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Development of stable cell lines expressing α1β2γ2 and α5β3γ2 GABAA receptors

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Development of stable cell lines expressing α1β2γ2 and α5β3γ2 GABAA receptors

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DEVELOPMENT OF STABLE CELL LINES EXPRESSING α1β2γ2 AND α5β3γ2
GABA
A
RECEPTORS

By

Chethana Armbya

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(Biochemistry and Molecular Biology)

December 2012

Copyright 2012 Chethana Armbya

ii

ACKNOWLEDGEMENTS

I would like to take this opportunity to express my gratitude towards the people whose
contributions have guided my journey through graduate school and my research. First and
foremost, I would like to thank my thesis advisor, Dr. Daryl Davies. His teaching and
contributions, along with his good-natured support, have been instrumental to my progress. I
would also like to thank Dr. Zoltan Tokes and Dr. Frank Markland for serving on my committee
and providing me with valuable insights.

I owe my deepest gratitude to Dr. Liana Asatryan for being an excellent mentor through my
research endeavors. I cannot thank her enough for her constant guidance and encouragement at
every stage of the research project. I am grateful to Miriam Fine for being a great teacher and
assisting me throughout this phase.

I am also indebted to all my fellow lab members whose support and friendly nature has always
created a healthy environment for conducting research and also to stimulate my personal growth.

Finally, I am eternally grateful to my parents and family members for their unconditional support
and enthusiasm and for making this journey possible.

iii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ............................................................................................................ ii
LIST OF FIGURES ........................................................................................................................ v
LIST OF TABLES ......................................................................................................................... vi
LIST OF ABBREVIATIONS ....................................................................................................... vii
LIST OF SYMBOLS ................................................................................................................... viii
ABSTRACT ................................................................................................................................... ix
CHAPTER 1 - INTRODUCTION .................................................................................................. 1
1.1 Receptors to the neurotransmitter γ-aminobutyric acid ................................................. 1
1.2 GABA
A
Receptors ......................................................................................................... 1
1.2.1 Structure and Function .............................................................................. 1
1.2.2 Role of GABA
A
Rs in pathophysiology of diseases .................................. 4
1.2.3 GABA
A
Rs as therapeutic targets ............................................................... 5
1.3 GABA
A
Rs as tools for drug discovery: Stable cell lines ............................................... 8
CHAPTER 2 - DEVELOPMENT AND VALIDATION ............................................................. 10
2.1 Specific Aims .............................................................................................................. 10
2.2 Background .................................................................................................................. 13
2.2.1 Expression of ion channels for drug discovery: approaches in generating
stable cell lines ................................................................................................. 13
2.2.2 Functional validation of GABA
A
Rs via pharmacological approaches.... 16
2.3 Methods ....................................................................................................................... 18
2.3.1 Generation of α1β2γ2L and α5β3γ2L GABA
A
R expressing stable cell
lines ........................................................................................................................ 18
2.3.2 Validation of functional expression of GABA
A
R ................................... 21
2.4 Results ......................................................................................................................... 24
2.4.1 Generation of stable cell lines expressing α1β2γ2L and α5β3γ2L
GABA
A
R ................................................................................................................ 24
2.4.2 Validation of the functional activity of GABA
A
Rs in the stable cells .... 28
2.5 Discussion .................................................................................................................... 36
2.6 Conclusion ................................................................................................................... 40
iv

CHAPTER 3 - FUTURE DIRECTION ........................................................................................ 41
BIBLIOGRAPHY ......................................................................................................................... 45
v

LIST OF FIGURES

Figure 1 The Structure of GABA
A
Receptor– Membrane Topology 2
Figure 2 GABA
A
Receptor Subunit Structure 3
Figure 3 Working of tetracycline inducible gene expression system 15
Figure 4 CHO Cells transfected with α1β2γ2L and α5β3γ2L GABA
A
R 25
Figure 5 Co-transfection: T-REx CHO cells co-transfected with the α1β2γ2L and
α5β3γ2L GABA
A
R
27
Figure 6 Sequential transfection: T-REx CHO cells transfected with the α5β3 27
Figure 7 Sequential transfection: T-REx CHO cells sequentially-transfected with the
α1β2γ2L and α5β3γ2L GABA
A
R
28
Figure 8 Whole-cell patch clamp recordings: GABA induced inward currents in T-
REx CHO expressing GABA
A
receptors.
29
Figure 9 Whole-cell patch clamp recordings: Effect of diazepam on GABA-evoked
currents in T-REx CHO expressing GABA
A
receptors.
29
Figure 10 A representative Western blot for α5 subunit in different clones of T-REx
CHO cells co-transfected with GABA
A
R subunits.
30,
31
Figure 11 A representative Western blot for α1 subunit in different clones of T-REx
CHO cells co-transfected with GABA
A
R subunits.
32
Figure 12 [
3
H]-flumazenil saturation binding curves for α1β1γ2 and α5β3γ2 33
Figure 13 [
3
H]-flumazenil binding inhibition curves with diazepam (A), L655,708 (b)
and zolpidem
34
Figure 14 Lentiviral-mediated transduction process 42
vi

LIST OF TABLES

Table 1 The characteristics and dilutions of primary antibodies used to test for GABA
A
α, β,
and γ subunits in Western Immunoblotting.
Table 2. K
i
values for respective BZ compounds in α1β1γ2 and α5β3γ2 GABA
A
receptors.

vii

LIST OF ABBREVIATIONS

AD Alzheimer Disease
BZ Benzodiazepine
CHO Chinese Hamster Ovary
CMV Cytomegalovirus
CNS Central Nervous System
DS Down Syndrome
FITC Fluorescent isothiocyanate
GABA Gamma(γ) aminobutyric acid
GABA
A
R Gamma(γ) aminobutyric acid A Receptor
HEK Human Embryonic Kidney
LGIC Ligand-Gated Ion Channel
MTA Material Transfer Agreement
PD Parkinson Disease
TM Transmembrane
T-REx Tetracycline-Regulated Expression
Tta Tetracycline transactivator

viii

LIST OF SYMBOLS

IC
50
– Inhibition constant; Concentration of unlabeled ligand that inhibits 50% of [
3
H] ligand
binding at 50%
K
i
- Equilibrium dissociation constant of the unlabeled ligand
K
d
- Equilibrium dissociation constant of the radioactive ligand
S - Concentration of radioligand

ix

ABSTRACT

Neurotransmitter gamma (γ)-aminobutyric acid (GABA) type A (GABA
A
) receptors are the
primary ligand-gated inhibitory ion channel group of receptors found in the Central Nervous
System (CNS). These are ionotropic receptors allow for the flux of Cl
-
upon activation of the
receptor by GABA binding. GABA
A
receptors are known to have a diverse role in the CNS with
implications in epilepsy, responses to drugs and alcohol and other disease states. GABA
A
Rs
form a heteropentameric structure which can be a combination of any of the 19 subunits that
make up the GABA
A
Rs. Distinct isoforms of GABA
A
Rs serve functionally distinct circuits due
to their domain-specific distribution and subtype-specific complex pharmacology. Specifically,
the alpha (α) subunit of GABA
A
Rs defines their distribution and the role in pathophysiology.
Specific GABA
A
R α subunits differentially respond to benzodiazepines, major GABA
A
R
allosteric modulators, thus are used to pharmacologically separate a wide range of actions such
as anxiolysis, sedation, and amnestic effects. α1-containing GABA
A
Rs mediate sedative action
as well as the anterograde amnestic action but not the anxiolytic-like action of benzodiazepines
like diazepam. Inverse agonists selective for α5-containing GABA
A
Rs provide memory
enhancement and agonists selective for α3-containing GABA
A
Rs might be suitable for the
treatment of deficits in sensorimotor processing in psychiatric disorders. The identification of
physiological and pharmacological functions of GABA
A
R subtypes defined by their α subunits is
a major area of investigation by the scientific community for the development of novel drugs
linked to the GABA
A
R system.

A significant number of compounds have now been developed that display GABA
A
R subtype
selectivity by affinity or efficacy, or by both. However, there is an increased need to identify
x

compounds with improved selectivity. One of the important tools that is becoming well
recognized as a necessary aid in the development of GABA
A
R subtype selective agents are cell
lines that stably express the GABA
A
R of interest. Having the means to produce GABA
A
R
subtype selective cell lines at the academic level would largely expand the research questions
and/or drug discovery projects that could be undertaken in an academic research environment.
The present work is focused on developing stable cell lines that express GABA
A
Rs; specifically
α1β2γ2 and α5β3γ2.

Stable cell lines expressing α1 and α5-containing GABA
A
Rs could be used for the identification
of drugs that act as allosteric modulators for the evaluation of their potential as a therapeutic
treatment for learning and memory disorders or cognitive deficiencies involved in
neurodegenerative pathologies such as Alzheimer Disease (AD) and Parkinson Disease (PD).
Further, these cell lines can be used to screen compounds and determine their selectivity for α5
vs. α1. The primary goal of this project was to develop stable cell lines expressing α1β2γ2L
and α5β3γ2L GABA
A
receptors. Specifically, my thesis focused on the initial steps of the
development and validation of stable cell lines expressing the GABA
A
α1β2γ2 and α5β3γ2
receptor subunit combinations. The experimental studies to support my thesis were done in two
primary steps that are presented in the following sections.

Step 1 of my project concentrated on development of stable CHO/ T-REx CHO cells
expressing α1β2γ2 and α5β3γ2 GABA
A
receptors. We introduced genes for the specific
subunits into T-REx CHO cells and isolated clones that expressed the desired proteins with the
xi

aid of selection antibiotics. We were able to obtain many clones that expressed the GFP reporter
gene, indirectly indicating successful transfection of the genes of interest.

Step 2 was aimed at validation of the presence and proper functioning of the transfected
GABA
A
R subunits. To achieve this, we used three different approaches. First, whole-cell patch-
clamp recordings from α1β2γ2 and α5β3γ2 clones showed GABA-induced currents suggesting
the presence of functional receptors. However, there were challenges with validation of the
presence of the γ subunit. Second, immunoblotting studies attempted to demonstrate the presence
of the protein expression of α or γ subunits. Number of clones demonstrated the presence of
either α or γ subunits.

And thirdly, in an attempt to validate the functionality of the generated stable cell lines
expressing GABA
A
R complexes, we developed a binding assay using a radioligand, [
3
H]-
flumazenil, that efficiently binds to the benzodiazepine site at GABA
A
Rs. There were several
outcomes associated with the findings of the binding assay. Our findings verified the presence of
functional GABA
A
Rs in Ltk cells that were known to express different combinations of
GABA
A
Rs. Further, we were able to determine the various pharmacological constants that
characterize the GABA
A
R complexes. Accordingly, our findings from the binding assay
demonstrated that the assay could be successfully used to validate the stable cell lines that we
already generated or will generate in the future in our laboratory settings as well as for programs
that are aimed at development of specific drugs that target α5 vs α1 containing GABA
A
Rs.

1

CHAPTER 1 - INTRODUCTION

1.1 Receptors to the neurotransmitter γ-aminobutyric acid
Neurotransmitter receptor systems have been the focus of intensive pharmacological research for
more than 30 years for basic and applied scientific reasons, but only recently has there been a
better understanding of their key features (Korpi et al., 2002). Gamma (γ)-aminobutyric acid
(GABA) is a major inhibitory neurotransmitter in the Central Nervous System (CNS) which
exerts its action by binding to the GABA binding region on the pentameric GABA receptor
system. The inhibitory actions of GABA are mediated by three receptor classes: GABA
A
,
GABA
B
and GABA
C
/GABAA-ρ (Rissman and Mobley, 2011). GABA
A
receptors, or
GABA
A
Rs, are ligand-gated ion channels (ionotropic receptors), whereas GABA
B
receptors are
G protein-coupled receptors. A subclass of slow-acting ionotropic GABA receptors found in the
human eye was designated GABA
С
/GABAA-ρ receptor.

1.2 GABA
A
Receptors
1.2.1 Structure and Function
GABA
A
Rs are members of the Cys-loop superfamily of Ligand-Gated Ion Channels (LGICs).
Members of this superfamily, which includes also nicotinic acetylcholine receptors (nACh),
glycine and 5-hydroxytriptamine 3 receptors (5HT
3
), possess a characteristic loop formed by a
disulphide bond between two cysteine residues (Fig. 1). The receptor proteins are composed of
pentameric clusters of homomeric or heteromeric composition that form a central ion pore. There
is similarity in the individual membrane subunit topology such that they possess an intracellular
domain, four transmembrane α-helical segments (TM1-4) and a large extracellular ligand-
2

binding domain made up of β sheets. Agonist binding to these receptors leads to ion channel
activation (Brejc et al., 2001; Langosch et al., 1988; Ortells et al., 1997; Tang et al., 2002).

Figure 1. Structure of GABA
A
R Subunit. The figure shows membrane topology of an individual GABA
A

receptor subunit.TM1-3 are indicated in blue and TM2 in yellow (Charycha et al., 2009).

Specifically, GABA
A
Rs form a heteropentameric structure assembled from five subunits (Fig. 2).
Molecular heterogeneity in the GABA
A
R subtypes is evident by the presence of 19 subunits
distributed into different classes of α , β, γ, δ (delta), ρ(rho) and ε (epsilon) which dictate specific
actions depending on the combinations in which they come together to form the pentameric
receptor complex (Korpi and Sinkkonen, 2005). The receptor subunit combination likely to exist
in the mammalian brain are two α subunits, two β subunits and one γ subunit (Mckernan and
Whiting, 1996) with two GABA binding sites formed by α and β subunits. Distinct isoforms of
GABA
A
Rs have different developmental, physiological and pharmacological properties and are
localized to specific brain regions and subcellular compartments (Martin et al., 2009). Binding of
GABA molecules to their binding sites in the extracellular part of the GABA
A
R triggers the
opening of a chloride ion-selective pore, resulting in inhibitory responses.
3

Figure 2. GABA
A
R structure. A. GABA
A
receptors are known to be large protein complexes consisting of five
subunits encircling a central ion conducting pore. Each subunit consists of two almost equally large domains:
an N-terminal extracellular domain responsible for agonist binding and a C-terminal trans-membrane
domain responsible for ion-channel pore formation. This figure shows the subunit arrangement of the most
common GABA
A
R as viewed from the synaptic cleft. The ‘‘+’’ refers to the ‘‘plus’’ asymmetric side of each
subunit, ‘‘G’’ indicates GABA binding sites and ‘‘B’’ indicates the benzodiazepine binding site. (Munro et al.,
2009) B. The tertiary structure of assembled GABA
A
R. Receptor α subunits are illustrated in blue, β subunits
in pink and δ/γ in green. The benzodiazepine binding pocket is formed between α and γ subunits (orange
square) and the GABA binding pocket is formed between α and β subunits (pink pentagon) (Charycha et al.,
2009).

Genetic, molecular and pharmacological tools have enabled understanding of activities of
distinct types of receptors, as judged by their mediation of benzodiazepine activities. The binding
site for benzodiazepines is formed by one of α subunits (α1, α2, α3 or α5) and a γ subunit
(typically the γ2 subunit, which is present in approximately 90% of GABA
A
R). GABA
A
Rs
containing the α4 or α6 subunit do not bind clinically used classical benzodiazepines (Rudolph
4

and Knoflach, 2011). The α1β2γ2 GABA
A
R combination is the most abundantly available
subunit combination (approximately 60%) throughout the brain region (Mckernan and Whiting,
1996). Approximately 15–20% have the α2β3γ2 combination, approximately 10–15% have the
α3βnγ2 combination, approximately 5% have the α4βnγ or α4βnδ, combination, less than 5%
have the α5β2γ2 combination (Rudolph and Knoflach, 2011). It is noteworthy that in
recombinant receptors, α subunit adjacent to the γ2 subunit determines the sensitivity to
benzodiazepines.

1.2.2 Role of GABA
A
Rs in pathophysiology of diseases
GABA
A
Rs are known to have a diverse role in the CNS and implications in epilepsy, responses
to drugs and alcohol and other disease states (Rissman and Mobley, 2011). GABA
A
R-mediated
events have two effects on the postsynaptic membrane: an increase of the postsynaptic
membrane conductance (shunting inhibition) and a change in the membrane potential owing to
movement of Cl
-
ions through the membrane (hyperpolarizing inhibition). Synaptic receptors that
detect millimolar concentrations of GABA mediate fast inhibitory postsynaptic potentials
(IPSPs), whereas extrasynaptic receptors that detect micro molar concentrations of GABA
mediate slower IPSPs and also tonic conductances (Rudolph and Knoflach, 2011). The tonic and
phasic conductances underlie different physiological and behavioral processes.

The roles of a large number of subtype permeations of GABA
A
subtypes expressed throughout
the brain are an area of intense investigation. Both genetic and medicinal chemistry approaches
have been used to identify the pharmacological relevance of these subtypes (Rudolph and
Möhler, 2005). A limited number of mutations have been found in GABA
A
R subunit genes.
5

However, the functional consequences of this genomic variation are not fully understood. These
include point mutations in the α1 and γ2 subunits in patients with genetic epilepsies (Baulac et
al., 2001). Genetic association studies indicate single nucleotide polymorphisms (SNPs) in the
gene encoding the α2 subunit in alcohol dependence and illicit drug dependence. Studies have
also helped in establishing a clear link between the modulation of α5-containing GABA
A
Rs and
associative memory (Rudolph and Möhler, 2005). Converging lines of evidence suggest that
dysfunction of the GABA system contributes to the pathophysiology of schizophrenia, mainly
via α3-containing GABA
A
Rs (Rudolph and Möhler, 2005; Ahn et al., 2011). In a similar manner,
other GABA
A
genes encoding the α1, α6, β2 and π subunits have been linked to schizophrenia
(Rudolph and Knoflach, 2011). Further, a high-affinity ethanol binding site on GABA
A
R that
contains a combination of α4 (or α6), β3, and δ subunits has been proposed and GABA
A
Rs are
implicated in the acute and chronic effects of alcohol including tolerance, dependence and
withdrawal (Wallner et al., 2003; Enoch, 2008). Significant changes in composition of various
GABA
A
Rs subunits, especially α5, β2/3 and γ2, have been reported in the progression of
pathology in Down Syndrome and AD animal models (Rissman and Mobley, 2011). It gradually
became evident that although these receptor subtypes are all expressed by the same cell type,
they serve functionally distinct circuits due to their domain-specific distribution.

1.2.3 GABA
A
Rs as therapeutic targets
One of the most significant characteristics of GABA
A
Rs is that they are allosterically modulated
by benzodiazepines, which are used for a wide range of actions (Rudolph and Knoflach, 2011).
Studies have revealed that different actions of benzodiazepines, such as anxiolysis, sedation, and
amnestic effects, are pharmacologically separable and can be functionally linked to specific α
6

subunits (Reynolds et al., 2012). α1-containing GABA
A
Rs mediate sedative action as well as the
anterograde amnestic action but not the anxiolytic-like action of benzodiazepines like diazepam
(Rudolph and Möhler, 2005). Receptors containing the α1, β2/3 and γ2 subunits are known to
mediate sedative, amnestic and anticonvulsant actions, whereas receptors containing subunits α2,
β2/3, and γ2 mediate anxiolytic and muscle relaxation (Olsen and Sieghart, 2009). Inverse
agonists selective for α5-containing GABA
A
Rs provide memory enhancement and agonists
selective for α3-containing GABA
A
Rs might be suitable for the treatment of deficits in
sensorimotor processing in psychiatric disorders (Rudolph and Möhler, 2005). Subsequently,
such findings stimulated interest in the development of subunit-specific compounds. In addition
to benzodiazepines, the GABA
A
R is the major target for the clinically used hypnotic drugs
zolpidem, zopiclone, (S)-zopiclone and zaleplone, for barbiturates and for many general
anesthetics (Rudolph and Knoflach, 2011). At low concentrations, barbiturates act as positive
allosteric modulators of GABA
A
Rs. At significantly higher concentrations, barbiturates can
directly activate the receptor (i.e. directly gate the channel even in the absence of GABA). It is
particularly interesting to compare benzodiazepines to barbiturates because both of these families
of drugs exhibit addictive properties and both modulate GABA
A
R (Tan et al., 2011b).

The alpha subunit of GABA
A
Rs defines their distribution and the role in pathophysiology. The
identification of physiological and pharmacological functions of GABA
A
R subtypes defined by
their α subunits continues to be an area of active research within the scientific community. For
instance, there continues to be investigations focusing on development of GABA
A
drugs with
fewer side effects than classical benzodiazepines (for example, non-sedating anxiolytics) and the
development of drugs with indications that are distinct from those of classical benzodiazepines
7

(for example, analgesics and cognition-enhancing drugs). A significant number of compounds
have now been developed that display GABA
A
R subtype selectivity by affinity or efficacy or by
both (Rudolph and Knoflach, 2011). More preclinical efforts are needed to identify compounds
with improved selectivity. This may be primarily achieved with higher affinity only at the
desired receptor subtype. In addition to the development of non-sedative anxiolytics and
cognition enhancing drugs, recent scientific discoveries provide hope for the development of
analgesic drugs for the treatment of chronic pain.

One interesting example of a GABA
A
R combination is the α5 containing GABA
A
Rs (e.g.,
α5β3γ2L) which has been implicated in learning, memory and cognitive dysfunction (Moser and
Paulsen, 1998), including AD (Cross et al., 1984). Consequently, a growing area of interest is the
identification and development of subtype selective agents that would aid in determining the
explicit roles of the α5 containing GABA
A
Rs (as well as others). Interestingly, the majority of α5
selective agents, developed to date, have been selective inverse agonists (Dawson et al., 2006b).
For instance, the treatment of cognition and memory related disorders such as AD and other
dementias may be done using selective α5 GABA
A
inverse agonists such as RO4938581 (Ballard
et al., 2009a), α5IA (Dawson et al., 2006) and L-655,708 (Atack et al., 2006; Quirk et al.,
1996a). But results from these studies have not achieved the desired outcomes (Navarro et al.,
2002). Even though drugs that are selective for certain subtypes of GABA
A
Rs have been under
development for a number of neurological disorders, focus on α5 selective drugs has mainly
revolved around agents that are negative allosteric modulators of GABA (Rudolph and Möhler,
2005; Atack, 2011). But recently, a growing body of evidence has suggested that augmenting
rather than reducing GABAergic controls is seen in conditions of over activity affecting
8

hippocampal circuits. Recent studies are suggesting that excessive activity in the hippocampal
region in the aging brain may be indicators of additional risk for AD (Koh et al., 2012).
Compounds that act as positive allosteric modulators at GABA
A
R α5 receptors might prove
handy in ameliorating this condition as GABA
A
α5 receptors cause tonic inhibition of neurons in
the affected network (Hauser et al., 2005). Thus, attention has turned towards positive allosteric
modulators of GABA that can potentiate GABA receptor function (Koh et al., 2012). This may
represent a favorable approach in the treatment of cognitive disorders such as AD, autism and
other learning and memory related disorders (Martin et al., 2010). Also, α5-selective compounds
can be characterized by either binding selectivity, efficacy selectivity or a mixture of both these
features. This advantage, in contrast to other α-selective compounds, most of which have been
restricted to efficacy selective compounds, suggests that the α5 receptor may be a more
chemically amenable target (Mirza et al., 2008; Atack, 2011).

1.3 GABA
A
Rs as tools for drug discovery: Stable cell lines

One of the important tools that is becoming well recognized as a necessary aid in the
development of GABA
A
R subtype selective agents are cell lines that stably express the
GABA
A
R of interest. Unfortunately, GABA
A
R expressing stable cell lines are difficult to
produce. Molecular cloning of the GABA
A
R has revealed the complexity of the receptor
complexes (Drewe et al., 1995). Although some of the common GABA
A
R subtypes are now
available commercially, these cell lines are very expensive, costing in many instances in excess
of fifty thousand dollars per cell line – thus making these cell lines mostly unavailable for
investigations conducted at the University level. Moreover, not all GABA
A
R combinations are
9

available. Therefore, having the capability to produce GABA
A
R subtype selective cell lines at
the academic level would greatly expand the research question and/or drug discovery projects
that could be undertaken in an academic research setting. In my thesis, we present the strategy
that we used and the experiments that we conducted in order to begin the development of
α1β2γ2L and α5β3γ2L GABA
A
Rs subtype selective stable cell lines.
10

CHAPTER 2– DEVELOPMENT AND VALIDATION

Modulation of GABA
A
Rs is potentiated by many drugs acting allosterically, including ethanol,
general anesthetics, benzodiazepines, barbiturates, and neuroactive steroids (Mihic et al., 1997;
Sieghart, 1995). GABA
A
Rs exist in a large number of different stoichiometric combinations and
many of these subtypes have distinct physiological actions. Therefore, the development of stable
cell lines expressing different GABA
A
R combinations would provide an important tool that
could be used to help tease apart the role of the various subunits. For example, in the
hippocampus, predominant expression of α5-containing GABA
A
Rs that are pharmacologically
distinct from α1-containing GABA
A
Rs, suggests their role in cognitive behaviors. Therefore,
stable cell lines expressing α5 and α1-containing GABA
A
Rs could be used for the identification
of drugs that act as allosteric modulators for the evaluation of their potential as a therapeutic
treatment for learning and memory disorders or cognitive deficiencies involved in
neurodegenerative pathologies such as AD and Parkinson Disease (PD). Moreover, these stable
cell lines could also be used to facilitate screening of libraries of compounds. With this in mind,
the main focus of this project was to begin the development of stable cell lines expressing
α1β2γ2L and α5β3γ2L GABA
A
Rs.

2.1 Specific Aims
The primary interest in developing stable cell lines expressing α1β2γ2L and α5β3γ2L GABA
A
subunit combinations is to enable screening of compounds to determine their selectivity towards
α5 containing GABA
A
Rs vs. α1 containing GABA
A
Rs. My thesis focused on the initial steps of
11

the development and validation of stable cell lines expressing the GABA
A
α1β2γ2L and α5β3γ2L
receptor subunit combinations.

Step 1. Development of stable CHO/ T-REx CHO cells expressing α1β2γ2 and α5β3γ2
GABA
A
Rs. Experiments in this step were designed and conducted to introduce the genes for
GABA
A
receptors into CHO or T-Rex CHO cell systems, selection of clones and their further
growth as stable cell lines. Lipid-mediated transfection methods were used for gene transfer.
Transfection efficiency was monitored by GFP fluorescence.

The first step was to transfect the individual genes for the α1, β2, α5, β3 and γ2 subunits into
Chinese Hamster Ovary (CHO) cells. Antibiotic selection strategy was used to isolate the clones
that were successfully transfected. Partial success led us to set up more focused experiments
directed at controlling the gene expression in cells. For this purpose, we used Tetracycline-
Regulated Expression in CHO (T-REx CHO) cells. Thus, utilizing precise control of gene
expression, experiments worked towards stable transfection of the GABA
A
R subunits into T-
REx CHO cells and subsequent induction of gene expression. We used two strategies for
transfection of the subunits: Sequential and Co-transfection of genes expressing the different
subunits.

Step 2.Validation of the presence of functional expression of GABA
A
Rs. For this, we used a
few different approaches: 1) To ensure the functional expression of the GABA
A
Rs, we
performed whole cell patch-clamp studies. 2) The presence of GABA
A
subunit protein
expression was analyzed using Western Immunoblotting. 3) Finally, we designed a ligand
12

binding assay that would confirm the presence of gamma subunit along with alpha and beta and
validate the proper functioning of the full receptor complex. To optimize this assay, the studies
were performed on Ltk cell lines expressing α1β1γ2L and α5β3γ2L GABA
A
Rs (from Merck Co.,
NJ, USA) using [
3
H]-flumazenil as the radioligand and tool compounds such as diazepam (non-
specific binding), Zolpidem (selective for α1) and L-655,708 compound (selective for α5) as
competitors for the GABA
A
Rs. Preliminary data from these experiments have been included.

13

2.2 Background
2.2.1 Expression of ion channels for drug discovery: approaches in generating stable cell lines
There are several challenges to the molecular drug discovery of ion channels. Ion channels are
typically complex, multimeric, transmembrane proteins that consist of separate pore-forming and
accessory subunits (Ashcroft, 2006). For high-throughput screening, abundant expression of the
target protein should be achieved in a heterologous expression system. Heterologous expression
of Cys-loop LGICs in mammalian cell lines has been achieved but rarely in the desired quantities
and purity. Especially in the GABA
A
R, much lower specific activities (1–6 pmol/mg), compared
to other receptors of the superfamily, have been reported (Dostalova et al., 2010).

Expressing ion channels in a form that resemble their native correlates has also been challenging.
Achieving native functional and pharmacological properties is dependent on efficient expression,
localization, and orientation of an appropriate combination of subunits, each of which may have
multiple transmembrane domains that fold in and out of the membrane. As a consequence there
is enormous potential for misfolding and mis-assembly when ion channels are over-expressed.
Furthermore, in many cases, the exact subunit composition of the target channel in the tissue of
interest is only poorly characterized (Clare, 2010).

Transiently transfected cell lines have a limited lifetime (24-48 hours) due to the loss of the gene
product via repeated mitosis and degradation cycles resulting also in unacceptable batch to batch
variations. Stable transfection ensures long-term, reproducible as well as defined gene
expression. Stable transfection occurs when the gene of interest introduced into the eukaryotic
cell is integrated into the nuclear genome of the cell and the cell, subsequently, continues to
14

propagate individual clones. Major applications for stable transfection are the analysis of gene
function and regulation, large scale protein production, drug discovery and gene therapy
(Brummelkamp et al., 2002; Srinivasakumar et al., 1997; Wurm, 2004). Stable expression can be
influenced by the transfection method used and the vector containing the gene of interest
(Brielmeier et al., 1998). After integration of the DNA, the level and time of expression of the
gene of interest depends on the promoter cloned upstream on the expression vector and on the
particular integration site. For constitutive expression, promoters such as the cytomegalovirus
(CMV) promoter are chosen. The final step in the development of stable cell lines is the selection
of cells that are stably transfected with the foreign gene of interest (Wurm, 2004).

Constitutive expression of ion channels frequently leads to functional toxicity leading to
deficiencies in processing of essential host cell proteins (Clare, 2010). As a result, overexpressed
ion channels are poorly tolerated. For a regulated expression inducible promoter systems are
available. The tetracycline/doxycycline-inducible gene regulatory system allows tight control of
transfected gene expression. The primary constituents of this system include the tetracycline
transactivator (tTA) driven by a promoter of choice and the polymeric tetracycline operator-
cytomegalovirus (CMV) minimal promoter (Tet [O]) driving expression of the desired gene (Fig.
3). tTA, a fusion protein, binds to the tetracycline operator sequence in the absence of
tetracycline or doxycycline and suppresses gene expression. When the fusion protein interacts
with doxycycline, it undergoes conformational change and releases from its DNA recognition
sequence, allowing gene transcription to proceed. tTA has been mutated to generate a promoter
molecule, designated the reverse tetracycline transactivator (rtTA), that binds to the tet (O)
sequence and induces gene expression when doxycycline is present.

15

Figure 3. The figure demonstrates the working of tetracycline inducible gene expression system. In the
presence of the effector molecule, doxycycline (dox), transcription of the target transgene is activated. In the
absence of dox, target transgene remains silent (Romano, 2004)

Transient expression of GABA
A
R subunits in mammalian cell lines or Xenopus oocytes results
in recombinant receptors with distinct pharmacological properties. Many transient expression
studies have been instrumental in supporting important theories such as the obligatory role of the
γ subunit for reconstituting the benzodiazepine binding site, etc. A permanent expression system
with stably transfected cell lines composed of various subunit combinations is necessary in order
to adequately characterize these receptors pharmacologically (Drewe et al., 1995).

GABA
A
R expressing cell lines are currently commercially available through a few companies
such as ChanTest (Cleveland, Ohio, USA) and Millipore (Billerica, MA, USA). However, the
two main drawbacks of commercially available cell lines expressing GABA
A
Rs are 1)
unavailability of all subtypes and 2) the extremely high selling prices. These disadvantages make
these cell lines mostly unavailable for academic research and thus form the main motivation to
generate stably transfected cell lines in our laboratory setting. Therefore, initially I focused on
generation of CHO cells stably expressing α1 and α5 subunit containing GABA
A
Rs. Due to
16

challenges that arose in the course of development, I then switched to an inducible T-REx CHO
system for generation of GABA
A
cell lines.

2.2.2 Functional validation of GABA
A
Rs via pharmacological approaches
One of the predominant characteristics of GABA
A
Rs that makes them a key interest as potential
drug targets is that they not only possess a binding site for GABA, the major inhibitory
neurotransmitter in the mammalian CNS, but also other recognition sites for a diverse range of
modulatory compounds, such as benzodiazepines (BZs), neurosteroids, barbiturates and some
anesthetics (Sieghart, 1995). The relationship between the GABA
A
R subunit composition and
the molecular pharmacology of the GABA
A
R modulating BZs has been extensively studied. To
date, most functional efficacy screening to find novel GABA subtype-selective pharmacological
agents has been performed using either measurement of radiolabelled Cl- flux or
electrophysiological methods (Simpson et al., 2000).

Ligand-binding studies are a reliable method to investigate the pharmacological characteristics of
expressed receptors (Hadingham et al., 1992(b)). The two main components involved in such
studies are the receptors and the ligands binding to the receptors. The majority of GABA
A
Rs in
the brain possesses a BZ binding site and has a combination of αβγ subunits in a 2:2:1 ratio,
arranged in a αβαβγ sequence (Minier and Sigel, 2004). The BZ binding site occurs at the
interface between α and γ subunits, with both contributing to the binding pocket pharmacology.
However, since the γ2 subunit is the predominant γ subunit in the brain, most of the variability in
the BZ pharmacology of native GABA
A
Rs is due to α subunit (Mckernan and Whiting, 1996a).
Hence, it has been shown that the classical BZ agonists, such as diazepam and lorazepam, have a
17

high affinity for GABA
A
Rs containing β and γ2 subunits in association with a α1, α2, α 3 or α5
subunit (Luddens et al., 1995; Sieghart, 1995). In contrast, GABA
A
Rs containing β and γ2 and
either a α4 or α6 subunit show no affinity for the BZ agonists (Hadingham et al., 1993(a);
Knoflach et al., 1996; Wisden et al., 1991).

In this context, functional expression of recombinant GABA
A
Rs can provide an appropriate
model to study the affinity of different subunit compositions of the GABA
A
R for selected
compounds as well as the intrinsic activity of drug interactions (Faure-Halley et al., 1993). Here,
we hoped to study the interaction of selective modulators with two different recombinant forms
of the GABA
A
receptor, namely α1β2γ2 and α5β3γ2. First, we validated the stable cell lines
using the whole cell patch clamp electrophysiology approach. This approach made use of the fact
that application of diazepam (a GABA receptor agonist) will enhance GABA-evoked currents.
Also, using the benzodiazepine modulator antagonist, [
3
H]flumazenil, as a probe, we designed
high affinity competition binding studies to determine the affinity of the compounds to the
GABA
A
R subtypes. Preliminary data from saturation and optimization experiments and some
test runs have been presented in this section.

18

2.3 Methods
2.3.1 Generation of α1β2γ2L and α5β3γ2L GABA
A
R expressing stable cell lines
 Mutagenesis and plasmid construction of GABA
A
R subunit cDNAs
The human α1, γ2L and rat β2 GABA
A
R subunit cDNAs in the pcDNA 3.1(+) expression
vectors were a kind gift of Dr. Adam Hall, PhD (Associate Professor, Dept. of Biological
Sciences, Smith College Northampton, MA, USA). The human β3 and α5 GABA
A
R subunit
cDNAs in the pcDNA 3.1-MycHis (-) was provided by AgeneBio Inc. (Carmel, IN). The human
β2 GABA
A
R subunit cDNA in PCR-Blunt II-TOPO was obtained from ATCC (Manassas, VA,
USA). cDNAs encoding the human α1 and α5 GABA
A
R subunits were sub-cloned into
pIRESpuro vector, and the cDNAs encoding human β3 and rat β2 GABA
A
R subunits were sub-
cloned into pIRESneo vector, while the human γ2L GABA
A
R subunit cDNA was sub-cloned
into pIREShygro vector (Clontech, Mountain View, CA, USA).For the T-REx system, the
human α, rat β and human γ subunit cDNAs were cloned into pcDNA5/TO hygro vector,
pcDNA3.1 zeo(-) vector and pcDNA 3.1(+) neo vectors (Clonetech, Mountain View, CA, USA).

 Cell line cultures
The Chinese hamster ovary (CHO) cells are the most widely used mammalian cells for
transfection, expression, and large-scale recombinant protein production. Due to various
characteristics of CHO cells such as the low chromosome number (2n=22) and stable and
accurate glycosylation, they offer a post-translationally modified product and thus a more
accurate in vitro rendition of the natural protein (Darroudi et al., 1982; Sheeley et al., 1997).
Tetracycline (Tet)-inducible CHO(T-REx CHO) stable cells were obtained from (Invitrogen,
Carlsbad, CA, USA). The cells were cultured and maintained in minimal essential medium
19

(Invitrogen, Carlsbad, CA, USA) supplemented withF12, 10% FBS and 100U/ml penicillin/ 100
µg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C under humidified air containing
5% CO2. Cells were passaged every 3-4 days as the confluence reached ~80%. The expression
of the GABA
A
R subunits in T-REx CHO cells was induced by treatment with 1 μg/ml
doxycycline for 24 h. The expression of genes was expected to peak from 24h to 48h.

 Transfection and selection of clones
Generation of stable cell lines containing α1β2γ2L and α5β3γ2L GABA
A
Rs were derived by
transfecting vectors containing the desired genes along with GFP into the cells. We used
Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for stable transfections in both cell lines.
In general, the transfection efficiency is usually low and hence, the transfected cells must be
amplified by selection. This process takes up to 3-4 weeks. Up scaling of the process parameters
requires about 3 weeks more. Overall, it takes 4-7 weeks on an average.

A) For selection of stable clones in CHO cells, we added 5 µg/ml puromycin (Invitrogen,
Carlsbad, CA, USA), in addition to 300 µg/ml hygromycin B (Roche Diagnostics,
Mannheim, Germany), and 200 µg/ml G418 (Invitrogen, Carlsbad, CA, USA). Selection
was typically done three days post-transfection. The cells were allowed to grow in
selection medium for three to four days. The cells were then re-plated followed by
picking colonies to ensure stable transfections. After several passages, a portion of the
cells were lysed using ice-cold lysis buffer supplemented with protease inhibitors.

20

B) In the T-REx CHO system, we tried two different strategies to achieve successful
transfection of the GABA
A
R subunits. The strategies were 1) Co-transfection of all three
subunits simultaneously and 2) Sequential transfection of the subunits. For the first
method, we simultaneously transfected vectors containing the desired genes for α, β and γ
in ratios of 1:1:2 and 1:1:5. In sequential transfection, we transfected only α and β subunit
expressing genes, along with GFP. After establishing stable expression of α and β by
antibiotic selection over many passages, the gene for γ subunit was introduced to the
cells. In both cases, the transfected cells were allowed to grow for 2-3 days before being
re-plated. On the second day following re-plating, antibiotics were introduced into the
medium for selection of stable clones. We added 400µg/ml each of Zeocin (Invitrogen,
Carlsbad, CA, USA), hygromycin B (Roche Diagnostics, Mannheim, Germany), and
G418 (Invitrogen, Carlsbad, CA, USA). Once the cells reached ~80% confluence, they
were re-plated at varying dilutions to allow individual clones to grow into colonies. The
media used to grow these cells always contained the three antibiotics at 400µg/ml, each.
Once the colonies began to grow, about 10-12 colonies for each combination of receptor
subunits were picked manually by using sterile pipette tips and grown in separate plates.
After several passages, a portion of cells from each colony/clone was induced for 24h
with 1µg/ml of doxycycline. The cells then were used for validation of the expression of
GABA
A
R subunits using Western Blot analysis.

21

2.3.2 Validation of functional expression of GABA
A
R
 Whole cell Patch Clamp Recordings
Transfected CHO and T-REx CHO cells (24h-48h after induction with 1.5mg/ml doxycycline)
were voltage clamped at -50 mV at room temperature, and GABA induced currents were
acquired using Axopatch 200B amplifier, Digidata 1320interface and pClamp 9.0 software
(Molecular Devices, Union City, CA). Data was digitized at 5 kHz and filtered at 1 kHz.
Composition of the external solution was (in mM): NaCl 135, KCl 5.4, CaCl
2
1.8, MgCl
2
1,
HEPES 10, and glucose 10with pH of 7.4 adjusted with NaOH. Patch electrodes (2-6 MΩ) were
filled with (in mM): KCl 140; MgCl
2
2; EGTA 2.5; TEA-Cl2; K
2
ATP 4; HEPES 10; pH of 7.3
was adjusted with KOH. Time between applications was 15-120 s depending on cell recovery
from desensitization. GABA applications were performed through a 3-barrel delivery system
using a Warner SB-77B Fast Perfusion apparatus and VC-8ValveController (Hamden, CT).
Patch clamp studies were conducted by Kaixun Li, PhD (Electrophysiologist, Dr. Davies Lab,
School of Pharmacy, USC).

 Western Immunoblotting
Non-induced and induced cells from each clone were lysed using ice-cold RIPA lysis buffer
supplemented with protease inhibitors. The lysates were cleared by centrifugation at 10,000 g for
10min at 4°C and stored in -20°C for further analyses. Protein concentrations were determined
using the Bradford protein assay with Bovine Serum Albumin (Sigma Aldrich, St.Louis, MO,
USA) as a standard. Ten micrograms of protein extracts were separated using 10% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to Polyvinylidene
fluoride (PVDF) membranes. Membranes were blocked with a 5% solution of non-fat dry milk
22

dissolved in a Tris-HCl-buffered solution (TBS-Tween, pH 7.5) and were then probed with
primary antibodies (1:1000 dilution) specific to each subunit followed by a secondary antibody
(1:10000 dilution). Bands were visualized using enhanced chemi-luminescence (Pierce
Biotechnology). The list of primary antibodies used for the subunits are shown in Table 1.

Table 1. The characteristics and dilutions of primary antibodies used to test for GABA
A
α, β, and γ subunits
in Western Immunoblotting.
Primary
Antibody
Specificity
Dilution Secondary Antibody Manufacturer
α1 1:1000 Anti-rabbit ThermoScientific (Waltham, MA, USA)
α5 1:1000 Anti-rabbit PhosphoSolutions (Aurora, CO, USA)
γ2 1:1000

1:750
Anti-rabbit ThermoScientific (Waltham, MA, USA)
Anti-rabbit PhosphoSolutions (Aurora, CO, USA)

 Membrane Preparation and Ligand Binding
Three to four days after dexamethasone (Invitrogen, Carlsbad, CA, USA), 500nM, induction of
expression, Ltk cells stably expressing α1β1γ2 and α5β3γ2 GABA
A
Rs (Provided to Dr. Davies
laboratory under an MTA agreement from Merck Co., NJ, USA) were scraped into Dulbecco’s
Phosphate buffered saline (Invitrogen, Carlsbad, CA, USA) and centrifuged at 150 x g for 10
min. The pellets were washed twice in the same buffer by resuspension and centrifugation. Then,
the pellets were homogenized in hypotonic solution (10 mM Tris HCI; 1mM MgCl2; 0.32M
Sucrose, pH 7.4) containing proteinase inhibitors and then centrifuged at 48,000 x g for 30 min
at 4°C. Finally, the pellets were resuspended in assay buffer (50mM KH2PO4; 1mM EDTA;
0.2M KCl). After subjection to brief sonication using a Branson Sonifier 150 (G.Heinmann,
23

Germany), protein content was measured using the BCA protein assay (Bio-Rad Laboratories,
Reinach, Switzerland) with Bovine Serum Albumin (Sigma Aldrich, St.Louis, MO, USA) as the
standard.

Saturation binding curves were obtained by incubating membrane with various concentrations of
[
3
H]flumazenil (75-85Ci/mmol, PerkinElmer, MA, USA). The inhibition of the radioligand
binding by competitive drugs such as (diazepam, L-655, 708 or zolpidem - all dissolved in
DMSO) was performed at concentrations of [
3
H]flumazenil at or lower than its K
d
values in the
cells expressing α1 and α5-containing GABA
A
Rs (obtained from the saturation curves). All
binding assays were performed for 30min at 4°C in assay buffer. The total assay volume was 0.5
ml, containing 200ug of protein. Incubations were terminated by filtration through Whatman
GF/B filters (Brandel, Gaithersburg, MD) followed by three 3-ml washes with ice-cold assay
buffer in a Cell Harvestor (Brandel, Gaithersburg, MD). The radioactivity was read after 24h
using a Beckman Coulter (Brea, CA, USA) radioactivity counter.

 Data Analysis
Saturation and inhibition curves were obtained using GraphPad Prism software (GraphPad
Software, Inc., CA, USA). K
i
values which refers to the equilibrium dissociation constant of the
unlabeled ligand were determined using Cheng-Prusoff equation K
i
= IC
50
/ (1+S/K
d
), where IC
50

is the concentration of unlabeled ligand that inhibits 50% of [
3
H] ligand binding, S is the
concentration of radioligand and K
d
is the equilibrium dissociation constant of the radioactive
ligand (Cheng, 2004). The K
d
and IC
50
values were obtained from inhibition curves. All points
24

on binding curves were derived from duplicate or triplicate assays. Non-specific binding was
defined using 10 µM of the drug (diazepam, K655,708 or zolpidem).

2.4 Results
2.4.1 Generation of stable cell lines expressing α1β2γ2L and α5β3γ2L GABA
A
R
1. Lipid based co-transfection of receptor subunits in CHO cells
We first attempted to develop the stable cell lines by performing simultaneous transfection of
each individual subunit (α: β: γ:: 1: 1: 1) and co-transfection of the reporter GFP using
Lipofectamine 2000. We found that the transfection efficiency was pretty high, as observed by
fluorescence microscope. Although the initial results were encouraging, this effort represented
only the first step in the process in the development of stable cell lines expressing GABA
A
Rs.
The next step was selection of transfected clones by antibiotic selection. Appropriate
concentrations of the antibiotics had been determined by titrating an antibiotic concentration
gradient against cell survival. After several passages, all maintained in medium with optimum
concentration of antibiotics, only 50% of the cells showed fluorescence (Fig. 4).

25

Figure 4. A, B: show FITC and Phase contrast images of CHO cells transfected with α1β2γ2L GABA
A
R
cloned into pIRESpuro, neo, hygro vector respectively using lipofectamine, three days post transfection. C, D:
FITC and Phase contrast images of CHO cells transfected with the α5β3γ2L GABA
A
R cloned into
pIRESpuro, neo, hygro vectors, respectively, using lipofectamine, three days post transfection. These images
were taken under a 10X objective of fluorescence microscope (Zeiss, Oberkochen, Germany).

The cells started to develop an unhealthy conformation when exposed to the selection medium
for more than three days. This might have been due to the concentration of one or more
antibiotics or the formation of receptor complex adversely affecting the cell system. Based on
these outcomes and other factors, we decided to try using the Tet-regulated CHO inducible
system to perform subunit transfections.

26

2. Lipid based transfection of receptor subunits in T-REx CHO cells
As mentioned earlier, we employed two strategies of transfection in T-REx CHO cells. For Co-
transfection, we introduced the subunit genes in a 1:1:5 and 1:1:10 ratios. We used higher
amounts for the γ subunit to ensure its proper expression. Antibiotic selection was initiated with
appropriate antibiotics (zeocin, hygromycin and G418, 400ug/ml each for respectively alpha,
beta and gamma subunits). The cells showed good transfection efficiency (over 50%). After
manual selection of grown colonies, about 90% of the cells were positive for GFP and
demonstrated normal morphology and growth rate (Fig. 5).

For sequential transfections, the first step was to transfect α and β subunits into the cells. Many
different colonies were picked by employing a combination of manual and antibiotic selection.
Approximately 98% of the cells were positive for GFP and showed normal morphology and
growth (Fig. 6). These select clones were then subjected to re-transfection with γ2 subunit gene,
culminating in a subunit gene ratio of 1:1:5. Following repeated selection, the fluorescence was
observed to be significantly lower in the α1β2γ2 cells (Fig. 7), indicating that not all the cells that
had the αβ complex were transfected with the γ subunit. After several passages, the cells were
assumed to be stably expressing the α1β2γ2 and α5β3γ2 receptor complexes.

27

Figure 5. A, B: FITC and Phase contrast image of T-REx CHO cells co-transfected with the α1β2γ2L (1:1:5)
GABA
A
R cloned into pcDNAzeo, neo, hygro vector respectively. C, D: FITC and Phase contrast image of T-
REx CHO cells co-transfected with the α5β3γ2L (1:1:5) GABA
A
R cloned into pcDNAzeo, neo, hygro vector
respectively. These images were taken under a 10X objective of fluorescence microscope (Zeiss, Oberkochen,
Germany).

Figure 6. A, B: FITC and Phase contrast image of T-REx CHO cells transfected with the α5β3 (1:1) GABA
A
R
cloned into pcDNAzeo and hygro vector respectively. These images were taken under a 10X objective of
fluorescence microscope (Zeiss, Oberkochen, Germany).

28

Figure 7. A, B: FITC and Phase contrast image of T-REx CHO cells sequentially transfected with the
α5β3γ2L (1:1:5) GABA
A
R cloned into pcDNAzeo, neo, hygro vector respectively. C, D: FITC and Phase
contrast image of T-REx CHO cells transfected with the α1β2γ2L (1:1:5) GABA
A
R cloned into pcDNAzeo,
neo, hygro vector respectively. These images were taken under a 10X objective of fluorescence microscope
(Zeiss, Oberkochen, Germany).

2.4.2 Validation of the functional activity of GABA
A
Rs in the stable cells
1. Functional expression of GABA
A
Rs
We used patch-clamp electrophysiology to demonstrate the presence of functional GABA
A
Rs.
GABA induced inward Cl
-
currents in T-REx cells expressing both α1β2γ2 and α5β3γ2
GABA
A
Rs (Fig. 8). We used 10µM and 3µM GABA respectively to induce currents in
respectively α1 and α5 expressing cells.
29

Figure 8. GABA induced inward currents in T-Rex CHO cells stably transduced with α1β2γ2 (A) and α5β3γ2
(B) GABA
A
R. All 3 subunits (α, β and γ) for both combinations were co-transfected simultaneously into the
cells followed by doxycycline induction. Currents were recorded using patch clamp.

Further, to prove the presence of γ subunit, we tested the effect of diazepam, a benzodiazepine
known to robustly bind to GABA
A
Rs and potentiate the receptor function. Unexpectedly,
diazepam inhibited GABA-induced currents in α5 expressing T-Rex CHO cells (Fig. 9). We also
obtained similar result using the α1 containing cells (data not shown). It is plausible that the γ2
subunit did not correctly incorporate into the pentameric GABA
A
R complex or this may be a
result of misfolding of the receptor complex.

Figure 9. Diazepam inhibited GABA (3 µM)-induced currents in α5-containing GABA
A
R. As in Fig. 8 the
cells were co-transfected with all subunits simultaneously. Data were generated using patch clamp.

250ms
200pA
GABA 1 2 2
100pA
250ms
GABA 5 3 2
A. B.
100 pA
250 ms
T-Rex CHO, 5 3 2
Diaz, 0  M
Diaz, 1 M
30

Since our findings were inconclusive regarding the presence of γ2 subunit in the receptor
complex, we sought to further validation techniques such as Western Immunoblotting.

2. Expression of GABA
A
R subunits in T-REx CHO cells
The presence of the GABA
A
R subunits was validated using Western Immunoblotting techniques.

A) CO-TRANSFECTION of subunits into T-REx CHO cells: Western Blotting
experiments did not reveal bands for α1 in the tested clones. With the α5β3γ2 cell
samples, there were several clones that showed positive bands when probed with
anti-α5 from PhosphoSolutions (Fig. 10A). The rest of the clones showed positive
bands when probed with anti-γ2 from PhosphoSolutions (Fig. 10B).

Figure 10 A. The image shows a band for a5 in one of the clones of doxycyclin-induced α5β3γ2 cell lysates.

31

Figure 10 B. The image shows γ2bands in two clones of α5β3γ2cell lysates, induced and non-induced. In both
A and B, antibodies used for probing were from PhosphoSolutions. 1:1:2 and 1:1:5 are the ratios in which the
three GABA
A
R subunit expressing genes were introduced into the cells.

B) SEQUENTIAL Gamma2 transfections on α1β2 and α5β3 clones: Western Blots of
cell lysates showed α1 bands in several clones after induction with doxycycline (Fig.
11). The same samples did not show specific bands for γ2 as tested using
Phosphosolution antibody. In addition, it did not appear that the β2/β3 antibodies
were very effective in the identification of the GABA
A
β subunits. The α5 bands did
not show up in the blots even though almost all the cells expressed the GFP.

32

Figure 11. The image shows bands for α1 in different clones of doxycycline-induced α1β2γ2 cell lysates. Anti-
α1 from ThermoScientific was used for probing.

3. Validation of [
3
H]-flumazenil binding assay
There have been differences in α1 vs α5 in the sensitivities to number of drugs that bind to the
BZ site (Ballard et al., 2009). Therefore, to further validate the presence of functional α1 vs α5
containing GABA
A
Rs in the cell lines that we generated, we set up a radioligand binding assay.
My studies focused on the initial steps of optimization and validation of the binding assay.
Binding of BZ-like compounds to the BZ binding site is independent of the GABA binding;
therefore, this assay is normally performed in the absence of GABA. For validation of the
binding assay, we used Ltk cells expressing α1β1γ2 and α5β3γ2 GABA
A
Rs (provided to Dr.
Davies laboratory by Merck Co., NJ, USA, under an MTA agreement). Saturation binding curves
(presented in Fig.12) demonstrated comparable affinities of α1β1γ2 and α5β3γ2 GABA
A
Rs for
[
3
H]-flumazenil. K
d
values obtained from these saturation were 0.37 ± 0.045nM and 0.23 ± 0.021
nM respectively for α1 and α5 containing complexes.

33

Figure 12. [
3
H]-flumazenil saturation binding curves for α1β1γ2 and α5β3γ2. Specific binding was obtained
by subtracting non-specific binding at each concentration of radioligand. Data normalized on the protein
amount used.

We then tested for the competitive antagonism of some known BZ-compounds. First, we tested
effects of diazepam at a range of concentrations from 1 nM to 10 uM (Fig.13). Both α1 and α5
containing GABA
A
Rs demonstrated a comparable sensitivity to inhibition of [
3
H]-flumazenil
binding with diazepam as observed from the respective K
i
values (Fig. 13A, Table 2).

0 1 2 3 4
0
100
200
300
400
1 1 2
3H-Flumazenil, nM
Bmax= 85.6  3.07 fmol/mg prot
Kd= 0.37  0.045 nM
Bmax= 398  10 fmol/mg prot
Kd= 0.23  0.021 nM
    2
Specific Binding
(fmol/mg protein)
34

Figure 13. [
3
H]-flumazenil binding inhibition curves with diazepam (A), L655, 708 (B) and zolpidem (C). A
concentration range of 1 nM to 10 uM for the drugs was tested. IC
50
values obtained from the curves
demonstrated differential sensitivity of α5 containing GABA
A
Rs to L655,708 (α5-sensitive) and zolpidem (α5-
insensitive).

To distinguish between the two receptor complexes, we also used α5 selective L-655,708 and α1
selective zolpidem in the inhibition studies at concentration ranges 0.1-10,000nM. As expected,
the α5 GABA
A
Rs had ~80-fold higher sensitivity to L-655,708 compound than the α1 containing
GABA
A
Rs (Fig. 13B, Table 2). In contrast, zolpidem was effective in inhibiting [
3
H]-flumazenil
binding in α1 containing receptors whereas in α5 GABA
A
Rs there was no effect (Fig. 13C, Table

1 10 100 1000 10000
-10
10
30
50
70
90
110
    2
    2
Log L655,708 (nM)
3H-Flumazenil, Binding
% Normalized Response
B.

10 100 1000 10000
-10
10
30
50
70
90
110
    2
    2
Log Zolpidem (nM)
3H-Flumazenil Binding,
% Normalized Response
C.

10 100 1000 10000
-10
10
30
50
70
90
110
    2
    2
Log Diazepam (nM)
3H-Flumazenil Binding,
% Normalized Response
A.
35

2). Taken together, these findings sufficiently validated the binding assay in that it can
effectively distinguish between the α1 and α5-containing GABA
A
Rs. Therefore, this assay can
be used to validate the stable cell lines that we have generated based on T-REx CHO cells.

Table 2. K
i
values for respective BZ compounds in α1β1γ2 and α5β3γ2 GABA
A
Rs expressed in Ltk cells. K
i

values were calculated using Cheng-Prussof equation and IC
50
values obtained in the inhibition assays.

Ligand α1β1γ2 α5β3γ2
Diazepam 20.2 ± 7.3 8.02 ± 1.7
L655,708 31.7 ± 6.3 0.39 ± 0.06
Zolpidem 25 ± 1.7 473 ± 15

36

2.5 Discussion
The primary goal of developing stable cell lines expressing α1β2γ2L andα5β3γ2L GABA
A
R
subunit combinations is to enable screening of commercially available compounds to determine
their selectivity towards α5 containing GABA
A
Rs vs. α1 containing GABA
A
Rs. As presented in
the results section, we developed the procedures for stably expressing two combinations of the
target GABA
A
R subunits, in mammalian cells. We first adopted a method of simultaneous
transfection/co-transfection of CHO cells with the GABA
A
receptor subunits. We observed that
over time, the morphology of the cells changed following antibiotic selection. Their changed
appearance and gradual deterioration suggested that the subunits were forming a complex that
was adversely affecting cell growth or the over expression was toxic for the cells. Our findings
from these experiments led us to look for a more efficient method to achieve our goal.

Evidence from recent studies propose that GABA
A
R can be produced in much higher yields than
previously reported by using an induction strategy in HEK cells (Dostalova et al., 2010a).
However, our previous experiments with HEK cells had been unsuccessful in that the cells did
not survive antibiotic selection after transfections. Even at very low concentrations of antibiotics,
the HEK cells did not survive for more than 3-4 days. This led us to conclude that expressing
subunit(s) were somehow causing toxicity to host cells. Therefore, we attempted to extend our
strategy using an inducible T-REx CHO system and a stepwise developmental approach. We
used strategies of co-transfection and sequential transfection on these cells. In both cases, 90% of
the cells showed fluorescence, indicating that the transfection was successful. Also, the cells
survived and grew normally in a medium that was regularly replenished with selection
antibiotics. The absence of change in morphology and general deterioration indicated that the T-
37

REx CHO cells were not suffering the same fate as the CHO cells. We went on to validate the
presence of transfected subunits by various methods.

To validate the T-REx CHO cells that were co-transfected with all three subunit combinations,
patch-clamp recordings fromα1β2γ2L and α5β3γ2L showed GABA-induced currents suggesting
the presence of functional receptors. However, GABA binding site is located between α and β
subunits (Tan et al., 2011), suggesting that α and β subunits can form functional receptors.
Therefore, the initial patch-clamp findings demonstrating GABA-induced currents may not
reflect the incorporation of the γ subunit into GABA
A
R complex. Since the benzodiazepine
binding site is formed at the interface of α and γ subunits (Drewe et al., 1995), we expected to
see potentiating of GABA-induced currents upon applying diazepam. Surprisingly, in our
experiments, diazepam inhibited the GABA-induced currents. This unexpected finding could be
a result of a few possibilities. If the γ2 was not successfully transduced and was absent, there
would have been no change in the GABA-induced currents. It is possible that the γ2 subunit did
not correctly incorporate into the pentameric GABA
A
R complex or this may be a result of
misfolding of the receptor complex. To gain insight into this inexplicable anomaly, we decided
to further validate the presence of the three subunits using Western Immunoblotting.

Some clones from co-transfection of all three subunits, i.e. α5, β3 and γ2 were positive for the
presence of α5 subunit in samples induced with doxycycline. The bands we obtained in Western
Blotting were approximately at 55kDa, which was very close to the expected molecular weight
for α5 (~50kDa). The presence of the β subunit in these transfected clones could not be
successfully established due to the lack of good antibodies for the same. When the same clones
38

were probed with antibodies for γ2, they did not show bands at the expected molecular weight
(~47kDa). However, there were other clones that showed bands at ~50kDa. Since, the gene
encoding for the γ2 subunit was not under the control of the tetracycline operator sequence, the
γ2 presence was demonstrated in both induced and non-induced samples of these clones. On the
whole, we observed that the presence of α and γ subunits in the clones were mutually exclusive.
Clones that were positive for the α5 subunit did not show γ2 and vice versa.

Outcomes from the sequential transfection experiments were much the same as seen in the co-
trasfection strategy. The first step in which only α and β subunits were introduced to the cells
provided many clones that showed bands for α subunit (~55kDa) in Western Blots. Again, the β
subunit could not be confirmed due to lack of efficient antibodies. Considering that the cells
were growing in a medium that had selective antibiotics for the β subunit, we went on to the
second step of introducing the γ subunit gene. Subsequent Western Blots failed to show bands
for γ. Together; these studies demonstrated that we were successful in generating cell lines that
upon induction of α subunit express the αβ combination but not the αβγ complex. Studies are in
progress to investigate the possible reasons for the absence/misfolding of the γ2 subunit in the T-
REx CHO cells expressing GABA
A
Rs. Therefore, those are still awaiting more conclusive
results and are currently beyond the scope of my thesis.

Stable cell lines expressing α1β2γ2L and α5β3γ2L GABA
A
R complexes with correct
conformation and benzodiazepine site would be necessary for screening of commercially
available compounds for their selectivity towards α5 vs. α1 containing GABA
A
Rs. Binding
assays using a radioligand that efficiently binds to the BZ site at GABA
A
Rs are very informative
39

in this respect. Flumazenil, also known as Ro 15-1788, is an imidazobenzodiazepine antagonist
with virtually no intrinsic activity (File and Pellow, 1986; Mohler and Richards, 1981(a)). Its
binding affinity is unaffected by other modulators of the GABA
A
receptor complex such as
GABA agonists and barbiturates (Mohler and Richards, 1981(a)). Because of its high specific
binding and lack of intrinsic activity, flumazenil is often used as a tracer compound in invitro as
well as in vivo receptor studies (Bertz et al., 1995; Koe et al., 1987; Miller et al., 1988; Mohler et
al., 1981(b); Tricklebank et al., 1990).

Therefore, part of my thesis focused on validating an in vitro [
3
H]-flumazenil binding assay in
two Ltk cell lines that are known to express different combinations of GABA
A
Rs, specifically
α1β1γ2 and α5β3γ2. There are several outcomes associated with the findings of the binding
assay. First, these findings verified the presence of functional GABA
A
Rs in the studied cells.
Second, they revealed that the protein expression of these receptor complexes is sufficient to
provide information on the pharmacological properties of the GABA
A
Rs. In this regards, we
were able to determine K
d
values from saturation binding curves that combined with the IC
50
of
the drugs tested that bind competitively to BZ binding site provided us with their K
i
values. The
latter values are essential when comparing the effects of different compounds and thus are
important for drug development programs. In addition, the obtained numbers were in agreement
with previously published results (Ballard et al., 2009; Hadingham et al., 1992; Quirk et al.,
1996) suggesting the validity of the binding assay. And finally, these findings demonstrated the
differential sensitivity of α1 vs α5 containing GABA
A
R complexes towards various
pharmacological tools (L566,708 and zolpidem). Collectively, our findings from the binding
assay demonstrate that this binding assay can be successfully used to validate the stable cell lines
40

that we generated in our laboratory settings as well as for programs that are aimed at
development of specific drugs that target α5 vs α1 containing GABA
A
Rs.

2.6 Conclusion
Together, the outcomes of my studies demonstrated the successful generation of cell lines that
upon induction of α subunit expressed the αβ combination. Less success was observed upon the
stable expression of αβγ complex. Our group is continuing to investigate the lack of successful
expression. As suggested in my thesis, the lack maybe due to the absence/misfolding of the γ2
subunit in the T-Rex CHO cells expressing GABA
A
Rs. Future studies should provide conclusive
information regarding this issue. If successful, our group should be able to demonstrate and
should lead to conclusive functional validation via the binding assay that we developed (and
demonstrated in my Thesis) using the Ltk cells provided to us by Merck. Having the capability,
at the academic level, to produce GABA
A
Rs subtype selective cell lines and screen specific
drugs that target these receptors would greatly expand the research question and/or drug
discovery projects that could be undertaken in an academic research setting.

41

CHAPTER 3 - FUTURE DIRECTION

Stable cell lines
As we found out during the course of our experiments, generation of stable cell lines by classical
transfection techniques is a time-consuming and labor-intensive process with an unpredictable
outcome. Integration of the heterologous DNA occurs randomly in the cellular genome (Gorman
and Bullock, 2000). Therefore, recombinant cells obtained by standard transfection are
heterogeneous in terms of the integrated transgene copy number and the site of integration,
leading to a broad range in the level of recombinant protein productivity. These reasons
necessitate the genetic selection step, such as antibiotic selection, to eliminate non-recombinant
and low-producing recombinant cells. Even then, a large number of clones must be screened to
find a few which express the protein of interest as desired (Matasci et al., 2008). Hence, the
chances of finding a stably-producing cell line are low (Browne and Al-Rubeai, 2007).
Additionally, the clones must be monitored over several months in the absence of selection
antibiotics to assess the stability of protein production overtime because gene silencing is a
frequent phenomenon (Kwaks and AP., 2006; Oberbek et al., 2010). Also, the unpredictability of
the integration process implies a considerable limitation to the reproducibility of the process. In
view of these limitations that we encountered, we began exploring the alternative method for
gene delivery that was readily available to us.

Viral gene delivery systems help in the transduction of genes of interest by integration of the
viral genome into the target cell genome, leading to long term expression of the desired gene.
Recombinant viral vectors have been derived from di ff erent viruses (Kay et al., 2001; Pfeifer,
2004; Pfeifer et al., 2001; Somia and Verma, 2001). The majority of integrating vectors are
42

derived from retrovirus, lentivirus or adeno-associated virus. Retroviral vectors have been used
for delivery of genetic material into cells for over 30 years. However, interest and use of
lentiviral vectors was popularized when researchers showed that human immunodeficiency virus
type 1 (HIV)-based vectors were capable of transducing non-dividing cells, thus overcoming one
of the important limitations of conventional retroviral vectors (Naldini et al., 1996; Quinonez and
Sutton, 2002). Sophisticated lentiviral gene delivery techniques have been used to successfully
transfect a wide range of dividing and non-dividing cells for over a decade now (Naldini et al.,
1996). The lentiviral-mediated transduction process is illustrated in the figure below (Fig.14)

Figure 14. Lentiviral-mediated transduction process (Amado and Chen, 1999)

Upon entering the host cell, the viral genome undergoes reverse transcription into DNA. This is
then incorporated into the host genome (provirus). The provirus acts as a template for the
production of many virions. Persistent infection and transmission of the integrated provirus to the
progeny is based on the integration of the viral and host cell genome (Pfeifer, 2004).

43

Currently, our lab is working towards developing the lentiviral DNA constructs for the GABA
A
R
subunits. We have well-tested packaging systems for transducing HEK 293T cells. Lentivirus
production and handling will be carried out in our Lenti-core facility (USC School of Pharmacy)
that follows all the rules applied by the NIH, institutional and regional organizations. Once we
have the lentivirus for all the subunits, we plan to subject HEK cells and CHO cells to lentiviral
infection. Cells that have been successfully integrated with the GABA
A
R DNA will exhibit
resistance to puromycin. Further validation of presence and function of the receptor subunits will
be done using Western Immunoblotting and radioligand binding studies.

Binding studies
To date, we have validated 3[H]-flumazenil binding assay for selectivity of a few compounds
towards α1 and α5 containing GABA
A
receptors using Ltk cell lines with known GABA
A
R
expression (Step 2 of this project). Currently, we are expanding these binding studies for testing
the stable GABA
A
R expressing cells that we developed and are still developing using the
recombinant lentiviral transduction approaches. Depending on the results from such studies, the
stable cell lines can be put to further use such as identification of drugs that act as allosteric
modulators of the GABA
A
subunits, screening of libraries of compounds, and for other research
purposes in an academic setting.

The identification of GABA
A
R subtype-specific functions has set the stage for the development
of novel subtype-selective agents (Mohler and Hanns, 2006). Mounting evidence from
pharmacology, neurology and molecular genetics implicates the GABA
A
R α5 subtype in at least
certain aspects of cognition (Atack, 2010; Ballard et al., 2009; Dawson et al., 2006; Fritschy and
44

Möhler, 1995; Sur et al., 1999). Not surprisingly, this receptor has evolved as a promising
potential target for the treatment of cognitive disorders (Dawson et al., 2006; Maubach, 2003).

45

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Abstract (if available)

Abstract Neurotransmitter gamma (γ)-aminobutyric acid (GABA) type A (GABAA) receptors are the primary ligand-gated inhibitory ion channel group of receptors found in the Central Nervous System (CNS). These are ionotropic receptors allow for the flux of Cl- upon activation of the receptor by GABA binding. GABAA receptors are known to have a diverse role in the CNS with implications in epilepsy, responses to drugs and alcohol and other disease states. GABAARs form a heteropentameric structure which can be a combination of any of the 19 subunits that make up the GABAARs. Distinct isoforms of GABAARs serve functionally distinct circuits due to their domain-specific distribution and subtype-specific complex pharmacology. Specifically, the alpha (α) subunit of GABAARs defines their distribution and the role in pathophysiology. Specific GABAAR α subunits differentially respond to benzodiazepines, major GABAAR allosteric modulators, thus are used to pharmacologically separate a wide range of actions such as anxiolysis, sedation, and amnestic effects. α1-containing GABAARs mediate sedative action as well as the anterograde amnestic action but not the anxiolytic-like action of benzodiazepines like diazepam. Inverse agonists selective for α5-containing GABAARs provide memory enhancement and agonists selective for α3-containing GABAARs might be suitable for the treatment of deficits in sensorimotor processing in psychiatric disorders. The identification of physiological and pharmacological functions of GABAAR subtypes defined by their α subunits is a major area of investigation by the scientific community for the development of novel drugs linked to the GABAAR system. ❧ A significant number of compounds have now been developed that display GABAAR subtype selectivity by affinity or efficacy, or by both. However, there is an increased need to identify compounds with improved selectivity. One of the important tools that is becoming well recognized as a necessary aid in the development of GABAAR subtype selective agents are cell lines that stably express the GABAAR of interest. Having the means to produce GABAAR subtype selective cell lines at the academic level would largely expand the research questions and/or drug discovery projects that could be undertaken in an academic research environment. The present work is focused on developing stable cell lines that express GABAARs

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Asset Metadata

Creator Armbya, Chethana (author)

Core Title Development of stable cell lines expressing α1β2γ2 and α5β3γ2 GABAA receptors

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 10/08/2012

Defense Date 08/27/2012

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

Tag binding assay,GABAA receptors,OAI-PMH Harvest,stable expression

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Tokes, Zoltan A. (committee chair), Davies, Daryl L. (committee member), Markland, Francis S., Jr. (committee member)

Creator Email armbyachethana@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-102478

Unique identifier UC11290614

Identifier usctheses-c3-102478 (legacy record id)

Legacy Identifier etd-ArmbyaChet-1235.pdf

Dmrecord 102478

Document Type Thesis

Rights Armbya, Chethana

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