GABA
A
R USERs remained similar (Fig. 2.3A). The ethanol concentration response
followed a bimodal pattern for γ2 GABA
A
R USER 2 similar to that of γ2 GABA
A
R USER
1.

54

FIGURE 2.3
Figure 2.3 γ2 GABA
A
R USERs 1, 2 and 3 have increased ethanol sensitivity and bimodal 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 (denoted
by black curves). The first curve followed an inverse U pattern that extended from 0.1 Mm to 3 Mm
ethanol, and the second curve extended across higher concentrations beginning at 3 Mm ethanol with
concentration-dependent increases in potentiation until 50 Mm. Values for ethanol potentiation are
presented as percentage of GABA EC
2
control. The GABA EC
2
concentrations utilized were 5 µM for
USERs and 10 µM for WT. There was a significant decrease in threshold for ethanol sensitivity and
increase in the magnitude of ethanol response compared to WT. γ2 GABA
A
R USER 4 was insensitive to
ethanol across the tested concentration range. Each data point represents the mean ± SEM from at least
4 – 9 oocytes.
[B] Manipulation of Loop 2 partially restores the agonist concentration response for γ2 GABA
A
R USER 3.
GABA-induced Cl
−
currents were normalized to the maximal current activated by a saturating
concentration of GABA (1,000 µM – 10,000 µM). The curves represent non-linear regression analysis of
the GABA concentration responses in γ2 GABA
A
R USERs and WT. There was a significant left shift in the
agonist concentration response curve for γ2 GABA
A
R USERs 1, 2 and 4 relative to WT. The agonist
concentration response curve for γ2 GABA
A
R USER 3 was partially restored to WT. However, there was
a statistically significant difference between the EC
50
values of γ2 GABA
A
R USER 3 and WT (Table 1).
Each data point represents the mean ± SEM from at least 4 – 23 oocytes.

55

TABLE 2.3

Lower Ethanol Threshold (mM) Upper Ethanol Threshold (mM)
α1β2γ2 GABA
A
R WT n/a 50
γ2 GABA
A
R USER 1 0.25 10
γ2 GABA
A
R USER 2 0.25 10
γ2 GABA
A
R USER 3 0.25 10
γ2 GABA
A
R USER 4 n/a n/a
α1 GABA
A
R USER 1 0.25 10

Table 2.3 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. The threshold for ethanol sensitivity was reduced from 50
mM ethanol in α1β2γ2 GABA
A
R WT to 0.25 mM and 10 mM for γ2 GABA
A
R USERs 1, 2 and 3, and α1
GABA
A
R USER 1. The γ2 GABA
A
R USER 4 produced ethanol-insensitive receptors. All GABA
A
R USERs
represent the α1β2γ2 isoform. Each data point represents the mean ± SEM from at least 4 - 13 oocytes.
All threshold values were statistically significant with p values < 0.05.

Agonist sensitivity of α1 GlyR and γ2 GABA
A
R USERs 1 and 2 were modestly
altered compared to respective WT
Glycine produced inward Cl
−
currents in a concentration-dependent manner (Fig.
2.2B). α1 GlyR WT values for glycine EC
50
, Hill slope and I
max
are shown in Table 2.1
and were consistent with results previously reported (Crawford et al., 2007; Perkins et
al., 2009). In both α1 GlyR USERs (USERs 1 and 2), Loop 2 mutations produced a left
shift in the agonist concentration response curve relative to α1 GlyR WT with a
significant decrease in EC
50
(Fig. 2.2B, Table 2.1). There were no significant
differences in Hill slope and I
max
between α1 GlyR USERs 1 and 2 and α1 GlyR WT
(Table 2.1).
GABA produced inward Cl
−
currents in a concentration-dependent manner (Fig.
2.3B). Values for EC
50
, Hill slope and I
max
of α1β2γ2 GABA
A
R WT are shown in Table 1
and are consistent with results previously reported (Perkins et al., 2009). In both γ2
GABA
A
R USERs 1 and 2, Loop 2 mutations produced a left shift in the agonist
56

concentration response curve relative to GABA
A
R WT with a significant decrease in
EC
50
(Fig. 2.3B, Table 2.1). There were no significant differences in Hill slope and I
max

between γ2 GABA
A
R USERs 1 and 2 and GABA
A
R WT (Table 2.1).
Since receptor characteristics of GlyR and GABA
A
R USERs 1 and 2 were modestly
altered compared to WT, we next restored additional Loop 2 residues in GlyR and
GABA
A
R USER 1 back to WT to determine if these changes would normalize EC
50
and
restore WT-like agonist sensitivity.
Manipulation of the Loop 2 structure results in GlyR and GABA
A
R USERs with
normalized receptor characteristics
α1 GlyR USER 3
The Loop 2 sequence of α1 GlyR USER 1 spans across both Exons 3 (positions 50-
55) and 4 (positions 56-59) of the human GLRA1 (glycine receptor, alpha 1) gene
(Table 2.1). To avoid possible complications for gene incorporation and functional
expression of USERs in vivo, we developed a mutant receptor that limited the mutations
in α1 GlyR USER 1 to Exon 3 (Table 2.1).
There was no significant change in agonist sensitivity in α1 GlyR USER 3 compared
to α1 GlyR WT, as indicated by similar EC
50
values (Fig. 2.2B, Table 2.1). Thus, the
agonist concentration response curve for α1 GlyR USER 3 was right-shifted relative to
α1 GlyR USERs 1 and 2 (Fig. 2.2B, Table 2.1). The other receptor characteristics of α1
GlyR USER 3 including Hill slope and I
max
did not differ significantly from α1 GlyR WT
(Table 2.1).
α1 GlyR USER 3 demonstrated an increase in ethanol sensitivity compared to α1
GlyR WT and α1 GlyR USERs 1 and 2, by decreasing the ethanol threshold to 0.05 mM
57

(Fig. 2.2A and Table 2.2). At ultra-low ethanol concentrations, α1 GlyR USER 3
demonstrated an increase in the magnitude of ethanol response compared to α1 GlyR
WT and USERs. Remarkably, at 0.1 mM ethanol, the magnitude of response in this
USER was equivalent to that of 30 mM ethanol in α1 GlyR WT (Fig. 2.2A). Across high
ethanol concentrations, the magnitude of ethanol response was lower than that of α1
GlyR USERs 1 and 2, but comparable to α1 GlyR WT (Fig. 2.2A). As with the other α1
GlyR USERs, the ethanol concentration response for α1 GlyR USER 3 was bimodal.
γ2 GABA
A
R USER 3
Crawford et al. 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 (Crawford et al., 2008). Therefore, we
hypothesized that substituting the charged glutamate at position 67 (homologous to
position 53 in α1 GlyRs) with the neutral WT alanine in GABA
A
R USER 1, would right
shift and thus normalize agonist sensitivity compared to GABA
A
R WT.
As predicted, the GABA concentration response curve for this USER was right-
shifted compared to γ2 GABA
A
R USERs 1 and 2 (Fig. 2.3B). However, γ2 GABA
A
R
USER 3 was significantly more sensitive to agonist compared to GABA
A
R WT (Fig 2.3B,
Table 2.1). Thus, these Loop 2 manipulations in γ2 GABA
A
R USER 3 partially restored
agonist sensitivity. Hill slope and I
max
did not differ significantly from GABA
A
R WT in γ2
GABA
A
R USER 3 (Table 2.1).
The ethanol threshold in γ2 GABA
A
R USER 3 was significantly reduced to 0.25 mM
compared to 50 mM in GABA
A
R WT (Table 2.3). The magnitude of ethanol response at
58

ultra-low concentrations was higher than GABA
A
R USER 1. 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. 2.3A).
γ2 GABA
A
R USER 4 (Null Mutant)
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 (Table 2.1). However, these mutations left-shifted the agonist concentration
response and thus did not normalize agonist sensitivity (Fig. 2.3B, Table 2.1). Hill slope
and I
max
of γ2 GABA
A
R USER 4 did not differ significantly from GABA
A
R WT (Table 2.1).
In contrast to the expected increase in ethanol sensitivity, this USER was insensitive to
ethanol across all concentrations tested (Fig. 2.3A, Table 2.3).
Loop 2 manipulations in multiple subunits of GlyRs and GABA
A
Rs result in
USERs
To demonstrate the applicability of USERs across multiple subunits and receptors of
the Cys-loop superfamily, we developed USERs in two new subunits: α2 GlyR and α1
GABA
A
R. 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). 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
59

ethanol-induced behaviors (Borghese et al., 2006; Kumar et al., 2009a; Werner et al.,
2006).
α2 GlyR WT
Ethanol produced concentration-dependent potentiation of glycine-induced Cl
-

currents in α2 GlyR WT with significant effects starting at 50 mM ethanol (Fig. 2.4A,
Table 2.2). There were no significant effects of ethanol on these WT receptors at
concentrations below 50 mM.
Glycine produced inward Cl
−
currents in a concentration-dependent manner (Fig.
2.4B). Values for glycine EC
50
, Hill slope and I
max
of α2 GlyR WT are shown in Table
2.1.
α2 GlyR USER 1
Since α1 GlyR USER 3 demonstrated increased ethanol sensitivity without altering
agonist response, we applied the same rationale to limit Loop 2 mutations to Exon 3 in
α2 GlyR USER 1 to develop USERs with similar characteristics.
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. 2.4A,
Table 2.2). At higher concentrations, ethanol produced a concentration-dependent
effect with significant potentiation in α2 GlyR USER 1 beginning at 10 mM (Fig. 2.4A,
Table 2.2). As with other GlyR USERs, the ethanol concentration response followed a
bimodal pattern (Fig. 2.4A). No changes in agonist sensitivity relative to α2 GlyR WT
were observed, as indicated by similar EC
50
values (Fig. 2.4B, Table 2.1). The other
receptor characteristics of α2 GlyR USER 1 including Hill slope and I
max
did not differ
significantly from α2 GlyR WT (Table 2.1).
60

FIGURE 2.4

Figure 2.4 α2 GlyR USERs have increased ethanol sensitivity and bimodal 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 (indicated by black curves). The first curve
followed an inverse U pattern that extended from 0.1 mM to 3 mM ethanol, and the second curve
extended across higher concentrations beginning at 3 mM ethanol with concentration-dependent
increases in potentiation until 50 mM. Values for ethanol potentiation are presented as percentage of
glycine EC
2
control. The glycine EC
2
concentrations utilized ranged from 5 - 10 µM for USERs and WT.
There was a significant decrease in the threshold for ethanol sensitivity and increase in the magnitude of
ethanol response in α2 GlyR USERs compared to WT. Each data point represents the mean ± SEM from
at least 4 - 16 oocytes.
[B]
Manipulation of Loop 2 produces normal agonist concentration response in α2 GlyR USER 1. Glycine-
induced Cl− currents were normalized to the maximal current activated by a saturating concentration of
glycine (1,000 µM - 10,000 µM). The curves represent non-linear regression analysis of the glycine
concentration responses in α2 GlyR USERs and WT. There was no significant difference in the agonist
concentration response curves for α2 GlyR USER 1 and WT. Each data point represents the mean ±
SEM from at least 4 - 5 oocytes.

α1 GABA
A
R USER 1
To explore the development of α1 GABA
A
R USERs, we first produced the homolog
of γ2 GABA
A
R USER 1. As expected, α1 GABA
A
R USER 1 had markedly increased
ethanol sensitivity compared to GABA
A
R WT (Fig. 2.5A), with ethanol sensitivity
threshold at 0.25 mM compared to 50 mM in GABA
A
R WT (Fig 2.5A, Table 2.3). At
ultra-low ethanol concentrations, α1 GABA
A
R USER 1 demonstrated an increase in the
61

magnitude of ethanol response compared to WT (Fig. 2.5A). The ethanol concentration
response exhibited a bimodal pattern, consistent with other GlyR and GABA
A
R USERs
(Fig. 2.5A, Table 2.3).
Importantly, 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. 2.5B, Table 2.1). 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 2.1).

62

FIGURE 2.5

Figure 2.5 α1 GABA
A
R USERs have increased ethanol sensitivity and bimodal response to
ethanol.

[A] Ethanol-induced potentiation of GABA EC
2
-activated Cl
−
currents in Xenopus oocytes expressing α1
GABA
A
R USER 1 with α1β2γ2 composition exhibited a bimodal response to ethanol (indicated by black
curves). The first curve followed an inverse U pattern that extended from 0.1 mM to 3 mM ethanol, and
the second curve extended across higher concentrations beginning at 3 mM ethanol with concentration-
dependent increases in potentiation until 50 mM. Values for ethanol potentiation are presented as
percentage of GABA EC
2
control. The GABA EC
2
concentrations utilized was 8 - 10 µM for USERs and
WT. There was a significant decrease in the threshold for ethanol sensitivity and increase in the
magnitude of ethanol response in α1 GABA
A
R USER 1 compared to WT. Each data point represents the
mean ± SEM from at least 4 - 13 oocytes.
[B] Manipulation of Loop 2 produces normal agonist concentration response for α1 GABA
A
R USER 1.
GABA-induced Cl
−
currents were normalized to the maximal current activated by a saturating
concentration of GABA (1,000 µM - 10,000 µM). The curves represent non-linear regression analysis of
the GABA concentration responses in α1 GABA
A
R USER 1 and WT. There was no significant difference
in the agonist concentration response curves for α1 GABA
A
R USER 1 and WT. Each data point
represents the mean ± SEM from at least 4 - 13 oocytes.

63

γ2 and α1 GABA
A
R USERs do not have altered sensitivity to the neurosteroid,
Allopregnanolone (3α,5α-THP)
Allopregnanolone is an active metabolite of progesterone and selectively enhances
GABA
A
receptor function (Evers et al., 2010; Smith et al., 1998; Turner and Simmonds,
1989). To determine whether Loop 2 mutations in the γ2 subunit and/or the α1 subunit
of the GABA
A
R interferes with the effects of 3α,5α-THP we tested the effects of 20 nM
and 100 nM 3α,5α-THP against two GABA
A
R USERs that demonstrated the greatest
increase in ethanol sensitivity relative to WT – γ2 GABA
A
R USER 3 and α1 GABA
A
R
USER 1. As illustrated, 3α,5α-THP significantly potentiated GABA-induced Cl
-
currents
in WT GABA
A
Rs and γ2 GABA
A
R USER 3 and α1 GABA
A
R USER 1 at 20 nM and 100
nM (Fig. 2.6). Notably, there were no significant differences between 3α,5α-THP
sensitivity in USERs relative to WT GABA
A
Rs as determined by measuring changes in
the magnitude of receptor potentiation (Fig. 2.6), and in threshold of activation (p >
0.05).

64

FIGURE 2.6

Figure 2.6 Loop 2 mutations do not alter sensitivity to 3α,5α-THP for γ2 GABA
A
R USER 3 and α1
GABA
A
R USER 1.
3α,5α-THP potentiated EC
2
GABA-induced Cl
-
currents in α1β2γ2 GABA
A
R WT and γ2 GABA
A
R USER 3
and α1 GABA
A
R USER 1. There was no significant difference in the magnitude of 3α,5α-THP potentiation
between WT and mutant receptors. Each data point represents the mean ± SEM from 6 - 13 oocytes.

65

DISCUSSION
The present study demonstrated that manipulation of the physical-chemical
characteristics of extracellular Loop 2 produced a library of homomeric α1 and α2 GlyR
USERs and heteromeric α1 and γ2 GABA
A
R USERs of the α1β2γ2 isoform. These
USERs were over 100-fold more sensitive to ethanol compared to WT receptors, with
increased magnitude of ethanol response and decreased threshold for ethanol
sensitivity (Tables 2.2 and 2.3, Figs. 2.1 – 2.5). We also developed an ethanol-
insensitive receptor in the γ2 subunit of GABA
A
Rs (Table 2.3, Fig. 2.3). Notably, we
demonstrated that Loop 2 manipulations that increase ethanol sensitivity in USERs do
not necessarily alter general receptor characteristics such as EC
50
, I
max
and Hill slope
relative to WT (Table 2.1). In addition, we found that Loop 2 mutations did not
significantly alter sensitivity to the neurosteroid, allopregnanolone (3α,5α-THP) (Fig.
2.6). Finally, our findings indicate that USERs can be produced across multiple receptor
subunits of GlyRs and GABA
A
Rs (Figs. 2.4 and 2.5).
The significance of these findings is attributable to the fact that the threshold
concentrations for ethanol sensitivity of USERs were ≤ 0.25 mM (BEC ≤ 0.001%).
Behavioral effects of ethanol in humans can be detected at BECs as low as 0.03% (w/v)
(7 mM) (Kumar et al., 2009a; Ogden and Moskowitz, 2004). Thus, USERs are sensitive
to ethanol concentrations that are far below those that produce known responses in vivo
or in vitro (Aguayo and Pancetti, 1994; Davies et al., 2003; Hanchar et al., 2006;
Hanchar et al., 2005; Mascia et al., 1996b; Ogden and Moskowitz, 2004; Olsen et al.,
2007; Perkins et al., 2009). Testing at low concentrations would minimize the probability
66

of activating multiple targets of ethanol and associated behaviors, thus allowing
activation of specific receptor subunits.
Notably, exposure to ultra-low ethanol concentrations can produce responses in
both GlyR and GABA
A
R USERs that are similar in magnitude to those produced in WT
receptors at higher ethanol concentrations (Figs. 2.2A – 2.5A). These results suggest
the exciting possibility that KI animals expressing USERs should respond similarly to
ultra-low dose ethanol as if they received higher ethanol doses in the 30 mM - 50 mM
range. Furthermore, the development of KI animals expressing the ethanol-insensitive
γ2 GABA
A
R USER 4 would help validate findings from current KI strategies that express
ethanol-insensitive receptors in vivo by direct inactivation of a single receptor subunit
population. Results from our studies would complement and add to the findings
identified in KI and KO studies of GlyRs and GABA
A
Rs, which utilize higher, intoxicating
ethanol concentrations that affect multiple targets and downstream cascades (Werner et
al., 2006). Thus, USERs in conjunction with current transgenic approaches can be
employed to increase our understanding of the pharmacological and physiological
effects of ethanol action by establishing precise links between specific receptor subunits
and behavioral outcomes.
In addition to GlyRs and GABA
A
Rs, other members of the Cys-loop superfamily
of LGICs, including the neuronal nicotinic cholinergic (nACh) and serotonergic (5-HT
3
)
receptors, have been linked to a number of behavioral effects of ethanol administration
(Hodge et al., 2004; Kamens et al., 2010a, b; Knapp and Pohorecky, 1992). Since the
sequence homology among subunits of Cys-loop receptors is on the order of 30%
67

(Olsen and Sieghart, 2009), this suggests that the applicability of USER technology can
be extended to other receptors within the Cys-loop superfamily.
USER technology will also add to recent advances in brain circuitry research
strategies including optogenetics and designer receptors exclusively activated by
designer drugs (DREADD). Optogenetics involves microbial opsins that allow photo
activation or inhibition of defined neuron populations, axonal pathways or brain regions,
while DREADD involves pharmacological activation of DREADD-expressing neurons
using clozapine N-oxide (Aston-Jones and Deisseroth, 2013). Similarly, USERs rely on
ethanol as a pharmacological probe, analogous to a specific receptor agonist or
antagonist, to directly activate receptor populations sensitized to ultra-low ethanol
concentrations or inactivate receptor populations without affecting other targets. Thus,
USERs could serve as an advantageous brain-mapping tool with direct, non-invasive
and bi-directional capabilities to provide further insight into specific neural cascades at
the resolution of the receptor subunit level. Taken together, USERs along with
optogenetics and DREADD provide researchers with a novel tool to determine the
interplay between neural pathways, thereby enhancing our understanding of the
neurobiological basis of behaviors.
Mutational studies in extracellular Loop 2 of GlyRs and GABA
A
Rs already have and
will continue to inform the construction of molecular receptor models that increase our
understanding of the targets of ethanol action, and structure-function relationships
underlying receptor activation and ethanol-induced modulation (Crawford et al., 2007;
Perkins et al., 2010; Perkins et al., 2009). The bimodal pattern of ethanol response
observed in GlyR and GABA
A
R USERs (Figs. 2.5A), combined with findings from prior
68

studies (Crawford et al., 2007; Davies et al., 2004), support the existence of multiple
potentiating and inhibitory sites of ethanol action. A potential explanation for this
bimodal effect could be that these Loop 2 mutations lead to the creation of an ultra-
sensitive ethanol site that produces robust potentiation of the receptor at sub-millimolar
concentrations. At concentrations between 1 mM - 3 mM, ethanol could act at inhibitory
sites that potentially reduce and/or mask the contribution of the ultra-sensitive site.
Then, at higher concentrations (> 3 mM), ethanol could act at WT potentiating sites. The
interplay between inhibitory and potentiating sites observed in USERs are similar to
those reported for other Cys-loop allosteric modulators such as zinc, propofol,
pentobarbital and neurosteroids (Bloomenthal et al., 1994; Evers et al., 2010; Laube et
al., 1995; Maksay and Biro, 2002; Morrow et al., 1990). Thus, future studies could
explore the actions of other allosteric agents on these USERs.
The selectivity of the GABA
A
R USERs is further strengthened by the lack of
significant changes in 3α,5α-THP potentiation and change in threshold activity on
GABA
A
Rs. Specifically, in this investigation we demonstrated that neither the highly
ethanol-sensitive γ2 GABA
A
R USER 3, nor the α1 GABA
A
R USER 1 showed any
significant difference in sensitivity to 3α,5α-THP compared to α1β2γ2 GABA
A
R WT (Fig.
2.6). The lack of significant change in sensitivity to 3α,5α-THP suggests that the
extracellular Loop 2 region may not be a critical site of neurosteroid action. Further
studies are necessary to determine if these findings extend to all neurosteroids.
Based on the findings, we propose new molecular models that highlight the
structural differences between Loop 2 of the ethanol ultra-sensitive γ2 GABA
A
R USER 1
and ethanol-insensitive γ2 GABA
A
R USER 4. Mutation of positions 71 and 73 in Loop 2
69

of γ2 GABA
A
R USER 4 eliminated ethanol sensitivity. This may be explained by the
distortion of the β hairpin structure of Loop 2 (indicated in yellow) in the γ2 GABA
A
R
USER 4 compared to that of γ2 GABA
A
R USER 1 (Fig. 2.7). This distortion could be
due to the formation of new electrostatic interactions between the flanking residues of
Loop 2 (H64 and R73). Specifically, hydrogen bond interaction(s) may form between the
arginine at position 73 and the histidine at position 64. Furthermore, additional
interactions may also form between positions 71 and 73 with the TM region, adding to
the change in the overall Loop 2 structure. These new electrostatic interactions, which
are not present in γ2 GABA
A
R WT, could increase the rigidity of the Loop 2 structure
and change its shape (Perkins et al., 2009). As a result, these changes may affect the
actions of ethanol molecules on Loop 2 and its surrounding regions. Thus, the
differences in ethanol sensitivity between γ2 GABA
A
R USERs 1 and 4 may be
explained by the introduction of new electrostatic interactions.

70

FIGURE 2.7

Figure 2.7 A molecular model of the α1β2γ2 GABA
A
R with mutations in Loop 2 of the γ2 subunit.
(A) A model of the full receptor α1β2γ2 GABA
A
R, but with the foreground two subunits removed to reveal
Loop 2 in the γ2 subunit with the USER 1 mutation (see Table 2-1 for sequences). The view is at the
plane of the membrane looking outward from the center of the ion pore. The α helices are rendered as
cylinders and the β strands as ribbons. The ribbon for Loop 2 in γ2 is highlighted in yellow. [B] A zoomed
view of Loop 2 from panel A. The residues that differ between the ethanol ultra-sensitive USER 1 and
ethanol-insensitive USER 4 are rendered with a space-filling surface whereas the other residues are
rendered in stick; carbon, hydrogen, oxygen, nitrogen, and sulfur are colored grey, white, red, blue, and
orange, respectively. [C] The two mutations that remove sensitivity to ethanol, E71D and T73R are
highlighted. Since USERs 1 and 4 have relatively similar sensitivity to GABA (Table 2-2), it is likely that
mutations in this region of Loop 2 influence ethanol potentiation of GABA, rather than directly influencing
agonist sensitivity and/or receptor gating.

Interestingly, γ2 GABA
A
R USERs 1 and 4 demonstrate similar agonist sensitivities
(Fig. 2.3B), thus it is likely that mutations at the terminal region of Loop 2 influence
ethanol potentiation of GABA, rather than agonist sensitivity. These findings represent
new evidence suggesting that the physical, chemical and structural properties of Loop 2
that regulate ethanol and agonist sensitivity are different. In addition, the findings
distinguish regions that mediate ethanol sensitivity from those that mediate agonist
sensitivity. Thus, we propose that residues in Loop 2 play a role in the activation
pathway from ligand binding to opening of the ion pore. We suggest that these
71

mutations do not alter agonist binding, but rather provide a site for ethanol modulation
by the amino acid side chains involved in the transition between the resting, open, or
desensitized states of the receptor. However, the factors that influence these properties
may not be completely independent of each other. Rather, the factors that mediate
ethanol and agonist sensitivity likely depend on the overall structure of Loop 2 and the
interplay between different residues that are within and surrounding this region.
Taken together, these Loop 2 models, in conjunction with those proposed earlier
(Crawford et al., 2007; Perkins et al., 2009; Perkins, 2012), will aid in defining the key
drivers that will determine the architecture of ethanol action sites of GlyRs and
GABA
A
Rs. While these studies focus primarily on the structural mechanisms of GlyR
and GABA
A
R USERs in response to ethanol, future studies may characterize channel
kinetics to determine the functional elements of GlyR and GABA
A
R USER-mediated
synaptic currents in response to ethanol. In addition, since these studies address USER
technology for the α1β2γ2 GABA
A
R isoform, future studies should investigate additional
isoforms of Cys-loop receptors that are known to mediate ethanol-induced behaviors.
In conclusion, USER technology will reveal the roles played by specific receptor
subunits in AUDs, thus providing new targets for the development of novel drugs to
prevent and/or treat alcohol-related problems. The development of our new molecular
model will provide additional insights regarding the specific sites and mechanisms of
ethanol action and identify potential pharmacophores for drug development by
mimicking, blocking or otherwise modulating the actions of ethanol on key receptor
subunits identified by USER technology.

72

73

Chapter 3 Manipulations of Extracellular Loop 2 in α1 GlyR Ultra-Sensitive
Ethanol Receptors (USERs) Enhance Receptor Sensitivity to Isoflurane, Ethanol,
and Lidocaine, but not Propofol

CHAPTER 3 ABSTRACT
We recently developed Ultra-Sensitive Ethanol Receptors (USERs) as a novel tool for
investigation of single receptor subunit populations sensitized to extremely low ethanol
concentrations that do not affect other receptors in the nervous system. To this end, we
found that mutations within the extracellular Loop 2 region of glycine receptors (GlyRs)
and γ-aminobutyric acid type A receptors (GABA
A
Rs) can significantly increase receptor
sensitivity to micro-molar concentrations of ethanol resulting in up to a 100-fold increase
in ethanol sensitivity relative to wild type (WT) receptors. The current study investigated:
1) Whether structural manipulations of Loop 2 in α1 GlyRs could similarly increase
receptor sensitivity to other anesthetics; and 2) If mutations exclusive to the C-terminal
end of Loop 2 are sufficient to impart these changes. We expressed α1 GlyR USERs in
Xenopus oocytes and tested the effects of three classes of anesthetics, isoflurane
(volatile), propofol (intravenous), and lidocaine (local), known to enhance glycine-
induced chloride currents using two-electrode voltage clamp electrophysiology. Loop 2
mutations produced a significant 10-fold increase in isoflurane and lidocaine sensitivity,
but no increase in propofol sensitivity compared to WT α1 GlyRs. Interestingly, we also
found that structural manipulations in the C-terminal end of Loop 2 were sufficient and
selective for α1 GlyR modulation by ethanol, isoflurane, and lidocaine. These studies
are the first to report the extracellular region of α1 GlyRs as a site of lidocaine action.
Overall, the findings suggest that Loop 2 of α1 GlyRs is a key region that mediates
74

isoflurane and lidocaine modulation. Moreover, the results identify important amino
acids in Loop 2 that regulate isoflurane, lidocaine, and ethanol action. Collectively,
these data indicate the commonality of the sites for isoflurane, lidocaine, and ethanol
action, and the structural requirements for allosteric modulation on α1 GlyRs within the
extracellular Loop 2 region.

75

INTRODUCTION
Glycine receptors (GlyRs), one of the major inhibitory neurotransmitter receptor
systems in the adult mammalian central nervous system (CNS) (Dutertre et al., 2012),
are fast-acting ligand-gated ion channels (LGICs) that belong to the Cys-loop
superfamily, whose members also include the closely related γ-aminobutyric acid-type A
(GABA
A
), nicotinic acetylcholine (nACh), and 5-hydroxytryptamine
3
(5-HT
3
) receptors
(Ortells and Lunt, 1995; Xiu et al., 2005). GlyRs mediate inhibitory neurotransmission in
the spinal cord, brain stem, cerebral cortex, ventral tegmental area, nucleus
accumbens, dorsal raphe, and amygdala, and are considered a site of action for
allosteric modulators including, alcohols, general anesthetics (volatile and intravenous),
neuroactive steroids, endocannabinoids, divalent cations, and avermectins (Downie et
al., 1996; Dutertre et al., 2012; Lynch, 2004; Maguire et al., 2014; Rajendra et al.,
1997). α1 subunit-containing GlyRs are widely expressed in the spinal cord and brain
stem and have been implicated in playing a role in the immobilizing effects of ethanol
and anesthetic action by inhibiting motor responses to noxious stimuli and mediating
ethanol-induced loss of righting reflex (Aguayo et al., 2014; Antognini and Schwarz,
1993; Legendre, 2001; Williams et al., 1995; Ye et al., 2009).
A large body of evidence implicates sites within the transmembrane (TM),
extracellular (EC), and intracellular (IC) domain of GlyRs and GABA
A
Rs as important
targets of ethanol and anesthetic action. Site-directed mutagenesis and radioligand
binding studies, for example, have identified both inter- and intra-subunit regions within
the TM domains of α1 GlyRs, and α2 and β1 subunits of GABA
A
Rs as critical sites of
action for ethanol as well as volatile and general anesthetics (Krasowski et al., 1998;
76

Mascia et al., 2000; Mihic et al., 1997). Current evidence suggests that some of these
anesthetics act, in part, via binding at different regions within the TM domain or the
central pore region of the receptors (Cummins, 2007). For example, ethanol and volatile
anesthetics (isoflurane, and its structural isomer, enflurane) are reported to share
overlapping sites of action in the TM2 and TM3 domain that are distinctly different from
the site of action of the intravenous anesthetic, propofol (Krasowski et al., 1998;
Lingamaneni et al., 2001; Mascia et al., 1996a; Mihic et al., 1997; Nury et al., 2011;
Sauguet et al., 2013). Mutations in the TM domain of GABA
A
Rs at positions 270 or 291
in α2, or at positions 265 or 286 in β2 rendered these receptors insensitive to isoflurane,
but maintained sensitivity to propofol (Krasowski et al., 1998). Thus, these findings
suggest that the sites of ethanol and isoflurane action on GABA
A
Rs differs from those of
propofol action.
The structural homology among Cys-loop receptors implies that the sites of
ethanol and anesthetic action identified in GABA
A
Rs may also correlate to GlyRs.
Isoflurane and propofol enhance glycine-induced chloride currents of GlyRs, suggesting
their involvement in the actions of these anesthetics (Downie et al., 1996; Krasowski
and Harrison, 1999). Prior studies found that a point mutation at position 52 in the EC
Loop 2 region of α1 GlyRs altered ethanol sensitivity (Davies et al., 2004, Crawford et
al., 2007, Crawford et al., 2008, Perkins et al., 2008) but did not affect propofol
sensitivity (Mascia et al., 1996a). These differences have also been reported in
GABA
A
Rs (Bali and Akabas, 2004). In addition, recent studies have characterized
position 380 in the large IC loop between TM 3 and TM 4 of α1 GlyRs as an important
site for propofol action, but not for other anesthetics (alcohols, etomidate,
77

trichloroethanol and isoflurane) (Moraga-Cid et al., 2011). Furthermore, lidocaine, a
local anesthetic, potentiates GlyRs, implicating their involvement in lidocaine action
(Hara and Sata, 2007). Recent studies report that lidocaine-induced potentiation of
GlyR currents was abolished when the serine S267 in the TM domain of α1 GlyRs, a
previously reported target site for ethanol (Mihic et al., 1997), was mutated (Hara and
Sata, 2007). Taken together, these studies indicate that ethanol and certain anesthetics
have overlapping sites of action on α1 GlyRs that are distinct from those of propofol.
Interestingly, previous studies indicate that GABA
A
Rs containing the δ subunit
are especially sensitive to potentiation by intoxicating (10-20 mM) concentrations of
ethanol (Wallner et al., 2003). With this in mind, we studied the differences in the amino
acid sequences between GABA
A
Rs containing the δ subunit and GlyR α1 subunits and
found major differences within the Loop 2 regions of the EC domain of these two
receptors. We therefore conducted a series of investigations that focused on the EC
domain of α1 GlyRs. Notably, we identified EC Loop 2 (positions 50-59) as a critical site
of ethanol action that mediates ethanol sensitivity of GlyRs and GABA
A
Rs (Crawford et
al., 2008; Crawford et al., 2007; Naito et al., 2014; Olsen et al., 2007; Perkins et al.,
2012; Perkins et al., 2008; Perkins et al., 2009).
Over the course of these aforementioned investigations, we found that
manipulation of the structural features of EC Loop 2 conferred a significant increase in
ethanol sensitivity of up to 100-fold as compared to homomeric recombinant WT α1
GlyRs and resulted in the discovery of Ultra-Sensitive Ethanol Receptors (USERs)
(Naito et al., 2014; Perkins et al., 2009). When expressed in oocytes and mammalian
cells in vitro, USERs respond to extremely low micro-molar ethanol concentrations –
78

concentrations too low to affect native receptors. Remarkably, despite this significant
increase in ethanol sensitivity, these USERs featured minimal changes relative to WT
α1 GlyRs in general receptor characteristics including the maximum current amplitude
(I
max
), Hill slope, and agonist sensitivity measured by the half maximal effective
concentration (EC
50
). In addition, similar Loop 2 manipulations also produced USERs in
homomeric α2 GlyRs and heteromeric α1 and γ2 subunits of α1β2γ2 GABA
A
Rs (Naito
et al., 2014). Interestingly, reversion of the residues in the C-terminal region of Loop 2 in
both α1 and α2 GlyR USERs (positions 55-59) back to WT, significantly increased
ethanol sensitivity by reducing the threshold and increasing the magnitude of response,
without altering agonist EC
50
sensitivity relative to WT α1 GlyRs (Naito et al., 2014).
Structural manipulation of the C-terminal region of Loop 2 in γ2 GABA
A
Rs eliminated
ethanol sensitivity; thus implicating this region as an important regulator of ethanol
sensitivity (Naito et al., 2014). Overall, these findings demonstrate the importance of the
EC Loop 2 region of ethanol action across multiple subunits of GlyRs and GABA
A
Rs
and could be useful in elucidating the role that these receptor subunits play in mediating
the behavioral manifestations of ethanol.
While USERs are ultra-sensitive to the effects of the general anesthetic, ethanol,
their selectivity to other anesthetics has not been investigated. The present study tested
the hypothesis that structural manipulations of Loop 2, particularly the C-terminal end, of
human α1 GlyRs are important for transducing the potentiating effects of three classes
of anesthetics – volatile, intravenous, and local. Based on prior studies that identify
common sites among ethanol and isoflurane, we expect that the Loop 2 manipulations
that increased ethanol sensitivity of α1 GlyR USERs would similarly increase receptor
79

sensitivity to the volatile anesthetic, isoflurane, but would not significantly alter the
effects of the intravenous anesthetic, propofol. Similarly, since earlier studies have
demonstrated that an ethanol site in the TM domain of α1 GlyRs modulated the effects
of the local anesthetic, lidocaine, we tested the effects of this drug in α1 GlyR USERs to
elucidate its sites of action on α1 GlyRs. We investigated the potentiating effects of
isoflurane, lidocaine, and propofol on α1 GlyR USERs using two-electrode voltage
clamp electrophysiology.
A long-term goal of our ongoing USER investigations is the creation of knock-in
mice that would allow us to evaluate the effectiveness of the Loop 2 mutations in vivo.
Expression of Loop 2 in GlyRs is controlled by two exons: exon 3 for the N-terminal end
and exon 4 for the C-terminal end. Since mutations exclusively contained within a single
exon would greatly simplify the development of knock-in mice, we developed a new α1
GlyR USER (i.e., USER 4) to investigate whether the C-terminal region of Loop 2 is
sufficient to impart changes in α1 GlyR sensitivity to ethanol and other anesthetics.
Overall, the findings supported the hypothesis and provide additional insight into the
structure-activity relationship of anesthetics on α1 GlyRs.

80

MATERIALS AND METHODS
Materials
Stage V or VI Xenopus oocytes were purchased commercially from EcoCyte
Bioscience (Austin, TX). Gentamycin, 3-aminobenzoic acid ethyl ester, glycine, and
propofol were purchased from Sigma (St. Louis, MO). Isoflurane (USP grade) was
purchased from Abbott Laboratories (North Chicago, IL), lidocaine was purchased from
Tocris Bio-Techne (Minneapolis, MN), and ethanol was purchased from Decon Labs,
Inc. (King of Prussia, PA). 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 a final
pH of 7.5 (Davies et al., 2003). 1 mM stock solutions of isoflurane, lidocaine, and
propofol were prepared in DMSO and serially diluted with buffer (DMSO ≤ 0.1%)
immediately prior to testing. Pilot studies found that DMSO at this concentration, with or
without glycine, had no appreciable effect on α1 GlyR currents in WT and USERs.
Mutagenesis and Expression of α1 GlyR in Oocytes
Amino acid sequences of the Loop 2 regions of human α1 GlyR wild type (WT)
and USERs are shown in Table 1. Loop 2 is defined as positions 50-59 in the α1 GlyR
subunit (Mihic et al., 1997, Perkins et al., 2009), and spans exon 3 (positions 50-56) and
exon 4 (positions 57-59) (Table 3.1). α1 GlyR USERs were developed according to
methods described previously (Perkins et al., 2009). Briefly, we replaced the Loop 2
region of human α1 GlyRs with that of the ethanol-sensitive Loop 2 of δ GABA
A
Rs using
site-directed mutagenesis to produce α1 GlyR USER 1 (Table 3.1). Using Loop 2 of α1
81

GlyR USER 1 as a template, we reverted specific non-conserved amino acid residues
back to WT α1 GlyR residues to generate USERs 2, 3 (Naito et al., 2014) and 4. α1
GlyR USER 4 represents Loop 2 mutations of USER 1 at positions 57 and 59 in exon 4
only (Table 1). Site-directed mutagenesis was performed by subcloning human α1 GlyR
into mammalian vector pCIS2 or pBK-CMV using Quick Change Site-Directed
Mutagenesis kit (Stratagene, La Jolla, CA) and verified by partial sequencing (DNA
Core Facility, University of Southern California) as described previously (Davies et al.,
2003). Stage V or VI Xenopus oocytes were injected with α1

GlyR WT or USER cDNA
(1 ng/32 nl). Oocytes were stored in incubation medium [ND96 supplemented with 2
mM sodium pyruvate, 50 mg/ml of gentamycin and 10 ml heat inactivated HyClone®
horse serum (VWR, San Dimas, CA), adjusted to pH 7.5] in Petri dishes (VWR, San
Dimas, CA). All solutions were sterilized by passage through 0.22-µM filters. Injected
oocytes were stored at 18°C and used in electrophysiological experiments 24 - 48 hours
after injection for a period of 1 week.

82

TABLE 3.1
α1 GlyR
Loop 2
Sequence Mutations
WT 50 SIAETTMDYR

USER 1 50 HISEANMEYT
S50H, A52S, T54A, T55N, D57E, R59T
USER 2 50 HIAEANMEYT
S50H, T54A, T55N, D57E, R59T
USER 3 50 HISEANMDYR
S50H, A52S, T54A, T55N
USER 4 50 SIAETTMEYT
D57E, R59T
Table 3.1 Loop 2 sequence alignment and mutations for the human WT and α1 GlyR USERs.
Loop 2 of α1 GlyR spans exon 3 (indicated by solid line) and exon 4 (indicated by dotted line). Mutations
present in each USER are indicated in bold. Loop 2 of USER 1 represents the Loop 2 sequence of δ
GABAARs. [A: Alanine, D: Aspartate, E: Glutamate, H: Histidine, I: Isoleucine, M: Methionine, N:
Asparagine, R: Arginine, S: Serine, T: Threonine, Y: Tyrosine].
Whole Cell Two-Electrode Voltage Clamp Recordings
Two-electrode voltage clamp recording was performed using techniques
according to those previously reported (Davies et al., 2003, Perkins et al., 2009). Briefly,
oocytes were voltage clamped at a membrane potential of -70 mV using oocyte clamp
OC-725C (Warner Instruments; Hamden, CT) and the oocyte recording chamber was
continuously perfused with MBS ± anesthetic (isoflurane, lidocaine, or propofol), ethanol
and/or glycine using a Dynamax peristaltic pump (Rainin Inst Co., Emeryville, CA) at 3
ml/min using an 18-gauge polyethylene tube (Becton Dickinson, Sparks, MD) and
resultant currents were recorded.
Anesthetic Concentration Responses
Concentration response curves for isoflurane, lidocaine, and propofol were
generated with oocytes expressing α1 GlyR WT or USERs by measuring the Cl
-

currents elicited by agonist concentrations producing 2% of the maximal effect (EC
2
) ±
5%. EC
2
glycine concentrations were applied until Cl
−
currents were within 10% of each
other. We studied EC
2
concentrations because GlyR responses are quite sensitive to
potentiation at this concentration, whereas, they are much less sensitive at EC
50
glycine
concentrations and potentiation is not observed at I
max
concentrations. EC
2
was used as
a control pre- and post-anesthetic application. Once stable, oocytes were tested for
potentiation by anesthetics. Oocytes were pre-incubated with either isoflurane,
lidocaine, or propofol for 60 s followed by co-application of the tested anesthetic and
glycine for 30 s at a perfusion rate of 3 ml/min (Davies et al., 2004). Washout periods (5
- 15 min depending on anesthetic concentration tested) were allowed between glycine
and anesthetic applications to ensure complete receptor re-sensitization. WT and USER
responses were measured across anesthetic concentrations ranging 1 - 400 µM for
isoflurane and 0.1 - 50 µM for propofol. Holding currents were not significantly affected
during pre-incubation with isoflurane, lidocaine, or propofol alone, i.e. in the absence of
glycine. As indicated in previous studies, the final bath concentration of isoflurane is
approximately 50% of the prepared concentration due to losses during delivery
(Krasowski and Harrison, 2000; Lopreato et al., 2003). To maintain the intended
concentration of isoflurane, solutions were prepared immediately prior to application.
Furthermore, to minimize loss of isoflurane due to volatilization, containers were sealed
with parafilm during perfusion. The anesthetic concentrations tested roughly correspond
84

to physiologically relevant concentrations that cause anesthesia (immobility) in
mammals (the human MAC for isoflurane is 260 - 320 µM; and EC
50
concentrations for
lidocaine, and propofol are 1 -1.9 µM, and 0.4 µM, respectively) (Franks and Lieb, 1994,
Downie et al., 1996, Krasowski and Harrison, 1999, Miller, 2010). Although studies have
reported direct activation of GlyRs by volatile and intravenous anesthetics, these effects
typically occur at concentrations beyond clinical relevance and have been observed to a
greater extent in GABA
A
Rs (Krasowski and Harrison, 1999).Therefore, this effect was
not studied in detail.
Ethanol Concentration Response
Electrophysiological recordings were conducted according to the methods
described above for anesthetic application.
Data Analysis
Data for each experiment were obtained from a sample size (n) of 6-10 oocytes.
Results are expressed as mean ± SEM. Graphs and statistical analyses were generated
by Prism (GraphPad Software Inc., San Diego, CA). The percent potentiation of α1
GlyR currents were determined by calculating the percent change in glycine EC
2
-
induced Cl
-
currents in the presence of anesthetic at each concentration tested. The
threshold for anesthetic sensitivity was determined by comparing the α1 GlyR USER
percent potentiation at the lowest concentration against WT percent potentiation. EC
50

values were approximated using a non-linear, exponential, least squares (ordinary) fit
analysis with a 95% confidence interval. Statistical significance was determined using
85

one-way repeated measures analysis of variance (ANOVA), Bonferroni post hoc
analysis, and student’s t test. Statistical significance was defined as *p < 0.05.

86

RESULTS
α1 GlyR USERs 1 and 2 have increased sensitivity to isoflurane
α1 GlyR WT
We tested the effects of isoflurane on WT α1 GlyRs at concentrations ranging 1 -
400 µM. These concentrations encompass clinical relevance based on published
minimum alveolar concentration (MAC) values of inhaled anesthetics that cause
immobility in 50% of mammals (Krasowski and Harrison, 2000). We found that
isoflurane produced concentration-dependent potentiation of EC
2
glycine-induced Cl
-

currents in WT α1 GlyRs with significant effects starting at 100 µM isoflurane (Fig. 3.1).
There were no significant effects of isoflurane on these receptors at concentrations
below 100 µM. Representative glycine-activated Cl
-
current tracings are shown in Fig.
3.2. There was minimal direct activation by isoflurane at concentrations ≥ 100 µM (Fig.
3.2), however these effects were not statistically significant (< 1% of maximal GlyR
currents, p > 0.05). EC
50
values for WT α1 GlyRs are presented in Table 3.2.
α1 GlyR USER 1
We tested the effects of isoflurane on α1 GlyR USERs to establish whether the sites
responsible for increasing receptor sensitivity to ethanol would similarly increase
sensitivity to isoflurane. In agreement with our recent ethanol findings (Naito et al.,
2014), isoflurane produced concentration-dependent potentiation of EC
2
glycine-
induced currents in α1 GlyR USER 1 starting at 10 µM (Figs. 3.1 and 3.2). The left shift
in the isoflurane concentration response relative to WT α1 GlyRs (Fig. 3.1) is
corroborated by a significant reduction in EC
50
compared to WT (Table 3.2). There was
a significant reduction in the threshold for isoflurane sensitivity from 100 µM in WT α1
87

GlyRs to 10 µM in α1 GlyR USER 1. The magnitude of glycine response to isoflurane
significantly increased in a dose-dependent manner in α1 GlyR USER 1 relative to WT
α1 GlyRs (Fig 3.1).
α1 GlyR USER 2
Isoflurane produced concentration-dependent potentiation of EC
2
glycine-
induced currents in α1 GlyR USER 2 starting at 10 µM (Fig. 3.1), significantly reducing
the threshold for isoflurane sensitivity 10-fold from 100 µM in WT α1 GlyRs. There was
a significant decrease in the EC
50
of α1 GlyR USER 2 compared to WT (Table 3.2). At
concentrations ≥ 10 µM, the magnitude of isoflurane potentiation significantly increased
in α1 GlyR USER 2 relative to WT (Fig. 3.1). However, the magnitude of isoflurane
potentiation was lower than those produced by α1 GlyR USER 1 across all tested
concentrations (Fig. 3.1).
α1 GlyR USER 3
Isoflurane produced concentration-dependent potentiation of EC
2
glycine-
induced currents starting at 100 µM in α1 GlyR USER 3, indicating no change in the
threshold for isoflurane sensitivity compared to WT α1 GlyRs (Fig. 3.1). While there was
a degree of isoflurane-induced potentiation at 10 µM, these responses were not
significant (p = 0.1). In addition, there was no significant difference in the EC
50
of α1
GlyR USER 3 compared to WT (Table 3.2). The magnitude of glycine response to
isoflurane did not differ significantly compared to WT α1 GlyR (Fig. 3.1) except at 400
µM, and was lower than that of other α1 GlyR USERs.

88

FIGURE 3.1

Figure 3.1 α1 GlyR USERs 1 and 2 have increased sensitivity to isoflurane compared to WT.
Isoflurane-induced potentiation of glycine EC
2
-activated Cl
−
currents in Xenopus oocytes expressing α1
GlyR USERs are shown. Values for isoflurane potentiation are presented as percentage of glycine EC
2

control. The glycine EC
2
concentrations utilized ranged from 5 - 10 µM in USERs and 18 - 25 µM for WT.
The threshold for isoflurane sensitivity in α1 GlyR USERs 1 and 2 was 10 µM, and 100 µM for α1 GlyR
WT and USER 3. Significant increases in the magnitude of isoflurane response for α1 GlyR USERs
compared to WT are denoted by * p <0.05. There were no significant differences in the threshold for
isoflurane sensitivity in α1 GlyR USER 3 relative to WT. Each data point represents the mean ± SEM from
6 - 9 oocytes.

89

FIGURE 3.2

Figure 3.2 Representative tracings of α1 GlyR USERs in response to isoflurane.
Two-electrode voltage clamp electrophysiology tracings of homomeric α1 GlyR WT and α1 GlyR USERs
expressed in Xenopus oocytes in response to isoflurane. (A) α1 GlyR WT in response to 100 µM
isoflurane; (B) α1 GlyR USER 1 in response to 10 µM isoflurane; (C) α1 GlyR USER 2 in response to 10
µM isoflurane; and (D) α1 GlyR USER 3 in response to 10 µM isoflurane. Effects of isoflurane were tested
with EC2 glycine (5 - 25 µM) and the membrane potential was -70 mV.
α1 GlyR USERs 1 and 2 have increased sensitivity to lidocaine
α1 GlyR WT
We tested the effects of lidocaine on WT α1 GlyRs at concentrations ranging 0.1
- 100 µM. The tested concentrations were based on clinical data, in which plasma
toxicity in humans is found at concentrations as low as 4.5 mg/kg (Miller, 2010).
Lidocaine potentiated EC
2
glycine-induced Cl
-
currents in WT α1 GlyRs with significant
effects starting at 1 µM (Fig. 3.3). There were no significant effects of lidocaine on these
receptors at concentrations below 1 µM.
α1 GlyR USER 1
Lidocaine potentiated EC
2
glycine-induced currents in α1 GlyR USER 1 starting
at 0.1 µM (Fig. 3.3), significantly reducing the threshold for lidocaine sensitivity from 1
µM in WT α1 GlyRs to 0.1 µM in α1 GlyR USER 1. The pattern of glycine response to
lidocaine followed an inverted-U profile with a peak response at 1 µM (Fig. 3.3). Similar
to our previous ethanol findings (Naito et al., 2014) and our current isoflurane findings,
α1 GlyR USER 1 demonstrated increased sensitivity to lidocaine with respect to
threshold and magnitude of response (up to 100 µM) relative to WT α1 GlyRs.
α1 GlyR USER 2
Lidocaine potentiated EC
2
glycine-induced currents in α1 GlyR USER 2 starting
at 0.1 µM (Fig. 3.3), significantly reducing the threshold for lidocaine sensitivity 10-fold
from 1 µM in WT α1 GlyRs. At concentrations below 0.1 µM, there were no significant
effects of lidocaine on these USERs. The magnitude of lidocaine potentiation was
significantly greater than WT α1 GlyRs at 0.1 and 1 µM. However, the magnitude of
lidocaine potentiation did not significantly differ from that of WT at lidocaine
concentrations ≥ 10 µM.
α1 GlyR USER 3
Lidocaine produced concentration-dependent potentiation of EC
2
glycine-induced
currents starting at 1 µM in α1 GlyR USER 3 (Fig. 3.3), indicating no change in the
threshold for lidocaine sensitivity compared to WT α1 GlyRs. The magnitude of glycine
response to lidocaine across all tested concentrations also did not differ significantly
compared to WT α1 GlyRs (Fig. 3.3), and was lower than that of other α1 GlyR USERs.

91

FIGURE 3.3

Figure 3.3 α1 GlyR USERs 1 and 2 have increased sensitivity to lidocaine compared to WT
Lidocaine-induced potentiation of glycine EC2-activated Cl− currents in Xenopus oocytes expressing α1
GlyR USERs are shown. Values for lidocaine potentiation are presented as percentage of glycine EC2
control. The glycine EC2 concentrations utilized ranged from 5 - 10 µM in USERs and 18 - 25 µM for WT.
The threshold for lidocaine sensitivity for α1 GlyR USERs 1 and 2 was 0.1 µM, and 1 µM for α1 GlyR WT
and USER 3. Significant increases in the magnitude of lidocaine response for α1 GlyR USERs compared
to WT are denoted by * p <0.05. Each data point represents the mean ± SEM from at least 6 - 9 oocytes.
α1 GlyR USERs exhibit no significant changes in propofol sensitivity compared to
WT
We tested a range of clinically relevant propofol concentrations ranging from 0.1 -
50 µM. While propofol appeared to inhibit EC
2
glycine-induced Cl
-
currents in α1 GlyR
WT and USERs 1, 2 and 3 at 0.1 µM (Fig. 3.4), these effects were not statistically
significant. Propofol potentiated WT α1 GlyR currents starting at 0.5 µM in a
concentration-dependent manner (Fig. 3.4). Representative tracings are shown in Fig.
3.5. In the current study, no significant effects of direct receptor activation by propofol
were observed across tested concentrations. There were no significant changes in the
92

threshold for propofol sensitivity relative to WT for all α1 GlyR USERs. The magnitude
of glycine response to propofol in α1 GlyR USER 1 did not significantly differ from that
of WT across all concentrations tested (Fig. 3.4). However, in α1 GlyR USERs 2 and 3,
the magnitude of glycine response to propofol increased significantly from that of WT α1
GlyRs and USER 1 at concentrations ≥ 50 µM (Fig. 3.4). EC
50
values indicate no
significant differences among α1 GlyR WT and all USERs (Table 3.2).
FIGURE 3.4

Figure 3.4 α1 GlyR USERs show no changes to propofol sensitivity compared to WT
Propofol-induced potentiation of glycine EC2-activated Cl− currents in Xenopus oocytes expressing α1
GlyR USERs are shown. Values for propofol potentiation are presented as percentage of glycine EC2
control. There were no significant changes in the threshold for propofol sensitivity between α1 GlyR
USERs and WT (0.5 µM). However, there was a significant increase in the magnitude of propofol
response in α1 GlyR USERs 2 and 3 compared to WT at 50 µM (denoted by * p <0.05). Each data point
represents the mean ± SEM from at least 6 - 9 oocytes.

93

FIGURE 3.5

Figure 3.5 Representative tracings of α1 GlyR USERs in response to propofol
Two-electrode voltage clamp electrophysiology tracings of homomeric α1 GlyR WT and α1 GlyR USERs
expressed in Xenopus oocytes in response to propofol. (A) α1 GlyR WT in response to 0.5 µM propofol;
(B) α1 GlyR USER 1 in response to 0.5 µM propofol; (C) α1 GlyR USER 2 in response to 0.5 µM propofol;
and (D) α1 GlyR USER 3 in response to 0.5 µM propofol. Effects of propofol were tested with EC2 glycine
(5 - 25 µM) and the membrane potential was -70 mV.

TABLE 3.2
α1 GlyR Isoflurane EC
50
(uM) Propofol EC
50
(uM)
WT 228.3 ± 21.5 19.3 ± 6.3
USER 1 78.3 ± 13.9* 17.1 ± 14.2
USER 2 174.2 ± 15.7* 10.6 ± 15.5
USER 3 242.4 ± 11.6 15.2 ± 17.3
USER 4 89.4 ± 9.7* 15.1 ± 8.2

Table 3.2 Approximate EC50 values for isoflurane and propofol in WT and α1 GlyR USERs.
EC50 values ± standard error of the mean (SEM) were approximated using standard curve-fitting analysis
for isoflurane and propofol. *Represent statistically significant differences in EC50 values compared to
WT α1 GlyRs.
94

Loop 2 mutations exclusive to exon 4 of α1 GlyRs are sufficient to increase
ethanol, isoflurane and lidocaine sensitivity
The Loop 2 region of α1 GlyRs spans both exons 3 and 4 (Table 3.1). Although
Loop 2 mutations spanning exons 3 and 4 of α1 GlyR USERs 1 and 2 significantly
increased isoflurane and lidocaine sensitivity, Loop 2 mutations in the N-terminal region,
exclusive to exon 3, in α1 GlyR USER 3 did not significantly alter sensitivity to these
anesthetic agents with regards to the threshold and magnitude of response relative to
WT α1 GlyRs (Fig. 3.1). Thus, we tested whether mutations exclusive to exon 4 in the
C-terminal end of Loop 2 (Table 3.1) are sufficient to alter ethanol and anesthetic
sensitivity in α1 GlyR USER 4.
As illustrated in Fig. 3.6A, ethanol significantly potentiated EC
2
glycine-induced
currents starting at 0.25 mM. The magnitude of response of α1 GlyR USER 4 in
response to 0.25 mM ethanol did not significantly differ from the response of WT α1
GlyRs exposed to 50 mM ethanol (Fig. 3.6A).

95

FIGURE 3.6

Figure 3.6 α1 GlyR USER 4 demonstrates increased sensitivity to ethanol, isoflurane, and
lidocaine, but not propofol
α1 GlyR WT and USER 4 glycine EC2-activated Cl− currents in response to (A) 0.25 mM and 50 mM
ethanol; (B) isoflurane; (C) lidocaine; (D) propofol. The threshold for ethanol sensitivity was reduced from
50 mM in α1 GlyR WT to 0.25 mM in α1 GlyR USER 4. The threshold for isoflurane sensitivity was
reduced from 100 µM in WT to 10 µM in α1 GlyR USER 4. The threshold for lidocaine sensitivity was
reduced from 1 µM in WT to 0.1 µM in α1 GlyR USER 4. There were no significant changes to the
threshold for propofol sensitivity and the magnitude of propofol response in α1 GlyR USER 4 relative to
WT. Each data point represents the mean ± SEM from at least 6 - 9 oocytes.
In addition, isoflurane produced concentration-dependent potentiation of EC
2

glycine-induced currents starting at 10 µM (Fig. 6B), indicating a decrease in the
threshold for isoflurane sensitivity relative to WT α1 GlyRs. Furthermore, EC
50
significantly decreased in α1 GlyR USER 4, compared to WT (Table 3.2). Thus, α1
GlyR USER 4 exhibited a similar increase in isoflurane sensitivity as that seen in α1
GlyR USERs 1 and 2 with regards to the threshold, but not the extent of the magnitude
96

of response. Although there was no incremental increase in the magnitude of isoflurane
potentiation relative to α1 GlyR USERs 1 and 2, the responses remained significantly
greater than that of WT α1 GlyRs at 10 µM and 100 µM isoflurane (Figs. 3.1 and 3.6B).
Lidocaine produced concentration-dependent potentiation of EC
2
glycine-induced
currents in α1 GlyR USER 4 starting at 0.1 µM (Fig. 3.6C). Similar to α1 GlyR USERs 1
and 2, the threshold for lidocaine sensitivity was reduced 10-fold relative to WT α1
GlyRs. At concentrations below 0.1 µM, there were no significant effects of lidocaine on
these receptors. While the magnitude of lidocaine potentiation increased in α1 GlyR
USER 4 relative to WT, these results were not significantly greater than those produced
by USER 1 (Figs. 3.3 and 3.6C). In agreement with our initial findings with propofol, the
threshold for propofol sensitivity, the EC
50
(Table 3.2), as well as the magnitude of
propofol response in α1 GlyR USER 4 did not significantly differ from that of WT α1
GlyRs and other α1 GlyR USERs (Fig. 3.6D).
97

DISCUSSION
The present study tested the hypothesis that structural manipulations of EC Loop
2 that affect ethanol sensitivity of human α1 GlyR USERs would also affect sensitivity to
the volatile anesthetic, isoflurane, but would not significantly alter the effects of the
intravenous anesthetic, propofol. We also tested the effects of lidocaine on these
USERs to understand the possible sites and mechanisms of this local anesthetic on α1
GlyRs. This was accomplished by characterizing the selectivity of α1 GlyR USERs to
volatile (isoflurane), local (lidocaine) and intravenous (propofol) anesthetics. In
agreement with earlier findings, ethanol, isoflurane, and lidocaine sites of action do not
appear to overlap with those of propofol action on α1 GlyRs, as indicated by the lack of
change in the threshold for propofol sensitivity of α1 GlyR USERs with respect to WT α1
GlyRs (Figs. 3.1, 3.3, 3.4 and 3.6). These findings suggest that the sites of propofol
action in α1 GlyRs are distinct from those of ethanol and isoflurane, reinforcing similar
findings reported in the TM domain of GABA
A
Rs (Bali and Akabas, 2004; Krasowski et
al., 1998). Overall, our findings confirm that Loop 2 plays an important role in mediating
ethanol, isoflurane, and lidocaine action by decreasing the threshold for sensitivity and
increasing the magnitude of α1 GlyR response to these agents. Furthermore, we
identified the C-terminal region of Loop 2 as a key site responsible for the enhancement
of GlyR function by ethanol, isoflurane, and lidocaine.
The Loop 2 mutations spanning both exons 3 and 4 (positions 50 – 59) in α1
GlyR USERs 1 and 2 that significantly increased ethanol sensitivity in prior studies
(Naito et al., 2014), also increased isoflurane and lidocaine sensitivity relative to WT α1
GlyRs by demonstrating a 10-fold decrease in threshold (Figs. 3.1 and 3.3). In contrast,
98

Loop 2 mutations that are exclusive to the N-terminal region (exon 3) in α1 GlyR USER
3 shared similar characteristics in the threshold and magnitude of potentiation to WT α1
GlyRs in response to isoflurane and lidocaine (Figs. 3.1 and 3.3). Interestingly, the
same mutations in exon 3 of α1 GlyR USER 3 in earlier studies imparted ultra-sensitivity
to ethanol and normalized the agonist sensitivity back to WT levels (Naito et al., 2014).
These findings suggest that certain regions within Loop 2 may differentially regulate
allosteric modulation of α1 GlyRs. In fact, merely two point mutations, D57E and R59T,
at the C-terminal end of Loop 2 in exon 4 of α1 GlyR USER 4 were sufficient to
significantly increase receptor sensitivity to ethanol, isoflurane, and lidocaine (Figs.
3.6A, B, C). Furthermore, the difference between maintaining the WT residues at
positions 57 and 59 of the C-terminal end of Loop 2 (USER 3), and mutating this region
(USER 4), seems to control isoflurane and lidocaine enhancement of α1 GlyRs.
Altogether, these results suggest that structural changes at the C-terminal end of Loop
2 appear to be a key determinant that is both sufficient, and importantly, selective to
increase ethanol, isoflurane, and lidocaine sensitivity of α1 GlyRs. Furthermore, this
study is the first to report an extracellular site of action for lidocaine on α1 GlyRs.
Interestingly, there were no significant differences in the threshold for propofol
sensitivity between WT and USER α1 GlyRs (Fig. 3.3). While propofol enhanced the
magnitude of potentiation of α1 GlyR USERs at concentrations ≥ 0.5 µM relative to WT,
there were no significant differences in the magnitude of propofol-induced potentiation
of α1 GlyR USERs compared to WT at clinically relevant concentrations between 0.5
and 10 uM (Fig. 3.3). These findings indicate that the mutations in Loop 2 that increase
ethanol, isoflurane, and/or lidocaine sensitivity in α1 GlyRs do not influence propofol
99

sensitivity, and further reinforce findings by Krasowski and colleagues that the sites in
the α2 or β1 subunits of GABA
A
Rs that abolish isoflurane potentiation do not affect
propofol potentiation (Krasowski et al., 1998). More importantly, these results highlight
the selective nature of Loop 2 mutations to increase α1 GlyR sensitivity to certain
anesthetic agents, but not all. Recent studies have reported propofol sites at the
interface between the EC and TM 2 domains, specifically between adjacent TM 2
helices in the β3 subunit of GABA
A
Rs (Franks, 2015). Our current findings suggest that
EC Loop 2 is not a critical region for propofol action and modulation of α1 GlyRs.
The distinguishing features that account for the increase in sensitivity to
isoflurane and lidocaine in α1 GlyR USERs 1, 2, and 4 (relative to WT and USER 3)
appear to be attributed to the differences between the C-terminal regions of Loop 2. As
reported earlier, the charged residue arginine R59 at the C-terminal end of Loop 2
increases the propensity to form salt-bridge interactions with N-terminal residues across
Loop 2, for instance, with the histidine H50 in α1 GlyR USER 3 (Naito et al., 2014).
Thus, the introduction of charged residues at the C-terminal end may contribute to
conformational changes to the structure of Loop 2, preventing accessibility of allosteric
modulators from acting at this site. Further studies should explore the gating properties
as a result of these Loop 2 mutations on USERs to determine the functional changes
induced in α1 GlyR USERs.
Through α1 GlyR USER 4, we demonstrate that mutations within a single exon
create receptors with high sensitivity to several anesthetic molecules. These findings
could potentially provide a discrete DNA sequence for searching databases for other
receptors that might share high sensitivity to alcohol and anesthetics. This goal is
100

important because, so far, only GABA
A
Rs containing delta subunits have exhibited
sensitivity to alcohol in the range of mild intoxication (18 mM) (Olsen et al., 2007,
Wallner et al., 2003). It is possible that there are undiscovered receptors that share the
high alcohol sensitivity of USERs; these receptors might help explain the constellation
of effects on coordination, balance, fear conditioning, and memory that corresponds to
mild intoxication.
In agreement with earlier studies, the in vivo expression of USERs in genetically
engineered animals may provide a novel tool for investigation of single receptor subunit
populations sensitized to extremely low anesthetic concentrations that do not affect
other receptors in the nervous system. While the current study tested α1 GlyR USER
sensitivity to anesthetics in the presence of EC
2
agonist concentrations, a recent study
reported that the effects of ethanol potentiation (30 mM) diminish in the presence of
higher agonist concentrations (EC
50
) in α4β1δ GABA
A
Rs (Bowen et al., 2015),
suggesting the possibility that the effects of USER potentiation may be diminished in
vivo. However, a recent investigation reported a glycine-dependent mechanism of
regulating neuronal excitability in the orbitofrontal cortex (OFC) by ethanol exposure,
providing the first line of evidence that ethanol-induced potentiation of GlyRs are the
primary mediators of inhibitory projections in the OFC (Badanich et al., 2013).
Importantly, while both GABA
A
Rs and GlyRs are expressed in OFC neurons, GABA
A
Rs
were not sensitive to behaviorally relevant concentrations of ethanol. Thus, the
inhibitory projections induced by ethanol in the OFC are glycine-dependent and not
regulated through GABA
A
R activity (Badanich et al., 2013). Furthermore, application of
the selective GlyR antagonist, strychnine, did not significantly alter tonic glycine-
101

mediated currents; thus implicating the existence of extrasynaptic GlyRs, and GlyR
transporters, that maintain low levels of extracellular glycine (Badanich et al., 2013).
Additional work reported similar findings of extrasynaptic GlyRs in the dorsal raphe
nucleus that largely govern ethanol-induced tonic inhibition (Maguire et al., 2014). In
light of these findings, GlyR USERs have the potential to significantly affect excitability
and neurochemical cascades in certain brain regions in the presence of low anesthetic
concentrations. Thus, testing these USERs in vivo would provide unambiguous results
about the role of individual receptor subunits in transducing the effects of anesthetics
without concerns about collateral effects on other nervous systems that often confound
these studies.
Conclusions
Collectively, our results demonstrate that the Loop 2 region of α1 GlyRs is a site
of ethanol, isoflurane, and lidocaine action. Moreover, we demonstrate that subtle
structural manipulations (D57E, R59T) at the C-terminal end of Loop 2 are sufficient to
significantly increase ethanol, isoflurane, and lidocaine sensitivity in α1 GlyR USER 4.
Our results also add to the body of literature that sites of ethanol, isoflurane, and
lidocaine action do not overlap with those of propofol. Importantly, these findings
demonstrate the selectivity of α1 GlyR USERs in that increases in receptor sensitivity
are not solely limited to ethanol, but also apply to isoflurane and lidocaine. The common
structural homology among the Cys-loop superfamily of receptors implies that similar
increases in anesthetic sensitivity via Loop 2 may also apply across multiple receptors
and receptor subunits. Moreover, the advantage of harboring mutations to a single exon
in Loop 2 may promote genetic incorporation to develop genetically engineered USER
102

knock-in animals. Ultimately, USERs allow activation of specific receptor subunits at
concentrations that do not affect WT receptors, and could potentially provide a valuable
tool to understand the functional role of specific receptors in mediating the behavioral
effects of anesthetic action.

103

Chapter 4 Development and Initial Behavioral Testing of α2 GlyR USER Knock-In
(KI) in Mice

CHAPTER 4 ABSTRACT
Previous studies demonstrated that structural manipulation of extracellular Loop 2 in
Glycine and GABA
A
receptors (GlyR and GABA
A
R) produce ultra-sensitive ethanol
receptors (USERs). These USERs, when expressed in vitro, demonstrated an increase
in the threshold for ethanol sensitivity of up to 100-fold relative to wild type (WT)
receptors, as well as an increase in the magnitude of ethanol potentiation. USERs
provide a novel tool to investigate the effects of ethanol action on a single receptor
subunit using extremely low ethanol concentrations that do not affect other native
receptors. Thus, USERs would provide unambiguous results about the effects of
ethanol action on these receptors without concerns about collateral effects on other
nervous systems that often confound studies of the effects of ethanol in vivo. The
present study is the first to characterize the in vivo application of α2 GlyR USERs by
global knock-in (KI) expression in C57BL/6J mice. We developed α2 GlyR USER KI
mice based on the importance of α2 GlyR involvement in alcohol-induced reward
pathways. We tested the hypothesis that USER KI mice will exhibit increased sensitivity
to ethanol at lower ethanol doses that do not affect WT control mice. To determine
whether α2 GlyR USERs exhibit increased sensitivity to ethanol in vivo, we compared
the effects of intraperitoneal (ip) injection of 0.02 g/kg, 0.2 g/kg, or 2 g/kg ethanol in
three behavioral paradigms (accelerating rotarod, loss of righting reflex, and novel open
field). The findings support our hypothesis and demonstrate that α2 GlyRs mediate the
anxiolytic effects of ethanol. Overall, USERs provide a novel tool to investigate the
104

individual contribution of specific receptor subunits in causing the behavioral
manifestations of ethanol.

105

INTRODUCTION
Alcohol use disorder (AUD) affect over 18 million people, cause over 100,000
deaths and cost over $220 billion annually in the U.S. alone (Bouchery et al., 2011a;
Grant et al., 2004; Johnson, 2010; Naimi, 2011). Unfortunately, the success rate of
available drugs, even when combined with psychotherapy, has been limited with
approximately 70% of patients relapsing back into heavy drinking within the first year of
treatment (Johnson, 2008; Litten et al., 2012). Thus, it is imperative to develop new
medications that would more effectively treat patients suffering from AUD.
A critical barrier to the development of effective medications to prevent and treat
alcohol-related problems has been our lack of specific knowledge regarding where and
how ethanol acts in the brain and the resultant neurochemical cascades leading to
behavioral change. This paucity of knowledge largely reflects ethanol’s physical-
chemical mechanism of action and low potency that requires millimolar (mM)
concentrations to alter brain function. The resultant lack of high-affinity, structure-
activity relationship precludes the classical approach of using highly specific ethanol
receptor agonists and antagonists to identify the sites and mechanisms of ethanol
action and map cause-effect relationships (Davies and Alkana, 2001a; Deitrich et al.,
1989; Little, 1991). This problem is further complicated by the multiple receptors and
receptor 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 (Blednov et al.,
2013; Blednov et al., 2010; Chandra et al., 2008; Liang et al., 2008; Moore et al., 2010).
However, studies involving functional deletion or reduction in receptor sensitivity to
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ethanol require testing with high ethanol concentrations (10 to 40 mM) that affect other
native receptor systems (Chandra et al., 2008; Werner et al., 2006). Thus, although
one ethanol-sensitive target is eliminated, ethanol’s action on other receptors makes it
difficult to isolate and identify the changes in ethanol-induced behavioral responses
produced by the mutations. Moreover, KO studies can be complicated by complex
compensatory responses that often arise during development of the genetically modified
animals (Chandra et al., 2008; Findlay et al., 2003; Mihalek et al., 2001; Werner et al.,
2006).
The goal of the proposed work is to help fill these deficiencies in knowledge and
tools by developing a novel, potentially transformative strategy to identify the
physiological and behavioral effects caused by the actions of ethanol on specific subunit
combinations of glycine receptors (GlyRs) and gamma-aminobutyric acid receptors
(GABA
A
Rs). Our strategy involves applying ultra-sensitive ethanol receptors (USERs)
as research tools. USERs are based on our recent construction of a library of mutant
GlyRs and GABA
A
Rs that, when expressed in oocytes and mammalian cells in vitro,
respond to extremely low, micro-molar ethanol concentrations – concentrations too low
to affect native receptors, but otherwise have wild type-like receptor characteristics
(Naito et al., 2014). As a result, the application of ethanol, in conjunction with USERs,
could serve as a specific receptor agonist that could stimulate receptors that are
sensitized to ultra-low ethanol concentrations without affecting other receptors or
physiological processes.
The current work tests the hypothesis that KI animals that express USERs are
viable, and can be developed as novel tools to investigate the role of specific receptor
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subunit combinations in mediating the behavioral effects of ethanol. We tested this
hypothesis by developing a strain of KI animals that express α2 GlyR USERs by
employing Transcriptional Activator-Like Effector Nucleases (TALENs) to quickly and
efficiently create new gene KI mouse lines (Ferguson et al., 2013). This approach is
much faster, less expensive, and more efficient than the traditional gene targeting
approach using embryonic stem cells.
Unlike the α1 subunit of GlyRs, which are mostly localized as heteromers (α1β)
in the spinal cord, the α2 subunit is the primary GlyR found in the NAc and VTA
(Adermark et al., 2011; Chen et al., 2011; Eichler et al., 2009; Weltzien et al., 2012).
Thus, the rationale for developing α2 GlyR USER KI mice is based on the predominant
expression of homomeric α2 GlyRs in reward, motivation, and addiction brain regions.
We assessed whether α2 GlyR USERs expressed in KI mice demonstrate increased
ethanol sensitivity by conducting several behavioral experiments including loss of
righting reflex, accelerating rotarod, and novel open field.
In summary, these studies provide proof-of-concept that USERs represent a
novel tool that can be employed to map the cascade of events caused by direct
stimulation of specific GlyR populations by ethanol, and the corresponding behavioral
effects. The findings support our hypothesis and demonstrate that USERs expressed in
KI mice provide a novel tool to understand the individual contributions of specific
receptor subunits in mediating the behavioral effects of ethanol.

108

METHODS
Materials
Gold Shield Alcohol, a 200 proof USP solution (Gold Shield Chemical Company,
Hayward, CA) was diluted in a 0.9% sodium chloride solution (saline) to achieve 20%
v/v solution (20E). Alcohol solutions were then serially diluted to achieve 10% v/v (10E),
1% v/v (1E) and 0.1% v/v (0.1E). Solutions were prepared immediately prior to testing.
Alcohol solutions were diluted to a concentration that would allow for an injection
volume of 0.02 ml/g body weight.
Studies were performed on drug-naive, C57BL/6J (Jackson Laboratory, USA) wild-
type (WT) and α2 GlyR USER knock-in (KI) male and female mice, aged 8–12 weeks at
the time of testing. α2 GlyR USER KI mice were generated and genotyped according to
previous protocols (Ferguson et al., 2013). Animals were acclimatized to the housing
facility for a minimum of 1 wk and group-housed (2-4 mice per cage) in polycarbonate
cages, with ad libitum access to food and water. The holding room was maintained at
approximately 22 °C with a 12:12 h light:dark cycle (lights on 06:00 hours). All
behavioral testing was conducted in the dark cycle between the hours of 12:00 – 18:00.
All procedures were in compliance with the National Institute of Health guidelines and
the protocols were approved by the University of Southern California Institutional Animal
Care and Use Committee.
TALEN mediated α2 GlyR USER KI mouse development
α2 GlyR KI mouse lines were created using the recently optimized TALEN
technology (Bogdanove and Voytas, 2011; Ferguson et al., 2013; Joung and Sander,
2013; Mussolino and Cathomen, 2012). Briefly, KI animals were created by co-injecting
109

a DNA repair template that harbors the desired α2 GlyR USER mutation(s) along with
the TALENs. The TALENs create a double strand break in the DNA of the target gene
and the embryo uses homologous recombination between the endogenous locus and
the repair template to precisely mend the break (Ferguson et al., 2013). TALENs
targeting Exon 3 were commercially produced (Transposagen Inc.) and TALEN mRNA
and a repair template (single stranded oligo) were co-injected directly into the
pronucleus one-cell C57BL/6J mouse embryos. Upon weaning, offspring were
genotyped, separated by sex and maintained in groups of 2-4 in individually ventilated
cages with free access to food and water under a 12:12 h light/dark cycle at 26 ± 1 °C.
The behavioral studies conducted in this work involved first-generation (F
1
)
homozygous mice expressing X
KI
/X
KI

and X
KI
/Y.
Accelerating Rotarod
Accelerating rotarod was used to measure sensitivity to ethanol’s effects of motor
incoordination in WT and KI mice. Mice were trained on the rotarod (Columbus
Instruments, Columbus, OH, USA) using 3 trials per day with a 20 minute inter-trial
interval for 3 consecutive days using an acceleration paradigm (initial speed = 5
revolutions per minute (rpm) = 1.15 m/min, acceleration = 6 rpm/min = 1.38 m/min
2
). All
mice were handled with extreme care in a quiet behavioral testing room. Mice were
injected (ip) with saline, 0.02 g/kg, 0.2 g/kg, or 2.0 g/kg of ethanol and were returned to
their home cage for 15 minutes before being placed on the rotarod. The α2 GlyR USER
KI mice and WT mice were allowed to stand on a slowly rotating (5 rpm) rotarod for 10
seconds (s) before acceleration. Each trial ended when the mouse fell off the rotarod or
110

after 300 s had elapsed. The time that each mouse remained on the rotarod was
measured as the latency to fall (Xiong et al., 2014).
Loss of Righting Reflex
LORR was used to determine the sensitivity of the hypnotic effects of ethanol in WT
and KI mice. Mice were injected ip with 3.6 g/kg of ethanol and returned to their home
cage until they appeared ataxic. Upon exhibiting ataxia, each mouse was placed in a V-
shaped trough on its back, and the LORR latency and duration was measured. The time
from injection to LORR and the time from LORR to return of righting reflex were
recorded. Return of righting reflex was defined as the animal’s ability to right itself on all
4 paws, three times in 60 s.
Novel open field
Exploratory/locomotion activities in mice were assessed in an open-field paradigm
and followed methods described previously (Bortolato et al., 2013; Liang et al., 2014).
Animals were allowed to explore the arena for 10 min and performance was recorded
for analysis offline. On the floor, two concentric zones of equivalent areas were defined:
a central square quadrant and a peripheral frame directly adjacent to the walls. The
open field arena measured 72 x 72 cm surrounded by four walls (36 cm high).
Behavioral measures included the total distance traveled, percent activity in the center
(the distance traveled in the center divided by the total distance traveled) and number of
rearing episodes. Mice were injected with ethanol or saline and placed in a corner
quadrant of the field for observation.
Statistical Analysis
Since there were no sex-based differences observed in any of the behavioral
paradigms, the data represents both sexes. Graphs and statistical analyses were
generated by Prism (GraphPad Software Inc., San Diego, CA). Data is represented as
mean ± SEM and was analyzed using one-way analyses of variance (ANOVA), followed
by Bartlett’s correction for post-hoc comparisons. Significance between WT and USER
KI mice were detected using unpaired, two-tailed student’s t-test. Statistical significance
threshold was set at p < 0.05.
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RESULTS
TALEN-mediated genetic incorporation successfully produced first generation α2
GlyR USER KI mice. The parental (F
0
) generation of α2 GlyR USER KI mice
demonstrated normal breeding behavior relative to WT, and produced viable male and
female offspring. The α2 GlyR gene (Glra2) gene, which encodes for the α2 GlyR
protein, is located on the x chromosome. Thus, three male offspring carrying the
mutation (X
KI
Y) and five homozygous female mice (X
KI
X
KI
) were used in these
behavioral experiments.
To translate our in vitro USER findings in vivo, and provide proof-of-concept that
USERs can be expressed in animals to understand the contributions of receptor
subunits in the behavioral outcomes of ethanol, WT and α2 GlyR USER KI mice were
subjected to three behavioral paradigms: accelerating rotarod, LORR, and novel open
field. To confirm whether USERs definitively demonstrate increased ethanol sensitivity
in vivo, mice were subjected to a known motor-impairing dose (10%), and to a 10-fold
(1%) and 100-fold (0.1%) lower dose, for the accelerating rotarod and novel open field
tests. The results are described below.
α2 GlyR USER KI mice demonstrated no differences in motor incoordination
compared to WT on accelerating rotarod
The accelerating rotarod paradigm was used to assess whether USER mice
exhibit motor incoordination at lower ethanol doses that do not affect WT mice. There
was no significant difference between USER KI mice and WT controls in the latency to
fall off of the rotarod at all concentrations tested (0.1%, 1% and 10% EtOH).

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FIGURE 4.1

Figure 4.1 WT and α2 GlyR USER KI mice on the accelerating rotarod in response to 0.1% ethanol.
Accelerating (5-25 rpm over 180 s) rotarod performance during 7 consecutive training trials and after 15
minutes post-injection of 0.1% ethanol (0.02 g/kg).

FIGURE 4.2

Figure 4.2 WT and α2 GlyR USER KI mice on the accelerating rotarod in response to 1% ethanol.
Accelerating (5-25 rpm over 180 s) rotarod performance during 7 consecutive training trials and after 15
minutes post-injection of 1% ethanol (0.2 g/kg).

1 2 3 4 5 6 7 15' post-
0
50
100
150
200
250
Training Trials
Latency to fall (sec)
WT
KI
1 2 3 4 5 6 7 15' post-
0
50
100
150
200
Training Trials
Latency to fall (sec)
WT
KI
114

FIGURE 4.3

Figure 4.3 WT and α2 GlyR USER KI mice on the accelerating rotarod in response to 10% ethanol.
Accelerating (5-25 rpm over 180 s) rotarod performance 15 minutes post-injection of 10% ethanol (2
g/kg), and 7 consecutive trials at 15 minute increments.

Pre-trial15 30 45 60 75 90 120
0
50
100
150
200
Minutes Post-Injection
Latency to fall (sec)
WT
KI
115

α2 GlyR USER KI mice demonstrate no change in sensitivity to a hypnotic dose of
ethanol in LORR
We measured the latency to LORR and the duration of LORR to assess whether
USER mutations affect sensitivity to a hypnotic ethanol dose (3.6 g/kg). There was a
trend towards significance with regard to a faster onset of LORR in α2 GlyR USER KI
mice compared to WT controls (p = 0.1). However, there were no significant differences
between the duration of LORR between WT controls and α2 GlyR USER KI mice.
FIGURE 4.4

Figure 4.4 α2 GlyR USERs exhibit no significant differences to the hypnotic effect of 3.6 g/kg
ethanol compared to WT.
[A] There was a trend towards significance in the difference between the latency to loss of righting reflex
(LORR) in KI compared to WT controls (p = 0.1).
[B] There was no significant difference in the duration of LORR between KI and WT controls. Values
represent mean ± SEM for 6 WT and 8 KI mice.

116

α2 GlyR USER KI mice exhibit significance increase in anxiolytic effects at 1%
ethanol exposure
We used the novel open field assay to determine whether USER KI mice exhibit
differences in locomotor activity, exploratory behavior, and anxiolytic activity in response
to low ethanol doses that do not affect WT mice. There were no significant differences
between the USER KI mice and WT controls in locomotor activity, indicated by the total
distance traveled, (Fig. 4.5) and percent activity in the center (the distance traveled in
the center divided by the total distance traveled) (Fig. 4.6) at all ethanol doses tested.
However, at 0.2 g/kg ethanol (1% ethanol), USER KI mice exhibited a significantly
greater frequency of rearing behavior, an indication of anxiolysis, compared to WT
controls (Fig. 4.7). Grooming behavior was greater in USER KI mice compared to WT
controls at saline control treatment only (Fig. 4.8).
FIGURE 4.5

Figure 4.5 α2 GlyR USERs exhibit no significant differences in locomotor activity compared to WT.
Comparison of locomotor activity measured by total distance traveled between WT and α2 GlyR USER KI
mice in a 5 minute novel open-field activity assay in response to saline control, 0.1%, 1%, and 10%
ethanol.

Saline 0.1% EtOH 1% EtOH 10% EtOH
0
10
20
30
Total Distance Traveled (m)
WT Control
α2 GlyR USER KI
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FIGURE 4.6

Figure 4.6 α2 GlyR USERs exhibit no significant differences in thigmotaxic behavior compared to
WT.
Comparison of proportion of time spent in center square of test arena relative to total distance traveled
between WT and α2 GlyR USER KI mice in a 5 minute novel open-field activity assay in response to
saline control, 0.1%, 1%, and 10% ethanol.
FIGURE 4.7

Figure 4.7 α2 GlyR USERs exhibit increased rearing behavior compared to WT at 1% ethanol.
Comparison of rearing behavior between WT and α2 GlyR USER KI mice in a 5 minute novel open-field
activity assay. *p < 0.05 in response to saline control, 0.1%, 1%, and 10% ethanol. There was a
significant increase in the number of rearing episodes in α2 GlyR USER KI mice in response to 1%
ethanol, compared to WT.

Saline 0.1% EtOH 1% EtOH 10% EtOH
0
2
4
6
8
% Distance Traveled in Center
WT Control
α2 GlyR USER KI
Saline 0.1% EtOH 1% EtOH 10% EtOH
0
20
40
60
80
Rearings
WT Control
α2 GlyR USER KI
*
118

FIGURE 4.8

Figure 4.8 α2 GlyR USERs exhibit differences in grooming behavior compared to WT.
Comparison of grooming behavior between WT and α2 GlyR USER KI mice in a 5 minute novel open-
field activity assay. *p < 0.05 in response to saline control, 0.1%, 1%, and 10% ethanol. There was a
significant increase in grooming behavior in α2 GlyR USER KI mice compared to WT.

Saline 0.1% EtOH 1% EtOH 10% EtOH
0
5
10
15
Grooming
WT Control
α2 GlyR USER KI
*
119

DISCUSSION
The present study is the first to investigate the in vivo application of the global KI
of α2 GlyR USERs in C57BL/6J mice. We tested several ethanol-induced behavioral
effects to characterize whether USER KI mice demonstrate increased sensitivity to low
doses of ethanol that do not significantly affect WT control animals. In particular, α2
GlyR USER KI mice exhibited greater anxiolytic behavior in response to 0.2 g/kg
ethanol (1% ethanol) compared to WT control mice. These findings complement our in
vitro studies, and support our hypothesis that α2 GlyR USER subunits in C57BL/6J mice
produce ethanol-induced behavioral effects at concentrations that do not significantly
affect WT control animals. Moreover, these findings reveal that α2 GlyRs play a role in
mediating the anxiolytic effects of ethanol.
Earlier in vitro studies found that α2 GlyR USERs are sensitive to extremely low
ethanol concentrations (0.25 mM – 1 mM); in comparison, WT α2 GlyRs do not respond
to ethanol concentrations below 50 mM (Naito et al., 2014). In the current study, we first
determined the threshold of ethanol sensitivity of α2 GlyR USER KI mice using three
behavioral paradigms: accelerating rotarod, LORR, and novel open field. WT control
mice did not exhibit ethanol-induced behavioral effects at concentrations as low as 0.2
g/kg (1% ethanol) in any of the behavioral paradigms tested in this study. As reported in
prior in vitro studies, in the absence of ethanol, these USERs do not exhibit differences
in general receptor characteristics including Hill slope, maximum current amplitude,
EC
50
, and endogenous allosteric modulators such as the neurosteroid,
allopregnanolone (Naito et al., 2014). Thus, any differences in behavioral effects
exhibited upon administration of low ethanol doses in these KI mice could reasonably
120

be attributed to the up-regulation of α2 GlyR USER activity.
In the α2 GlyR USER KI mice, administration of low ethanol doses (0.1% and 1%
ethanol) did not produce overt differences in locomotor behavior (accelerating rotarod),
ataxia (LORR), exploratory behavior, and thigmotaxis (proportion of distance traveled in
outer squares of test arena) compared to WT control mice. The increase in rearing
behavior seen at 0.2 g/kg ethanol (1% ethanol) in the novel open field test in α2 GlyR
USER KI mice suggests an increase in anxiolytic activity. These findings align with the
expected effects of the up regulation of inhibitory projections of α2 GlyR activity in the
brain. Anxiolysis, or the reduction of anxiety, is regulated by the release of dopamine in
the CNS (Refojo et al., 2011). Since activation of GlyRs increases dopamine levels in
various brain regions including the striatum (Yadid et al., 1993), NAc (Ericson et al.,
2006), and the ventral tegmental area (Ye et al., 2004), it is likely that ethanol
potentiation of α2 GlyR USERs augments dopamine release, resulting in anxiolytic
effects.
Moreover, homomeric α2 GlyRs are the predominant GlyR subunit expressed in
reward-related brain regions including the amygdala and NAc (Jonsson (Delaney et al.,
2010; Jonsson et al., 2009; Jonsson et al., 2012). In contrast, α1 GlyRs are implicated
in mediating the hypnotic effects of ethanol-induced LORR (Quinlan et al., 2002) and
motor incoordination (Blednov et al., 2012; McCracken et al., 2013). Thus, the lack of
behavioral changes in these α2 GlyR USER KI mice in motor-related assessments –
accelerating rotarod and LORR, reinforce the specific function of the α2 subunit of
GlyRs in mediating anxiolytic pathways. Unlike other behavioral assessments of anxiety
that rely upon noxious stimuli such as acoustic startle, electric shock, food/water
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deprivation, exposure to predator odors, or fear-based anxiety such as the elevated plus
maze (Walf and Frye, 2007), the novel open field paradigm measures motivational
anxiety to explore a novel area. Thus, the increase in rearing behavior seen in this study
helps parse out a specific form of anxiety that is based on the mouse’s proclivity for
exploration in an open field, rather than fear- or shock-induced anxiety. These results
provide further justification that α2 GlyRs participate in anxiolysis that results from
ethanol-induced motivation and reward pathways.
Our laboratory has already demonstrated the production of USERs in multiple
subunits of GlyRs and GABA
A
Rs in vitro (Naito et al., 2014). The marked increases in
ethanol and anesthetic sensitivity seen in USER GlyRs and GABA
A
Rs are produced by
structural mutations in extracellular Loop 2 (Naito et al., 2014; Naito et al., 2015; Perkins
et al., 2012; Perkins et al., 2009). The sequence homology among the Cys-loop
superfamily of receptors suggests that USERs can also be produced for other ligand-
gated ion channels (LGICs) in the superfamily such as serotonergic (5HT
3
)

and nicotinic
cholinergic (nACh) receptors. Thus, once the individual receptor contribution is
identified, multiple KI expression of different USERs would help determine the
interactions between receptor subunit combinations in producing ethanol’s behavioral
effects. Therefore, USERs could help dissect out the roles played by ethanol action on
specific receptor subunit combinations in causing the behavioral effects responsible for
the acute and chronic effects of AUD.
The insights regarding the roles played by specific receptor subunits in AUD will
provide new, more specific targets, for the development of new drugs to prevent and/or
treat alcohol-related problems. Understanding the specific sites within receptor subunits
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that govern specific behavioral effects is imperative to developing more effective, and
targeted therapies with desired behavioral outcomes. Furthermore, USER KI mouse
models will continue to inform the construction of molecular receptor models that
increase our understanding of structure-function relationships underlying receptor
activation and modulation by ethanol as well as anesthetics. More importantly, USER
technology could help identify key receptor subunit sites that could help construct
potential pharmacophores for drug development to prevent and/or treat alcohol-related
problems by mimicking, blocking or otherwise modulating the actions of ethanol at these
specific sites.
While the results of these studies demonstrate that USERs expressed in vivo
have increased ethanol sensitivity to concentrations that do not affect WT animals,
further studies in second and third generation animals are necessary to validate these
results. Future studies should further characterize the role of α2 GlyRs in mediating the
reward and addiction pathways through additional behavioral paradigms including two-
bottle choice to determine craving and overall ethanol intake, conditioned place
preference to determine the effects of addiction, and operant behavior to determine the
rewarding effects of ethanol.
In summary, the present study provides early proof-of-concept that USERs
represent a novel tool that can be employed to map the cascade of events leading from
modulation by ethanol of specific populations of α2 GlyRs to behavioral change. More
importantly, we demonstrate that low doses of ethanol can be used as an agonist to
specifically activate a receptor subunit that has been sensitized to extremely low ethanol
concentrations to understand the role of an individual receptor subunit in causing
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behavioral effects. Thus, the findings set the stage for employing USER technology as a
brain-mapping tool to increase understanding of the biological basis for AUD, for
identifying new targets for drug development, and for dissecting the roles that these
LGICs play in behavior and disease.

124

Chapter 5 Overall Discussion and Conclusions
For centuries, alcohol (ethanol) has been consumed as part of social, religious,
and cultural customs. However, the psychoactive properties of ethanol are linked to
abuse and dependence that cause harm to not only the individual suffering from an
AUD, but also to society as a whole. One of the many critical obstacles for the
development of effective medications to mitigate and prevent the development of an
AUD is the lack of a clear understanding of the molecular sites and mechanisms of
ethanol action in the CNS. There are two major factors that contribute to this obstacle:
1) Lack of pharmacological tools to identify ethanol binding sites; and 2) Lack of
understanding regarding the role of individual receptors in mediating ethanol-induced
reward, addiction, and dependence. The studies presented in this dissertation introduce
a transformative tool to investigate the primary sites of ethanol action on individual
receptor subunits that are expressed in the reward and addiction brain regions.
Ultimately, these studies allow us to amplify our understanding about the direct cause
and effect role of individual receptor subunits in mediating ethanol-induced behaviors,
and identify key sites of ethanol action to develop potential drug targets.
The lack of classical pharmacologic agonists and antagonists specific to ethanol
present critical barriers to our understanding regarding the sites and mechanisms of
ethanol action. To date, the closest antagonist to ethanol is a physical antagonist –
atmospheric pressure, first reported by Alkana and Malcom in 1982 (Alkana and
Malcolm, 1982). The non-selective, non-specific pharmacological nature of ethanol to its
targets greatly challenges the identification of exact sites and mechanisms. Moreover,
the requirement of extremely high ethanol concentrations, equivalent to twice the legal
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driving under the influence (DUI) blood alcohol concentration, to cause behavioral
effects in animals implies the simultaneous activation of multiple receptor systems and
downstream pathways that complicate the understanding of the role of individual
receptor subunits in mediating the diverse behavioral effects of ethanol. In fact, ethanol
action in the CNS causes a plethora of behavioral effects such as sedation, hypnosis,
motor incoordination, memory loss, reward, addiction, dependence, anxiolysis, ataxia,
and motivation (Crabbe et al., 2006). Thus, the greatest challenge for studying the
molecular basis of ethanol reward and addiction is to be able to identify the initial sites
of ethanol action on specific receptors and distinguish which are responsible for the
consequential cascades that lead to each behavioral manifestation.
Emerging genetic technologies have prompted great strides into the
understanding of the sites and mechanisms of ethanol action at the molecular level. For
example, optogenetics make it possible to study the consequences of the activation of
brain region-specific neurons on animal behavior (Zhang et al., 2007; Zhang et al.,
2009). These are the first studies to provide a brain-mapping tool to directly probe the
cause and effect paradigm of the activation of a neuronal population and the resultant
behavioral effects. Indeed, these studies have provided important global brain mapping
insights. However, with the great diversity of ethanol-sensitive receptors (both
metabotropic and ionotropic), and the even greater diversity of receptor subunit
isoforms expressed in the adult human brain, the potential targets for ethanol remain at
large. Thus, the next step for AUD drug development is to identify tangible ethanol
targets that provide receptor subunit-specific resolution.
The studies presented in the current dissertation begin to fill this gap through the
126

discovery of USERs. This work includes findings that demonstrate that manipulations of
the physical-chemical properties of extracellular Loop 2 of GlyRs and GABA
A
Rs greatly
alter receptor sensitivity to ethanol with regard to the threshold and magnitude of
response. These studies, described in detail in Chapter 2 (Naito et al., 2014), found that
as few as four point mutations harbored within a single exon at the N-terminus of Loop 2
in α1 and α2 GlyRs are sufficient to increase ethanol sensitivity by more than 100-fold
relative to WT receptors. Importantly, in the absence of ethanol, these USERs did not
exhibit changes in general ligand-receptor characteristics including Hill slope, I
max
, and
EC
50
. A similar approach was used to produce GABA
A
R USERs in the γ2 and α1
subunits and the α2 subunit of GlyRs. These findings are in agreement with our
previous work in the EC domain, which showed that structural modifications of Loop 2
create highly ethanol sensitive GlyRs and GABA
A
Rs that respond to low ethanol
concentrations that do not affect WT receptors (Perkins et al., 2009). Importantly, the
GABA
A
R USERs did not exhibit increased sensitivity to other endogenous allosteric
modulators including the neurosteroid, allopregnanolone, suggesting a degree of
ethanol selectivity for the USERs. Moreover, the lack of alteration in general receptor
characteristics with these USERs thereby reduces the risk for compensatory
mechanisms that may arise during development of genetically engineered KI animals
that may potentially confound interpretation of behavioral tests. Taken together, these
studies provide strong evidence that USERs can be developed across multiple Cys-loop
receptors, and set the stage for the development of genetically engineered animal
models of USERs.
In Chapter 3, these studies were expanded to determine the selectivity of α1
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GlyR USERs to three different classes of anesthetic agents: volatile, local, and
intravenous. These studies revealed that α1 GlyR USERs demonstrated a 10-fold
increase in sensitivity relative to WT α1 GlyRs to the volatile anesthetic, isoflurane, and
the local anesthetic, lidocaine, but not to the intravenous anesthetic, propofol. The
findings are consistent with prior studies reported by Krasowski et al., who showed that
the sites of action for isoflurane and ethanol in the TM region overlap, but are distinctly
different from the site of propofol action (Krasowski et al., 1998). Importantly, the current
findings are the first to report common sites of action in the EC region (Loop 2) among
ethanol, isoflurane, and lidocaine, which are not shared by propofol action (Naito et al.,
2015). We further explored the sites within Loop 2 that are responsible for the alteration
of receptor sensitivity to these anesthetic agents and identified two key amino acid
residues in the C-terminal region of Loop 2 (D57 and R59) that govern GlyR sensitivity
to these agents. Thus, the findings indicate that the physical and chemical structure of
the C terminus of Loop 2 is sufficient and selective to increase α1 GlyR sensitivity to
ethanol, isoflurane, and lidocaine.
Finally in Chapter 4, the α2 GlyR USER was expressed in vivo through the
development of genetically engineered KI mice. This initial behavioral work represented
the first steps in translating our in vitro findings to animals. As presented, the goal of
these initial in vivo studies was to demonstrate that USER KI constructs can be
expressed in viable animals and that global KI expression of α2 GlyR USERs could
increase the sensitivity of the animals to the behavioral effects of ethanol. α2 GlyR
USER KI mice were sensitive to ethanol doses as low as 0.2 g/kg by demonstrating an
increase in rearing behavior (an indicator of increased dopamine activity), not seen in
128

WT control mice in the novel open field test. These findings represent the first results to
implicate the up-regulation of GlyR activity in response to a low ethanol dose that may
amplify GlyR-mediated dopamine release pathways in various reward/addiction brain
regions including the striatum, NAc, and VTA (Ericson et al., 2009; Yadid et al., 1993;
Ye et al., 2004). Furthermore, the results implicate the importance of the α2 GlyR
subunit in mediating ethanol-induced reward and addiction pathways, and identify Loop
2 as a key site of ethanol action. Therefore, these studies provide proof of concept that
USERs expressed in vivo have increased sensitivity to ethanol and reinforce the in vitro
findings that EC Loop 2 is an important target of ethanol.
In conclusion, the development of USERs presents a novel tool to determine the
role played by specific receptor subunit combinations in causing the behavioral effects
of ethanol leading to development of an AUD. This work is the first to provide
unambiguous results about the role of individual receptor subunits in transducing the
effects of ethanol without concerns about collateral effects on other nervous systems
that often confound these studies. The findings from these studies open the possibility
to understanding the sites and mechanisms that are not only limited to ethanol, but
other anesthetic agents such as isoflurane and lidocaine. In addition, the fact that
merely two point mutations Loop 2 are sufficient increase ethanol sensitivity 100-fold
over WT receptors implies the possible existence of genetic polymorphisms within Loop
2 that could predict the propensity of an individual to develop an AUD. For example,
current genome wide association studies have found significant associations among
mutations that produce ethanol-insensitive receptors and single nucleotide
polymorphisms (SNPs) (Borghese and Harris, 2012), suggesting the existence of
129

genotypic markers that could serve as predictors of efficacy for current drug treatments
and aid in the development of new drug candidates. Thus, these findings represent
major advances in our knowledge regarding the sites and mechanisms underlying
ethanol and anesthetic action for GlyRs and GABA
A
Rs. Overall, this work provides
additional insight for the development of pharmacophores that could potentially mimic or
block ethanol action within Loop 2, to prevent and/or treat alcoholism.

130

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Abstract (if available)

Abstract A critical obstacle to developing effective medications to prevent and/or treat an alcohol‐use disorder (AUD) is the lack of specific knowledge regarding the role of individual receptors in mediating ethanol‐induced reward, addiction, and dependence. The studies presented in this dissertation introduce a transformative tool to investigate the primary sites of ethanol action on individual receptor subunits that are expressed in the reward and addiction brain regions. Here, we developed a novel class of Ultra‐Sensitive Ethanol Receptors (USERs) that allow activation of a single receptor subunit population sensitized to extremely low ethanol concentrations. USERs were created by mutating as few as two residues in the extracellular Loop 2 region of glycine receptors (GlyRs) or γ-aminobutyric acid type A receptors (GABAARs), which are implicated in causing many behavioral effects linked to ethanol abuse. USERs, expressed in Xenopus oocytes and tested using two‐electrode voltage clamp electrophysiology, demonstrated an increase in ethanol sensitivity of 100‐fold over wild‐type receptors by significantly decreasing the threshold for ethanol sensitivity and increasing the magnitude of ethanol response, without altering general receptor properties including sensitivity to the neurosteroid, allopregnanolone. These profound changes in ethanol sensitivity were observed across multiple subunits of GlyRs and GABAARs. We also demonstrated the selectivity of these USERs by reporting that the extracellular Loop 2 region is a common site of action for anesthetics including ethanol, isoflurane, and lidocaine in α1 GlyRs. Interestingly, Loop 2 was not a site of action for the general anesthetic, propofol. We further employed USER technology in genetically engineered knock‐in mice to demonstrate proof‐of‐concept that USERs expressed in vivo can be activated by extremely low ethanol concentrations. Ultimately, USERs provide a unique tool to increase understanding of the individual role of receptors in mediating the reward and addiction pathways of ethanol. Thus, USERs could help identify key sites of ethanol action and potential drug targets that could treat and/or prevent alcohol addiction.

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Asset Metadata

Creator Naito, Anna (author)

Core Title Development of glycine and GABAA ultra-sensitive ethanol receptors (USERs) as novel tools for alcohol and brain research

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Molecular Pharmacology and Toxicology

Publication Date 07/15/2017

Defense Date 05/11/2015

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

Tag Addiction,Alcohol,GABA,glycine,OAI-PMH Harvest,reward

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Davies, Daryl L. (committee chair), Okamoto, Curtis Toshio (committee chair), Asatryan, Liana (committee member), Brinton, Roberta (committee member)

Creator Email anaito@usc.edu,anna.naito@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-596209

Unique identifier UC11300074

Identifier etd-NaitoAnna-3616.pdf (filename),usctheses-c3-596209 (legacy record id)

Legacy Identifier etd-NaitoAnna-3616.pdf

Dmrecord 596209

Document Type Dissertation

Format application/pdf (imt)

Rights Naito, Anna

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