Using TBPS to Identify Functionally Distinct GABAA Receptors in the Rodent CNS

Using TBPS To Identify Functionally Distinct GABAA Receptors In The Rodent CNS

by Nidaa A. Othman

B.S. in Biopsychology, May 2006, Wagner College

A Dissertation submitted to

The Faculty of The Columbian College of Arts and Sciences of The George Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

May 15, 2011

Dissertation directed by

Timothy G. Hales Adjunct Professor of Pharmacology & Physiology The George Washington University & Professor of Anaesthesia University of Dundee, Scotland

The Columbian College of Arts and Sciences of The George Washington University certifies that Nidaa A. Othman has passed the Final Examination for the degree of Doctor of Philosophy as of March 15th, 2011. This is the final and approved form of the

dissertation.

Using TBPS To Identify Functionally Distinct GABAA Receptors In The Rodent CNS

Nidaa A. Othman

Dissertation Research Committee:

Timothy G. Hales, Adjunct Professor of Pharmacology & Physiology, The

George Washington University; Professor of Anaesthesia, University of

Dundee, Dissertation Director

David C. Perry, Professor of Pharmacology & Physiology, Committee

Member

Linda L. Werling, Professor of Pharmacology & Physiology, Committee

Member

iii Dedication

I would like to dedicate my dissertation to everyone who has ever expressed the curiosity, courage and willingness to question the world around them, never settling for less than what their dreams could imagine.

iv Acknowledgments

First and foremost, I would like to thank Tim G. Hales for his support and mentorship over the last five years. I am truly lucky to have had a supervisor who showed as much dedication to his students as he did to his research. Without his guidance, brainstorming sessions, musical influence, and unique humor, this dissertation would not have come to fruition. From my first day in his laboratory, I knew that my standards had to be high, my work had to be thorough and that in the end, I would never settle for anything less. Tim, I would like to thank you for raising the bar higher than anyone else ever would.

I would also like to thank my committee members for their influence on my research over the years. Dr. David C. Perry, Dr. Linda L. Werling, Dr. Vincent A.

Chiappinelli were essential to the progression of my dissertation. I would like to thank Dr.

Kenneth J. Kellar for accepting the position as my outside examiner and Dr. Katherine A.

Kennedy for presiding over my defense as well as for all of her astute questions at each of my departmental seminars. I would also like to thank all of the faculty and staff of the

Institute for Biomedical Sciences, the Department of Pharmacology at the George

Washington University and the University of Dundee. Dr. Jerry J. Lambert, Dr. John A.

Peters, Dr. Delia Belelli, Dr. David Balfour, Dr. Christopher N. Connolly, Dr. Michelle A.

Cooper and Mrs. Karen A. Bollan were essential to the completion of my experiments at the University of Dundee. I would like to thank all of the students and staff that have rotated through the Hales lab over the years, especially Dr. Tarek Z. Deeb, who provided vital guidance during my early years as a graduate student, and Lisa Wright, who helped me retain my composure during my final year as a graduate student.

v I cannot forget my fellow graduate students, especially the entering class of 2006.

We held each other together whenever there was a need to do so and I would not have survived the roughest times of the last five years without your support.

I would like to thank my family for all of their support throughout my education.

They always encouraged me to push forward and never give up hope, no matter how difficult the circumstances. My mother, who always believed in me, my father, who always said I was “the greatest”, and my two brothers, who have always encouraged me to strive for something better, I thank you for all of your loving support.

Lastly, but by no means least, I would like to thank my fiancé, Aaron, who was strong enough to stand by my side when I felt I could stand no longer. He listened when I needed him to, smiled when I could no longer smile, and helped carry my burdens when I could bear them no more. Without his support, I might not have made it to the end.

vi Abstract of Dissertation

Using TBPS To Identify Functionally Distinct GABAA Receptors In The Rodent CNS

The GABAA receptor is a pentameric anion permeable Cys-loop receptor that can

be formed from several combinations of α(1-6), β(1-3), γ(1-3), ρ(1-3), δ, ε or θ subunits

and is the site of action for treatment of epilepsy, anxiety, insomnia and anesthesia.

Mutations in the GABAA receptor result in deficits of channel function and loss of

inhibitory neurotransmission, resulting in seizures. GABAA receptor-mediated inhibition is

either phasic, mediated by synaptic αβγ receptors or tonic, mediated by GABA-activation

of high affinity extrasynaptic αβδ receptors or spontaneous agonist-independent gating of

different subtypes. The ultimate goal of this study was to develop the GABAA receptor-

specific radioligand [35S]t-butylbicyclophosphorothionate ([35S]TBPS) as a probe to

identify functionally distinct GABAA receptors.

TBPS and picrotoxin (PIC) are non-competitive GABAA receptor antagonists that

bind within the second transmembrane domain (TM2). Accessibility to their binding sites is

dependent on whether the receptor is resting, activated or desensitized. Putative activation

and desensitization “gates” within the TM2 control the flow of ions through the GABAA

receptor’s ion channel. GABA modulation of [35S]TBPS binding differs between receptor

subtypes, indicating that their distinctive channel properties may influence accessibility to

35 the TM2 binding site. [ S]TBPS is primarily used to localize GABAA receptors in the

brain and I hypothesized that the differential binding of [35S]TBPS to various brain regions vii is dependent on the functional channel properties dictated by the expression of specific

subunits in each brain region and that [35S]TBPS could be used as a tool to detect functional deficits associated with neurological disorders such as epilepsy.

The in vivo mutant γ2(K289M) subunit, associated with generalized epilepsy with

febrile seizures and corresponding synthetic α1(K278M) mutant cause deficits in GABA

efficacy and diminish spontaneous gating. Using patch-clamp electrophysiology, I

examined the ability of TBPS and PIC to block wild-type and mutant receptors containing

the epilepsy subunits and established that the binding site for TBPS lies above the GABAA receptor activation gate and below the desensitization gate, indicating that biphasic modulation of [35S]TBPS binding by GABA represents channel activation (enhancement of

binding) and desensitization (inhibition of binding). Together, these findings demonstrated

that the modulation of [35S]TBPS binding to different receptor populations by GABA

represented the functional properties attributed by distinct subunits.

I have also demonstrated that [35S]TBPS can be used to detect receptors containing

different wild-type α, β and auxiliary γ, δ or ε subunits on the basis of their sensitivities to

selective ligands that preferentially modulate specific GABAA receptor subtypes.

Experiments with the mutant γ2(K289M) and α1(K278M) subunits demonstrated that

35 [ S]TBPS binding enables the detection of functional deficits in GABAA receptors

affected by mutations associated with epilepsy. Taken together, my findings provide a

greater understanding of the contribution of GABAA function in the differential binding of

[35S]TBPS. These findings pave the way for the use of [35S]TBPS binding to detect

functionally distinct GABAA receptors in different brain regions and in the brains of

individuals suffering from epilepsy. viii Table of Contents

Dedication. iv

Acknowledgments. v

Abstract of Dissertation. vii

List of Figures. xi

List of Tables. xiv

List of Nomenclature. xviii

Chapter 1: General Introduction. 1

I. Inhibitory neurotransmission. 1

II. General Structure and Function. 4

III. Subunit composition contributes to receptor function. 22

IV. The GABAA receptor is the target of a wide variety of

pharmacological compounds. 28

V. Role of GABAA receptors in epilepsy. 46

VI. Localizing GABAA receptors. 53

Overarching hypothesis. 60

Chapter 2: Experimental Methods. 62

A. Laboratory reagents. 62

B. Preparation of cDNAs. 63

C. Cell culture and transient transfection. 64

D. Electrophysiology recording equipment. 65

E. Whole Cell Patch Clamp Experiments. 66

F. Rapid agonist application. 67

G. Electrophysiology data acquisition and analysis. 68

H. Preparation of Rodent Brains. 69

I. [35S]TBPS Autoradiography Experiments. 70

J. [35S]TBPS Homogenate Binding Experiments. 72

K. Data Analysis and Statistical Procedures. 73

Chapter 3: Results: GABAA receptor activation and TBPS/PIC blockade. 74

Background and significance. 74

Inhibition of GABAA receptors by TBPS and PIC. 77

Blockade by TBPS and PIC occurs independently of GABA-induced

channel activation. 78

Modulation of [35S]TBPS binding by GABA. 83

Bicuculline affects GABA-independent [35S]TBPS binding to

α1β2γ2 receptors. 85

GABA-independent blockade by TBPS is reduced by BIC. 87

A mutation that reduces spontaneous GABAA receptor gating reduces

the GABA-independent block by TBPS and PIC. 89

The mutant α1(K278M) subunit affects [35S]TBPS binding. 91

The α1(K278M) substitution enhances the proportion of

GABA-dependent stimulation of [35S]TBPS binding. 91

The γ2(K289M) epilepsy mutation alters GABA modulation of

[35S]TBPS binding. 92

Conclusions. 96

Chapter 4: Results: GABAA receptor desensitization and TBPS/PIC

blockade. 98

Background and significance. 98

Steady-state desensitization of recombinant α1β2γ2 receptors. 102

The α1(K278M) subunit affects steady-state desensitization. 103

The γ2(K289M) subunit also affects steady-state desensitization. 107

The role of GABAA receptor desensitization in blockade by TBPS

and PIC. 110

Blockade by 100 μM PIC is not affected by desensitization. 111

Rapid application of GABA induces less desensitization in

α1(K278M)β2γ2 receptors. 113

Accessibility to the TBPS binding site in desensitized α1β2γ2

receptors. 117

The effects of desensitization on blockade by non-saturating

concentrations of TBPS in α1β2γ2 receptors. 119

Blockade of mutant α1(K278M)β2γ2 receptors by TBPS. 123

The potency of blockade by TBPS is reduced in α1(K278M)β2γ2

receptors. 125

Conclusions. 128

Chapter 5: Results: Identifying functionally distinct GABAA Receptors. 131

Background and significance. 131

Detecting the functionally unique ε subunit using [35S]TBPS

binding. 134

GABA modulation of [35S]TBPS binding to different brain

regions. 143

GABA modulation of [35S]TBPS binding established by

autoradiography. 147

Propofol modulation of [35S]TBPS binding to brain membranes. 151

Propofol modulation of [35S]TBPS binding to recombinant α1β2/3γ2

receptors. 154

Gabazine competes with endogenous GABA to modulate [35S]TBPS

binding. 156

Modulation of [35S]TBPS binding by bicuculline. 161

[35S]TBPS binding to receptors containing the δ subunit. 166

The α subunit contributes to GABA affinity. 173

Benzodiazepine ligands modulate [35S]TBPS binding in the

cerebellum. 176

The β subunit influence GABA-evoked modulation of [35S]TBPS

binding. 184

Conclusions. 188

Chapter 6: Discussion. 196

35 The role of GABAA receptor activation in [ S]TBPS binding. 197

35 The role of GABAA receptor desensitization in [ S]TBPS binding . 204

xii

The development of [35S]TBPS as a probe for functionally distinct

GABAA receptors. 212

The future of [35S]TBPS binding as a tool to detect functionally

distinct GABAA receptors. 220

References. 223

xiii

List of Figures

Figure 1. The TM2 domain of GABAA receptors. 9

Figure 2. Models of gating of ligand gated ion channels. 16

Figure 3. The location of the conserved residue, Lys289. 51

Figure 4. The K289M mutation reduced spontaneous gating of GABAA receptors. 52

Figure 5. The effects of TBPS and PIC on recombinant α1β2γ2 receptors. 78

Figure 6. Inhibition of GABA-evoked currents recorded from recombinant α1β2γ2 receptors by TPBS & PIC. 81

Figure 7. Inhibition of GABA-evoked currents recorded from recombinant α1β3γ2 receptors by TBPS & PIC. 82

Figure 8. GABA modulation of [35S]TBPS binding to recombinant α1β2γ2 receptors. 84

Figure 9. BIC reduces GABA-independent [35S]TBPS binding to recombinant α1β2γ2 receptors. 86

Figure 10. BIC reduces GABA-independent blockade by TBPS. 88

Figure 11. Inhibition of GABA-evoked currents recorded from α1(K278M)β2γ2 receptors by TBPS and PIC. 90

Figure 12. [35S]TBPS binding to mutant α1(K278M)β2γ2 receptors. 94

Figure 13. [35S]TBPS binding to mutant α1β2γ2(K289M) receptors. 95

Figure 14. Steady-state desensitization of α1β2γ2 receptors by GABA. 105

Figure 15. The mutant α1(K278M) subunit increases the fraction of available steady-state

GABA-evoked current. 106

xiv

Figure 16.The mutant γ2(K289M) subunit reduces steady-state desensitization. 109

Figure 17. Rapid agonist application of GABA to α1β2γ2 receptors. 112

Figure 18. Rapid agonist application of GABA to mutant α1(K278M)β2γ2 receptors. 115

Figure 19. Blockade of desensitized α1β2γ2 receptors by 10 μM TBPS. 118

Figure 20. Blockade of desensitized α1β2γ2 receptors by 1 μM TBPS. 121

Figure 21. Blockade of desensitized α1β2γ2 receptors by 100 nM TBPS. 122

Figure 22. Blockade of desensitized α1(K278M)β2γ2 receptors by 100 nM TBPS. 124

Figure 23. Blockade of GABA-evoked currents recorded from α1β2γ2 or

α1(K278M)β2γ2 receptors by TBPS. 126

Figure 24. Modulation of [35S]TBPS binding to recombinant α1β3ε receptors by

inhibitors of spontaneous gating. 138

Figure 25. Modulation of [35S]TBPS autoradiography by pregnenolone sulfate. 149

Figure 26. GABA modulation of [35S]TBPS binding to recombinant α1β2γ2 and α1β2ε

receptors transiently expressed in HEK293 cells. 142

Figure 27. GABA modulation of [35S]TBPS binding to homogenate brain regions. 145

Figure 28. Representative [35S]TBPS autoradiography images obtained in the presence of

exogenous GABA. 148

Figure 29. Quantification of [35S]TBPS autoradiography in the presence of GABA. 149

Figure 30. Propofol modulation of [35S]TBPS binding to homogenized rodent brain

regions. 152

Figure 31. Propofol modulation of [35S]TBPS binding to recombinant α1β2γ2 and

α1β3γ2 receptors in the absence of GABA. 155 xv

Figure 32. Representative [35S]TBPS autoradiography images in the presence of GBZ.158

Figure 33. Quantification of [35S]TBPS autoradiography in the presence of GBZ. 159

Figure 34. GBZ modulation of [35S]TBPS binding to homogenate membranes. 162

Figure 35. [35S]TBPS autoradiography in the presence of BIC. 164

Figure 36. Modulation of [35S]TBPS binding by BIC. 165

Figure 37. Representative [35S]TBPS autoradiography images in the presence of DS2. 169

Figure 38. Modulation of [35S]TBPS binding using a δ subunit-selective ligand, DS2. 170

Figure 39. Specific binding of [35S]TBPS is dependent on subunit composition. 172

Figure 40. [35S]TBPS binding to recombinant α1β2γ2, α1β3γ2 or α1β3γ2 receptors

transiently expressed in HEK293 cells. 175

Figure 41. Representative images of [35S]TBPS autoradiography in the presence of flunitrazepam. 181

Figure 42. Quantified [35S]TBPS autoradiography in the presence of flunitrazepam. 182

Figure 43. Representative [35S]TBPS autoradiography images in the presence of

Ro 15-4513. 183

Figure 44. Quantified of [35S]TBPS autoradiography in the presence of Ro 15-4513. 184

Figure 45. [35S]TBPS binding to receptors containing mutant β2(GKER) or β3(DNTK)

subunits. 188

xvi

List of Tables

Table 1. Summary of the desensitization induced using the rapid agonist application

protocol. 116

35 Table 2. IC50 values and Hill coefficients for the reduction of [ S]TBPS binding by

GABA. 146

35 Table 3. IC50 values and Hill coefficients for the reduction of [ S]TBPS binding by GABA

or propofol. 153

xvii

List of Nomenclature

1. [35S]TBPS t-butylbicyclophosphorothionate ([35S]TBPS)

2. 5HT 5-hydroxytryptamine

3. 5HT3 5-hydroxytryptamine type 3

4. ACHBP acetylcholine binding protein

5. BIC bicuculline

6. CGL cerebellar granule layer

7. CGN cerebellar granule neurons

8. CNS central nervous system

9. CRB cerebellum

10. DA dopaminergic

11. DMEM Dulbecco’s Modified Eagle Medium

12. DS2 4-chloro-N-[2-(2-thienyl)imidazo[1,2a]pyridine-3-yl benzamide

(δ-selective ligand type 2)

13. FC frontal cortex

14. GABA γ-amino butyric acid

15. GABAA γ-amino butyric acid type A

16. GABAAR γ-amino butyric acid type A receptor

17. GABAB γ-amino butyric acid type B, G-protein coupled receptor

18. GBZ gabazine

19. HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

20. HEK293 human embryonic kidney 293 cells xvii i

21. nH Hill slope

22. H hippocampus

23. HA amphipathic intracellular domain

24. % IGABA percent of GABA-evoked current

25. % IGABA max percent of GABA-evoked current normalized to maximal response

26. LC locus coeruleus

27. MA membrane associated intracellular domain

28. nACh nicotinic acetylcholine

29. PKC protein kinase C

30. PKCε epsilon isoform of protein kinase C

31. PIC picrotoxin

32. PS pregnenolone sulfate

33. SCAM substituted cysteine accessibility methods

34. S.E.M standard error of mean

35. TBPS t-butylbicyclophosphorothionate

36. Th thalamus

37. TM transmembrane

38. Tris-HCl Tris-buffer supplemented with hydrochloric acid

39. ZAC zinc activated channel

xix

Chapter 1: General Introduction

I. Inhibitory neurotransmission

In the central nervous system (CNS), neurotransmission is governed by either

slow metabotropic G-protein coupled receptors linked to second messenger systems, or

fast transmission that occurs via an influx of ions through ion channels. G-protein

coupled receptors often modulate nearby ion channels (Wickman and Clapham, 1995).

The neurotransmitter γ-aminobutyric acid (GABA) activates two types of receptors in the

CNS: GABAA, a ligand-gated ion channel, and GABAB, a G-protein coupled receptor.

The GABAB receptor is primarily located on presynaptic neurons (Bettler et al., 2003,

Stephens, 2009). This class of GABA receptors mediates slow neuronal inhibition

+ through several mechanisms. Coupling of the GABAB with inwardly rectifying K channels (e.g. GIRK) and high threshold voltage activated Ca2+ channels are two of the

main ways presynaptic neurons are modulated by GABA. Efflux of K+ from a

presynaptic neuron via GIRK channels results in hyperpolarization (Bettler et al., 2003).

Action potentials in a presynaptic neuron stimulate the activation of voltage gated Ca2+

channels (normally N, P or Q type in the CNS) resulting in neurotransmitter release into

the synapse. Inhibitory coupling of GABAB receptors to high-threshold voltage activated

Ca2+ channels reduces neurotransmitter release (Takahashi et al., 1998). While these

mechanisms are slower than ligand-gated neurotransmission, the GABAB receptor acts as

an important regulator of neuronal transmission and a therapeutic target in the CNS.

By contrast, activation of ligand-gated ion channels selectively allows rapid,

direct flux of either cations or anions across the cell membrane. Ligand-gated

neurotransmission in the CNS is either excitatory, which is mediated by cation channels,

or inhibitory, which is mediated by anion channels. The focus of my dissertation work

was on the GABAA receptor. The GABAA receptor is the major inhibitory ligand-gated

ion channel in the CNS (Luscher and Keller, 2004).

Two types of inhibitory transmission are governed by GABAA receptors: phasic

and tonic inhibition (Farrant and Nusser, 2005). Phasic inhibition is direct synaptic

transmission, where GABAergic neurons release GABA into the synapse, which then

activates GABAA receptors on postsynaptic neurons. Upon activation of GABAA receptors, anions (mainly Cl-) cross the cell membrane through the integral ion channel.

The influx of anions causes hyperpolarization and reduces the probability of postsynaptic

action potentials.

Patch clamp electrophysiology has demonstrated that phasic inhibitory currents

have a rapid rise time, during which several channels open simultaneously, and a

relatively slower decay time, during which channels close through desensitization and/or

deactivation. The rates at which GABAA receptors are activated or desensitized during

phasic inhibition depends on subunit composition (Jones and Westbrook, 1995; 1996;

Bianchi and MacDonald, 2002). Activation and desensitization will be described further

in section III of this chapter, where the attributes of GABAA receptor subunits are more

thoroughly described.

Unlike phasic inhibition, tonic inhibition results in a persistent Cl- current. Tonic

inhibition is most often attributed to the activation of high-affinity, extrasynaptic GABAA 2

receptors by low levels of GABA (Bai et al., 2001; Hamann et al., 2002). A level of

“ambient” GABA accumulates through spillover from synapses into the peri- and

extrasynaptic space, escaping reuptake by neuronal and/or glial GABA transporters (Hog

et al., 2006). However, tonic inhibition can also occur independently of activation by

agonists. Some GABAA receptor subtypes exhibit constitutive gating, enabling anions to pass through the channel in the absence of GABA (Neelands et al., 1999; Davies et al.,

2001; Birnir et al., 2002; Wagner et al., 2005; McCartney et al., 2007; Jones and

Henderson, 2007). The majority of GABAA receptors in the CNS mediate phasic

inhibition at the synapse (Luscher and Keller, 2004; Farrant and Nusser, 2005). Tonic

inhibition activated by ambient GABA is primarily associated with extrasynaptic

receptors containing the δ subunit, which will be discussed in section III of this chapter.

In contrast, several GABAA receptors consisting of different subunit combinations

exhibit low levels of constitutive gating (Yueng et al., 2003; Semyanov et al., 2004).

The role of phasic inhibition in synaptic transmission is clear. However, the

physiological function of tonic inhibition is less certain. Some hypothesize that tonic

inhibition provides a means of maintaining a persistent, low level of inhibitory control that prevents hyperexcitability in the CNS (Semyanov et al., 2004, Bieda and MacIver,

2004, Mody, 2005). GABA transporters are the main GABA reuptake mechanism in the

CNS and prevent overexposure of synaptic GABAA receptors to GABA. Several

subtypes of GABAA receptors in the CNS are therapeutic targets for treatment of diseases

such as epilepsy, anxiety and insomnia (see section V for more information). Not

surprisingly, some therapies target GABA transporters in hopes of enhancing the ability

of GABA receptors to regulate hyperexcitability (Hog et al., 2006).

Overexcitation of neurons can lead to necrosis. Excessive release of glutamate

into synapses has been shown to cause toxicity in the brain (Sattler and Tymianski, 2001;

Matute et al., 2002; Mody, 2005). As such, tonic current provides a means of maintaining

low levels of inhibitory control that may prevent overexcitation in the event of

dysregulation of synaptic ion channels or transporters. Overall, the localization of

GABAA receptors on presynaptic, postsynaptic or extrasynaptic regions of neurons provides several avenues for the regulation of excitatory neurotransmission throughout the CNS. I have outlined the role of functionally distinct GABAA receptor subtypes in

disease in greater detail in sections IV and V.

II. General Structure and Function

The GABAA receptor is a member of the Cys-loop receptor family of pentameric

ligand gated ion channels. This family also includes the anion permeable glycine

receptor, and the cation permeable nicotinic acetylcholine (nACh) receptor, 5-

hydroxytryptamine type 3 receptor (5HT3) and the zinc activated channel (ZAC).

Subunits forming the various Cys-loop receptors share structural resemblance and

sequence homology (Unwin, 1989; Ortells and Lunt, 1995; Davies et al., 2003, Connolly

& Wafford, 2004).

The GABAA receptor is formed by various combinations of α(1-6), β(1-3), γ(1-3),

ρ(1-3), δ, ε, θ or π subunits, which will be described in greater detail in section III

(Whiting et al., 1999). The subunit structure consists of a large extracellular N-terminal domain, four transmembrane domains (TM1-4) connected by either extracellular (TM2-

TM3) or intracellular (TM1-TM2, TM3-TM4) loops, and a small extracellular C-terminal

domain (Unwin, 2005). While the amino acid sequences for subunits forming GABAA receptors contain homologous strings of residues, distinct differences between the gene sequences create functionally distinct subunits that can alter the overall function of the receptor. Furthermore, each of the regions of these subunits contributes to overall function of Cys-loop receptors and several mutations in each of these domains have been shown to alter channel function (Sine and Engel, 2006). To date, much of the structural information for the GABAA receptor is based on cryoelectron microscopy published by

Nigel Unwin on the nACh receptor isolated from the electric ray, Torpedo marmorata

(2005). However, the published structure is incomplete. It includes the N-terminal agonist

binding domain, four transmembrane domains and the membrane associated (MA) or amphipathic (HA) intracellular region contained within the TM3-TM4 cytoplasmic loop.

Since then, more complete structures of bacterial pentameric ligand gated ion channels

related to Cys-loop receptors have been solved and will be described in greater detail

throughout this section (Hilf and Dutzler, 2008, Hilf et al., 2010, Corringer et al., 2010).

Extracellular domain

As mentioned earlier, much of what we know of the structure of Cys-loop receptors can be attributed to studies examining nACh receptors. Specifically, the acetylcholine binding protein (AChBP) provides a high resolution structure that can be used to model the N-terminal extracellular domain of Cys-loop receptors. This structure is a soluble protein released in response to the release of acetylcholine into the synapse from the glial cells of mollusks such as lymnaea stagnalis and aplysia Californica (Brejc et al., 2001; Yakel, 2010; Tsetlin et al., 2011). In addition to binding acetylcholine, the

AChBP also binds other agonists or antagonists of the nACh receptor. Sequence homology studies have demonstrated that the AChBP shares conserved residues

(including the Cys-loop) within the N-terminal domain of nACh receptor subunits as well as other Cys-loop receptors. As such, the AChBP has been the basis for several structural studies in the Cys-loop receptor field, particularly those examining ligand binding to the orthosteric agonist binding site.

The extracellular N-terminal domain of Cys-loop receptors is composed of a series of α-helices and β-sheets and contains a disulfide bond formed between two cysteines which form the “Cys-loop”, for which the Cys-loop receptor family is named

(Brejc et al., 2001; Unwin et al., 2002; Unwin, 2005). Six other loops in the extracellular domain are labeled A-F (Miller & Smart, 2010) and form the agonist binding pocket.

Several studies have focused on deciphering the importance of each extracellular loop in agonist binding, efficacy, and dissociation of ligands from the orthosteric agonist

(GABA) binding site. This binding site is formed at the interface of adjacent α and β

6 subunits, where loops A, B and C of the principle subunit (α) and loops D, E and F of the complimentary subunit (β) overlap to form the agonist binding pocket (Tsetlin, et al.,

2010; Miller & Smart, 2010). Several competitive agonists and antagonists bind at this interface and will be discussed in greater detail later in this chapter. The diverse pharmacology of the GABAA receptor also encompasses the benzodiazepine binding site, which is formed at the corresponding extracellular interface of adjacent α and γ subunits, as well as allosteric modulation which occurs outside of the extracellular domain.

A phenomenon referred to as “agonist capping” is thought to result from a conformational change that occurs in Loop C (Mukhtasimova et al., 2009; Silviotti, 2010;

Miller and Smart, 2010) when a ligand binds in the extracellular domain. The capping mechanism seems to occur differently for ligands exhibiting partial efficacy (Silviotti,

2010). In addition, agonist capping refers to a phenomenon during which one of the extracellular loops forms a “cap” over the ligand binding domain. These conformational transitions in the ECD have led to investigation of the relationship between extracellular agonist binding and gating, which occurs in the transmembrane domains described below. The differences in gating properties across different Cys-loop receptors as well as within the GABAA receptor are a major theme of this dissertation. Most of the results presented here will be in the context of the different states of channel gating.

Transmembrane domains

The TM domains of Cys-loop receptors are embedded in the cell membrane and are numbered TM1-4. They are also the site of action for many allosteric modulators,

including neurosteroids, ethanol, intravenous and inhalation anesthetics, as well as noncompetitive channel antagonists (Korpi and Sikkonen, 2006; Olsen and Sieghart,

2009). These binding sites will be discussed in greater detail later in this chapter. The

TM1 domain is directly connected to the extracellular N-terminal domain and is linked to the TM2 domain by a small intracellular loop which is thought to participate in

determining ion selectivity (Jensen et al., 2005). The TM2 domains of the five subunits

face the inner lumen of the channel, while TM1, TM3 and TM4 face away from the

channel (Unwin, 1989; Unwin, 2005). The TM2 has become of particular interest to

groups studying Cys-loop receptors due to its involvement in gating kinetics and ion

conductance. The TM2 is also an important site of interaction with several non-

competitive antagonists and allosteric modulators which will be described in detail in

section IV. Based on work using the homologous nACh receptor, the TM2 of Cys-loop receptors has been assigned a prime numbering system, where the inner most residue

(closest to the cytoplasm) is designated 0’, and the outermost residue (facing the extracellular region) is designated as 20’ (Fig 1 and Miller, 1989). Although much of the

work examining the structure of Cys-loop receptors is based on the nACh receptor

isolated from the ray, Torpedo marmorata, recent studies have demonstrated that the transmembrane domains of prokaryotic ligand gated ion channels are closely related to those of Cys-loop receptors.

Figure 1

20’

16 13

9’ 6’ PIC TBPS 2’

0’

Figure 1. Ribbon diagram of the GABAA receptor transmembrane domain with key TM2 residues labeled. The innermost residue of the TM2 is designated as the 0’ position and the outermost residue is the 20’ position. The proposed binding sites for TBPS and PIC are emphasized with black bars. The 9’ position represents the activation gate proposed by Nigel Unwin (2005). The 2’ and 9’ positions correspond to the activation and desensitization gates, respectively, as proposed by Wilson and Karlin (2001). The 13’ and 16’ residues were implicated in channel gating in the GLIC receptor (Corringer et al., 2010).

X-ray crystallography structures of a prokaryotic ligand gated ion channels isolated from the cyanobacterium Gloeobacter violaceous (GLIC) and Erwinia chrysanthemi (ELIC) provide further insight into the relationship between structure and function in Cys-loop receptors (Hilf and Dutzler, 2008; Bocquet et al., 2009; Hilf et al.,

2010, Corringer et al., 2010). While these receptors are not part of the Cys-loop family, they are members of a larger pentameric ligand gated ion channel (pLGIC) family and share many similarities to the known structure of Cys-loop receptors. The extracellular domain is similar to that of Cys-loop receptors, but lacks the Cys-loop. Also, the TM3-

TM4 intracellular loops are shorter in both GLIC and ELIC than in Cys-loop receptors

(Corringer et al., 2010). The structure of the GLIC was resolved in an open pore conformation, while the ELIC structure was resolved in a closed pore conformation. The comparison of these two structurally related channels revealed a hydrophobic constriction towards the extracellular region of the channel at residues 9’, 13’ and 16’ that may govern channel gating (Fig 1 andCorringer et al., 2010).

The TM2 region forms the pore of the ion channel, and is connected to the TM3 via an extracellular loop. In addition to controlling channel gating, the TM2 is also responsible for determining ion selectivity and rectification. While sequence homology is highly conserved across the different Cys-loop receptors, there are key determinants of anion or cation selectivity present in the TM2 domain. Mutations at the 1’ and 19’ are sufficient to convert the cation selective 5HT3 receptor to an anion channel (Thompson and Lummis, 2003; Barnes et al., 2009). Residues lining the channel pore also influence current rectification (Imoto et al., 1988; Moorhouse et al., 2002; Miller and Smart, 2010)

The TM2 and TM3 domains are connected by an extracellular loop that is

thought to transduce agonist binding in the ECD to gating in the TM2 domains (O’Shea

and Harrison, 2000, Wilkins et al., 2005). Studies examining the TM2-TM3 loop have

demonstrated that gating kinetics and transduction of agonist binding to the channel are

altered when mutations are present in this region (O’Shea and Harrison, 2000, Macdonald

et al., 2003, Hales et al., 2006, Wang et al., 2010). The TM3 region has been implicated

in gating kinetics and receptor assembly. The mutation of a single Cys residue (312) to an

Ala in the 5HT3A subunit causes impaired receptor function while maintaining surface

membrane expression (Wu et al., 2010). Mutations in the TM3 region of GABAA receptors have also demonstrated a role for this region in the function of allosteric modulators such as ethanol and anesthetics (Krasowski and Harrison, 2000).

Finally, the TM3 and TM4 regions are connected with a large intracellular loop involved in ion conductance and gating kinetics (Fisher, 2004). The length of the TM3-

TM4 cytoplasmic loop varies across subunits within the GABAA receptor family, as well

as between subunits across all members of the Cys-loop receptor family. A conserved

aspartate residue in the TM3-TM4 loop is required for GABAA receptor assembly, (Lo et

al., 2008). The loop also contributes to ion conductance, and is involved in associations

with intracellular scaffolding/signaling proteins (see below).

Ligand gated ion channels exist in closed, open or desensitized states. Ion conductance in Cys-loop receptors is primarily governed by the TM2 domain (Karlin,

2002; Peters et al., 2010). Comprehensive studies using X-ray crystallography and the substituted cysteine accessibility method (SCAM) have revealed that receptors exhibit

physically distinct conformations during the three different channel states (Auerbach and 11

Akk, 1998; Wilson & Karlin, 2001; Unwin, 2005; Gay and Yakel, 2007). Much of the

evidence for these gates comes from studies of the nACh receptor. Nigel Unwin proposed

that a hydrophobic barrier prevents Na+ from passing through nACh receptor channels in

the absence of acetylcholine (Unwin 2005). Based on his work using cryoelectron

microscopy, a closed channel gate was proposed at the 9’ level of the TM2 (Miyazawa et

al., 2003; Unwin, 2005).

Recent studies examining prokaryotic GLIC and ELIC channels have

demonstrated that lidocaine and divalent cations can cause open channel block (Hilf et

al., 2010). Anesthetics are known to have sites of action on various Cys-loop receptors,

including the nACh and GABAA receptors. Resolution studies of open channel block at

GLIC and ELIC channels demonstrates that lidocaine and divalent cations bind at

residues located below the activation gate in these receptors. These data support a gating

model that describes the activation gate in the upper TM2 region. However, the exact location of the activation gate in different ligand gated ion channels is under debate. The gating model proposed by Nigel Unwin is not currently accepted by all researchers in the field.

There may be more than one gate within the TM2 that governs the transition between different channel states. Voltage-gated Na+ channels are known to contain of

both activation and inactivation gates that control function of the ion channel. These gates

control activation, which is dependent on the membrane reaching threshold during

depolarization and inactivation after prolonged depolarization (Hodgkin and Huxley,

1952, Marban et al., 1998). These gates appear to behave independently to control

conductance through the channels. The TM2 of Cys-loop receptors may also contain 12

independent gates that control ion conductance during agonist induced activation and

desensitization. SCAM has been used to investigate the accessibility of individual

residues in different channel states. In these experiments, individual TM2 residues are

mutated to a cysteine, and the ability of methanethiosulfonate (MTS) reagents to covalently modify each residue is established by observing changes in receptor function.

The rate at which functional modification occurs provides a measure of relative

accessibility of the MTS reagent to the Cys residue in a given channel conformation.

Wilson and Karlin (2001) mutated individual residues of the nACh receptor TM2 to a

cysteine and measured the rate of accessibility of MTSEA, applied either extracellularly

or intracellularly, to each residue in resting, open or desensitized receptors. During these

studies, they demonstrated that accessibility to residues below the 9’ position depended

on whether the channel was open, closed, or desensitized. They proposed that the

activation gate was located at the 2’ residue, not the 9’ as Unwin had proposed (Fig 1).

This work was supported by other groups examining the location of the activation

gate in GABAA receptors (Xu and Akabas, 1996; Bali and Akabas, 2007) and the nACh

receptor (Akabas et al., 1994), where the accessibility to known binding sites of

noncompetitive antagonists provided clues to the location of the closed-channel gate. In

experiments published by Bali and Akabas (2007), the ability of non-competitive

antagonists, such as the channel blockers picrotoxin (PIC) and penicillin to reach their

binding site in the closed, activated, or desensitized states aided in the localization of

functional gates in GABAA receptors. Using this approach in combination with SCAM,

they demonstrated that PIC could block channels in the closed conformation, suggesting

that the activation gate lies below the PIC binding site, but this blockade was prevented 13

by penicillin, whose binding site lies higher in the TM2 than the PIC binding site. While

these studies can be quite complex, they provide functional evidence for the possible

locations of barriers that prevent ion conductance through different ligand-gated ion channels.

Furthermore, Wilson and Karlin demonstrated that desensitization, not channel closure, rendered residues between 9’ and 2’ inaccessible, and proposed that the 9’ residue was the location of a desensitization gate in the nACh receptor. This work was supported by recent studies examining the nACh receptor during gating and desensitization (Yamodo et al., 2010). Similar studies have been published on the closed channel and desensitization gates in GABAA receptors using both MTS reagents and non-

competitive channel blockers to access residues along the TM2 in different channel states

(Bali and Akabas, 2007). The overlap of these studies across different Cys-loop receptors confirms the structural homology across this family of ligand-gated ion channels.

The conflicting evidence regarding the location of the activation gate and the

existence of desensitization gates in Cys-loop receptors poses a requirement for further

investigation throughout the field. Work dedicated to locating the gates has employed

several techniques, including computational modeling, single residue mutagenesis,

SCAM, and using the activity of channel blocking drugs, to localize the gates with

reference to known binding sites in the channel. The diverse pharmacology of GABAA

receptors provides a means of describing these gates by functionally measuring their

effects using patch-clamp electrophysiology. Throughout my dissertation, I used two

non-competitive channel blockers, t-butylbicyclophosphorothionate (TBPS) and PIC, to examine the location of activation and desensitization gates in GABAA receptors, based 14 on their ability to access their proposed TM2 binding sites. I will revisit these ligands in greater detail in section IV and VI.

In addition to contrasting hypotheses for the location of different channel gates in

Cys-loop receptors, several mechanistic models of Cys-loop receptor gating currently exist. Agonist binding in the extracellular domain induces channel opening through mechanisms coupling the orthosteric binding site to the TM2 domain, which contains the channel gate (Kash et al., 2003; Bartos et al., 2009; Lee et al., 2009). The induced fit model (del Castillo & Katz, 1956) implies that the ion channel remains closed until an agonist binds and induces channel opening (Fig 2A). In contrast, the allosteric model

(Monod, Wyman and Changeux, 1965) accounts for the tendency of ligand-gated ion channels to open spontaneously in the absence of agonist. In this model, the constitutively open channel has a higher affinity for an agonist than does the closed channel, therefore agonist binding increases the probability of channel opening through allosteric modulation (Fig 2B). Recently, constitutive gating of GABAA receptors has been incorporated in some contemporary models based on the original concepts proposed by Monod, Wyman and Changeux (Scheller and Foreman, 2002) (Fig 2C).

One of the main goals of my dissertation research was to utilize information available describing the location of activation and desensitization gates in other Cys-loop receptors to help localize these gates in GABAA receptors. Much of what we know about

GABAA receptor gating has been inferred from experiments studying the nicotinic acetylcholine receptor. As such, I have used these studies as preliminary background for the dissertation work I have presented here.

Figure 2 Induced Fit Model A R AR AO* Castillo and Katz (1956)

B O* AO* Allosteric Model Monod, Wyman and Changeux (1965)

R AR

C AR AO* Modified Allosteric Model incorporating agonist- independent gating and a R desensitized state. O* Scheller and Foreman (2002) AD

Figure 2. Schematic diagrams of proposed gating models of ligand gated ion channels. Each of these models incorporates the possibility for an agonist-bound closed state (AR). A. The induced fit model indicates that the channel remains in the resting state (R) until an agonist binds (AR) and induces channel opening while bound (AO). B. The allosteric model incorporates constitutive channel gating in the absence of agonist (O). In this schematic, the agonist bound open state AO occurs with greater probability (bold lines) when the channel is already open (O) in the absence of agonist. C. A recently modified version of the Monod-Wyman-Changeux model that includes desensitization (AD) from the agonist bound open state (AO*), agonist bound shut state (AR), or desensitization in the absence of agonist (D). * Indicates a channel state that is permeable to ions.

Intracellular domain

Ion conductance

The intracellular domains of Cys-loop receptors govern several properties

including ion conductance, receptor trafficking and expression. Conserved residues in

this region are responsible for distinguishing between the anion permeable GABAA or glycine receptors, and the cationic nACh receptor, 5HT3 receptor and ZAC (Jensen, 2005,

Peters et al., 2006). Although these receptors are classified based on their ion selectivity,

there is substantial sequence homology across different subunits within each receptor

class, as well as across the Cys-loop family. The TM1-TM2 intracellular loop and

residues in the lower TM2 domain have been shown to govern ion selectivity of the

nACh receptor (Corringer et al., 2000), 5HT3 receptor (Peters et al., 2004; Livesey et al.,

2008) and the GABAA receptor (Wang et al., 1999). Studies substituting residues from

TM1-TM2 loops and TM2 in cationic receptors have demonstrated a cationic nACh

receptor can be converted to an anionic channel (Galzi et al., 1992), suggesting ion

permeability and selectivity is governed by residues in these locations. Furthermore,

studies examining the 5HT3 and nACh receptors (Hales et al., 2006; Deeb et al., 2007;

Peters et al., 2006) and the glycine receptor (Carland et al., 2009) have demonstrated that

in addition to the TM2 region, single channel conductance and ionic selectivity, in the

case of the 5HT3 receptor (Livesey et al., 2008), is influenced by conserved residues in

the intracellular MA stretch.

The GABAA receptor has recently been implicated in dopaminergic systems,

introducing a possible role for the regulation of psychomotor disorders such as

Parkinson’s disease or schizophrenia. This relationship is evident by direct coupling of 17

the GABAA receptor with nearby D5 dopamine receptor via the intracellular loop (Liu et

al., 2000, Lee et al., 2005). This communication is not surprising, since the GABAA receptor is the primary mediator of fast inhibitory neurotransmission in the CNS.

Modulation of GABAA receptor expression by intracellular proteins

In addition to ion permeability, the intracellular domains of Cys-loop receptors

are targets for post-translational modification and participate in trafficking to and from

the membrane. Over the years, several proteins that associate with the TM3-TM4

intracellular loop have been identified. One of the most studied proteins is the GABAA

receptor-associated-protein, or GABARAP (for review, see Chen & Olsen, 2007).

GABARAP binds to γ1, and both short and long isoforms of the γ2 subunit (γ2S and

γ2L). Pentameric receptors are formed in the endoplasmic reticulum (ER). When the γ2

subunit is present, GABARAP aids in a variety of post-translational modifications in the

ER. Some studies have demonstrated that GABARAP is structurally similar to ubiquitin,

and can catalyze signaling cascades similar to those initiated by ubiquitination

(Hochstrasser, 2000).

Studies using immunochemistry have demonstrated that GABARAP is

colocalized with receptors clustered at the synapse (Leil et al., 2004). Coexpression of

GABARAP with recombinant α1β2γ2 receptors has been shown to increase mean

channel open time as well as single channel conductance compared to recombinant αβγ

receptors in the absence of GABARAP (Luu et al., 2006). The increase in single channel

conductance by GABARAP demonstrates that the protein interaction at the intracellular

level modulates the function of individual GABAA receptors. Coexpression of

GABARAP has also been shown to increase the rate of activation and deactivation by of recombinant α1β2γ2 receptors by GABA (Chen et al., 2000).

GABARAP also associates with gephyrin, another membrane associated protein involved in trafficking of GABAA and glycine receptors to the plasma membrane

(Luscher & Keller, 2004). Inhibition of gephyrin expression has been shown to reduce

GABAA receptor clustering at the synapse (Kneussel et al., 2002). It is likely that the

interaction between GABARAP, gephyrin, and other anchoring and membrane associated

proteins facilitates the clustering or tethering of receptors already expressed in the plasma

membrane. Interestingly, γ2 knockout mice exhibit significantly reduced gephyrin

clusters, suggesting the specificity of the actions of gephyrin for the receptors containing

the γ2 subunit (Essrich et al., 1998).

GABARAP also has a postsynaptic density protein (PDZ)-binding domain in its

C-terminus, which is the region that associates with gephyrin (Kneussal M, 2002, Chen &

Olsen, 2007). PDZ-proteins interact with the intracellular domain of some ion channels,

and are thought to promote clustering of receptors within the cell membrane. However,

while GABARAP is evidently important for surface expression and clustering of GABAA receptors containing a γ subunit, studies using GABARAP knockout mice have demonstrated that GABARAP is not required for surface expression of all GABAA

receptors (O’Sullivan et al., 2005). Furthermore, GABARAP knockout mice are

phenotypically normal, suggesting that while GABARAP may accelerate or promote

trafficking and surface expression of αβγ receptors, it is not the sole membrane

associated protein involved in this process.

Modulation of GABAA receptor function by receptor recycling

Inhibitory synapses in the CNS are known to contain clusters of GABAA receptors, which, as described above, are formed with the assistance of intracellular regulatory proteins. The intracellular M3-M4 domain has been implicated in recycling or restoration of internalized receptors to the cell membrane. However, in addition to the formation of synapses, clustering can also be a mechanism of targeting receptors for degradation. Receptors are internalized and either immediately stored by endosomes, degraded by lysosomes, or recycled back into the plasma membrane. GABAA receptor

internalization can either result from constitutive recycling (Herring et al., 2003; Kittler

et al., 2004) or as a result of prolonged agonist exposure (Calkin and Barnes, 1994;

Barnes, 1996; Klein and Harris, 1996).

One proposed mechanism of internalization of GABAA receptors is via clathrin-

dependent processes (Barnes, 2000; Kittler et al., 2000, Bogdanov et al., 2006).

Specifically, internalization of clustered GABAARs has been shown to involve clathrin

adapter protein 2, (AP2) (Kittler et al., 2004; Herring et al., 2003, Chen and Olsen,

2007). Immunoflourescence studies have demonstrated that GABAA receptors co-localize

with clathrin coated pits and AP2 (Kittler et al., 2000). The internalization of inhibitory

receptors would enhance the excitation by temporarily reducing inhibitory control. As

such, the relationship between internalization and the regulation of inhibitory synapses

suggests that the intracellular domains of the GABAA receptor play a significant role in maintaining the balance between excitation and inhibition in the CNS. Blocking AP2 interactions increases the amplitude of miniature inhibitory postsynaptic currents

(mIPSCs) (Kittler et al., 2005; Smith et al., 2008), demonstrating a significant role for

AP2 in the regulation of GABAergic function.

Besides AP2, some evidence suggests that Huntingtin associated protein-1

(HAP1) (Li and Li, 1995) also prevents internalized GABAA receptors from being

degraded. Immunohistochemistry has demonstrated that HAP1 co-localizes with

internalized GABAA receptors (Kittler et al., 2004; Chen and Olsen, 2007). Furthermore,

over-expression of HAP1 has been shown to enhance the stability of receptors that have

been internalized, suggesting that perhaps, they are less likely to enter a degradation

pathway. In contrast, siRNA knockdown of HAP1 has been shown to reduce intracellular

movement of vesicles containing internalized GABAA receptors. Recently, it has been

shown that HAP1 interacts with other intracellular proteins to recruit vesicles containing

internalized receptors back to the cell surface (Twelvetrees et al., 2010).

Taken together, these data suggest that the majority of regulation of GABAA receptor expression, trafficking and degradation is dependent on key residues located on the M3-M4 intracellular loops. In addition to linking several transmembrane domains and adding structural integrity to the receptor, the intracellular loops play a vital role in regulating GABAA receptor mediated inhibitory control in the CNS.

III. Subunit composition contributes to receptor function

Most GABAA receptors are heteromeric combinations of α(1-6), β(1-3), γ(1-3), δ,

ε, θ or π subunits (Whiting et al., 1999). In the retina, there is a subset of homomeric

GABAA receptors formed entirely by ρ subunits (ρ1-3) referred to historically as GABAC receptors. The insensitivity of ρ receptors to the GABAA receptor antagonists bicuculline

(BIC) and gabazine (GBZ) initially led to their classification as GABAC receptors; however, the International Union of Pharmacology adopted ρ subunits into the GABAA family on the basis of amino acid sequence identity and a predominance of shared function (Collingridge et al., 2009). The regional distribution of GABAA subunits in the

rodent brain has been described by several groups (Pirker et al., 2000; Moragues et al.,

2002). Some evidence exists suggesting the incorporation of more than one subunit

derivative, α1α6βxγ2 (Baur et al., 2010), multiple β subunits (Fisher and Macdonald,

1997) or multiple auxiliary subunits, such as γ and δ (Saxena and Macdonald, 1994).

Virtually all GABAA receptors contain a combination of α and β subunits, with a third

subunit in the auxiliary position, which does not appear to contribute to the orthosteric

binding pocket. For most GABAA receptors, the subunit stoichiometry is thought to be

2α, 2β and 1γ (Chang et al., 1996, Whiting et al., 1999, Baumann et al., 2003; Baur et

al., 2006).

The agonist binding pocket is located at the extracellular interface between α and

β subunits. The incorporation of a γ subunit is required for benzodiazepine binding.

Benzodiazepines are a large class of GABAA receptor selective ligands used in the 22

treatment of a variety of disorders, including insomnia, epilepsy and anxiety. The

presence of the γ subunit reduces the potency of GABA compared to receptors containing

both α and β subunits, or containing either δ or ε subunits in place of the γ subunit

(Whiting et al., 1999). The heterogeneity and distinct pharmacology of the GABAA

receptor is defined by the expression of specific subunit combinations in different brain

regions. The remainder of this section will focus on the properties of auxiliary subunits and their contribution to GABAA receptor function.

The γ2 subunit

The majority of GABAA receptors in the brain consist of α, β and γ subunits. The

most common GABAA receptor is the α1β2γ2 combination, which accounts for the

majority of GABAA receptors in the rodent brain (Chang et al., 1996; Pirker et al., 2000).

These receptors are sensitive to modulation by benzodiazepines, as well as other allosteric modulators described in section IV. The α1β2γ2 subtype is widely distributed

throughout the brain. The α1β3γ2 is less common than the α1β2γ2 subtype, but has been

localized to the cortex and parts of the cerebellum. Less common GABAA receptor

subtypes are formed by differing α or β subunits (e.g. α1β1γ2 or α6β3γ2). Studies using

mutant mouse models examining the action of benzodiazepine ligands at various receptor

subtypes have demonstrated the co-localization of specific α subunits with the γ2

throughout the CNS (Sinkkonen et al., 2004; Rudolph and Mohler, 2004; Halonen et al.,

2009). Subunit-specific benzodiazepine pharmacology will be described later in section

IV of this chapter. Studies using recombinant receptors have demonstrated that several

other subtypes can be formed, but not all subtypes are expressed in the brain (Korpi and

Luddens, 1993; Luddens and Korpi, 1995).

The γ2 subunit exists in two isoforms due to alternative splicing. The long isoform, or γ2L subunit contains an additional eight residues in the intracellular domain compared to the shorter γ2S isoform (Barnes 2000). Several studies have demonstrated that clusters of GABAA receptors at the synapse contain the γ2L isoform (Essrich, 1998;

Kneussal, 2002; Meier and Grantyn, 2004; Chen and Olsen, 2006). Furthermore, unique

amino acids in the γ2L subunit insert confer a phosphorylation site that is sensitive to

protein kinase C (PKC) (Proctor et al., 1992, Chapell et al., 1998, Meier and Grantyn,

2004). Internalization of GABAA receptors mediated by PKC has also been linked to the

γ2L isoform (Chapell et al., 1998). Section IV describes allosteric modulators of GABAA receptor function. For the purposes of this dissertation, all wild-type γ2 subunits discussed will be the γ2L isoform.

The δ subunit

The αβδ subunit combination is primarily found in the cerebellum, hippocampus and thalamus (Nusser et al., 1996; Pirker et al., 2000, Moragues et al., 2002, Herd et al.,

2008). These brain regions are also populated with other subtypes, including the canonical α1β2γ2 receptor. Receptors containing the δ subunit are thought to be primarily located extrasynaptically (Nusser et al., 1998; Fritschy and Brunig, 2003). At the synapse, GABAA receptors are exposed to saturating (mM) concentrations of GABA

(Maconochie et al., 1994). It is known that GABA spills over from the synapse into the 24

extrasynaptic space in the cerebellum leading to a background ambient level of GABA

(Saxena & Macdonald, 1994; Hamann et al., 2002; Waller et al., 2003). Extrasynaptic

αβδ receptors in the cerebellum are thought to respond to ambient GABA by virtue of their higher affinity (Brickley et al., 1999). The δ subunit attributes high affinity, but lower efficacy in response to GABA than receptors containing the γ subunit (Bianchi and

Macdonald, 2003, Mortenson et al., 2010). Of the brain regions thought to express

αβδ receptors, the extrasynaptic receptors in the cerebellum are the most extensively characterized. The agonists gaboxadol (THIP) and muscimol have a higher efficacy at

αβδ receptors compared to GABA. Activation by THIP often referred to as supramaximal, because the efficacy of these ligands is higher than the maximal efficacy of GABA, the native ligand for the receptor (Mortenson et al., 2010). Furthermore, these ligands are often used in radioligand binding assays to isolate high affinity αβδ receptors in different brain regions.

In addition to exhibiting increased affinity for some agonists, receptors containing the δ subunit exhibit different desensitization kinetics compared to αβγ receptors. For several years, there have been conflicting reports on the role of the δ subunit in desensitization. Some have argued that the δ subunit causes prevents desensitization.

Others groups have argued that the gating kinetics of desensitization exerted by αβδ receptors are different than those containing the γ2 subunit, but that desensitization still occurs. As mentioned earlier, synaptic GABAA receptors are thought to exhibit a rapid

rise time, slow decay time, and rapid desensitization in the presence of GABA (Farrant

and Nusser, 2005). In contrast, several groups have proposed that extrasynaptic GABA 25

receptors containing the δ subunit desensitize slowly and do not exhibit complete loss of current despite prolonged exposure to GABA (Haas and Macdonald, 1999; Bianchi and

Macdonald, 2002; Yueng et al., 2003). The fact that some current is evident from desensitized αβδ receptors, while the majority of current is lost from αβ or

αβγ receptors, accounts for earlier studies concluding that the δ subunit caused insensitivity to desensitization. Slow desensitization may be a physiological characteristic of extrasynaptic GABAA receptors that enables them to mediate persistent tonic current in response to low levels of GABA. One recent study has demonstrated that low levels of

ambient GABA are sufficient to cause desensitization that renders extrasynaptic-like

recombinant αβδ receptors insensitive to activation by synaptic GABA (Bright et al.,

2011). There is an increasing consensus that extrasynaptic receptors containing the δ

subunit do desensitize, but in a different manner than receptors containing the γ2 subunit.

Immunohistochemical experiments have demonstrated that α4, α6, β2 or β3

subunits colocalize with the δ subunit, implicating the presence of these subunits in

extrasynaptic αβδ receptors (Pirker et al., 2000; Moragues et al., 2002). There is also

some evidence to support the combined expression of δ and γ subunits in recombinant

receptors (Saxena and MacDonald, 1994). Radioligand binding to recombinant receptors

containing αβγδ subunits demonstrate that the δ subunit reduces specific binding of

radiolabeled compounds specific for GABAA receptors, including the agonist

[3H]muscimol, and non-competitive antagonist [35S]t-butylbicyclophosphorothionate

(TBPS) (Hevers et al., 2000). This concept will be revisited in section VI and chapter 5

of the results section. 26

The ε subunit

The αβε combination is a rare GABAA subtype that exhibits unique properties

compared to αβγ receptors. The ε subunit confers insensitivity to benzodiazepines and anesthetics (Davies et al., 1997a; Kasparov et al., 2001). Several studies have shown that the ε subunit is highly localized in very specific brain regions, including the locus coeruleus (Sinkkonen et al., 2000), hypothalamus (Moragues et al., 2003, Sergeeva et al.,

2005) and thalamus (Moragues et al., 2002). Receptors containing the ε subunit exhibit high levels of GABA-independent gating that can be inhibited by non-competitive ligands like picrotoxin, TBPS and furosemide, but not BIC (Neelands et al., 1999;

Whiting et al., 1999; Davies et al., 2001; Thompson et al., 2002; McCartney et al., 2007).

The ε subunit is less understood than other GABAA subunits, but to date, more is known

about the human form than the two rodent isoforms (short and long) (Davies et al., 2002).

Recombinant expression studies

Recombinant expression of GABAA receptors can be advantageous when

studying receptor subtypes that may not be easily isolated in vivo. Many studies using

recombinant receptors have contributed to understanding subunit stoichiometry,

pharmacology, and gating kinetics of individual receptor subtypes. Furthermore,

recombinant studies provide a means of examining receptor subtypes that may not be

present in the brain, as well as synthetic mutations that help elucidate the importance of

specific residues or binding sites in a given subunit.

One drawback to using recombinant expression of GABAA receptors is the

possibility of forming stoichiometries that may not be present in vivo. One primary

example of this phenomenon is the ability of the β3 subunit to form homomeric receptors

that can be transported to the membrane (Davies et al., 1997b). In a recombinant setting,

heteromeric receptors can also be formed, consisting of β3 and γ2 subunits that elicit Cl-

currents in response to activation by GABA or pentobarbital (Taylor et al., 1999). It is

unclear whether these unique homomeric β3 receptors are present in vivo, however, there

is evidence to support the existence of α1β3 receptors in the CNS (Mortenson and Smart,

2006). These receptors may contribute to tonic current in the hippocampus, are highly

sensitive to inhibition by Zn2+, and exhibit low conductance compared to αβγ and αβδ

receptors. In contrast to the β3 subunit, β2 or β1 subunits contain a retention signal that

prevents the subunits from exiting the endoplasmic reticulum unless they are combined

with an α subunit (Connolly et al., 1996; Taylor et al., 1999). As mentioned earlier, the majority of GABAA receptors in the CNS consist of 2α, 2β and an auxiliary subunit. I

will revisit the role of different auxiliary (δ, ε, γ), α or β subunits in Chapter 5 of the

results section.

IV. The GABAA receptor is the target of a wide variety of pharmacological

compounds.

The native GABAA neurotransmitter is GABA, which is metabolized from

glutamic acid by the enzyme glutamic acid decarboxylase (GAD) (Erlander et al., 1991).

GABAergic neurons can be identified by labeling with antibodies to GAD. Non-native

agonists at the orthosteric site not only displace GABA, but in some cases also activate

the receptor with a comparable efficacy. Two of the most widely studied agonists are

muscimol and THIP. Agonist binding at the orthosteric site is thought to cause a

conformational wave that passes through the transmembrane domain leading to opening

of the activation gate (Kash et al., 2003; Unwin, 2005). Partial agonists, such as

piperidine-4-sulphonic acid (P4S), 5-(4-piperidyl)-3-isoxazolol (4-PIOL) and Thio-4-

PIOL, activate GABAA receptors, but do so with less efficacy and potency than the full

agonists, GABA and muscimol (Mortensen et al., 2004).

Competitive antagonists of the GABAA receptor include the widely used ligands

gabazine and BIC. While gabazine is thought to be a neutral competitive antagonist,

some evidence exists suggesting it is a weak inverse agonist, which promotes channel

closure (Chang and Weiss, 1999). Recent evidence indicates that BIC is an inverse

agonist that is able to inhibit tonic currents caused by constitutive, GABA-independent

gating (McCartney et al., 2007). The diverse pharmacology of GABAA receptors makes it

a target for many therapeutic ligands as described below.

Benzodiazepines

The central site of action of benzodiazepine ligands is found on GABAA receptors that contain a γ subunit (Mohler et al., 2002). The GABAA receptor is the only Cys-loop receptor that binds benzodiazepine ligands with high affinity. Benzodiazepine ligands modulate GABAergic neurotransmission by binding at the interface between α and γ

subunits. Furthermore, the benzodiazepine family of pharmacological compounds contains a diverse range of full or partial agonists, as well as neutral antagonists and inverse agonists (Korpi and Sinkkonen, 2006; Da Settimo et al., 2007).

Benzodiazepines agonists exhibit positive efficacy that enhances GABAergic neurotransmission by enhancing GABA binding. Behaviorally, benzodiazepine agonists tend to be hypnotic, sedative and anxiolytic. When both submaximal concentrations of

GABA and these positive modulators bind, the channel opens more frequently, allowing more anionic current to cross the membrane than in the presence of GABA alone.

However, some benzodiazepines can prevent channel opening when bound to the receptor. These inverse agonist benzodiazepines exhibit negative efficacy that prevents the receptor from responding fully to the effects of GABA (Da Settimo et al., 2007).

Inverse agonist benzodiazepines are not used therapeutically due to their ability to cause convulsions by inhibiting GABAergic function. In my experiments, I employed the benzodiazepines ligands flunitrazepam and Ro 15-4513 to isolate GABAA receptors with different α subunits within the rodent CNS (see Chapter 5).

It is important to note that GABAA receptors are not directly activated by benzodiazepine ligands in the absence of GABA. Positive efficacy of a benzodiazepine ligand simply refers to its ability to enhance the activation caused by GABA or another orthosteric agonist (Downing et al., 2005). Benzodiazepine agonists increase the rate of association of GABA by increasing affinity, thereby increasing the probability that a

channel will open in response to GABA (Rogers et al., 1994).

The selectivity of benzodiazepine ligand binding and efficacy is governed by the contribution of different α subunits (α1-6) or γ subunits (γ1-3) (Wingrove et al., 1997;

Renard et al., 1999; Kloda & Czajkowski, 2007; Da Settimo et al., 2007). The development of several mutant mouse models has led to the definition of several benzodiazepine ligands based on their selectivity for different α subunits (Rudolph and

Mohler, 2004). These studies will be outlined throughout this section. Benzodiazepine ligands with selective affinity bind with higher affinity to one subtype over another, but exhibit the same efficacy. In contrast, benzodiazepine ligands that exhibit subtypeselective efficacy bind to different subtypes, potentially with similar affinities, but have differing efficacies (Da-Settimo et al., 2007). For years, receptors containing α4 or α6 subunits were considered to be resistant to benzodiazepine ligands. A single amino acid substitution, (A101H), confers sensitivity to benzodiazepine ligands to GABAA receptors containing mutant α4(A101H) or α6(A101H) subunits (Wieland et al., 1992). However, recent studies have demonstrated that some benzodiazepines act at receptors containing the α6 subunit, but exhibit different efficacies at this subtype compared to others (Hauser et al., 1997).

The physiological effects of benzodiazepine ligands are governed by the presence of specific α subunits. Behavioral studies have been published over the past several years that demonstrate the role of distinct GABAA receptor subtypes in mediating the behavioral effects of these drugs. The use of autoradiography with radiolabeled benzodiazepines has contributed much of what we know about the localization of different α subunits throughout the brain. Benzodiazepine agonists that bind selectively

to receptors containing the α1 subunit result in sedation and hypnosis and are often used

in the treatment of sleep disorders (Ebert et al., 2006).

In contrast, benzodiazepine ligands that bind selectively to receptors containing the α2 or α3 subunits are nonsedative and anxiolytic (Dawson, et al., 2005). Mutations in the GABAA receptor have also been identified in different types of anxiety (Belzung,

2001; Korpi and Sinkkonen, 2006). Several mutant animal models have been developed

that demonstrate the role of GABAA receptors in anxiety (Rudolph and Mohler, 2004).

Behavioral experiments studying the role of deleted subunits or reduced

expression of subunits, particularly various α subunits, have demonstrated impaired

cognitive behaviors in mice. Point mutations on specific subunits, such as the α2 subunit,

have confirmed the selectivity for some anxiolytic benzodiazepines for specific α

subunits (Morris et al., 2006; Dixon et al., 2008). Since many benzodiazepines produce

multiple effects, such as sedation and anxiolysis, it is important to understand the genetic

implications of GABAA receptors in anxiety in order to develop selective therapeutic

ligands that only produce the desired effects without unwanted consequences.

The GABAA receptor has also been implicated as a target for analgesia (Zeilhofer

et al., 2009). This led to the investigation of which GABAA receptor subtypes could be

involved in the perception of pain. Pathological pain can involve a loss of synaptic

inhibitory transmission in the spinal cord (Takazawa and MacDermott, 2010), which is

largely mediated by the glycine receptor (Webb and Lynch, 2007; Lynch, 2009).

However, in such models of pathological pain, it seems plausible that enhancing GABAA

receptor mediated inhibitory transmission would compensate for the deficit in glycinergic

function (Knabl et al., 2008, Munro et al., 2009). As such, several groups have examined the ability of modulators of GABAA receptor function, like benzodiazepines, to help

localize the GABAA receptor subtypes involved in pain pathways.

Classical benzodiazepines such as diazepam have been implicated in

antinociception, or loss of pain sensitivity (Knabl, et al., 2009). Many of the classical

benzodiazepines are not subtype selective, which means they are likely to modulate

several neuronal pathways at once. Interestingly, some of these benzodiazepines are now

being used as potential treatments for chronic pain (Munro et al., 2009, Zeilhofer et al.,

2009). Recent findings suggest that benzodiazepines which cause muscle relaxation also

reduce hyperalgesia through receptors containing the α2, α3 and α5, but not receptors

containing the α1 subunit, which primarily induces sedation and hypnosis (Zeilhofer et

al., 2009).

Furthermore, benzodiazepine inverse agonists that bind selectively to receptors containing the α5 subunit enhance cognition (Dawson et al., 2006; Atack, 2010). In

contrast, inverse agonists binding to receptors containing the α3 subunit are anxiogenic,

the opposite physiological response produced by benzodiazepine agonists with positive

efficacy (Atack et al., 2005). Several studies examining knock-in mouse models have

demonstrated the role of different α subunits in the behavioral effects of benzodiazepines

(Rudolph et al., 1999; Rudolph and Mohler, 2004). To further complicate this

pharmacology, the efficacy of some benzodiazepines is often subunit dependent, giving

rise to ligands that may act as agonists at some subtypes and antagonists or inverse

agonists at others. Furthermore, mutations in the γ2 subunit have demonstrated that the

33 selectivity and affinity of different benzodiazepines is also governed by the contribution of the γ subunit to the benzodiazepine binding pocket (Ogris et al., 2004).

Knockin mouse models have provided more information about the regional distribution of different α subunits that mediate the diverse behavioral effects caused by subunit-selective benzodiazepines. Knockin mutant mouse models containing

α1(H101R), α2(H101R), α3(H126R), or α5(H105R) mutations have been used to describe benzodiazepine pharmacology and the distribution of various α subunits throughout the brain (reviewed by Rudolph and Mohler, 2004). However, benzodiazepines, like most prescription medications, have the potential for abuse (Licata and Rowlett, 2008). The development of newer, subtype-selective benzodiazepines has focused on reducing this abuse potential. Flumazenil is a neutral benzodiazepine antagonist and is often given therapeutically to counteract benzodiazepine overdose

(Weinbroum et al., 1996; Seger, 2004). Recently, a study examining the role of the

α1(H101R) mutant subunit demonstrated that receptors containing the α1 subunit are located in the ventral tegmental area (VTA), alongside dopaminergic (DA) neurons where they participate in the addicitive properties of some benzodiazepines. The authors hypothesized that benzodiazepines cause disinhibition by GABAA receptors in this area, thereby enhancing DA neurotransmission that is known to be involved in addiction (Tan et al., 2010). These studies have proven useful in determining which GABAA subtypes are responsible for mediating certain behavioral effects of benzodiazepine ligands, such as sedation, anxiolysis, hypnosis and more recently, addiction and overdose.

Neurosteroids

In addition to endogenous neurotransmitters, the GABAA receptor is modulated

by neurosteroids. Endogenous neurosteroids include allopregnanolone, 3α-hydroxy-5α- pregnan-20-one or 3α,5α-tetrahydroprogesterone (3α,5α-THP) and tetrahydrodeoxycorticosterone (3α,21-dihydroxy-5α-pregnan-20-one, 3α,5α-THDOC.

Endogenous neurosteroids are produced in the brain and secreted by glial cells.

Neurosteroids can either enhance or inhibit the efficacy of GABA at various GABAA

receptor subtypes (Lambert et al., 2003; Belelli & Lambert, 2005). Cleavage of

cholesterol in glial cells produces pregnenolone, a precursor for inhibitory neurosteroids

such as pregenolone sulphate (sulfate) and dihydroepiandrosterone sulphate (DHEAS)

(Hosie et al., 2007). Furthermore, positive modulators can also directly activate the channel in the absence of GABA at binding sites within the TM1 and TM2 domains

(Hosie et al., 2006a). To date, many general anesthetics target the GABAA receptor via

the steroid binding site. For years, the exact location of the neurosteroid binding site was

under debate. Some studies have shown that steroids bind at an allosteric binding site

within the transmembrane domains (Sousa and Ticku, 1997; Park-Chung et al., 1999;

Hosie et al., 2007). Residues within the TM1 domain are thought to be responsible for

neurosteroid-evoked positive modulation of GABAA receptors (Hosie et al., 2009)

Sulfated and unsulfated steroids modulate GABAergic currents via different binding sites

(Park-Chung et al. 1999). Inhibitory neurosteroids such as pregnenolone sulfate inhibit

channel function in the presence and absence of GABA through a site within the TM2

35 domain. In my experiments, I used pregnenolone sulfate to inhibit spontaneous, GABA- independent gating in receptors containing the ε subunit.

The subunit composition of a GABAA receptor can influence the modulation by neurosteroids. In particular, modulation of synaptic receptors, typically αβγ, differs compared to extrasynaptic αβδ receptors (Feng et al., 2004). There was an early report that the presence of the δ subunit inhibits modulation by 3α,21-dihydroxy-5α-pregnan-

20-1 (THDOC) (Zhu et al., 1996). However, it is now widely accepted that neurosteroids cause supramaximal potentiation at αβδ receptors, while being unable to surpass maximal GABA efficacy at synaptic αβγ receptors (Lambert et al., 1993; Korpi and

Sinkkonen, 2006).

Intravenous and inhalation anesthetics

In addition to modulation by neurosteroids, the activity of the GABAA receptor can also be modulated by general anesthetics such as barbiturates and propofol (Belelli et al., 1999). Several general anesthetics modulate GABA-evoked currents, and directly activate GABAA receptors in the absence of GABA (Hales and Lambert, 1991; Sanna et al., 1996, Davies et al., 1997b, Ueno et al., 1997). Several general anesthetics inhibit nACh receptor function, and recent studies examining prokaryotic pentameric ligand- gated ion channels demonstrate that anesthetics such as propofol also block GLIC channels by binding to specific residues (Hilf et al., 2010; Nury et al., 2011). As mentioned above GABAA receptors and the nACh receptor have evolved from these pentameric prokaryotic ion channels.

While some general anesthetics modulate most GABAA receptors regardless of

subunit composition, the β2/3 subunits confer selectivity for some modulators such as the

intravenous anaesthetic etomidate (Wafford et al.; 2004) and the anticonvulsant

loreclezole (Sanna et al., 1996; Lambert et al., 2003). In contrast, the barbiturate anesthetic pentobarbital and the intravenous anesthetic propofol show less selectivity for different β subunits (Smith et al., 1994). SCAM experiments performed on mutated TM2 and TM3 domains of recombinant GABAA receptors have identified a putative propofol binding site near the extracellular portion of the TM3 of the β2 subunit (Bali and Akabas,

2004). The use of mutant mouse models has demonstrated that the binding sites of

different anesthetics within the same class (e.g. intravenous) may differ.

In vitro mutagenesis studies have demonstrated that the one residue, N289, in the

TM2 domain is responsible for selectivity of etomidate for β2 or β3, but not β1 subunits

(Belelli et al., 1997). However, since the populations of GABAA receptors in the CNS are

heterogeneous, it has been difficult to determine the selectivity of these anesthetics for

certain subunits in in vivo studies (for review, see Franks, 2008). As such, mutant mouse

models have been developed in order to examine the role of different β subunits in

anesthetic action at GABAA receptors. Behavioral experiments examining mutant mouse models expressing mutated β2 or β3 subunits, rendered insensitive to etomidate reveal that receptors containing β2 subunits mediate the sedative effects of etomidate, while

those containing the β3 subunits participate in the immobilizing effects of this drug (Jurd et al., 2002; Reynolds et al., 2003; Zeller et al., 2007). Furthermore, mutant β2 N265S subunits exhibit reduced sensitivity to etomidate, but not propofol (Reynolds et al.,

2003), while β3N265M mutations cause reduced sensitivity to both etomidate and

propofol (Jurd et al., 2002). This residue has also been implicated in the effects of

inhalation anesthetics (see below), demonstrating the role of the upper TM2 domain of

GABAA receptors in anesthesia.

The effects of inhalation anesthetics may also be mediated through GABAA receptors. The sensitivity to these anesthetics seems to involve residues on α subunits.

Several studies have identified a residue in the upper TM2 domain (α1 S270) that is required for sensitivity to volatile anesthetics (Mihic et al., 1997; Borghese et al., 2006).

Mutations at position S277 of α1 subunits eliminated sensitivity to isoflurane, but not halothane. Interestingly, corresponding mutations on the α2 subunit also reduce sensitivity to isoflurane (Werner et al., 2011). Mutations on the β2 subunit (N265) have also demonstrated a potential binding site for volatile anesthetics. Mutant receptors expressing α1β2(N265C)γ2 receptors in Xenopus oocytes have demonstrated that this residue is important for the binding of volatile anesthetic isoflurane, but not the neurosteroid alphaxalone or thebarbiturate pentobarbital (McCracken et al., 2010).

Taken together, these studies have implicated the role of not only different

GABAA receptor subtypes, but also specific residues present on different subunits, in

mediating anesthetic action in the CNS.

Alcohol

Like other Cys-loop receptors, the GABAA receptor contains a binding site for

ethanol (Dopico & Lovinger, 2009). Mutagenesis studies by Harrison and colleagues

(Mihic et al., 1997) have described key residues in the TM2 (α1 S270, β1 S265 or β3

S265) and TM3 (α2 A291) regions required for ethanol activity at human GABAA receptors. These residues correspond to the upper portion of the TM2 and TM3 that are nearest to the extracellular domain. Ethanol causes potentiation, or enhancement of

GABA-activated responses, resulting in an increase in anion flow through the channel

(Kumar et al., 2009). There are several lines of evidence to support the modulation of

GABAA receptors by alcohols, mainly ethanol. Behavioral studies using animal models

have demonstrated drugs that modify GABAA receptors also modulate behavioral and locomotor responses to ethanol (Atack, 2010).

Recently, mutant knockin mouse models have been used to examine the behavioral and electrophysiological effects of ethanol at different GABAA receptor subtypes. Knockin mice containing α2 subunits with S270H and L277A mutations

(homozygous HA/HA mice) have demonstrated that the effects of ethanol at GABAA

receptors may be mediated by multiple residues. Blednov et al. demonstrated that

recombinant α2(HA)β2γ2 receptors expressed in Xenopus oocytes lost their sensitivity to ethanol potentiation. Based on these studies, it was expected that mutant knockin mice

would exhibit reduced sensitivity to the behavioral effects of ethanol. Interestingly, while

benzodiazepine actions at α2 receptors are anxiolytic, mutant HA/HA mice showed no

change in the anxiolytic effects of ethanol. These same mutations on the α1 subunit have

been previously shown to increase the anxiolytic effects of ethanol (Werner et al., 2006).

Other behavioral effects such as loss of righting reflex, ethanol consumption and bitter-

taste aversion, increased. These behavioral effects are also associated with high

consumption of alcohol in humans, suggesting that the GABAA α2 subunit may mediate

some of the behavioral effect of ethanol in humans (Blednov et al., 2011).

Some GABAA antagonists and benzodiazepine inverse agonists block the

electrophysiological effects of ethanol (Atack, 2010). In particular, Ro 15-4513 is

considered a “behavioral antagonist” because it blocks the effects of ethanol in animal

studies (Olsen et al., 2007). Studies using electrophysiology suggested that the γ2L

isoform is required for ethanol sensitivity, whereas the γ2S isoform lacks sensitivity to

modulation by ethanol (Wafford et al., 1991). However, this is no longer accepted, as studies have since demonstrated that the responses to alcohol of the γ2L and γ2S isoforms

are indistinguishable (Homanics et al., 1999). The γ2L isoform contains an intracellular

target for phosphorylation by PKC. PKC-inhibitors prevent potentiation of GABA-

activated responses by ethanol (Wafford and Whiting, 1992; Weiner et al., 1997; Song

and Messing, 2005). Upon phosphorylation of Ser-313 by PKC, it is likely that a

conformational change occurs in the transmembrane domain, causing the site of ethanol

activity to become available. Specifically, the epsilon isoform of PKC (PKCε) is thought

to regulate ethanol potentiation of GABAA receptors through phosphorylation of this

intracellular target on γ2L subunits (Qi et al., 2007).

Some believe that ethanol preferentially modulates extrasynaptic α6βxδ in the

cerebellum (Carta et al., 2004; Wallner et al., 2003; Botta et al., 2007). Locomotor

control originates from various nuclei in the cerebellar granule neurons (CGNs) and it is

possible that the impaired motor function caused by excessive alcohol consumption is

mediated through GABAA receptors expressed on these neurons. However, conflicting

evidence supporting or refuting whether extrasynaptic GABAA receptors are hypersensitive to ethanol question the relevance of extrasynaptic GABAA receptors in

this regard (Crabbe et al., 2007).

The exact mechanism behind potentiation of GABAA receptor activity by ethanol

is not fully understood. Some groups believe alcohol enhances desensitization of Cys-

loop receptors, including the GABAA receptor (Dopico and Lovinger, 2009). Several

studies have demonstrated that ethanol increases the decay rate of GABA-evoked

currents (Homanics et al., 1999). This increase in decay rate is usually associated with

enhanced desensitization. It is likely, however, that the enhanced channel opening caused

by positive allosteric modulators like ethanol could promote desensitization by increasing

the probability of the channel transitioning into the desensitized state in the presence of

agonist.

Modulation of GABAA receptors by cations

In addition to being a target for many types of pharmacological ligands, the

GABAA receptor is also modulated by mono- and bivalent cations (Wilkins et al., 2002;

Huang et al., 2004; Wilkins et al., 2005).

+ Proton (H ) modulation of GABAA receptors has been widely studied over the

past few decades. The role of H+ in neurotransmission has been derived from the fact that

ion channels and the ligands that modulate them behave differently at non-physiological

pH levels (Uusi-Oukari et al., 2004). As with many allosteric modulators, the activity of

protons at GABAA receptors is also subunit dependent. Feng and Macdonald

demonstrated that incorporation of the δ subunit with α1 and β3 subunits increases the

sensitivity to lower [H+] compared to α1β3γ2L receptors (2004). Other studies using

recombinant GABAA receptors have demonstrated that pH levels < 7.3 reduce, while pH

levels > 7.3 increase the potency of GABA and BIC. These same pH changes had no

effect on the direct activation of α1β2γ2 receptors by the barbiturate pentobarbital, which

binds at a site different from the extracellular binding site for GABA and BIC (Amin and

Weiss, 1993). Interestingly, in related experiments, spontaneously active mutant

α1(L264)β2γ2L receptors were not affected by changes in pH (Huang et al., 2004).

Mutagenesis studies have identified a single residue, H267 on GABAA receptor β

subunits (Wilkins et al., 2002) and other residues in the TM2-TM3 loop (Wilkins et al.,

2005) that are responsible for H+ modulation of GABAergic function.

2+ Zinc (Zn ) has been shown to inhibit all GABAA receptor subtypes tested with subunit dependent potency. Interestingly, the residue responsible for modulation of

+ 2+ GABAA receptors by H also mediated modulation of GABAergic function by Zn

(Wilkins et al., 2002). Receptors comprised of α and β subunits alone are most sensitive to inhibition by Zn2+, while the γ subunit attributes marked insensitivity, and the δ subunit

causes moderate, but detectable, sensitivity (Hosie et al., 2003; Lees and Edwards, 1998).

2+ Recordings from cultured neurons and brain slices have demonstrated that Zn inhibits

GABAA receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs, Barberis

et al., 2000) and action potential dependent spontaneous inhibitory postsynaptic currents

(sIPSCs, Strecker et al., 1999). Ultra rapid agonist application, which allows examination

of the discrete kinetics of different receptor subtypes, has demonstrated that Zn2+ may

inhibit GABA-evoked mIPSCs by slowing the rate of receptor activation (Barberis et al.,

2000). However, Zn2+ can also inhibit spontaneously open homomeric β3 receptors

(Wooltorton et al., 1997) and spontaneous activity mediated by the ε subunit (Neelands et al., 1999). Compared to α1β3γ2 receptors, α1β3ε receptors exhibit increased sensitivity

to Zn2+, but reduced sensitivity compared to α1β3 receptors (Davies et al., 2001).

The non-competitive antagonists TBPS and picrotoxin

Several non-competitive antagonists bind in the TM2 region of the GABAA receptor. These antagonists are often open channel blockers. Some antagonists are charged, such as the positively charged penicillin, resulting in a voltage-dependent block.

Penicillin causes a more profound block at negative membrane potentials when the molecule is attracted into the channel lumen (Tywman et al., 1992; Lindquist et al., 2004;

Feng et al., 2009).

Many uncharged non-competitive GABAA channel blockers also interact with the

channel pore to prevent ion conduction. Two channel blockers that are the focus of this

dissertation are the plant toxin picrotoxin (PIC), and t-butylbicyclophosphorothionate

(TBPS) (Casida and Lawerence, 1985; Im et al., 1994). Both PIC and TBPS have been

used to induce pharmacological models of epilepsy in rodents, since they can block

GABA-evoked currents and therefore cause convulsions (Olsen, 1982; Jonker et al.,

2007). In fact, almost all inhibitors of GABAA receptors have been used to model

different types of epilepsy in animals (Jonker et al., 2007).

TBPS is a synthetic bicyclophosphorus ester derivative that belongs to a family of

ligands primarily used as insecticides (Casida and Lawrence, 1985; Law and Lightstone,

2008). TBPS and other similar anion channel blockers are often referred to as cage- convulsants. These ligands cause convulsions in animals, but require a symmetrical

“cage” or binding pocket for full efficacy. In the case of convulsant ligands such as

TBPS, this cage is simply the ion channel (Casida and Lawrence, 1985). These ligands

are known for their selectivity for GABAA receptors and their ability to successfully

cause convulsions by blocking ion channels. A large proportion of early work was done

using Drosophila RDL receptors, the insect homologues of mammalian GABAA

receptors, as well as mutant Drosophila strains that became resistant (ffrench-Constant et

al., 1993a, b) to the effects of the cage-convulsants, picrotoxin, or penicillin (Casida and

Lawrence, 1985; Hosie et al., 2006b; Law and Lightstone, 2008). TBPS more potently

inhibits GABAA receptors and binds with higher affinity than picrotoxin (Havoundjian et al., 1986). Unlike other convulsant ligands, TBPS appears to bind specifically to GABAA

receptors (Squires et al., 1983). This may be due to very specific residues forming the

binding site for TBPS that may only be found in the GABAA receptor. Alternatively this

could be due to a unique pattern of GABAA receptor gating that stabilizes TBPS binding.

35 35 The [ S]-labeled ligand ([ S]TBPS) has been used to localize GABAA receptors in the

CNS.

Picrotoxin is an equimolar mixture of two non-competitive antagonists, picrotin

and the cage convulsant picrotoxinin (Jarboe et al., 1968) and has been a widely used

antagonist of inhibitory currents for decades. When examined independently,

picrotoxinin is more potent and exhibits a rapid blocking rate compared to picrotin (Yang 44

et al., 2007). Both components, as well as other related compounds such as α-

dihydroypicrotoxinin, have also been used as [3H]-labeled radioligands for the

localization of inhibitory receptors (Ticku et al., 1978).

The majority of work examining the “convulsant” binding site in GABAA

receptors revolves around studies using picrotoxin. Mutagenesis experiments have

demonstrated that in GABAA receptors, the 6’ residue is essential for full inhibition of

αβγ receptors (Fig 1; Buhr et al., 2001; Sedelnikova et al., 2006; Kalueff, 2007; Erkkila et al., 2008). Picrotoxin has often been referred to as an open channel blocker, because

GABA enhances the rate of accessibility to its binding site. These studies also demonstrated that PIC blocks channels in the absence of GABA application (Newland and Cull Candy 1992; Dillon et al., 1995). Furthermore, prolonged exposure to GABA appears to overcome blockade by TBPS and PIC, perhaps indicating that desensitization

compromises accessibility to their binding sites (Bloomquist et al., 1991).

Both TBPS and PIC are thought to bind to the same site within the open channel

(Sigel et al., 1989; Zhang et al., 1994). The ability of PIC to fully displace [35S]TBPS in

radioligand binding studies (Sinkkonen et al., 2001) provides strong evidence that the

two molecules bind at the same or at least overlapping sites. PIC-displaceable [35S]TBPS binding in autoradiography studies is used to localize GABAA-receptor specific activity

within various brain regions. The bulk of the work presented in this dissertation examines

the relationship between the binding sites for TBPS and PIC and channel activation,

closure and desensitization.

V. Role of GABAA receptors in epilepsy

Several treatments for idiopathic generalized epilepsies enhance GABAergic

function, which implicates a role for GABAA receptors in this type of neurological

disease. Normal neurotransmission in the CNS is maintained by a balance of inhibitory

and excitatory circuits. Since epilepsies result from dysregulation of neurotransmission in

the CNS, it is not surprising that dysfunctional GABAA receptors are associated with

some types of epilepsy. A reduction in GABAergic inhibitory function results in an

increase in excitatory function, which is the common pathophysiological event leading to an epileptic seizure (McCormick and Contreras, 2001; Noebels, 2003). Furthermore, several point mutations and deletions affecting different parts of α, β, γ or δ subunits have been identified that each result in specific types of epilepsy, suggesting that the involvement of the GABAA receptor in dysregulation of neurotransmission is complex

(Macdonald et al., 2003; Macdonald et al., 2004; Macdonald and Kang, 2009). Some of

the most common forms of epilepsy resulting from mutant GABAA receptor subunits are myoclonic epilepsy of infancy (SMEI or Dravet Syndrome), childhood absence epilepsy

(CAE), juvenile myoclonic epilepsy (JME), febrile seizures (FS) and generalized epilepsy with febrile seizures (GEFS). .

Interestingly, the location of these mutations (N-terminus, transmembrane domains, intracellular loops or C-terminus) does not correspond with the type of epilepsy that occurs. For example, several mutations have been identified that result in CAE, including the N-terminal β3(G32R), γ2(R43Q), γ2 (R139G) point mutations, the N-

terminal γ2 (IVS6->G) intron splice donor, and TM3 α1(S326fs328X) frameshift

(Macdonald and Kang, 2009). Clearly, these different mutations occur on three major subunits, in various regions, suggesting that no single subunit or region on a subunit is solely responsible for the involvement of GABAA receptors in epilepsy. Similar mutant profiles are also evident for SMEI-Dravet Syndrome and GEFS. However, one common result from the above mentioned mutations, as well as γ2(K289M), γ2(Q351X), and

α1(A322D) is a loss of some GABAA receptor function, through altered gating and/or

reduced levels of surface expression.

Most of the GABAA receptor mutations involved in epilepsy result in reduced

function, which is evident by higher EC50 values, reduced maximal GABA currents,

and/or reduced channel opening (Macdonald et al., 2003). Mutations on the γ2 subunit

are particularly detrimental to GABAA receptor function, due to its involvement in

trafficking of fully formed receptors to the cell surface (Keller et al., 2004; Kang et al.,

2006; Hales et al., 2005). In the case of febrile seizures, one study examined the effects of elevated temperatures on various receptors containing mutant γ2 subunits, in order to understand why fever triggers seizures in GEFS (Kang et al., 2006). The authors found that elevated temperatures (37°C to 40°C) significantly reduced surface expression of mutant GABAA receptors containing either γ2(K289M) subunits, but not wild-type γ2 subunits. The increase in temperature caused increases in intracellular retention of mutant receptors, either by accelerating internalization or reducing trafficking of receptors to the cell surface.

Interestingly, elevated temperatures had no significant effect on receptors containing the α1(A322D) subunit, which does not occur in patients with febrile seizures, suggesting there are multiple avenues of GABAergic dysregulation among the various types of epilepsy. Although the α1(A322D) mutant is not temperature sensitive, it does cause significant deficits in GABAA receptor function. Gallagher et al. (2004)

demonstrated that the effects of this mutation are most prominent in homozygous individuals (where no wild-type α1 subunit is present. Heterozygous phenotypes result in

less significant deficits in peak current amplitude, EC50, and total and surface expression

of mutant receptors. Although this mutation does not affect the γ2 subunit, it seems that

the stoichiometry or ratio of mutant to wild-type subunits in receptors plays a role in the

severity of the resulting phenotype. It is possible that the α1(A322D) subunit is unable to

form an α−γ interface with wild-type γ2 subunits, which prevents γ2 incorporation into

the pentamer. This inability would result in increased expression of αβ receptors (Fisher

and MacDonald, 1997). In contrast, when the α1(A322D) subunit is located at the α-β

interface, in the presence of a wild-type α subunit, the γ2 subunit is able to successfully

traffic the receptor to the cell membrane.

While the majority of epilepsy mutations identified seem to affect synaptic

GABAA receptors, some mutations have also been identified on the δ subunit, which is in

receptors that are localized extrasynaptically. Recently, two mutations on the δ subunit,

E177A and R220H have been described with respect to generalized epilepsy and juvenile

myoclonic epilepsy. Both mutations caused a reduction in current amplitude. However,

interestingly, neither mutation causes a shift in potency, which is seen for other mutations 48

(e.g. γ2(K278M)), nor a shift in macroscopic gating kinetics, such as desensitization and deactivation rates. However, the reduction in current amplitude measured from mutant

α4β2δ(E177A) or α4β2δ(R220H) receptors was correlated with reduced surface expression and reduced single channel conductance (Dibbens et al., 2004; Feng et al.,

2006). In a mouse model of temporal lobe epilepsy, expression of the δ subunit was markedly reduced, yet some tonic current remained in dentate granule cells of the hippocampus, suggesting possible compensation for altered expression by other subtypes

(perhaps αβ receptors) (Zhang et al., 2007). Taken together, these findings demonstrate that tonic inhibition mediated by extrasynaptic αβδ receptors could play a role in minimizing hyperexcitability in the CNS.

Using epilepsy mutants as functional tools

For my dissertation work, I employed the γ2(K289M) epilepsy and synthetic

α1(K278M) mutant subunits, in order to understand the role of disrupted gating in the binding and blockade of GABAA receptors by TBPS and PIC. Figure 3 depicts two structural models of the GABAA receptor based on the pentameric ligand-gated ion channel isolated from GLIC and the nACh receptor model obtained through studies examining the electric ray, Torpedo marmorata. The proposed location of the K289M mutation is in the TM2-TM3 loop and is thought to affect gating (O’Shea and Harrison,

2000, Macdonald et al., 2003, Hales et al., 2006, Wang et al., 2010). Previously, we have demonstrated that the TM2-TM3 lysine to methionine mutation (equivalent to K289M in the γ2 subunit) results in reduced surface expression when present on the β2 subunit or

49 reduced channel mean open time when present on α1 or γ2 subunits (Hales et al., 2006).

Unpublished work from our laboratory demonstrates that the γ2(K289M) and the equivalent mutation of the α1 subunit also caused a reduction in spontaneous gating (Fig.

4). Spontaneous gating was measured by the amount of leak current recorded in the absence of GABA that could be blocked by the noncompetitive channel blocker, picrotoxin. I hypothesized that spontaneous, GABA-independent gating would enable accessibility to the TM2 TBPS/PIC binding site in the absence of agonist activation. As part of this dissertation, I used the γ2(K289M) epilepsy mutant and the synthetic

α1(K278M) mutant as tools to disrupt normal channel function. The goal of these experiments was to gain insight into the location and accessibility of the TBPS/PIC binding sites the resting, open, and desensitized states of the channel.

Figure 3

Figure 3. The location of the Lys (K) to Met (M) substitution. A. Sequence alignment of GABAA α1, β2 and γ2 subunit TM2 domains. The highlighted residues represent the conserved lysine located in the TM2-TM3 linker. Prime numbers (0’, 9’ and 19’) frame the residues that line the channel pore. B. Partial ribbon homology diagrams of T. marmorata and G. violaceus. Only the extracellular ligand binding domain and part of the membrane spanning domains are shown here. The hypothesized location of the K289M mutation in each model is designated by “Lys 289”.

Figure 4 α1β2γ2 α1(K278M)β2γ2 A B PIC (100 μM) PIC (100 μM)

GABA (1 mM) GABA (1 mM)

1nA 2 nA

10 s 10 s

C 0.30 A 0.25

0.20

0.15 ontaneous/GAB I

p 0.10

% I % I S 0.05

0 ∗ 2 2 2 2 2 γ γ γ γ ∗ 2 β 2 2 2 1 β β β ∗ β α 1 1 1 1 α α α α

Figure 4. Whole cell patch clamp electrophysiology demonstrates that the K289M epilepsy mutation reduces spontaneous, GABA-independent gating when transiently expressed in HEK293 cells. A. Spontaneous leak currents recorded from recombinant α1β2γ2 receptors are inhibited by picrotoxin (100 μM) in the absence of GABA (top panel). GABA (1 mM) evoked a typical whole-cell response in the same experiment, demonstrating the presence of functional GABAA receptors. B. Mutant α1(K278M)β2γ2 receptors have diminished spontaneous gating, as evident by the lack of current blockade in the presence of picrotoxin (100 μM). Mutant receptors also exhibit a typical whole cell response to GABA (1 μM). C. Graphical summary of spontaneous leak current recorded from different GABAA receptor subtypes. Spontaneous current is expressed as a percentage of whole cell current evoked by GABA. Mutant subunits containing the K289M substitution caused a reduction in spontaneous current compared to wild type receptors. * Indicates mutated subunits. These data were obtained by Tarek Deeb.

VI. Localizing GABAA receptors

Mammalian GABAA receptors can be formed from several combinations of α(1-

6), β(1-3), γ(1-3), ρ(1-3), δ, ε, θ, π, and ρ subunits (Whiting et al., 1999). Due to the different functional properties attributed by differing subunit combinations it is important to localize GABAA receptor subtypes to specific brain regions. The location of specific subunits, especially those with unique properties that stand apart from the canonical

α1β2γ2 receptor, would provide evidence for the involvement of certain subunits in different neurological processes, such as sleep, drug addiction, anxiety, memory and so on. Over the past several decades, many techniques have been used in an effort to identify the specific subunit distribution throughout the various brain regions of rodent and mouse brains. This section will focus on the advantages and disadvantages of each of the main techniques used, and how this dissertation provides valuable support of the current knowledge of GABAA receptor distribution in the rodent brain.

In situ hybridization

In situ hybridization involves the detection of mRNA encoding different proteins within a tissue sample using oligonucleotide probes designed to specifically target distinct mRNA sequences. Film autoradiographs of thin-sliced tissue samples reveal the distribution of targeted mRNA. As such, in situ hybridization has been used for several decades to detect possible GABAA receptor subtypes in different brain regions. However, designing probes that discriminate between the many GABAA receptor subunits has

proven difficult. If probes are not specific enough to detect a single subunit, they can

cross-hybridize to closely related gene family members (Wisden et al., 1992).

Nonetheless, several groups have used this technique to localize different populations of

GABAA receptors in the CNS (Mohler et al., 1990; Laurie et al., 1992; Wisden et al.,

1992, Stephenson, 1995). In situ hybridization also proved useful in detecting altered

mRNA distribution in mouse models of disease (Luntz-Leybman et al., 1995; Liu and

Glowa, 1999; Petri et al., 2005).

However, one caveat that arises from using in situ hybridization is that the

presence of mRNA detected is not proportional to the amount of protein, in this case,

GABAA receptors expressed on the cell surface. Furthermore, several cell types contain

transcripts for all isoforms of each subunit, yet we know from functional assays that not

all subunits are expressed in all brain regions. Another disadvantage of this technique is

that mRNA is often located in the cell body, while protein subunits are largely expressed

within the postsynaptic and perisynaptic membrane, which may not be within close

proximity of the neuronal cell body. However, despite these disadvantages, in situ

hybridization experiments have helped to demonstrate that the α6 subunit is primarily

localized in the cerebellar granule layer and the ρ subunits are segregated in the retina

(Stephenson, 1995). Recent studies over the last decade have demonstrated that the ε

subunit is localized in discrete brain regions, including the locus coeruleus (Sinkkonen et

al., 2000; Belujon et al., 2009) and is expressed in cholinergic, serotoninergic,

norepinephrinergic and some dopaminergic neurons (Moragues et al., 2002).

Antibodies and Immunohistochemistry

The regional distribution of GABAA receptors is diverse throughout the brain.

Early immunohistochemical studies enabled researchers in the field to make inferences

about the subunit combinations that were localized to specific brain regions (Pirker et al.,

2000; Moragues et al., 2002). In contrast to in situ hybridization, which targets mRNA,

the use of immunohistochemical antibodies allowed researches to target expressed

protein. However, while distinguishing between different subtypes (i.e, α versus β) was

relatively straightforward, detecting different subsets of each subunit (i.e. β2 versus β3)

proved to be difficult due to considerable sequence homology (Miralles et al., 1999).

Immunohistochemistry has become a useful tool in detecting altered expression

levels of different subunits associated with various diseases of the nervous system.

GABAA receptor expression is most often examined in the case of different forms of

epilepsy, and several groups have demonstrated that the distribution of various subunits is

altered in different varieties of the disorder. In a model of temporal lobe epilepsy, the α4

subunit was found to be localized synaptically, as shown by colocalization with GAD and

gephyrin in mutant mice compared to the extrasynaptic localization in wild type mice

(Sun et al., 2007). Immunohistochemistry has also been used on patient brain samples in

therapeutic studies examining markers for neurodegenerative diseases. Decreased α3

subunit expression has been observed in the cortex of patients diagnosed with focal

epilepsy, but not temporal lobe epilepsy (Loup et al., 2006).

In a recent study of hippocampal degeneration in Alzheimer’s disease patients,

upregulation of γ subunit expression was observed. Interestingly, the expression of these

upregulated subunits did not colocalize with markers for neurofibrillary tangles and

plaques, which indicate progression of Alzheimer’s disease (Iwakiri et al., 2009). This

study proposed that upregulation of receptors containing γ subunits may be a

neuroprotective response in neurodegenerative diseases. Taken together, the use of

immunohistochemistry in experiments examining neurological disorders has provided insight into the role of GABAA receptors. The contribution and altered expression of

distinct GABAA subunits further supports the idea that functionally distinct GABAA

receptor subtypes are diversely distributed in different brain regions in order to provide

greater control over specific neurological functions, such as locomotor control, memory,

sedation and so on.

Immunohistochemistry has also been used to describe the distribution of various

GABAA receptors in the spinal cord, where glycine receptors primarily govern neuronal

inhibition (Waldvogel et al., 2009). However, while immunohistochemstry has proven

useful in the localization of different GABAA subunits throughout the CNS, this technique also provides a means of detecting surface expression of different receptors with other proteins. The colocalization of the γ2 subunit with intracellular proteins gephyrin and GABARAP has given rise to the idea that αβγ receptors are located at the synapse (Chen et al., 2000, Leil et al., 2004), while αβδ receptors are located extrasynaptically.

Immunohistochemistry has provided much of the basis for localization of GABAA receptors in discrete brain regions today. Using in situ hybridization and antibody staining has helped to bridge the gap between mRNA expression within a neuron and

protein expression on the cell body. Furthermore, the majority of this work has also been confirmed by radioligand binding assays, which can take advantage of the subunit-

specific pharmacological characteristics of different GABAA receptors.

Radioligand binding

Radiolabeled ligand binding is a tool that can be used to examine different

receptor subtypes based on their ability to bind a radiolabeled ligand. A radiolabeled

ligand binds to receptors in the same way that a nonradiolabeled (cold) ligand, providing

a means of measuring receptor expression based on the level of radioactivity detected.

The displacement of radioligands by non-radioactive or “cold” ligands gives insight into

the affinity and selectivity of a given ligand, which may relate to receptor subtype. In

autoradiographic radioligand binding studies, cryostat sectioned tissue slices are

examined to determine the regional distribution of different receptor subtypes. The level

of radioligand binding is quantified based on the relative intensity of labeling of exposed film. This type of assay is advantageous when using subunit specific ligands to localize distinct receptor subtypes in different brain regions.

In studies using homogenized tissue or recombinant receptors, isolated brain regions or receptor subtypes can be examined independently of endogenous factors that would otherwise be found in a whole brain slice. The use of recombinant receptors minimizes the contribution of endogenous ligands that would likely affect ligand binding.

Several radiolabeled benzodiazepine ligands are available including

[3H]flunitrazepam (Shin et al., 1985), [3H]diazepam (Tallman and Gallager, 1979),

[3H]Ro 15-4513, [3H]zolpidem, and [3H]Ro 15-1788 (McKernan et al., 1998).The use of 57

radiolabeled benzodiazepines to examine a subunit distribution in knockin mouse models has also provided the majority of evidence for the localization of distinct benzodiazepine sensitive subtypes throughout the brain (Rudolph and Mohler, 2004). The availability of these radioligands has aided in understanding the distribution of different α subunits and

benzodiazepine insensitive receptors throughout the brain. Radiolabeled agonists

[3H]muscimol (Nobrega et al., 1995), [3H]GABA (Lloyd et al., 1983) and the competitive antagonist [3H]SR-95531 (gabazine), bind at the orthosteric agonist binding

site. However, radioligands that bind at the extracellular domain do not provide

information about the functional state of the receptor. In this respect, isotopes of ligands that depend on channel activity for their binding, such as allosteric modulators binding in

the TM2, may provide insight into the state of the receptor when modulated by ligands that bind elsewhere.

35 Radiolabeled [ S]TBPS has been used extensively to localize GABAA receptors

in the brain (Squires et al., 1983). The advantage of using [35S]TBPS as a tool is that it

provides the ability to simultaneously screen multiple brain regions within a slice, for

GABAergic activity (Edgar and Schwartz, 2000; Sinkkonen et al., 2001). Drugs that

target specific receptor subtypes are often used in conjunction with [35S]TBPS to identify

regions high in a given receptor subtype (Korpi and Luddens, 1993; Sinkkonen et al.,

2004). Isolated brain regions are often used in homogenate binding studies to examine

35 differences in [ S]TBPS modulation in a group of GABAA receptor subtypes that are

specific to certain brain regions, i.e, comparing the hippocampus to the cerebellum (Gallo et al., 1985; Srinivasan et al., 1999; Atack et al., 2007). In addition, [35S]TBPS binding to

recombinant GABAA receptors provides a means to examine receptor activity in the

absence of additional confounding factors in the brain, such as endogenous GABA, other neurotransmitter systems or allosteric modulators (Luddens and Korpi, 1995; Davies et al., 1997; Srinivasan et al., 1999).

The diversity of radioligands available provides several avenues for selectively isolating certain receptor subtypes, such as those with unique sensitivities to benzodiazepines. Furthermore, brain slice autoradiography allows several brain regions to be examined within one experiment, which provides an advantage over experiments involving single cells or isolated brain regions. In contrast, electrophysiological assays target one cell at a time, or isolated cell circuits. Using this approach, rare subtypes often evade detection. Binding assays provide a method for finding less prevalent receptor combinations, which may be important for targeting new therapeutic agents. However, at this stage, modulation of TBPS binding by GABA is complex, and the relationship between binding and receptor function is not well understood. Thus far, functional studies using unlabelled TBPS suggest there may be a correlation between channel gating and the binding of TBPS in the channel, but these are scarce (Bloomquist et al., 1991; Dillon et al., 1995; Behrends, 2000; Jursky et al., 2000). Agonists, allosteric modulators and

35 antagonists at the GABAA receptor alter [ S]TBPS binding through poorly understood mechanisms (Im et al., 1994; Luddens and Korpi, 1995).

In my dissertation work, I set out to examine the role of channel activation, desensitization and subunit composition in the diverse distribution of [35S]TBPS binding

throughout the brain. In Chapter 3, I will describe the role of the GABAA receptor

activation and spontaneous, GABA-independent gating in 1) binding of both TBPS and 59

PIC and 2) blockade of receptor function by both agents, using a combination of patch-

clamp electrophysiology and [35S]TBPS binding. Chapter 3 will focus entirely on the role

of desensitization. I used whole-cell patch clamp electrophysiology to examine

desensitization of different recombinant GABAA receptor subtypes. Chapter 5 focuses on

the role of subunit composition in the differential binding of [35S]TBPS throughout the

rodent CNS. I used a combination of both autoradiography and homogenate binding, as

well as recombinant receptors expressed in HEK293 cells and the adult rodent brain to

examine the contribution of α, β, δ, ε and γ2 subunits to channel function and binding of

[35S]TBPS.

The principal questions I sought to answer during my dissertation work were:

1. Does GABAA receptor activation affect the accessibility of TBPS and PIC to the

channel?

2. Does GABAA receptor desensitization affect the accessibility of TBPS and PIC to

the channel?

35 3. Can [ S]TBPS binding distinguish between functionally distinct GABAA receptors in the rodent CNS?

The ultimate overarching goal of this dissertation was to use [35S]TBPS as a tool

to detect functionally distinct GABAA receptors in the rodent CNS. Based on the current

knowledge describing the binding sites for TBPS and PIC in the TM2 domain of GABAA

receptors, I hypothesized that the activity of these antagonists at different receptor

subtypes could provide insight into the location of activation and desensitization gates

with reference to the convulsant binding site. Furthermore, I hypothesized that the

contribution of functionally distinct subunits to different brain regions is responsible for

35 the differential binding of [ S]TBPS to GABAA receptor subtypes. My dissertation has

focused on developing [35S]TBPS as a functional probe to detect unique subtypes based on knowledge of the role of activation, desensitization and the influence of different

subunits expressed throughout the CNS.

Chapter 2. Experimental Methods

A. Laboratory Reagents

[35S]-t-butylbicyclophosphorothionate ([35S]TBPS) was obtained from Perkin

Elmer (Waltham, PA) and stored at -20°C. Dilutions of [35S]TBPS were made

immediately prior to each experiment in either ice-cold incubation buffer. For homogenate binding assays, incubation buffer contained 20 mM Tris-HCl and 1 M NaCl, pH 7.5 with HCl. For autoradiography assays, incubation buffer contained 50 mM Tris-

HCl and 120 mM NaCl, pH 7.5 with HCl. Buffers were maintained at 4°C for no more than 2 weeks.

All non-radiolabeled ligands (GABA, propofol, picrotoxin, TBPS, bicuculline, gabazine, THIP, pregnenolone sulfate, furosemide), antibiotics (tetracycline, ampicillin and kanamycin) and general laboratory chemicals were obtained from Sigma-Aldrich (St.

Louis, MO). Stock solutions of drugs stored either in -20°C freezers or in 4°C

refrigerators for up to 3 months. Working concentrations of these ligands were made

fresh daily from these stocks as needed for each experiment in either Saline A for

electrophysiology (140 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 10 mM

HEPES, and 10 mM glucose at a pH of 7.4 with NaOH) or ice-cold incubation buffers for

[35S]TBPS homogenate binding or autoradiography.

B. Preparation of cDNAs

Wild-type and mutant human, murine or rodent GABAA receptor and GFP cDNAs were cloned using ampicillin, tetracycline or kanamycin resistant E. coli vectors obtained from InVitrogen (Carlsbad, CA). Briefly, DH5α, Top10/P3 or MC1063 bacteria were transformed as outlined in manuals provided by Invitrogen and grown overnight on sterile 150 mm Corning dishes (Corning, NY) filled halfway with LB-Agar (10 g/l

Tryptone, 5 g/l yeast extract, 10 g/l sodium chloride, and 15 g/l bacto-agar (all from

Sigma Aldrich (St. Louis, MO), pH 7.4 ). LB-Agar plates were supplemented with 100

μg/ml ampicillin (mutant GABAA subunits), 100 μg/ml ampicillin & 10 μg/ml tetracycline (wild-type GABAA subunits) or 50 μg/ml kanamycin (GFP). For each cDNA preparation, a single bacterial colony was first amplified under antibiotic selection in an orbital shaker at 300 RPM, 37°C for 4-6 hours in 5 ml LB-media containing 10g/l tryptone, 5 g/l yeast extract, 10 g/l sodium chloride (pH 7.4), then transferred to 500 ml

LB-media for amplification overnight. Plasmid cDNA was isolated from bacteria using a

Plasmid Maxi Prep kit (Qiagen, Valencia, CA) and function was tested by transient expression in HEK293 cells and using whole cell electrophysiology as described in the following sections.

C. Cell culture and transient transfection

HEK293 cells obtained from ATCC were maintained in Corning T-25 or T-75

flasks containing 89 % Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented

with 10 % bovine serum and 1% penicillin/streptomycin (HEK media) at 37°C and 5%

CO2. All experiments involving recombinant receptors were performed using transient

transfection of HEK293 cells. Preparation of recombinant receptors expressed in

HEK293 cells was performed over a 4-5 day timeline.

On day 1, cells were subcultured in sterile tissue-culture pre-treated 35-mm

diameter dishes for electrophysiology, or 60-mm diameter dishes for use in binding

assays. Corning dishes (Corning, NY) used for electrophysiology were plated at a low

density of approximately 10-20% confluence, while dishes used for binding assays were

plated using a higher cell density of approximately 50-60% confluence. Cell density was

estimated visually using light microscopy.

On day 2, HEK293 cells were transiently transfected using the Ca2PO4

precipitation method (Chen and Okayama, 1987). Briefly, for transfection cDNAs were

pipetted at equal ratios to achieve a total concentration of 1 μg of cDNA per 35 mm dish,

or 7 μg of cDNA per 60 mm dish. GFP was used as a marker for successful transfection

and detected on an inverted Nikon eclipse TE2000-S fluorescent microscope (Nikon,

Melville, NY). Transfection solutions were made by adding GFP and cDNAs to sterile

ddH20 in sterile 1.5 ml microcentrifuge tubes (Fisher Scientific, Suwanee, GA), followed by precipitation of cDNA by adding 2.5 M CaCl2 solution. Then, the entire volume of

cDNA, ddH20 and CaCl2 was mixed slowly by vortexing and adding an equal volume of

2X HEPES-phosphate buffered saline to the transfection solution (containing, in mM:

280 NaCl, 10 KCl, 1.5 Na2PO4, 12 Glucose, 50 HEPES pH ~7.0 for low density 35 mm

dishes, 280 mM NaCl, 50 mM HEPES, 1.5 mM Na2PO4 pH ~7.3 for high density 60 mm dishes). The transfection cocktail was allowed to form a precipitate at room temperature for 10 minutes prior to vortexing and then adding 50 or 500 μl to each 35 or 60 mm dish, respectively. Dishes were stored in incubators maintained at 37°C and 3% CO2 for 15-19

hours overnight.

On day 3, cells were carefully washed with fresh HEK media warmed to 37°C,

halting the transfection reaction. On days 4 & 5, cells were ready to use for

electrophysiology experiments. For binding assays, confluent cells were harvested on day

5 in room temperature Hank’s balanced salt solution (HBSS), centrifuged at 3000 g and

cell pellets were frozen at -80°C for later use. Visual quantification of expression of GFP

by fluorescence was used to determine relative expression of GABAA receptor subunits.

DMEM, bovine serum, penicillin/streptomycin and HBSS were obtained from

Gibco/Invitrogen (Carlsbad, CA).

D. Electrophysiology Recording Equipment

Patch clamp electrophysiology recordings were obtained on an inverted Nikon eclipse TE2000-S fluorescent microscope (Nikon, Melville, NY) fitted with standard electrophysiological equipment. Whole cell recordings were obtained and quantified

using pClamp 8.0 software (Axon Instruments, Sunnyvale, CA) on the hard drive of a

PC. Data were acquired by a Digidata 1320 A digitizer and amplified using an Axopatch

200B amplifier (both from Axon Instruments, Sunnyvale, CA). Recording electrodes

were lowered onto single fluorescent cells using a MXC-45DR manipulator attached to a

MC1100e electronic controller (SD instruments, San Diego, CA).

Recording electrodes were fashioned from thin-walled glass capillary tubes with

filaments (World Precision Instruments, Sarasota, FL) using a P-87 horizontal

microelectrode puller (Sutter Instruments, Novato, CA) to create two recording electrodes

with tapered tips. Electrodes were fire polished under microscopic inspection by exposing

the tapered tip to a glass coated platinum filament heated by means of an electrical

current to remove imperfections and obtain electrode resistances between 2.0 and 4.0

mΩ. Series resistances in the whole-cell configuration ranged from 2.0 to 9.0 mΩ and

experiments falling outside of this range were excluded.

E. Whole Cell Patch Clamp Experiments

For all experiments, recording electrodes were partially filled with CsCl recording

solution, containing (in mM) 140 CsCl, 2 MgCl2, 10 HEPES, 11 EGTA and 3 ATP at a pH of 7.4 (with CsOH). During recordings, individual fluorescent cells were voltage clamped at a -60 mV and maintained in an extracellular solution (Saline A) containing (in

mM) NaCl 140, KCl 4.7, MgCl2 1.2, CaCl2 2.5, HEPES buffer 10, and glucose 10 at a pH

of 7.4 (with NaOH). For some experiments, GABA was locally applied through thin

walled glass electrodes using a Narishige IM 300 microinjector (Narishige International,

East Meadow, NY) while modulatory drugs were perfused through the bath system at a

rate of 5 ml/min.

F. Rapid agonist application

For experiments where the kinetics of macroscopic currents were examined,

GABA, PIC or TBPS were applied rapidly. Rapid solution exchanges were achieved using an SF-7713 fast step perfusion system (Warner Instruments, Hamden, CT). Ligands dissolved in saline were perfused through a Cole Parmer syringe pump (Cole Parmer

Instrument Company, London UK), fitted with 60 ml BD falcon syringes (Becton,

Dickinson and Company, Franklin Lakes, NJ). Solutions were perfused at a rate of 300

μl/min through three-barrel square glass tubes (Warner Instruments, Hamden, CT)

connected to individual syringes. In these experiments, whole cells were carefully lifted

off of the bottom of the culture dish and placed in front of the glass tubes to ensure

perfusion of the entire cell surface. Rapid solution exchange times were measured by

determining the junction potential rate of exchange of Saline A to 90% Saline A + 10%

ddH20 over an open tip electrode containing Saline B. Complete solution exchange rates

averaged between 2.5 to 5 ms. Experiments performed in the laboratory with the same

system, using bicuculline to rapidly inhibit propofol-evoked GABAA receptor-mediated

currents, achieved cessation of macroscopic current with a rate (τ) of 12 ms (Gallacher

and Hales, unpublished). Therefore complete solution exchange around the whole-cell

occurs within 2.5-12 ms.

G. Electrophysiology Data acquisition and Analyisis

For all electrophysiology experiments, data was acquired using Clampex 8.0 and

traces were analyzed using Clampfit 8.0 (pClamp 8.0, Axon Instruments, Sunnyvale,

CA). To determine current density for each trace, recordings were normalized to remove

leak current, and the peak response in the presence of GABA was measured.

For experiments measuring the ability of TBPS or PIC to block GABAA receptors

in the presence of GABA, the maximal level of % inhibition was measured by subtracting the current remaining, if any, in the presence of GABA from the peak GABA response obtained prior to exposure to antagonists. GABA-independent blockade was determined by comparing the current amplitude remaining in the presence of GABA after prolonged exposure to TBPS or PIC in the absence of GABA, to the control peak response prior to exposure to antagonists. During experiments where bicuculline was used to prevent spontaneous channel openings, the peak responses were measured in the presence of both

GABA and bicuculline.

During experiments measuring the steady-state desensitization of different

GABAA receptors, GABA was bath applied at various concentrations (300 nM to 1 mM),

while maximal concentrations of GABA (1 or 3 mM) were locally applied. The level of

steady state desensitization was determined by measuring the current amplitude evoked

by locally applied maximal GABA concentrations after cumulative desensitization 68

caused by increasing levels of GABA in the bath solution. These data were plotted as % of control GABA-evoked current. Concentration-activation curves were obtained by locally applying various concentrations of GABA (100 nM to 1 mM). At any given

concentration of GABA, a proportion of receptors exist in the activated or desensitized

conformation, and the current passing from these receptors is depicted by the “window

current”, which is obtained by superimposing activation and desensitization curves.

For experiments using rapid agonist application, peak responses to GABA were

measured as described above for experiments using locally applied GABA. The rates of

block and unblock by TBPS or PIC were determined by fitting the rise time for each trace

with either mono- or biexponential functions. The rates of desensitization and recovery

from desensitization were also fitted to mono- or biexponential functions. Where

appropriate, data were plotted against time in the desensitized state to determine if the

level of desensitization affected the ability of TBPS or PIC to block the channel. All

calculations were done using Clampfit 8.0 and exported for further analysis as described

in section K.

H. Preparation of Rodent Brains

Whole brains were isolated from adult male Sprague-Dawley rats weighing 200-

225 g. For some experiments, live animals were obtained from Hilltop Lab Animals

(Scottdale, PA), whereas others utilized whole brains ordered directly from

Bioreclaimation (Liverpool, NY), where tissue was isolated from similar animals. Data

obtained using brains from both sources were indistinguishable. For homogenate assays, 69

whole brains were thawed in petri dishes placed on ice and whole cerebella, frontal cortices and hippocampi were isolated and weighed, then homogenized in incubation buffer at a final concentration of 20 mg/ml.

For autoradiography, fresh tissue was frozen on dry ice and either wrapped in parafilm and stored at -20°C prior to use, or immediately mounted onto cryostat chucks using Tissue-Tek optimum temperature cutting (O.C.T) embedding media (Fisher

Scientific). Brains mounted in O.C.T. medium could be stored at -20°C for several weeks without tissue deterioration. For sagittal sections, brains were sliced down the midline and mounted with the medial side facing downward. Using a Clinicut 3020 cryostat

(Bright instruments, Cambridgeshire, UK) held at a constant temperature of -19°C, 14-16

μm thick sections were taken from approximately lateral 2.1 to lateral 1.4 for sagittal sections, or intraneural – 1.32 (Bregma 10.32) to intraneural 0.48 (Bregma -9.48) for coronal sections. Individual sections were thaw mounted on Thermoscientific Superfrost

Plus microscope slides and stored at -20°C when not in use. Tissue-Tek O.C.T, microscope slide holders and microscope slides were obtained from Fisher Scientific

(Suwanee, GA or Leicestershire, UK).

I. [35S]TBPS Autoradiography Experiments

[35S]t-butylbicyclophosphorothionate ([35S]TBPS) was obtained from Perkin

Elmer (Waltham, MA). For autoradiography experiments, slides containing single

sections of rodent brains were pre-incubated in ice-cold autoradiography buffer (50 mM

Tris-HCl and 120 mM NaCl, pH 7.4) for 15 minutes. Slides were then lightly dried using compressed air fed through Drierite (Sigma Aldrich, St. Louis, MO) and vacuum suction to remove excess buffer. 400 μl of 6 nM [35S]TBPS buffer containing agonists or antagonists was applied to each slide to completely cover and surround the surface of each brain slice. Slides were incubated with [35S]TBPS for 90 minutes at room temperature (~ 25°C). The incubation was halted by vacuuming excess fluid off of slides, followed by three 15 s washes in ice-cold autoradiography buffer. Finally, slides were quickly dipped in room-temperature distilled water and air dried using compressed air fed through Drierite. Slides were stored in a vacuum sealed desiccator overnight (Fisher

Scientific, Suwanee, GA) prior to exposure to Kodak BioMax MR film (Sigma Aldrich,

St. Louis, MO) in the presence of 14C-plastic standards obtained from GE Amersham-

Healthcare (Buckinghamshire, UK). Typical exposures ranged from 42-49 hours in an

8°C cold room, in light-sealed autoradiography film cassettes from Cole-Parmer

(London, UK) or GE Amersham-Healthcare. Films were developed using an automated developer and digitized using a fluorescent lightbox (Eyebright, Cheshire, UK) and a

Moticam 2500 camera (Motic, Richmond, British Columbia). MCID Analysis software

(Cambridge, UK) was used to analyze digitized images and obtain radioactivity values

(nCi/g) based on standard curves obtained using exposures from 14C-plastic standards.

Non-specific binding was determined in the presence of 100 μM picrotoxin during incubation.

J. [35S]TBPS Homogenate Binding Experiments

To obtain a crude membrane fraction, HEK293 cell membranes containing

recombinant GABAA receptors, or rodent brain tissue were suspended in 10 ml T.E.N.

solution (10 mM Tris-HCl, 1 mM EDTA and 100 mM NaCl, pH 7.5 with HCl) using a

Tissue-Tearer hand homogenizer (Biospec Products, Racine, WI) and centrifuged for 30

min at 30,000 g. Brain tissue was washed by centrifugation in T.E.N an additional two

times for 10 min at 30,000 g to remove endogenous GABA. Membrane pellets were then

resuspended in incubation buffer (20 mM Tris-HCl and 1 M NaCl, pH 7.5 with HCl) for

a final concentration of 20 mg/ml for brain tissue, or a range of 3 to 6 mg/ml for cell

membranes, detected using an ND-1000 spectrophotometer (Nanodrop, Wilmington, DE)

or by Bradford standard protein curve programmed on an Eppendorf Biophotometer

(Eppendorf International, Cambridge, UK).

100 μl aliquots of membrane suspensions were incubated in 12 x 75 mm

borosilicate glass tubes (Fisher Scientific, Suwanee, GA) in the presence of 30 nM

[35S]TBPS (Perkin Elmer, Waltham, MA) at 25°C for 90 minutes, in the presence of

various agonists or antagonists, in a total volume of 200 μl. Non-specific binding was

determined in the presence of 100 μΜ PIC. The incubation was halted by perfusing 5 ml of ice-cold incubation buffer through a Brandel M-48 (Gaithersburg, MD) or Brandel M-

30T harvester (London, UK). Membranes bound to [35S]TBPS were collected on

Whatman GF/B filter paper (Brandel, Gaithersburg, MD or Alphabiotech, Glasgow, UK)

pre-soaked with 0.5 % polyethylenimine (Sigma Aldrich, St. Louis, MO). Filters were air

dried for 10-15 minutes, then counted for radioactivity in 4 ml of scintillation fluid using

a Beckman LS 6500 scintillation counter (Brea, CA).

K. Data Analysis & Statistical Procedures

Raw data values were maintained in spreadsheets and mean, S.E.M and S.D were

determined using Microsoft Excel 2003 or 2007. All figures and fitted curves were

produced using Slide Write Plus 5.0 (Encinitas, CA). Statistical analysis was performed

using Slide Write Plus 5.0 and Graphpad Prism 5.0 (La Jolla, CA). In all figures, data are

represented as mean ± S.E.M and statistical comparisons of means were made using

either Student’s t-test or one-way ANOVA with Tukey’s post-hoc test where appropriate.

Differences were accepted at p < 0.05.

Chapter 3. Results The role of channel activation in TBPS/PIC blockade

Background and significance

In all GABAA receptors, the orthosteric agonist (GABA) binding site lies at the

interface between α and β subunits (Whiting et al., 1999). Modulation by most other

allosteric GABAA receptor modulators involves interactions with the TM2, a locus that participates in anesthetic, neurosteroid and convulsant binding (Lambert et al., 2003;

Korpi & Sinkkonen, 2006). By convention, Cys-loop receptor TM2 residues are numbered from intracellular 0’ to extracellular 20’ (Miller, 1989). Pro-convulsant ligands, such as picrotoxin (PIC) and t-butylbicyclophosphorothionate (TBPS) inhibit

GABA neurotransmission by preventing Cl- current from passing through the ion pore

(Sigel et al., 1989). Mutations at the 2’, 6’ or 9’ residues reduce the potency of PIC,

suggesting that the convulsant site lies deep within the channel pore (Buhr et al., 2001,

Sedelnikova et al., 2006; Erkkila et al., 2008).

To date, the majority of work examining the convulsant binding site has been

35 done using PIC. However, PIC fully displaces [ S]TBPS binding from all native GABAA

receptors. As such, it is assumed that TBPS and PIC bind to overlapping sites (Squires et

al., 1983, Sinkkonen et al., 2001). Throughout this chapter, I will describe the activity of

TBPS with reference to evidence describing the residues known to bind PIC.

PIC is thought to require an open channel to access its binding site within the

GABAA receptor. GABA enhances the rate of association of PIC to the convulsant

binding site, suggesting that PIC binds more favorably to the open state (Newland &

Cull-Candy, 1992; Dillon et al., 1995). This idea is reinforced for both PIC and TBPS by

evidence for a common binding motif within the channel pore between the 9’ and 2’

residues within the TM2 (Fig 1; Chen et al., 2006; Sedelnikova et al., 2006; Kalueff,

2007). The 2’ residue lies below the proposed Cys-loop receptor closed-channel gate

(Wilson & Karlin, 2001; Bali & Akabas, 2007; Unwin, 2005). Furthermore, mutations

that affect gating alter inhibition by PIC (Buhr et al., 2001; Xu et al., 1995; Chang &

Weiss, 1999). These data suggest that channel activation is required for access to the

convulsant binding site.

35 However, PIC displaceable [ S]TBPS binding to recombinant GABAA receptors

occurs in the absence of GABA, suggesting that there is a component of blockade that is

independent of activation (Im et al., 1994; Luddens & Korpi, 1995). These conflicting

results demonstrate a need for a better understanding of the relationship between

convulsant blockade and GABAA receptor gating. In my experiments, I examined this relationship using recombinant wild-type and mutant GABAA receptors expressed in

HEK293 cells.

Recombinant GABAA receptors exhibit a low level of spontaneous channel

activity in the absence of GABA (McCartney et al., 2007). Constitutive, GABA-

independent gating, may provide accessibility to the TBPS binding site and be

responsible for [35S]TBPS binding to receptors in the absence of GABA. To test this

possibility, I used the inverse agonist bicuculline, which binds at the GABA binding site

to inhibit spontaneously active channels (McCartney et al., 2007; Bai et al., 2001; Birnir

et al., 2000; Ueno et al., 1997).

I also exploited the mutant α1(K278M) and γ2(K289M) subunits, which confer reduced spontaneous gating (Fig. 3 & 4), mean channel open time, and an increase in the

GABA EC50 for activation (the latter only applies to the α1(K278M) subunit; Hales et

al., 2006). The data presented in this chapter reveal a relationship between GABAA

receptor gating and TBPS binding and receptor blockade by both TBPS and PIC.

Experimental Results

Inhibition of GABAA receptors by TBPS and PIC

The potency of inhibition of GABAA receptors by the non-competitive antagonist

TBPS has been described by other groups using membrane preparations from the rodent brain (Squires et al., 2003; Casida and Lawrence, 1985) and cells expressing recombinant

α1β3ε receptors (Maksay et al., 2003). The majority of work examining the PIC binding site in GABAA receptors has been described using patch clamp electrophysiology (Chen et al., 2006; Sedelnikova et al., 2006; Erkkila et al., 2008). I examined the relationship between the concentration of PIC or TBPS and inhibition of recombinant GABAA receptors using the whole-cell patch-clamp technique to record GABA-evoked currents from HEK293 cells (voltage-clamped at -60 mV) transiently expressing recombinant

α1β2γ2 receptors (Fig. 5A). In these experiments, receptors were activated by local application of GABA (100 μM) in the absence or presence of bath-applied PIC (0.3 – 100

μM) or TBPS (0.01 – 10 μM). Inhibition was calculated at steady-state by expressing the current remaining after bath application of PIC or TBPS as a percentage of the current amplitude recorded in the presence of GABA alone (Fig. 5A). The IC50 values for TBPS

and PIC were 0.33 ± 0.05 μM and 2.0 ± 0.2 μM, respectively. Maximal inhibition of

GABA-evoked currents occurred in the presence of 10 μM TBPS and 100 μM PIC.

Figures 5B and 5C depict representative traces confirming the concentration dependence of inhibition by TBPS and PIC respectively. My findings agree with earlier publications that have demonstrated that TBPS binds to GABAA receptors with higher affinity than

PIC (Squires et al., 1983; Casida and Lawrence, 1985, Maksay et al., 2003).

Figure 5 A 100

60 GABA GABA

% I 40

0 0.003 0.01 0.1 1.0 10 100 [Convulsant] (μM)

10 μM TBPS B 3 μM TBPS 1 μM TBPS

300 nM TBPS 100 nM TBPS 1 nA 30 nM TBPS control 1 s

C 100 μM PIC 30 μM PIC 10 μM PIC 3 μM PIC 1 μM PIC 300 nM PIC 100 pA control 1 s

Figure 5. The effect of TBPS and PIC on recombinant α1β2γ2 GABAA receptors. A. Concentration-inhibition curves for TBPS (○) and PIC (●). Whole-cell currents recorded from α1β2γ2 receptors expressed in HEK293 membranes were activated by GABA (100 μM). GABA-evoked current amplitudes recorded in the presence of TBPS (n ≥ 4) or PIC (n ≥ 3) were expressed as a percentage of those recorded under control conditions (% IGABA). TBPS caused a more potent inhibition of GABA-evoked currents. IC50 values for PIC and TBPS were 2.0 ± 0.2 μM and 0.33 ± 0.05 μM respectively. Data are represented as mean ± S.E.M. of 3-10 recordings. B. Representative traces depicting inhibition of GABA-evoked (100 μM, control) currents by TBPS (0.03 to 10 μM). Recordings in the presence of 10 nM TBPS are not shown. C. Representative traces depicting inhibition of GABA-evoked (100 μM, control) currents by PIC (0.3 to 100 μM).

Blockade by TBPS and PIC occurs independently of GABA-activation

The concentration inhibition curves for TBPS and PIC were established using periodic local application of GABA (100 μM for 100 ms every 60 s, Fig. 5). Both TBPS and PIC have been referred to as open channel blockers, because GABA activation of the channel has been shown to enhance their rate of block (Newland and Cull-Candy, 1992;

Dillon et al., 1995). I investigated whether blockade by either TBPS or PIC was influenced by GABAA receptor activation. GABA (100 μM for 100 ms) was episodically

(every 60 s) locally applied to HEK293 cells expressing α1β2γ2 receptors. To determine the level of GABA-enhanced inhibition, TBPS (10 μM) or PIC (100 μM) were applied to the bath (Fig. 6A) in the presence of episodic stimulation by 100 μM GABA. Both PIC and TBPS caused abolition of GABA-evoked currents (98 ± 0.4% and 98 ± 0.6% inhibition, respectively) in the presence of GABA application (Fig. 6C). I measured the level of inhibition that occurred after a prolonged exposure to either TBPS or PIC in the absence of GABA. The time required to reach maximal inhibition with episodic GABA application was used to determine the duration of bath application of TBPS or PIC to the same cell in the absence of GABA for each cell (Fig. 6B). There was a significant (p <

0.05, Student’s t-test) reduction in the inhibition caused by both TBPS and PIC (86 ±

4.8% and 91 ± 3.1%, respectively) in the absence of episodic GABA application (Fig.

6C).

In addition to examining the α1β2γ2 receptor subtype, I also examined the ability of TBPS and PIC to block recombinant α1β3γ2 receptors. The rationale for these experiments was that the α1β3γ2 subtype is the second most common GABAA receptor

subtype in the CNS compared to α1β2γ2 (Pirker et al., 2000). Furthermore, several studies examining the TBPS binding site have used mutant β3 subunits (Jursky et al.,

2000, Chen et al., 2006) or homomeric wild type β3 receptors (Olsen, 2006; Davies et al., 1997b). The significance of the formation of homomeric β3 receptors will be revisited in Chapter 5.

In the presence of episodic local application of 100 μM GABA (every 60 s),

TBPS and PIC were fully able to block α1β3γ2 receptors (Fig. 7A). Both TBPS and PIC caused near abolition of GABA-evoked currents (95 ± 2.2%, and 98 ± 0.3% inhibition, respectively) in the presence of episodic GABA application (Fig. 7C). To determine the role of channel activation in blockade of α1β3γ2 receptors by TBPS and PIC, I measured the inhibition caused by prolonged exposure to TBPS or PIC in the absence of GABA

(Fig. 7B). As was seen for the α1β2γ2 subtype, the majority of inhibition of α1β3γ2 receptors occurred independently of GABA-activation (Fig. 7B). However, there was a significant (* p < 0.05, ** p < 0.01, Student’s t-test) reduction in the inhibition caused by both TBPS and PIC (75 ± 5.4%, and 91 ± 1.9% inhibition, respectively) in the absence of episodic GABA application (Fig. 7C).

For α1β2γ2 and α1β3γ2 receptors, the majority of blockade by both TBPS and

PIC was independent of GABA application. Consistent with a small use-dependent component of blockade, inhibition by TBPS or PIC grew somewhat by the second application of GABA (Fig. 6B and 7B). Taken together, these data confirm that while

GABA enhances block by TBPS and PIC, the majority is independent of GABA-evoked channel activation.

Figure 6 α1β2γ2 A TBPS

2 nA

4 s

PIC

800 pA 4s

B TBPS C 100

90 600 pA 80 1 s

% inhibition 70 PIC 60

200 pA 50 TBPS TBPS PIC PIC 1 s GABA GABA

Figure 6. Inhibition of GABA-evoked currents by PIC and TBPS recorded from α1β2γ2 receptors. A. Top traces, inhibition of episodic GABA (100 μM)-evoked currents by TBPS (10 μM) or, bottom traces, PIC (100 μM). The time required to reach maximal inhibition in the presence of GABA was used to determine the duration of bath application of TBPS or PIC to the same cell in the absence of episodic GABA application. B. The majority of block occurred after prolonged exposure to TBPS and PIC in the absence of GABA. C. Data were quantified as % inhibition and are represented as mean ± S.E.M. Black and grey bars represent inhibition with and without episodic GABA application, respectively. Time indicated in legends represents the scale for a single trace, not the entire experiment. * p < 0.05 obtained by Student’s t-test.

Figure 7 α1β3γ2 TBPS A

2nA

PIC

1 nA 6 s

B TBPS C 100

1 nA 90

2 s 80

PIC % inhibition 70

500 pA 50 TBPS TBPS PIC PIC 2 s GABA GABA

Figure 7. Inhibition of GABA-evoked currents by PIC and TBPS recorded from α1β3γ2 receptors. A. Top traces, inhibition of episodic GABA (100 μM)-evoked currents by TBPS (10 μM) or, bottom traces, PIC (100 μM). The time required to reach maximal inhibition in the presence of GABA was used to determine the duration of bath application of TBPS or PIC to the same cell in the absence of episodic GABA application. B. The majority of block occurred after prolonged exposure to TBPS and PIC in the absence of GABA. C. Data were quantified as % inhibition and are represented as mean ± S.E.M. Black and grey bars represent inhibitions with and without episodic GABA application, respectively. Time indicated in legends represents the scale for a single trace, not the entire experiment.* p < 0.05, ** p < 0.01, obtained by Student’s t-test.

Modulation of [35S]TBPS binding by GABA

I further investigated the ability of TBPS and PIC to reach their binding sites in

the absence of GABA using [35S]TBPS binding to homogenates of HEK293 cells

expressing α1β2γ2 receptors. The electrophysiological data described earlier in this

chapter demonstrate that the majority of blockade by both PIC and TBPS occurs independently of GABA activation of the channel (Fig. 6 and 7). Consistent with this,

[35S]TBPS binds to recombinant receptors in the absence of GABA (Luddens and Korpi,

1995, Davies et al.,1997b). I observed robust PIC-displaceable [35S]TBPS binding to

HEK293 membranes containing α1β2γ2 receptors. Addition of GABA (30 nM to 100

μM) caused a biphasic modulation of [35S]TBPS binding (Fig. 8). Low concentrations of

GABA (30 nM to 1 μM) enhanced [35S]TBPS binding. This enhancement corresponds to

the use-dependent component of inhibition by TBPS observed using the

electrophysiological assay (Fig. 6).

Although much binding occurs in the absence of GABAA receptor activation, it

seems that GABA is required to reach maximal levels of [35S]TBPS binding in α1β2γ2

receptors. Consistent with previous reports, higher concentrations of GABA (3 μM to

35 100 μM) reduced [ S]TBPS binding (Fig. 8) presumably through GABAA receptor

desensitization. These data demonstrate that channel activity induced by GABA binding

directly influences accessibility of [35S]TBPS to its binding site. In the next set of experiments, I continued to examine the role of spontaneous channel activation in blockade and binding of TBPS and PIC. I will revisit the role of desensitization in blockade and binding of TBPS and PIC in Chapter 4.

Figure 8

160

140

120

100

60 % control binding binding % control α1β2γ2 40

0.01 0.1 1.0 10 100

[GABA] (μM)

Figure 8. Modulation of [35S]TBPS binding to recombinant α1β2γ2 receptors by GABA is biphasic. [35S]TBPS binding to α1β2γ2 receptors in HEK293 membrane homogenates was normalized to PIC (100 μM)-displaceable GABA-independent binding (% control binding). Low concentrations of GABA (0.03 to 1 μM) enhanced binding, while high concentrations of GABA (3 to 100 μM) inhibited binding. Data are representative of the mean ± S.E.M of 5-8 independent experiments performed in triplicate.

Bicuculline affects GABA-independent [35S]TBPS binding to α1β2γ2 receptors

Several GABAA receptor subtypes exhibit a low level of spontaneous gating that can be inhibited by PIC or the inverse agonist bicuculline (BIC) (McCartney et al., 2007).

I hypothesized that TBPS and PIC enter spontaneously active channels in the absence of

GABA. This mode of entry could account for their apparent use-independent current blockade (Fig. 6 & 7). In the past, it has been shown that BIC slows the rate of association of TBPS to its binding site (Behrends, 2000). In my experiments, I examined the relationship between spontaneous gating and GABA-independent [35S]TBPS binding using bicuculline to inhibit spontaneous gating and examined its effect on [35S]TBPS

binding in the absence of GABA.

Bicuculline reduced PIC-displaceable [35S]TBPS binding in a concentration-

dependent manner (30 nM to 100 μM) (Fig. 9). A maximum inhibition of approximately

25% occurred with 3 μM BIC. The observation that bicuculline reduces, but does not entirely abolish [35S]TBPS binding suggests that spontaneous gating increases accessibility to the convulsant site but is not necessary for blockade in the absence of

GABA. These data are consistent with the majority of block by TBPS and PIC occurring

independently of channel opening, either spontaneous or induced by GABA (Fig. 6 & 7).

To evaluate this further, I examined the ability of bicuculline to prevent current blockade

by TBPS or PIC in the presence and absence of GABA.

Figure 9

125

100

50 α1β2γ2 % control binding binding % control

0.01 0.1 1.0 10 100 300 [Bicuculline] (μM)

Figure 9. Bicuculline reduces GABA-independent binding of [35S]TBPS to recombinant α1β2γ2 receptors in a concentration dependent manner. Bicuculline caused a peak inhibition of GABA-independent binding of approximately 25%. Control specific binding was determined in the presence of 100 μM PIC. Data are represented as mean ± S.E.M of 4-10 independent experiments performed in triplicate. * p < 0.05 compared to binding in the presence of 30 nM BIC. Statistics were obtained using one-way ANOVA and Tukey’s post-hoc test.

GABA-independent blockade by TBPS is reduced by bicuculline

BIC (100 μM) was bath applied to HEK293 cells expressing α1β2γ2 receptors to

inhibit spontaneous agonist-independent channel activity. GABA (10 mM) was locally

applied in order to activate GABAA receptors in the presence of BIC (Fig. 10A). This

high concentration of GABA was sufficient to compete with bath applied BIC in the

recording chamber, enabling phasic agonist-dependent channel activation in the absence

of tonic spontaneous gating. I used this approach to compare the amplitude of activity-

dependent and activity-independent blockade of GABAA receptor-mediated currents by

PIC and TBPS. Inhibition in the presence of episodic GABAA receptor activation was

determined using the same approach as described for Figures 6 and 7 with two

modifications. First, BIC (100 μM) was present in the recording chamber throughout the

experiment and second, GABA was applied at 10 mM, not 100 μM.

Bath application of either TBPS or PIC inhibited GABA-evoked current (Fig.

10A). I investigated use-independent GABAA receptor blockade by PIC or TBPS without

episodic GABA application (Fig. 10B). Compared to data in Figure 6, these experiments

revealed that prevention of spontaneous channel gating by BIC caused a significant (* p <

0.01, one-way ANOVA with Tukey’s post hoc Test) reduction in inhibition by TBPS, but not PIC (Fig. 10C). The activity-independent inhibition by TBPS in the presence of BIC was approximately 40% less than inhibition observed in the presence of both spontaneous and episodic GABA-evoked gating (Fig. 10C). These data suggest that spontaneous gating provides a significant means of entry for TBPS into the GABAA receptor channel

(** p < 0.001, Student’s t-test).

Figure 10 α1β2γ2

TBPS A Bicuculline

400 pA

8 s Bicuculline PIC

3 nA

4 s

B Bicuculline TBPS C 100 \

200 pA 80 2 s

70 % inhibition Bicuculline PIC 60

50 2 nA TBPS TBPS PIC PIC 2 s GABA GABA

Figure 10. Bicuculline reduces GABA-independent blockade by TBPS of currents recorded from α1β2γ2 receptors. A. Top traces, time-dependent inhibition of episodic GABA (10 mM)-evoked currents by TBPS (10 μM) or, bottom traces, PIC (100 μM) in the presence of bath applied bicuculline (100 μM). The time required for maximal inhibition with episodic GABA application was used to determine the duration of bath application of TBPS or PIC in the absence of GABA with bicuculline in the bath. B. Bicuculline prevented approximately 40% of GABA-independent blockade by TBPS, but not PIC. C. Data were quantified as % inhibition and compared to data obtained in the absence of bicuculline (black bars) taken from Figure 6C. Data are represented as mean ± S.E.M. * p < 0.01, ** p < 0.001 comparing inhibition in the presence (gray bars) or absence (black bars) of bicuculline obtained by one-way ANOVA with Tukey’s post-hoc test. Time indicated in legends represents the scale for a single trace, not the entire experiment.

A mutation that reduces spontaneous GABAA receptor gating reduces the GABA- independent block by TBPS and PIC

Replacement of a conserved Lys residue by Met in the TM2-TM3 loop at position

278 in the α1 subunit reduces the efficacy of GABA (Hales et al., 2006). This mutation also reduced spontaneous GABAA receptor gating. The proportion of spontaneous current mediated by the mutant receptors was <15% of that mediated by wild type α1β2γ2 receptors (Fig. 4).

I investigated the effects the mutation had on blockade by TBPS and PIC applied using the same protocols for use-dependent and use-independent inhibition described earlier (Fig. 6A,B, Fig. 7A,B and Fig. 10A,B). Current traces are shown in Figure 11A and 11B for recordings made with TBPS and PIC when bath applied in the presence and absence of local episodic GABA application, respectively. There was a significant reduction in the level of block by PIC in the absence of episodic GABA application of

α1(K278M)β2γ2 receptors compared to wild type receptors (Fig. 11C, p < 0.01, one-way

ANOVA with Tukey’s post hoc test). While the data for TBPS indicate a similar trend, the effect of the α1(K278M) mutant was not significant for TBPS. I also examined whether BIC would further reduce the use-independent blockade by TBPS and PIC by recording in the presence of BIC as described in Figure 10. There was no additive effect of these approaches for reducing spontaneous gating of α1β2γ2 receptors (Fig. 11D).

These data suggest that the mutation reduces the accessibility of TBPS and PIC to

GABAA receptor channel particularly in the absence of agonist activation.

Figure 11

TBPS A

200 pA

4 s PIC

100 pA 4 s

B C 100

90 80 PIC 70 % inhibition 60

100 pA 50 TBPS TBPS PIC PIC 2 s GABA GABA

D 100

60 α1β2γ2

40 α1(K278M)β 2γ2 % inhibition 20 α1(K278M)β2γ2 + BIC

0 TBPS TBPS PIC PIC GABA GABA Figure 11. Inhibition of GABA-evoked currents recorded from mutant α1(K278M)β2γ2 receptors by TBPS or PIC. A. Top traces, time-dependent inhibition of episodic GABA (100 μM) evoked currents by TBPS (10 μM) or, bottom traces, PIC (100 μM). The time required to reach maximal inhibition in the presence of episodic GABA was used to determine the duration of bath application of TBPS or PIC in the absence of GABA. B. Block after exposure to TBPS and PIC in the absence of episodic GABA. C. Data were quantified as % inhibition. Use-independent inhibition by PIC was significantly reduced in α1(K278M)β2γ2 receptors compared to α1β2γ2 receptors (n ≥ 5). Wild type data are reproduced here from Figure 6C. D. Bicuculline has no additional effect on the reduction of use-independent blockade by TBPS or PIC of α1(K278M)β2γ2 receptors. Data are represented as mean ± S.E.M and statistics were obtained by one-way ANOVA with Tukey’s post-hoc test. ** p < 0.01.

The mutant α1(K278M) subunit affects [35S]TBPS binding

Next, I examined the effect of the α1(K278M) subunit on [35S]TBPS binding to

α1β2γ2 receptors. Receptors containing the α1(K278M) subunit exhibited a reduced level of PIC-displaceable [35S]TBPS binding compared to wild type receptors (Fig. 12A).

This suggests that GABA-independent binding of [35S]TBPS is reduced by the

incorporation of the α1(K278M) subunit into the receptor. Unlike the reduction in

[35S]TBPS binding by BIC observed for wild-type α1β2γ2 receptors (Fig. 9), BIC had no

significant effect on [35S]TBPS binding to α1(K278M)β2γ2 receptors (Fig. 12B),

demonstrating that the mutant was sufficient to eliminate spontaneous gating in α1β2γ2

receptors.

The α1(K278M) substitution enhances the proportion of GABA dependent stimulation of

[35S]TBPS binding

The α1(K278M) subunit had a dramatic effect on the modulation of [35S]TBPS

binding by GABA (Fig. 12C). Low concentrations of GABA (0.03 to 3 μM) caused a much larger enhancement of [35S]TBPS binding to α1(K278M)β2γ2 receptors compared

to wild type receptors. Furthermore, at higher concentrations (> 3 μM), GABA caused

less of a reduction of [35S]TBPS binding to α1(K278M)β2γ2 receptors compared to wild

type α1β2γ2 receptors (Fig. 12C). Since these binding assays occur over a prolonged

exposure to GABA (90 min), I suspected that the abolition of [35S]TBPS binding by 100

μM GABA resulted from receptor desensitization, which I will examine in Chapter 4.

The γ2(K289M) epilepsy mutation alters GABA modulation of [35S]TBPS binding

Previous studies demonstrate that the effects of the Lys to Met substitution differ

when present on α1 or γ2 subunits (Hales et al., 2006). The γ2(K289M) mutation has

been associated with generalized epilepsy with febrile seizures (Macdonald et al., 2004),

however, its effects on channel function have not been fully described. In my

experiments, I used this mutant subunit to determine if the differences seen in GABA

modulation of [35S]TBPS binding to α1(K278M)β2γ2 receptors (Fig. 12) was due to

subunits contributing to the orthosteric (GABA) binding site. Incorporation of either the

α1(K278M) or γ2(K289M) subunits into α1β2γ2 receptors reduces both spontaneous

gating (Fig. 4C) and channel mean open time (Hales et al., 2006), however, these effects

are more substantial in the α1(K278M) subunit. Furthermore, the α1(K278M) subunit

causes reduced potency of GABA activation, whereas, the γ2(K289M) subunit does not

(Hales et al., 2006).

Interestingly, unlike the α1(K278M) subunit, the γ2(K289M) subunit does not

reduce PIC displaceable [35S]TBPS binding in the absence of GABA (Fig. 13A). This suggests that GABA-independent access to the PIC/TBPS binding site is not altered by deficits in channel function caused by the γ2(K289M) epilepsy mutation. However, presence of the γ2(K289M) mutant subunit significantly affects GABA modulation of

[35S]TBPS binding compared to wild type α1β2γ2 receptors (Fig. 13B), * p < 0.05, ** p

< 0.01, Student’s t-test. The rightward shift was also observed in α1(K278M)β2γ2

receptors (Fig. 12C). In contrast to α1(K278M)β2γ2 receptors, there was no upward shift

in the GABA modulation of [35S]TBPS binding to α1β2γ2(K289M) receptors.

I also examined the influence of bicuculline on [35S]TBPS binding to mutant

α1β2γ2(K289M) receptors. This Lys to Met substitution reduces spontaneous, GABA independent gating of α1β2γ2 receptors (Fig. 4). As such, I anticipated that bicuculline would have no significant effect on [35S]TBPS binding in the absence of GABA.

However, [35S]TBPS binding to α1β2γ2(K289M) receptors was reduced to a similar

extent as was observed for the wild-type α1β2γ2 receptors (Fig. 13C). Overall, these

results indicate that there is a component of spontaneous, GABA-independent gating that

remains despite the presence of the mutant γ2(K289M) subunit. These data also confirm

that the gating deficits caused by the Lys to Met substitution are subunit dependent. The

role of individual subunits in the pattern of [35S]TBPS binding will be revisited in

Chapter 6.

Figure 12

A 125 100 B 100 80 75 60 † 50 40

% control binding control % 25 20 0

% specific binding/total binding binding binding/total specific % 0

2 0.01 0.1 1.0 10 100 γ 2 2 γ β

2 [Bicuculline] (μM) β 1 α (K278M) α1

C # 300 #

250 α1β2γ2

200 α1(K278M)β2γ2 † 150

% control binding control % 100 †

0 0.01 0.1 1.0 10 100 500

[GABA] (μM) Figure 12. [35S]TBPS binding to mutant α1(K278M)β2γ2 receptors. A. The level of PIC-displaceable specific [35S]TBPS binding expressed as a percentage of total binding for mutant and wild type receptors (n ≥ 19 experiments performed in triplicate). Compared to α1β2γ2 receptors, the mutant α1(K278M)β2γ2 receptor showed significantly reduced binding of [35S]TBPS in the absence of GABA, B. Bicuculline has no significant effect on GABA-independent [35S]TBPS binding to mutant α1(K278M)β2γ2 receptors (n ≥ 4 experiments performed in triplicate). C. GABA modulation of [35S]TBPS binding to α1(K278M)β2γ2 receptor is enhanced compared to α1β2γ2 receptors. Wild type data are included here from Figure 6 for comparison. Low [GABA] (0.03 to 3 μM) enhance binding, while high [GABA] (> 3 μM) reduce binding (n = 3-15 experiments performed in triplicate). Data are represented as mean ± S.E.M. # p <0.001, † p < 0.0001 compared to α1β2γ2 by Student’s t-test.

Figure 13

A 100 B 160 140 80 120

60 100

40 80 60 20

% control binding 40 % specific binding/total binding 0 20 2 γ

2 0 β 1 α 2(K289M)

γ 0.01 0.1 1 10 100 2 β

1 GABA (μM) α

C 125

100

α1β2γ2 75 α1β2γ2(K289M) % control binding 50

0.01 0.1 1 10 [Bicuculline] (μM)

Figure 13. [35S]TBPS binding to mutant α1β2γ2(K289M) receptors. A. The level of PIC- displaceable specific [35S]TBPS binding expressed as a percentage of total binding for wild type and mutant receptors (n ≥ 11 experiments performed in triplicate). Compared to α1β2γ2 receptors, the epilepsy mutant α1β2γ2(K289M) receptor showed no significant difference in specific binding of [35S]TBPS in the absence of GABA. B. GABA modulation of [35S]TBPS binding to α1β2γ2(K289M) was significantly less potent compared to α1β2γ2 receptors. C. Bicuculline causes a similar reduction in [35S]TBPS binding to α1β2γ2(K289M) receptors. Data are represented as mean ± S.E.M. * p < 0.05, ** 0.01, by Student’s t-test.

Conclusions

In summary, in this chapter, I examined the mechanism of non-competitive

blockade of GABAA receptors by TBPS and PIC. I have demonstrated that the majority

of blockade by TBPS and PIC occurs independently of both spontaneous agonist-

independent channel gating, as well as GABA-evoked channel gating at wild type

α1β2γ2 and α1β3γ2 receptors (Fig. 5 and 6). Reducing spontaneous gating either with the competitive inverse agonist BIC or the α1(K278M) mutant subunit caused a significant reduction in activity-independent blockade by TBPS (Fig 10 and 11).

Furthermore, BIC caused a significant reduction in [35S]TBPS binding to wild type

α1β2γ2 receptors (Fig. 9), confirming a role for spontaneous gating in the accessibility of

[35S]TBPS to its binding site in the absence of GABA. Taken together, these data

demonstrate that the binding site for PIC, encompassed by the 2’ and 9’ residues (Buhr et al., 2001, Sedelnikova et al., 2006; Erkkila et al., 2008) and TBPS is most accessible in the open state.

GABA modulates PIC-displaceable GABA-independent [35S]TBPS binding to

α1β2γ2, α1(K278M)β2γ2 and α1β2γ2(K289M) receptors in a biphasic manner (Fig. 8,

12, 13). Low concentrations of GABA enhanced [35S]TBPS binding, consistent with the hypothesis that GABA-evoked channel activation enhances blockade by TBPS and PIC.

High concentrations of GABA caused a concentration-dependent displacement in all three receptor subtypes. However, the potency and efficacy of GABA modulation differed significantly between α1β2γ2, α1(K278M)β2γ2 and α1β2γ2(K289M) receptors.

GABA caused a much more prominent enhancement of [35S]TBPS binding to

α1(K278M)β2γ2 receptors compared to α1β2γ2 or α1β2γ2(K289M) receptors. In

addition, both mutant subunits caused a dextral shift in the potency of displacement of

[35S]TBPS binding by GABA.

Similarly, both the α1(K278M) and γ2(K289M) subunits cause deficits in GABA-

evoked gating compared to wild type receptors (Hales et al., 2006); however, only the

α1(K278M) subunit reduces the apparent potency of GABA. It is possible that the

potency of GABA in modulation of [35S]TBPS binding is not reflective of the potency for

activation measured using patch clamp electrophysiology. However, the deficits in channel gating caused by the Lys to Met mutations clearly affected accessibility to the

TBPS/PIC binding site. Taken together, these data suggest that [35S]TBPS binding may

reflect a summation of different channel properties. I will discuss these findings and their

contribution to the GABAA receptor field in Chapter 6.

In the next chapter, I will examine the effects of these mutations on

desensitization and whether it plays a role in the significant differences in GABA

modulation of [35S]TBPS binding observed for these two TM2-TM3 mutations.

Chapter 4: Results Contribution of GABAA receptor fractional availability to blockade by TBPS & PIC

Background and significance

In chapter 3, I examined the role of channel activation in the blockade of GABAA receptors by TBPS and PIC. Using patch clamp electrophysiology and [35S]TBPS binding

to recombinant wild-type and mutant GABAA receptors, I established that the majority of

blockade by TBPS and PIC is use-independent. In this chapter, I will outline experiments

examining the role of channel desensitization in the blockade of GABAA receptors by

TBPS and PIC. I focused on the recombinant wild-type α1β2γ2, and mutant

α1(K278M)β2γ2 or α1β2γ2(K289M) receptors to elucidate the relationship between

desensitization and fractional availability to blockade by TBPS/PIC.

Desensitization causes a ligand-gated ion channel to close in the presence of

agonist. For synaptic receptors, two phases of desensitization have been described. The

slow desensitization phase occurs over longer periods of time than the rapid phase of

desensitization. Some groups have examined the role of these different phases and have

suggested that slow desensitization prevents IPSCs in the presence of non-saturating

concentrations of GABA at the synapse (Overstreet et al., 2000). Similar studies have

demonstrated the same phenomenon in extrasynaptic receptors containing the δ subunit

(Lagrange et al., 2007; Bright et al., 2011). As such, desensitization may be a

physiological property conferred to these receptors to avoid over-activation during

prolonged exposure to an agonist, such as native neurotransmitters in the CNS. The

kinetics of desensitization of GABAA receptors are subunit dependent (Bianchi et al.,

2007). However, it is not known whether the location of a desensitization gate is different

among the various GABAA receptor subtypes or indeed among different Cys-loop

receptors.

Several groups have demonstrated that a conformational shift in the TM2 domain

of Cys-loop receptors occurs during desensitization. Mounting evidence suggests that the closed state of an ion channel during desensitization is different from the closed state following agonist unbinding and receptor deactivation (Auerbach and Akk, 1998; Wilson

& Karlin, 2001; Unwin, 2005; Gay and Yakel, 2007). Many groups have hypothesized that channel closure is mediated by hydrophobic barriers or “gates” that prevent ion conduction. Currently, there is contradictory evidence describing the location of such gates in Cys-loop receptors. The Wilson and Karlin SCAM-based model, positions the activation gate at the level of the 2’ residue in the muscle type α2βδγ nACh receptor

(Wilson and Karlin, 2001). This would leave residues above 2’ accessible in the closed state. By contrast, according to the 4 Å resolution cryo-electron microscopy model of the

T. marmorata αβδγ nACh receptor, the gate is located at the 9’ residue (Fig. 1 and

Unwin, 2005). This would render amino acids below this level inaccessible in the closed state. My data (Chapter 3) reveal that PIC and TBPS bind and block GABAA receptors in

the closed state. As mentioned in Chapter 1, the PIC/TBPS binding site lies deep within

the TM2 of GABAA receptor, overlapping a span of residues between the 2’ and 9’

positions (Buhr et al., 2001, Chen et al., 2006; Sedelnikova et al., 2006; Kalueff, 2007;

Erkkila et al., 2008). Specifically, substitution of the 6’ residue can substantially affect

the potency of PIC (Sedelnikova et al., 2006; Erkilla et al., 2008), while substitution of

the 3’ residue also affects blockade by TBPS (Jursky et al., 2000). Therefore my findings

are more in keeping with the SCAM-derived model in which the activation gate is below

the residues thought to be involved in PIC and TBPS binding. The SCAM model positions the desensitization gate at the 9’ residue based on reduced accessibility to Cys residues substituted into positions below 9’ (Wilson and Karlin, 2001). Should the gate lie at a similar position in GABAA receptors then its closure during desensitization would

likely render the channel inaccessible to PIC and TBPS (Fig. 1).

Prolonged activation by GABA increases the rate of unblocking by TBPS or PIC

(Bloomquist et al., 1991). Based on this evidence, I hypothesized that prolonged desensitization would reduce the accessibility of PIC and TBPS for their binding sites within the GABAA receptor.

[35S]TBPS binding is usually assayed for prolonged periods of exposure to

different ligands (Squires et al., 1983, Maksay and Simoni, 1986). Assays measuring

modulation of [35S]TBPS binding by GABA have demonstrated that concentrations of

35 GABA in the μM range cause displacement of [ S]TBPS binding from various GABAA receptor subtypes. The concentrations required to fully displace [35S]TBPS binding from different brain regions differs, presumably due to receptors exhibiting higher or lower affinities for GABA (Sinkkonen et al., 2001). Furthermore, some groups have described brain regions that appear “GABA-insensitive” because [35S]TBPS remains bound in the

presence of concentrations of GABA that caused displacement throughout the rest of the

brain (Sinkkonen et al., 2004; Halonen et al., 2009). Taken together, these findings

demonstrate a need for understanding the underlying cause of displacement of [35S]TBPS binding to different GABAA receptor subtypes by GABA. As such, I hypothesized that

the differential sensitivities to displacement of [35S]TBPS binding from various receptor

100

subtypes by an agonist could represent different sensitivities to desensitization of

GABAA receptor subtypes.

In this chapter, I will present data examining desensitization of functionally distinct GABAA receptor subtypes and the influence of desensitization on blockade by

TBPS/PIC.

101

Experimental Results

Steady-state desensitization of recombinant α1β2γ2 receptors

Using the patch clamp technique, I examined the relationship between GABAA receptor channel activation and steady-state desensitization. In these experiments, I sought to establish the concentration range of GABA required to desensitize α1β2γ2 receptors transiently expressed in HEK293 cells. I established current availability by locally applying GABA (300 μM) to single HEK293 cells transiently expressing α1β2γ2 receptors. Between recordings, GABA (0.1 to 100 μM) was perfused into the bath for several minutes in the absence of episodic GABA stimulation to induce steady-state desensitization (Fig. 14A). Receptors were then activated by locally applying GABA

(300 μM, 300 ms every 180 s) in the presence of bath applied GABA. Over time, the current activated by locally applied GABA decreased as the concentration of GABA reached steady-state in the recording chamber (Fig. 14B).

The relative fractional availability of GABAA receptors was established by

comparing the response to GABA (300 μM) after prolonged bath GABA application to

the control response. The EC50 for desensitization of α1β2γ2 receptors by GABA was

0.86 ± 0.1 μM (determined by fitting the data with the logistic equation). Activation

curves (previously recorded in the laboratory (Hales et al., 2006)) and desensitization

curves obtained using the protocol described above were superimposed to determine the

fractional availability of the GABAA receptor channel after prolonged application of a

range of concentrations of GABA (Fig. 14C). Using this relationship, I estimated the

fraction of GABA-evoked current mediated by α1β2γ2 receptors, with an increasing

102

proportion of receptor desensitization. The EC50 for desensitization of α1β2γ2 receptors

by GABA was 0.86 ± 0.1 μM. With 30 μM GABA in the bath, the maximal level of desensitization was achieved, leaving 8 ± 1.3 % of the maximal current remaining.

Increasing the concentration of GABA in the bath caused no further desensitization (Fig.

14C).

I examined the relationship between activation and desensitization to determine if the ability of [35S]TBPS to bind at different concentrations of GABA correlated with the fractional availability of current.

The α1(K278M) subunit affects steady-state desensitization

In the last chapter, I established that the synthetic TM2-TM3 Lys to Met (K278M)

substitution in the GABAA receptor α1 subunit caused a significant reduction in GABA-

independent [35S]TBPS binding and channel blockade by TBPS and PIC (Fig. 12A).

GABA caused a marked enhancement in [35S]TBPS binding to mutant α1(K278M)β2γ2

receptors compared to wild-type α1β2γ2 receptors (Fig. 12C) . Furthermore, the mutant

α1(K278M) subunit reduced the efficacy of the displacement of [35S]TBPS by high

concentrations of GABA (Fig. 12C).

The K-M α1 mutation reduces the EC50 for GABA through a reduction in efficacy

as evidenced by a diminished mean channel open time (Hales et al., 2006; Deeb and

Hales unpublished). I anticipated that the α1(K278M) mutant subunit would similarly

reduce the potency of GABA-evoked desensitization. To examine this, I used the same

steady-state protocol described for estimation of fractional availability to the α1β2γ2

103

receptor channel. I investigated the effect of the α1(K278M) subunit on the fraction of available current compared to α1β2γ2 receptors.

To compensate for the reduced potency of GABA, I used 3 mM GABA to maximally activate α1(K278M)β2γ2 receptors, a 10-fold higher concentration than that used to activate α1β2γ2 receptors. In order to achieve steady-state desensitization during whole-cell recording, GABA was bath applied at increasing concentrations (0.1 to 1000

μM) while recording episodic responses to GABA (3 mM) locally applied every 180 s to establish fractional current availability (Fig. 15A). The half maximal concentration for steady-state desensitization was higher for α1(K278M)β2γ2 receptors (20.3 ± 6.2 μM,

Fig. 15B) compared to α1β2γ2 receptors (0.86 ± 0.1 μM, Fig. 14B).

Superimposing activation and desensitization curves provides the fractional availability of the mutant α1(K278M)β2γ2 receptor channel (Fig. 15C). I anticipated that this corresponds to the relative accessibility of TBPS or PIC to the channel at steady state. Consistent with this idea the fraction of available current is smaller for α1β2γ2 receptors (dark shading) than α1(K278M)β2γ2 receptors (light shading, Fig. 15D). Based on these findings, I concluded that the increased fraction of available current in the presence of high concentrations of GABA in mutant α1(K278M)β2γ2 receptors correlated with the increased level of [35S]TBPS binding described in Chapter 3 (Fig.

12C).

104

Figure 14 α1β2γ2 A

[GABA] in bath 100 nM 300 nM 1 μM 3 μM 10 μM 30 μM 100 μM Sal A 0 pA

2 nA

5 s

B C

100 100

) M 80 80 μ 300 ( GABA 60 60 GABA GABA 40 40

% control I 20 20 % control I 0 0 0.03 0.1 1.0 10 100 1000 0.03 0.1 1 10 100 1000 [GABA] (μM) [GABA in bath] (μM)

Figure 14. Steady-state desensitization of α1βγ2 receptors by GABA. A. α1β2γ2 receptors transiently expressed in HEK293 cells were locally activated with 300 μM GABA in the absence or presence of increasing concentrations (0.1 to 100 μM) GABA in the bath. B. Cumulative desensitization was quantified as % control GABA-evoked current (300 μM). The EC50 value for desensitization by GABA was 0.86 ± 0.1 μM (n ≥ 4). C. Superimposing activation and desensitization curves gave us the window current. A fraction of receptors remained insensitive to desensitization over time, as indicated by the increase in baseline at high concentrations of GABA (A). The steady-state current conducted by these receptors was quantified as % control GABA-evoked current (300 μM). Data are presented as mean ± S.E.M.

105

Figure 15 α1(K278M)β2γ2

A [GABA] in bath 1 μM 3 μM10 μM 30 μM 100 μM 300 μM 1 mM Sal A 0 pA

500 pA

5 s B C 100 100

80 A

GAB 60

GABA max GABA 60

40 40

20 % control I % control I 20 0 0 0.03 0.1 1 10 100 1000 3000 0.03 0.1 1.0 10 100 1000 [GABA in bath] (μM) [GABA] (μM)

D 60

GAB 40 α1β2γ2

20 α1(K278M)β2γ2 % control I

0 0.03 0.1 1.0 10 100 1000 [GABA] (μM)

Figure 15. The mutant α1(K278M) subunit increases the fraction of available steady- state GABA-evoked current. A. Representative traces demonstrating that the α1(K278M)β2γ2 receptor is less sensitive to desensitization by GABA. B Relationships for GABA concentration-dependent desensitization α1β2γ2 (●) or α1(K278M)β2γ2 (○) receptors. Current amplitudes are expressed as a function of maximal current evoked by locally applied GABA (300 μM or 3mM for α1β2γ2 or α1(K278M)β2γ2 respectively) and steady-state desensitization was determined by bath applying GABA at the concentrations indicated. EC50 0.86 ± 0.1 μM (n ≥ 4) or 20.3 ± 6.2 μM (n ≥ 3) for α1β2γ2 or α1(K278M)β2γ2 receptors respectively. C. Superimposing activation and desensitization curves gave us the window current. D. The fraction of available current for α1β2γ2 (dark shading) and α1(K278M)β2γ2 (light shading) receptors is represented by the area bounded by fits to the activation and desensitization data. The graph indicates that there is a greater portion of available current for α1(K278M)β2γ2 receptors compared to wild type receptors. Data are presented as mean ± S.E.M.

106

The γ2(K289M) subunit also affects steady-state desensitization

The concentration-dependence for activation of α1β2γ2 and α1β2γ2(K289M) receptors is indistinguishable (Hales et al., 2006). I established current availability by locally applying GABA (300 μM) to single cells expressing α1β2γ2(K289M) receptors.

Between recordings, GABA (0.1 to 100 μM) was perfused into the recording chamber for several minutes in the absence of episodic GABA stimulation to induce steady-state desensitization (Fig. 16A). Receptors were then activated by locally applying GABA

(300 μM, 300 ms every 180s) in the presence of bath applied GABA. Over time, the current activated by locally applied GABA decreased as the concentration of GABA increased in the bath (Fig. 16B).

The relative fractional availability of GABAA receptor mediated current was established by comparing the response to GABA (300 μM) after prolonged GABA application to the control response in the absence of GABA. The EC50 for desensitization of α1β2γ2(K289M) was 1.97 ± 0.24 μM compared to 0.86 ± 0.1 μM for α1β2γ2 receptors. Activation curves (Hales et al., 2006) and desensitization curves obtained using the protocol described above, were superimposed to determine the fractional availability of the GABAA receptor channel (Fig. 16C). The maximal level of desensitization was achieved in the presence of 100 μM GABA, leaving a remainder of

14 ± 5.7 % of available GABA-evoked current. Using this relationship, I estimated the fraction of GABA-evoked current mediated by α1β2γ2(K289M) receptors (Fig. 16C).

The dextral shift in potency of desensitization by GABA, with no significant reduction in the EC50 for current activation, led to an increase in the fraction of available current for α1β2γ2(K289M) receptors (light shading) compared to α1β2γ2 receptors

107

(dark shading, Fig. 14D). These data suggest that at steady-state, there is greater

accessibility to the α1β2γ2(K289M) channel pore than there is in α1β2γ2 receptors. The

greater fractional availability of the mutant channel correlates well with the increased

binding of [35S]TBPS to α1β2γ2(K289M) receptors at high concentrations of GABA

(Fig. 13C).

However, these data were surprising, since the concentration response

relationships previously established for activation of the α1β2γ2(K289M) receptor

demonstrate that the mutant γ2(K289M) subunit did not affect GABA potency. It is

possible the efficacy for GABA may be altered in these mutant receptors despite no

significant changes in the potency of activation by GABA. Indeed, unpublished findings

(Deeb, Gallacher and Hales) suggest that incorporation of the γ2(K289M) subunit

reduces the efficacy of GABA, a phenomenon that can be counteracted by applying

propofol. Furthermore α1β2γ2(K289M) receptors exhibit altered gating kinetics that

reduce the mean GABAA receptor channel open time compared to wild type receptors a phenomenon that also implies reduced efficacy caused by the mutation (Hales et al.,

2006). For the remainder of this chapter, I will compare the wild-type α1β2γ2 receptor to the mutant α1(K278M)β2γ2.

108

Figure 16 α1β2γ2(K289M)

A [GABA] in bath 100 nM 300 nM 1 μM 3 μM 10 μM 30 μM 100 μM Sal A 0 pA

1 nA

5 s B 100 100 C

80 75 GABA GABA max α1β2γ2 60 α1β2γ2(K289M) 50 40 % control I % control I 25 20

0 0 0.1 1 10 100 1000 0.1 1 10 100 [GABA in bath] (μM) [GABA] (μM) D 50

GABA 30

% control I 10

0 0.03 0.1 1 10 100 100 0 [GABA in bath] (μM)

Figure 16. The mutant γ2(K289M) subunit reduces steady state-desensitization. A. Cumulative steady-state desensitization by GABA occurs in a concentration dependent manner. B. The concentration-response relationships for desensitization by GABA were expressed as % control of GABA evoked current measured from locally applied GABA (300 μM). EC50 values for α1β2γ2 (●) or α1β2γ2(K289M) (●) receptors were 0.86 ± 0.1 μM and 1.97 μM ± 0.24, respectively. C. Superimposing activation and desensitization curves gives us the window current. D. The fraction of available current for α1β2γ2 (dark shading) and α1β2γ2(K289M) (light shading) receptors is represented by the area bounded by fits to the activation and desensitization data. The graph indicates that there is a greater portion of available current for α1β2γ2(K289M) receptors compared to wild type receptors.

109

The role of GABAA receptor desensitization in blockade by TBPS and PIC

The data described earlier in this chapter demonstrate that both the α1(K278M)

and γ2(K289M) mutant subunits have a reduced sensitivity to desensitization when

expressed with wild type α1 and β2 subunits. This reduction caused an increase in fractional availability of current (Fig. 15 and 16D). Next, I examined the ability of TBPS or PIC to block desensitized receptors. As mentioned earlier, Wilson and Karlin hypothesized that a desensitization gate in Cys-loop receptors is located at the 9’ residue, above the proposed binding sites for PIC and TBPS (Wilson and Karlin, 2001).

Accordingly, I hypothesized that desensitization would reduce the blockade of GABAA

receptors by TBPS and PIC. If this is the case then these drugs should be better able to access their binding sites and achieve an enhanced blockade of mutant receptors in which

GABA causes less desensitization.

I used rapid agonist application and the whole cell patch clamp technique to reproducibly induce desensitization of recombinant α1β2γ2 or α1(K278M)β2γ2

receptors transiently expressed in HEK293 cells. I focused on the α1(K278M)β2γ2

receptor since this mutant exhibited the most significant disruption of both current

availability and [35S]TBPS binding. In contrast to the experiments examining steady-state

desensitization, single cells were lifted from the base of the recording chamber and

positioned in front of the rapid agonist application system fitted with three-barrel glass

tubing. GABA was applied for an increasing duration (0.2 to 25 s) to induce greater

levels of desensitization. Then, a fixed duration (5 s) of either TBPS (10 μM) or PIC (100

μM) in the presence of GABA (100 μM) was applied to induce maximal blockade,

110 followed by a recovery step into GABA alone (5 to 15s). In these experiments, 100 μM

GABA was used to mimic the highest concentration of GABA used in the [35S]TBPS binding assay comparing α1β2γ2 and α1(K278M)β2γ2 receptors (Fig. 12C).

Blockade by 100 μM PIC is not affected by desensitization

I measured the level of blockade of α1β2γ2 receptors by a maximally efficacious concentration of PIC (100 μM, Fig. 5A). As the duration of GABA application (100 μM) increased, the level of desensitization also increased as revealed by the reduction in current amplitude. (Fig. 17A, bottom trace). Desensitization was measured by taking the current remaining at the end of the first GABA application and expressing it as a percentage of the current amplitude recorded at the beginning of the experiment.

Blockade by PIC was determined by extrapolating the current remaining after 5 s of exposure of desensitized receptors to GABA and PIC simultaneously. I measured the current amplitude at several points during the initial GABA application as well as during the recovery after exposure to PIC. These values were then fitted with a linear regression to estimate the current amplitude that would have been observed in the absence of PIC.

Regardless of the level of desensitization, the % inhibition by PIC remained unchanged

(Fig. 17B). These data suggest that the binding site for PIC is equally accessible at different levels of wild type receptor desensitization.

111

Figure 17 α1β2γ2

A B

100 μM GABA + 100 μM PIC 100 100 μM GABA 200 ms 90 80 70

100 μM GABA + 100 μM PIC % Inhibition 100 μM GABA 60 1 s 50 1 s 5 s 25 s 15 s 0.2 s

100 μM GABA + 100 μM PIC 100 μM GABA 5s

100 μM GABA + 100 μM PIC 100 μM GABA 15 s 2 nA

5 s 100 μM GABA + 100 μM PIC 100 μM GABA 25 s

Figure 17. Prolonged rapid agonist desensitization does not affect blockade of α1β2γ2 receptors by picrotoxin. A. The duration of rapid GABA (100 μM) application varied from 0.2 to 25 s to induce desensitization. Blockade by PIC (100 μM) was measured by presenting the fraction of current remaining after 5 s of PIC application in the presence of GABA (100 μM) as a percentage of current remaining after desensitization. B. Blockade by 100 μM PIC was not significantly affected by increased proportion of desensitized receptors. Data are presented as mean ± SEM. n ≥ 4 cells.

112

This was surprising, since the 6’ residue, implicated in PIC binding (Sedelnikova et al., 2006; Erkkila et al., 2008), lies below the hypothesized Cys-loop receptor desensitization gate revealed by the SCAM model (albeit of the nACh receptor).

However, several lines of evidence exist for additional residues participating in PIC binding (discussed in Chapter 6). My data indicate that PIC (100 μM) is still able to block desensitized receptors.

Rapid application of GABA induces less desensitization in α1(K278M)β2γ2 receptors

Using the steady state desensitization protocol described earlier (Fig. 15), I established that the potency of desensitization by GABA was reduced by the α1(K278M) mutant subunit. This was consistent with the reduction in potency of activation by GABA established previously (Hales et al., 2006). The shift in the apparent potency of desensitization by GABA caused by the mutant α1(K278M) subunit is greater than the shift in the potency of activation leading to an increased fraction of available current mediated by α1(K278M)β2γ2 compared to α1β2γ2 receptors (Fig. 15D). This increased availability correlated with the increased GABA stimulation of [35S]TBPS binding (Fig.

11). I anticipated that the reduced level of α1(K278M)β2γ2 receptor desensitization observed in the presence of GABA would enable a greater blockade by TBPS or PIC in electrophysiological recordings. I initially used the α1(K278M)β2γ2 receptor as a tool to examine whether receptors with a reduced sensitivity to desensitization have altered levels of inhibition by PIC.

113

Unlike my previous experiments in which GABA was bath applied to mimic

steady state desensitization, the rapid application approach exploited the lack of

equilibrium conditions to sample desensitization after briefer GABA exposure. As the

duration of GABA application (100 μM) increased, the level of desensitization also

increased as revealed by the reduction in current amplitude (Fig. 18A, bottom trace).

Desensitization was measured by taking the current remaining at the end of the first

GABA application and expressing it as a percentage of the current amplitude recorded at

the beginning of the experiment (% control). The level of desensitization caused by 100

μM GABA was reduced in α1(K278M)β2γ2 receptors compared to α1β2γ2 receptors

(Table 1). I chose to use this concentration in order to compare the desensitization

induced by GABA during [35S]TBPS binding assays to the desensitization measured

using patch clamp electrophysiology. Blockade by PIC was determined by extrapolating

the current remaining after 5 s of exposure of desensitized receptors to GABA and PIC simultaneously (Fig. 18B) as described earlier. Regardless of the level of desensitization, the % inhibition by PIC remained unchanged. These data suggest that the binding site for

PIC is accessible at different levels of desensitization in the α1(K278M)β2γ2 receptor.

114

Figure 18 α1(K278M)β2γ2 A B 100 μM GABA + 100 μM PIC 100 μM GABA 100 200 ms 90

100 μM GABA + 100 μM PIC % Inhibition 60 100 μM GABA 50 1 s 5 s 1 s 25 s 15 s 0.2 s

100 μM GABA + 100 μM PIC 2 nA 100 μM GABA 5 s 5 s

100 μM GABA + 100 μM PIC 100 μM GABA 15 s

100 μM GABA + 100 μM PIC 100 μM GABA 25 s

Figure 18. Rapid agonist application to α1(K278M)β2γ2 receptors. A. The duration of rapid GABA (100 μM) application varied from 0.2 to 25 s to induce desensitization.100 μM GABA caused less desensitization of mutant receptors. Blockade by PIC (100 μM) was measured by presenting the fraction of current remaining after 5 s of PIC application in the presence of GABA (100 μM) as a percentage of current remaining after desensitization. B. Blockade by 100 μM PIC was not significantly affected by increased duration of GABA application. Data are presented as mean ± SEM. n ≥ 5 cells.

115

Table 1.

% control IGABA after different durations GABAA of GABA application receptor Time in 100 μM GABA (s) subtype 0.2 1 5 15 25

94 ± 0.8 77 ± 2.7 55 ± 3.9 37 ± 4.5 27 ± 3.8 α1β2γ2 n = 21 n = 24 n = 23 n = 19 n = 16

96 ± 2.6 87 ± 3.3 80 ± 6.7 72 ± 10.9 70 ± 4.7 α1(K278M)β2γ2 n = 8 n = 13 n = 7 n = 5 n = 8

Statistics NS p < 0.05 p < 0.01 p < 0.01 p < 0.0001

Table 1. Summary of the desensitization induced using the rapid application protocol. The mutant α1(K278M)β2γ2 receptor exhibited significantly lower desensitization than the wild type α1β2γ2 subtype. Lower n numbers for the mutant α1(K278M)β2γ2 receptor resulted from non-saturating efficacy of 100 μM GABA. Data are presented as mean ± SEM of % control of GABA-evoked current recorded at the start of the experiment. Statistics comparing the wild-type α1β2γ2 and mutant α1(K278M)β2γ2 receptor at each time point were obtained using Student’s t-test.

116

Accessibility to the TBPS binding site in desensitized α1β2γ2 receptors

35 Picrotoxin competitively displaces [ S]TBPS binding from GABAA receptors

(Luddens and Korpi, 1993; Sinkkonen et al., 2001). Earlier, I demonstrated that peak

blockade by PIC (100 μM) was unaffected by increased desensitization of wild type

α1β2γ2 and mutant α1(K278M)β2γ2 receptors. I sought to use the same rapid agonist

application protocol described earlier to directly compare the relationship between

desensitization and TBPS blockade of α1β2γ2 receptors.

I used a maximally efficacious concentration of TBPS (10 μM) to induce

blockade of α1β2γ2 receptors. On the basis of the [35S]TBPS binding data which indicated failure of binding at a high GABA (100 μM) concentration, I anticipated that

TBPS would be unable to access its binding site in desensitized receptors. Consistent with previous experiments using PIC (Fig. 16A), increasing the duration of GABA application rendered a greater proportion of receptors desensitized, as shown by the decrease in current amplitude after 25 s compared to shorter durations of activation by

100 μM GABA (Fig. 19A). Blockade by TBPS was determined by extrapolating the current remaining after 5 s of exposure of desensitized receptors to GABA and TBPS simultaneously (100 μM and 10 μM, respectively) as described earlier. As the duration of exposure to GABA increased, there was a trend towards a reduction in blockade (92 ± 1.8

% to 76 ± 8.9%, at 0.2 s and 25 s, respectively) caused by GABA induced desensitization

(Fig. 19B), however, the reduction was not statistically significant (Student’s t-test comparing each time point to blockade obtained after 0.2 s in GABA, n ≥ 5).

117

Figure 19 α1β2γ2 A B 100 100 μM GABA + 10 μM TBPS 100 μM GABA 90 200 ms 80

70 60 50

100 μM GABA + 10 μM TBPS % Inhibition 40 100 μM GABA 1 s 30 20 10 0

100 μM GABA + 10 μM TBPS 1 s 5 s 0.2 s 25 s 100 μM GABA 15 s 200 pA 5 s 2 s

100 μM GABA + 10 μM TBPS 100 μM GABA 15 s

100 μM GABA + 10 μM TBPS 100 μM GABA 25 s

Figure 19. Blockade of desensitized α1β2γ2 receptors by 10 μM TBPS. A. The duration of rapid GABA (100 μM) application varied from 0.2 to 25 s to induce desensitization. As the time in GABA increased, desensitization also increased (bottom trace). Blockade by TBPS (10 μM) was measured by presenting the fraction of current remaining after 5 s of TBPS application in the presence of GABA (100 μM) as a percentage of current remaining after desensitization. B. Increased desensitization caused an apparent decrease in blockade by 10 nM TBPS (92 ± 1.8 to 76 ± 8.9 %). However, this decrease was not statistically significant (Student’s t-test). Data are presented as mean ± SEM. n ≥ 5 cells.

118

The effects of desensitization on blockade by non-saturating concentrations of TBPS in

α1β2γ2 receptors

In binding assays such as those performed for Chapter 3 and 5 of my dissertation,

I used concentrations of [35S]TBPS ranging from 6 to 50 nM, which represent a value

close to the steady-state binding affinity for GABAA receptors (Squires et al., 1983,

Maksay and Simoni, 1986). Binding assays took place after a prolonged exposure of 90

minutes. In contrast, blockade was assessed using a maximally efficacious concentration

of TBPS (10 μM) for α1β2γ2 receptors according to my concentration-response

relationship established after much shorter periods of exposure to GABA (ms - s) and

TBPS (minutes). Concentrations of TBPS that reflect the steady-state binding affinity for

α1β2γ2 receptors were not saturating in the electrophysiological assay and caused

minimal blockade (Fig. 5A). In an attempt to more closely mimic the TBPS binding site

occupancy achieved in my binding assay I chose to examine two lower concentrations, 1

μM and 100 nM, ~IC75 and ~IC25, respectively. I hypothesized that the effects of

desensitization on non-saturating concentrations would more accurately describe the

relationship between receptors bound to [35S]TBPS at high concentrations of GABA.

As predicted by the concentration-response relationship for blockade by TBPS, 1

μM TBPS caused considerably less block than did 10 μM TBPS (Fig. 20 compared to

Fig. 19). Block by TBPS was determined by extrapolating the current remaining after 5 s of exposure of desensitized receptors to GABA (100 μM) and TBPS (10 μM) simultaneously as described earlier. As duration of exposure to GABA increased, block by 1 μM TBPS appeared maximal at 0.2 s and declined after 15 s of GABA exposure,

119

from 76 ± 2.9 to 68 ± 4.3 %, respectively (Fig. 20B). Even at these two points, the

apparent reduction of block by TBPS (1 μM) associated with more profound

desensitization lacked statistical significance (Student’s t-test, n ≥ 4).

I also examined blockade by 100 nM TBPS (Fig. 21A). This concentration of

TBPS used in my electrophysiological experiments is closest to that used in radioligand

binding. Using the rapid agonist application protocol described earlier, I found that 100

nM TBPS caused 42 ± 5% inhibition of GABA-evoked currents after 0.2 s of activation

by 100 μM GABA. As the duration of GABA application increased, desensitization also

increased and this was associated with a significant reduction in the inhibition measured,

from 42 ± 4.7% to 23 ± 4 % (Fig. 21B, n ≥ 3, P < 0.05, Student’s t-test). Inhibition by

100 nM TBPS was often difficult to distinguish from desensitization by GABA. Figure

21A shows an expansion of the recording trace showing recovery from block by 100 nM

TBPS after 5s of desensitization. Unbinding of TBPS from the channel restored the

current to the amplitude available after desensitization by GABA. Taken together these

data imply that the potency of TBPS at α1β2γ2 receptors is affected by desensitization.

Furthermore, the data support the idea that the displacement of [35S]TBPS by GABA in binding assays is due to desensitization (Fig. 8). Figure 21C is a graphical summary of blockade by the three concentrations of TBPS examined, 100 nM, 1 μM and 10 μM after either 0.2 s (black bars) or 25 s (gray bars) of exposure to 100 μM GABA. The effects of desensitization were significant for 100 nM, not other concentrations.

120

Figure 20 α1β2γ2 1 μM TBPS 100 A B

80 100 μM GABA + 1 μM TBPS 100 μM GABA 60 200 ms

40 100 μM GABA + 1 μM TBPS % Inhibition 100 μM GABA 1 s 20

100 μM GABA + 1 μM TBPS 0 100 μM GABA 0.2 s 1 s 2 s 25 s 5 s 1 nA 15 s

2 s 100 μM GABA + 1 μM TBPS 100 μM GABA 15 s

100 μM GABA + 1 μM TBPS 100 μM GABA 25 s

Figure 20. Blockade of desensitized α1β2γ2 receptors by 1 μM TBPS. A. The duration of rapid GABA (100 μM) application varied from 0.2 to 25 s to induce steady state desensitization. As the time in GABA increased, desensitization also increased, until receptors reached steady-state (bottom trace). Blockade by TBPS (1 μM) was measured by presenting the fraction of current remaining after 5 s of TBPS application in the presence of GABA (100 μM) as a percentage of current remaining after desensitization. B. Increased desensitization by GABA caused steady blockade of approximately 70-75 %. Data are presented as mean ± SEM. n ≥ 4 cells.

121

Figure 21 α1β2γ2 A B 100 μM GABA + 100 nM TBPS 50 100 μM GABA 200 ms 40

100 μM GABA + 100 nM TBPS 30 100 μM GABA 1 s 20 % Inhibition Inhibition % 1 nA 10 100 μM GABA + 100 nM TBPS 2 s 100 μM GABA 0 5 s 1 s 2 s 0.2 s 25 s 25 15 s 15

C 0.2 s 25 s

100

40 % Inhibition 100 μM GABA + 100 nM TBPS 100 μM GABA 20 15 s 0 100 nM 1 μM 10 μM [TBPS] 100 μM GABA + 100 nM TBPS 100 μM GABA 25 s

Figure 21. Blockade of desensitized α1β2γ2 receptors by 100 nM TBPS. A. The duration of rapid GABA (100 μM) application was increased from 0.2 to 25 s to induce steady state desensitization. As the time in GABA increased, the level of desensitization increased until receptors reached steady-state (bottom trace). Blockade by TBPS (100 nM) was measured by presenting the fraction of current remaining after 5 s of TBPS application in the presence of GABA (100 μM) as a percentage of current remaining after desensitization. The expanded trace depicts the recovery from blockade by 100 nM TBPS after 5 s of GABA application. B. Increased desensitization by GABA caused a significant decrease in blockade by 100 nM TBPS at 25 s compared to 0.2 s (92 ± 1.8 to 76 ± 8.9 %). C. Graphical summary of blockade of GABA-evoked currents by TBPS using rapid agonist application and the whole cell patch clamp technique. (* p < 0.05, Student’s t-test).Data are presented as mean ± SEM. n ≥ 5 cells. 122

Blockade of mutant α1(K278M)β2γ2 receptors by TBPS

Using the steady-state protocol I described earlier in this chapter, I observed significantly less steady-state desensitization of α1(K278M)β2γ2 receptors by GABA

(100 μM), as evidenced by the dextral shift in potency of desensitization by GABA (Fig.

15B). I examined blockade by 100 nM TBPS at mutant α1(K278M)β2γ2 receptors to further explore the role of desensitization on blockade by non-saturating concentrations of TBPS. I hypothesized that the reduced potency of desensitization would minimize the effects of desensitization on blockade by TBPS (100 nM). GABA (100 μM) caused significantly less desensitization of α1(K278M)β2γ2 receptors compared to α1β2γ2 receptors (Fig. 22A, Table 1). Block by TBPS was determined by extrapolating the current remaining after 5 s of exposure of receptors to GABA and TBPS simultaneously

(100 μM and 100 nM, respectively) as described earlier. An expanded trace in Figure

22A emphasizes the blockade of α1(K278M)β2γ2 receptors by 100 nM TBPS. These data were quantified as described earlier and presented in Figure 22B and compared to the effects of 100 nM TBPS at α1β2γ2 receptors in Figure 22C.

Desensitization was associated with an apparent reduction of blockade by 100 nM

TBPS from 18 ± 5.2 to 11 ± 3.7 % (Fig.22B), but this was not significant, suggesting that desensitization of α1(K278M)β2γ2 receptors by 100 μM GABA did not affect accessibility to the TBPS binding site within the channel. Taken together, these data confirm that receptors exhibiting reduced sensitivity to desensitization are more susceptible to blockade by TBPS.

123

Figure 22 α1(K278M)β2γ2 A B 100 μM GABA + 100 nM TBPS 30 100 μM GABA 200 ms 20 200 pA 10

2 s % Inhibition 100 μM GABA + 100 nM TBPS 100 μM GABA 1 s 0 0.2 s 1 s 2 s 25 s 15 s

100 μM GABA + 100 nM TBPS 100 μM GABA C 5 s 50

0.2 s 40 25 s 30

% Inhibition 20

0 α1β2γ2 α1(K278M)β2γ2 100 μM GABA + 100 nM TBPS 100 μM GABA 15 s

100 μM GABA + 100 nM TBPS 100 μM GABA 25 s

Figure 22. Blockade of desensitized α1(K278M)β2γ2 receptors by 100 nM TBPS. A. The duration of rapid GABA (100 μM) application varied from 0.2 to 25 s to induce steady state desensitization. As the time in GABA increased, desensitization also increased as receptors approached steady-state (compare upper traces to bottom trace). Blockade by TBPS (100 nM) was measured by presenting the fraction of current remaining after 5 s of TBPS application in the presence of GABA (100 μM) as a percentage of current remaining after desensitization. The expanded trace depicts the recovery from blockade by 100 nM TBPS after 5 s of GABA application . B. Increased desensitization by GABA had no significant decrease in blockade by 100 nM TBPS. C. Graphical summary depicting blockade of α1β2γ2 and α1(K278M)β2γ2 receptors by 100 nM TBPS. In both subtypes, there was a trend towards a reduction of % inhibition. Data are presented as mean ± SEM. n ≥ 5 cells (* p < 0.05, Student’s t-test).

124

The potency of blockade by TBPS is reduced in α1(K278M)β2γ2 receptors

As described earlier, 100 nM TBPS caused less blockade of α1(K278M)β2γ2

receptors than α1β2γ2 receptors. I examined the concentration-response relationship for

inhibition by TBPS using the whole cell patch clamp technique. α1β2γ2 or

α1(K278M)β2γ2 receptors were transiently expressed in HEK293 cells. As described

earlier in Chapter 3 (Fig. 5), I activated receptors with locally applied GABA (100 μM)

and bath applied various concentrations of TBPS (0.01 to 10 μM). Inhibition by TBPS

was determined by expressing the current remaining in the presence of TBPS as a

percentage of the maximal current evoked by GABA (Fig. 23). TBPS caused a

concentration dependent inhibition in both α1β2γ2 and α1(K278M)β2γ2 receptors, with

IC50 values of 0.33 ± 0.05 and 0.57 ± 0.06 μM, respectively. The potency of TBPS was significantly reduced by the mutant α1(K278M) subunit (* p < 0.05, *** p < 0.001,

Student’s unpaired t-test, n ≥ 4). At 100 nM TBPS, blockade of α1β2γ2 and

α1(K278M)β2γ2 receptors caused 25 ± 1.07 % and 9 ± 5.4 % inhibition, respectively.

125

Figure 23

120

100 α1β2γ2 α1(K278M)β2γ2

M ) 80

60 GABA (100 μ

% control I 20

0.003 0.01 0.1 1 10 [TBPS] (μM)

Figure 23. Blockade by TBPS of GABA-evoked currents recorded from wild type α1β2γ2 (●) or mutant α1(K278M)β2γ2 (○) GABAA receptors. A. Concentration- inhibition curves for TBPS. Whole-cell currents recorded from α1β2γ2 receptors expressed in HEK293 membranes were activated by GABA (100 μM). GABA-evoked current amplitudes recorded in the presence of TBPS (n ≥ 4) were expressed as a percentage of those recorded under control conditions (% IGABA). TBPS caused a slightly less potent inhibition of GABA-evoked currents in mutant α1(K278M)β2γ2 receptors compared to wild type α1β2γ2. IC50 values for TBPS were 0.33 ± 0.05 μM and 0.57 ± 0.05 μM for α1β2γ2 or α1(K278M)β2γ2 respectively. Data are represented as mean ± S.E.M. of n ≥ 4 recordings. (* p < 0.05, *** p < 0.001, Student’s unpaired t-test)

126

In turn, 100 nM was roughly the IC25 of α1β2γ2 receptors, but only an the IC10 of

α1(K278M)β2γ2 receptors. The difference in potency of TBPS at these GABAA receptor complicates the interpretation of data obtained using rapid agonist application to

α1(K278M)β2γ2. The data obtained from α1β2γ2 receptors described earlier in this chapter support the hypothesis that desensitization significantly reduces accessibility to the TBPS binding site. However, the effects of reduced sensitivity of α1(K278M)β2γ2 receptors to desensitization by 100 μM GABA on blockade by TBPS (100 nM) require further investigation. I will discuss potential future directions of this project in Chapter 6.

127

Conclusions

The data presented in this chapter suggest that desensitization significantly reduces accessibility to the TBPS binding site causing a reduction in the potency but not efficacy of block. Using the whole cell patch clamp technique, I established the concentration dependence of desensitization of wild type α1β2γ2 and mutant

α1(K278M)β2γ2 and α1β2γ2(K289M) receptors. These TM2-TM3 mutations caused significantly reduced potency of desensitization by GABA (Fig. 15B and 16B). The dextral shifts in the concentration-response relationships resulted in an increase in the fractional availability of current. I described fractional availability as the relative current that was conducted by a mixed population of activated, closed and desensitized receptors.

I hypothesized that these increases in fractional availability of current corresponded with the increased binding of [35S]TBPS to Lys-to-Met mutant receptors at

high concentrations of GABA (> 1 μM). Using rapid agonist application of GABA and

PIC or TBPS, I established the sensitivity of each noncompetitive ligand to

desensitization by 100 μM GABA. Blockade by PIC seemed to be unaffected by

increasing levels of desensitization, demonstrating that the PIC binding site remained

available despite the decrease in fractional availability to the channel caused by

desensitization (Fig. 17 and 18). These data indicate that the PIC binding site may

involve residues lying above the proposed desensitization gate (9’). Some studies have

shown that mutations in the upper TM2 region also reduce potency/efficacy of PIC,

however it is not understood if these residues participate directly in the PIC binding site

128

or simply influence accessibility by determining gating kinetics (Dibas et al., 2002,

Mortenson et al., 2003). At this time, a secondary binding site for picrotoxin in the upper

TM2 domain is not universally accepted throughout the GABAA receptor field.

Since the main goal of my dissertation was to describe the role of different

35 GABAA receptor channel properties in the differential binding of [ S]TBPS to

functionally distinct GABAA receptor subtypes, I examined the effects of desensitization

on three concentrations of TBPS in both wild type α1β2γ2 receptors and mutant

α1(K278M)β2γ2 receptors. I focused on the α1(K278M)β2γ2 receptor because the

mutation in the α1 subunit has the most substantial impact on [35S]TBPS binding

(Chapter 3, Fig. 12C). Compared to block by 10 μM (Fig 19) or 1 μM TBPS (Fig 20),

block by 100 nM TBPS (Fig 21) was significantly reduced by desensitization of α1β2γ2

receptors. This most closely corresponds to the low nM concentrations (6 to 50 nM for my assays) of [35S]TBPS used in binding assays where receptors undergo prolonged

exposures to GABA.

Furthermore, the reduced desensitization of α1(K278M)β2γ2 receptors by 100

μM GABA (Table 1) was associated with a similar block by TBPS (100 nM) after short

and long durations of GABA application (Fig. 22). TBPS (100 nM) caused approximately

20% inhibition of α1(K278M)β2γ2 receptors compared to ~ 40% inhibition of α1β2γ2

receptors. Concentration-response relationships for inhibition by TBPS revealed that the

potency of blockade was slightly reduced in α1(K278M)β2γ2 receptors (Fig. 23). This

may account for the spread in data depicted in Figure 22, since smaller changes in current

amplitude are more difficult to accurately detect. In future experiments, it would be

129

beneficial to compare the effects of desensitization with equally effective concentrations

of TBPS.

In the future, it would be interesting to see if desensitization caused by a

saturating concentration of GABA (3 mM) has any effects on blockade by either TBPS or

PIC. Nonetheless, the data described in this chapter are consistent with the hypothesis that desensitization reduces the accessibility to the TBPS binding site in wild type

α1β2γ2 receptors. In the next chapter, I will describe the experiments I conducted to

discriminate between functionally distinct GABAA receptor subtypes on the basis of

differential [35S]TBPS binding.

130

Chapter 5: Results 35 [ S]TBPS binding to identify functionally distinct GABAA receptors

Background and Significance

The distribution of GABAA receptor subunits in the CNS is diverse (Korpi and

Luddens, 1993; Pirker et al., 2000). Several groups have examined the regional

distribution of different GABAA receptor subtypes to determine if certain subunits are localized to distinct brain regions using subunit selective ligands, such as the benzodiazepines described in Chapter 1 (Rudolph and Mohler, 2004). Much of what we know about GABAA receptor distribution in the CNS is based on in situ hybridization

(mRNA studies) and antibody immunohistochemistry (Pirker et al., 2000; Sinkkonen et

al., 2000; Moragues, et al., 2002; Moragues et al., 2003; Sergeeva et al., 2005).

However, the disadvantage to both of these techniques is that they do not identify

functional receptors, but instead, suggest possible receptor subtypes. In situ hybridization

provides evidence for mRNA transcripts encoding subunits that may or may not be

expressed functionally, while antibodies are often not specific enough to distinguish

between subunits within a similar class, e.g. between the α1-6 subunits. A good example

of this is the often-used bd17 monoclonal antibody that recognizes both the β2 and the β3

subunit, making it difficult to distinguish between them in the CNS (Terai et al., 1998;

Zhang et al., 2003).

The radioligand [35S]TBPS has been used extensively to localize regions of the

brain that contain GABAA receptors (Squires et al., 1983; Korpi & Luddens, 1993). The

131

advantage to using [35S]TBPS as a tool is that it provides the ability to screen all brain

regions for GABAergic activity. Many other GABAA receptor specific ligands bind at the

extracellular domain, often at the orthosteric binding site ([3H]GABA, [3H]muscimol,

[3H]SR95531) or at the benzodiazepine binding site (e.g. [3H]zolpidem, [3H]diazepam,

[3H] Ro 15-1788) (Wingrove et al., 1997; Renard et al., 1999; Kloda & Czajkowski,

2007; Da Settimo et al., 2007). These ligands have often been used to localize receptor

populations for different agonist affinity or benzodiazepine subtype selectivity. In

contrast, [35S]TBPS, which is thought to bind in the TM2 domain when it has access to

the GABAA receptor channel (Jursky, 2000; Chen et al., 2006), provides a tool to target functionally active GABAA receptors depending on their channel activity. As described

earlier in Chapters 1 and 3, picrotoxin (PIC) competitively displaces [35S]TBPS from its

binding site. Throughout this chapter, I will describe [35S]TBPS experiments in the

context of picrotoxin-displaceable (control) binding. Also in Chapter 1, I described

GABA induced inhibition of [35S]TBPS binding at high concentrations (Fig. 8).

However, this displacement inhibition is not due to direct competition for binding, but

instead, is dependent on the efficacy of GABA as an activator of channel activity leading

to altered accessibility of the TBPS binding site in the TM2 domain.

As described in detail in Chapters 3 and 4, accessibility to the TBPS binding site

is significantly reduced when spontaneous, GABA-independent gating is reduced (Fig

10-11), as well as during desensitization (Fig. 21). Based on these findings, I proposed to

35 use [ S]TBPS as a tool to detect functionally distinct GABAA receptors in the CNS.

Furthermore, I hypothesized that the agonist-induced enhancement of [35S]TBPS binding

often observed in the presence of agonists was due to increased accessibility to the 132

channel. In contrast, noncompetitive displacement was due to decreased accessibility to

the channel by reduced spontaneous gating (Chapter 3) or channel constriction during

desensitization (Chapter 4). Taken together, these data demonstrated that the pattern of

modulation of [35S]TBPS binding by different ligands should provide insight into the

different gating properties of functionally distinct GABAA receptors throughout the CNS.

Throughout this chapter, I will describe a series of [35S]TBPS radioligand binding

assays examining both intact rodent brain slices (using autoradiography), as well as

homogenized isolated brain regions and transiently expressed recombinant receptors.

Drugs that target specific receptor subtypes (e.g. benzodiazepines) are often used in

conjunction with [35S]TBPS to screen for regions high in a given subunit or series of

subunit combinations (Makela et al., 1997; Sinkkonen et al., 2001; Halonen et al., 2009).

Each of these techniques provided an advantage in screening for functionally distinct

GABAA receptors. Autoradiography enabled me to examine intact receptors in their native environment, while isolating brain regions in homogenate assays allowed for manipulation of the microenvironment. In contrast, using recombinant receptors allowed me to target subtypes that might not be easily detected in the whole brain, as well as more common subtypes in the absence of endogenous modulators of GABAA receptor

function.

The modulation of TBPS binding by GABA is complex and is not well

understood. The data presented earlier in this dissertation have provided greater insight

into the mechanisms of action of TBPS at different GABAA receptor subtypes. Using this

knowledge, I employed [35S]TBPS binding as a tool to detect functionally distinct

GABAA receptors throughout the rodent brain. 133

Experimental Results

Detecting the functionally unique ε subunit using [35S]TBPS binding

As mentioned earlier in Chapter 1, the GABAA receptor auxiliary subunits include

the δ, γ or ε subunit. The ε subunit is sparsely distributed in the CNS and its role is less

understood compared to the δ or γ subunits. The recombinant human and rodent isoforms

of the ε subunit have been examined (Davies et al., 2002), but to date, little is known

about the localization or functional incorporation of the ε subunit into GABAA receptors

throughout the CNS. Upon incorporation of the ε subunit into recombinant receptors, the

affinity for GABA increases compared to α1β3γ2 receptors (Whiting et al., 1997;

Maksay et al., 2003) as does the level of tonic, agonist-independent current (Whiting et

al., 1997; Neelands et al., 1999; McCartney et al., 2007). In situ hybridization and

immunohistochemistry studies reveal that the ε subunit is enriched in the locus coeruleus,

hypothalamus and thalamus (Sinkkonen et al., 2000, Moragues et al., 2002; Moragues et al., 2003, Sergeeva et al., 2005).

In my experiments, I sought to distinguish between GABAA receptors containing

or lacking the ε subunit based on their affinities for GABA and sensitivity to inhibitors of

spontaneous, GABA-independent gating, such as the neurosteroid pregnenolone sulfate

and the antidiuretic, furosemide. Both ligands inhibit spontaneous current mediated by ε subunit containing receptors (Maksay et al., 2003; McCartney et al., 2007), but through

different means. Pregnenolone sulfate (PS) inhibits GABA-evoked currents by binding

primarily to the TM1 domain of GABAA receptor α or β subunits (Hosie et al., 2009; 134

Baker et al., 2010). Some studies have also implicated a role for residues in the TM2

domain in neurosteroids binding (Hosie et al., 2006) Furthermore, mutations in the TM1

of the β2 subunit decrease spontaneous gating and sensitivity to inhibition by PS or

furosemide (Baker et al., 2010). Furosemide more potently inhibits GABA-evoked currents at α1βε receptors compared to α1βγ receptors (Neelands et al., 1999), but its

effects on spontaneous, tonic current have only been shown using recombinant receptors

containing the ε subunit (Maksay et al., 2003; McCartney et al., 2007). The exact

mechanism of inhibition of GABAA receptor activity by furosemide is has not been

established. Based on these studies, I used pregnenolone sulfate and furosemide to inhibit

[35S]TBPS binding mediated by spontaneous, agonist independent gating in an attempt to

identify the αxβxε receptor combinations in situ.

As described in Chapter 3, the reduction of spontaneous gating by either the

inverse agonist BIC or the mutant subunit α1(K278M) caused a significant reduction in

GABA-independent binding of [35S]TBPS (Fig. 10-11). The ε subunit attributes a higher level of spontaneous gating compared to other receptor subtypes. I hypothesized that inhibition of spontaneous gating dependent on the ε subunit would localize regions high in αβε receptors in the rodent CNS. Since the tonic current evoked from αβε receptors is resistant to BIC (McCartney et al., 2007), I focused on pregnenolone sulfate and furosemide.

First, I examined the effects of these inhibitors on [35S]TBPS binding to

recombinant α1β3ε receptors to determine if the higher level of spontaneous gating

caused by the presence of the ε subunit could be detected. I hypothesized that inhibiting

135

spontaneous gating evoked by the ε subunit would inhibit [35S]TBPS binding in the same

manner that the inverse agonist BIC reduced [35S]TBPS binding to α1β2γ2 receptors

(Fig. 7). Both pregnenelone sulfate and furosemide caused a concentration dependent

reduction of [35S]TBPS binding in the absence of GABA (Fig 24). Pregnenolone sulfate

(Fig. 24A) caused a more potent reduction in [35S]TBPS binding than furosemide (Fig.

24B). A maximum reduction of ~50% was obtained in the presence of either ligand. I

hoped that by using these inhibitors, I would be able to identify brain regions expressing

αβε receptors. Since pregnenolone sulfate caused a more potent inhibition of [35S]TBPS binding to recombinant α1β3ε receptors than furosemide, I chose this ligand to identify spontaneous ε subunit-containing receptors in the rodent CNS.

In my experiments using recombinant receptors, I examined the human ε subunit, which is more thoroughly described than the rodent ε subunit isoforms. Two isoforms of the ε subunit exist in the rodent brain (Davies et al., 2002). It is unclear whether rodent ε subunits incorporate into functional receptors in vivo. Some studies have localized mRNA for both of these isoforms in the locus coeruleus (Sinkkonen et al., 2000). I chose to examine sagittal and coronal brain slices (16 μM) using [35S]TBPS autoradiography to determine if brain regions containing functional αβε receptors could be localized on the basis of their pregnenolone sulfate sensitive spontaneous gating. I chose to examine both sagittal and coronal slices. The latter was used in an attempt to identify the locus coeruleus.

[35S]TBPS binding to homogenized brain regions provides the advantage of

isolating individual brain nuclei and manipulating the microenvironment surrounding 136

GABAA receptors. In contrast, radioligand autoradiography is a useful technique because

it provides a means of monitoring brain regions in situ. This is especially advantageous

when examining GABAA receptors whose localization in the brain is not well known.

Since one of the main goals of the experiments outlined in this chapter was to develop

35 [ S]TBPS as a tool to detect functionally distinct GABAA receptors, I compared

[35S]TBPS binding to homogenized brain regions, whole brain slices, and recombinant

receptors transiently expressed in HEK293 cells. For most [35S]TBPS autoradiography

experiments outlined in this dissertation, I chose to examine sagittal sections (14 or 16

μM thick) in order to capture the largest number of brain regions in a single brain slice.

Figure 25 summarizes the effects of pregnenolone sulfate (PS) on [35S]TBPS

binding to the frontal cortex (FC), locus coeruleus (LC), hippocampus (H) and

cerebellum (CRB). Representative autoradiographs are presented in Figure 25A and B.

Pregnenolone sulfate caused minimal modulation of [35S]TBPS binding to the frontal cortex and resulted in a ~20% reduction in [35S]TBPS binding in the locus coeruleus (Fig.

25C). In contrast, PS caused a concentration-dependent reduction in [35S]TBPS binding

to both the hippocampus and cerebellum (Fig. 25D). These data were surprising, since I

was expecting to see modulation of receptors containing the ε subunit in the locus

coeruleus, but not other brain regions. However, pregnenolone sulfate is a neurosteroid,

and as such, its binding site lies mainly within the TM1 and TM2 domain of the GABAA receptor. Therefore it is likely that pregnenolone sulfate is acting at receptors lacking the

ε subunit (Chung et al., 1999).

137

Figure 24

A B 120 150 100

80 100 60

40 % control binding 50 % control binding 20

0 0 0.03 0.1 1 10 100 300 0.1 1 10 100 1000

[Pregnenolone Sulfate] (μM) [Furosemide] (μM)

Figure 24. Modulation of [35S]TBPS binding to recombinant α1β3ε receptors by inhibitors of spontaneous gating. A. Pregnenolone sulfate caused a concentration dependent reduction of [35S]TBPS binding. B. Furosemide caused a concentration dependent reduction of [35S]TBPS binding at high concentrations (> 30 μM). The reduction of [35S]TBPS binding by both ligands (within the concentrations range employed) reached a maximum of ~50% of control binding. Non-specific binding was determined in the presence of 100 μM PIC. Data are presented as mean ± SEM of n ≥ 4 independent experiments performed in triplicate.

138

Figure 25 A B

H 35 FC CRB Total [ S]TBPS binding

100 μM PIC

LC 100 μM PS

Frontal Cortex D C 120 Locus coeruleus 150 Hippocampus 100 Cerebellum 80 100 60

40 % control binding control % 50 20 % control binding control % 0 1 10 100 0 1 10 100 [Pregnenolone sulfate] (μM) [Pregnenolone sulfate] (μM) Figure 25. Modulation of [35S]TBPS autoradiography by pregnenolone sulfate (PS). Representative images of [35S]TBPS autoradiography to sagittal (A) or coronal (B) rodent brain slices. The regions of interest emphasized are the frontal cortex (FC), hippocampus (H), cerebellum (CRB) and locus coeruleus (LC). C. Pregnenolone sulfate caused minimal modulation of [35S]TBPS binding to the frontal cortex. ~20% of [35S]TBPS binding to the locus coeruleus was lost in the presence of high concentrations of PS (> 10 μM). D. PS caused a concentration dependent reduction in [35S]TBPS binding to hippocampal and cerebellar receptors. Data are presented as mean ± SEM of four independent experiments. Non-specific binding was determined in the presence of 100 μM PIC. 139

It is possible that pregnenolone sulfate might render the [35S]TBPS binding site

inaccessible, regardless of whether the channel gated spontaneously in the absence of

GABA. Furthermore, the locus coeruleus was difficult to locate in coronal slices due to

its small size and resemblance to other brainstem nuclei in the region. These confounding

factors led me to focus on other GABAA receptor subtype specific functional properties

that may be more readily detectable using [35S]TBPS binding.

In addition to spontaneous gating elicited by the GABAA ε subunit, I examined

the potency of GABA modulation of [35S]TBPS binding in recombinant α1β2γ2 or

α1β2ε receptors transiently expressed in HEK293 cells. GABA caused a biphasic

modulation of [35S]TBPS binding to both receptor subtypes (Fig. 26). Low concentrations of GABA enhanced [35S]TBPS binding to α1β2γ2 (< 1 μM) and α1β2ε (< 0.1 μM)

receptors. High concentrations of GABA reduced [35S]TBPS binding to α1β2γ2 (> 1 μM) and α1β2ε (> 0.1 μM) receptors. The incorporation of the ε subunit caused a sinistral shift in the potency of modulation by GABA (* p < 0.05, ** p < 0.01, Student’s unpaired t-test, n ≥ 3). These data are supported by earlier studies that demonstrated incorporation of the ε with α and β subunits into functional receptors increases the potency of GABA compared to α1β3γ2 receptors in Xenopus oocytes (Whiting et al., 1997; Maksay et al.,

2003).

140

Taken together, these data suggest that the presence of the ε subunit can be detected on the basis of GABA affinity using [35S]TBPS binding. It has already been

established that the affinity of GABAA receptors for GABA is also influenced by the

presence of the auxiliary γ and δ subunits. This is evident in the case of extrasynaptic αβδ

receptors that are thought to exert a higher affinity for GABA compared to synaptic αβγ

receptors (Farrant and Nusser, 2005). In my experiments, I sought to use [35S]TPBS to

detect functionally distinct GABAA receptors based on their affinity for GABA.

141

Figure 26

160

140

120

g α1β2γ2 100 α1β2ε 80

60 % control bindin % control

0.03 0.1 1 10 100

[GABA] (μM)

Figure 26. GABA modulation of [35S]TBPS binding in recombinant α1β2γ2 or α1β2ε transiently expressed in HEK293 cells. GABA causes a more potent modulation of [35S]TBPS binding in α1β2ε receptors compared to the α1β2γ2 subtype. In both subtypes, low concentrations of GABA (< 1 μM for α1β2γ2 or < 0.1 μM for α1β2ε) enhanced [35S]TBPS binding, while high [GABA] displaced [35S]TBPS binding. Complete displacement of [35S]TBPS binding occurred at 100 μM GABA in both subtypes. Non-specific binding was determined in the presence of 100 μM PIC. Data are presented as mean ± SEM of n ≥ 3 independent experiments performed in triplicate. * p < 0.05, ** p < 0.05, Student’s t-test.

142

GABA modulation of [35S]TBPS binding to different brain regions

Hippocampi, frontal cortices and cerebella were isolated from adult male

Sprague-Dawley rats and homogenized as described in Chapter 2. As expected,

[35S]TBPS bound in a picrotoxin-displaceable (100 μM) manner in the absence of exogenous GABA (Fig. 26). Addition of increasing concentrations of GABA primarily caused a concentration-dependent reduction of [35S]TBPS binding. Low concentrations of

GABA caused a modest enhancement of [35S]TBPS binding to hippocampal and cortical

membranes. I hypothesized that the marked enhancement of [35S]TBPS binding observed

in experiments using recombinant receptors (Fig. 5) is absent in brain homogenates due

to the presence of endogenous GABA in the latter that is not completely removed during the preparation and washing of membranes. The reduction of [35S]TBPS binding by

GABA was most potent in the cerebellum compared to the other brain regions examined

(* p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA with Tukey’s post-hoc test, n

≥ 4 experiments performed in triplicate).

Fitting the data with a logistic equation gave IC50 values for inhibition of

35 [ S]TBPS binding by exogenous GABA. The IC50 values were 2.0 ± 0.2, 1.9 ± 0.1 and

1.1 ± 0.2 μM for the frontal cortex, hippocampus and cerebellum, respectively (Table 2).

The Hill slopes (nH), which are influenced by the cooperativity of GABA binding, are

also listed in Table 2. The nH values were 2.6 ± 0.4, 1.4 ± 0.1 and 1.0 ± 0.1 for the frontal

cortex, hippocampus and cerebellum, respectively.

Consistent with my findings, the cerebellum is thought to contain a population of

high-affinity, extrasynaptic GABAA receptors containing the δ subunit (Nusser et al.,

143

1996; Pirker et al., 2000, Moragues et al., 2002, Halonen et al., 2009). I hypothesized that the higher potency of GABA modulation of [35S]TBPS binding to the cerebellum was due of the presence of these high affinity GABAA receptors in the cerebellum.

Furthermore, as mentioned above there was a slight enhancement of [35S]TBPS binding to membranes of hippocampi and frontal cortices at low concentrations of GABA (< 1

μM). This is smaller than the enhancement of [35S]TBPS binding observed in experiments examining [35S]TBPS binding to recombinant receptors in Chapter 3 (Fig. 8) as well as by others (Korpi and Luddens, 1995, Davies et al., 1997b). This enhancement of [35S]TBPS binding was not seen in the cerebellum (Fig. 27). These observations are consistent with the idea that low levels of endogenous GABA remaining in the cerebellar homogenates allow [35S]TBPS to bind maximally to high affinity receptors in the absence of added exogenous GABA.

144

Figure 27

125

Cerebellum 100 Frontal Cortex

75 Hippocampus

50 % control binding

0.003 0.01 0.1 1 10 100 [GABA] (μM)

Figure 27. GABA caused a concentration dependent modulation of [35S]TBPS binding in homogenized brain regions. Noncompetitive displacement of [35S]TBPS from the cerebellum occurred at lower concentrations of GABA than in the frontal cortex or hippocampus. In contrast, low concentrations of GABA (< 1 μM) enhanced [35S]-TBPS binding to receptors in the frontal cortex and hippocampus, but not cerebellum. Data are represented as mean ± S.E.M of n ≥ 4 independent experiments performed in triplicate. Statistics were obtained using one way ANOVA with Tukey’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to frontal cortex. Non-specific binding was determined in the presence of 100 μM PIC.

145

Table 2.

GABA

IC50 (μM) nH

Frontal Cortex 2.0 ± 0.2 2.6 ± 0.4

Hippocampus 1.9 ± 0.1 1.4 ± 0.1

Cerebellum 1.1 ± 0.1 1.0 ± 0.1

35 Table 2. IC50 values and Hill coefficients (nH) for the reduction of [ S]TBPS binding by GABA in the homogenized membranes of rodent frontal cortices, hippocampi or cerebella. GABA caused a more potent reduction of [35S]TBPS binding to the cerebellum. The nH values for each brain region demonstrate the presence of different receptor populations in each brain region, where receptors with high affinities for GABA were present in the cerebellum and hippocampus.

146

GABA modulation of [35S]TBPS binding established by autoradiography

Basal [35S]TBPS binding to various rodent brain regions occurs in the absence of exogenous GABA (Fig. 28A) and was competitively displaced by picrotoxin (100 μM,

Fig. 27B). However, binding occurs at different levels, as noted by the range of light or

dark intensities, presumably due to the distinct populations of receptors expressed

throughout the brain. GABA caused a concentration dependent reduction in [35S]TBPS

binding throughout the rodent brain (Fig. 28C). At different concentrations of GABA (0.1

to 100 μM), the distribution of [35S]TBPS binding varied in different brain nuclei. I

quantified the relative intensity of [35S]TBPS binding in the cerebellum, frontal cortex

and hippocampus from digitized film exposures of radiolabeled brain slices (as described

in Chapter 2).

The higher potency of GABA modulation of [35S]TBPS binding to cerebellar

receptors compared to those of hippocampus and cortex observed using homogenate

binding assays (Fig. 27) was not as obvious using autoradiography (Fig. 28). In contrast

to the data shown in Figure 27, [35S]TBPS autoradiography to cerebellar receptors was

only significantly different compared to cortical and hippocampal receptors at 1 μM

GABA (p < 0.01, one-way ANOVA with Tukey’s post-hoc test). Full noncompetitive

inhibition of [35S]TBPS binding occurred at 30 μM GABA.

147

Figure 28 A Basal binding B 100 μM PIC

H FC CRB

C Exogenous GABA

10 nM 10 μM

100 nM 100 μM

1 μM

Figure 28. Representative autoradiographs of [35S]TBPS binding to rodent brain slices in the sagittal plane. A. Basal binding of [35S]TBPS occurs independently of exogenous ligands. The distribution of basal binding varied across the different brain regions. B. 100 μM picrotoxin competitively displaced [35S]TBPS binding from all brain regions. C. Exogenous GABA caused a concentration dependent reduction in [35S]TBPS binding in all brain regions. 1 μM GABA significantly reduced [35S]TBPS binding in the cerebellum compared to other brain regions, presumably due to high affinity receptors expressed there. FC, frontal cortex; H, hippocampus; and CRB, cerebellum.

148

Figure 29

100 Cerebellum 80 Frontal Cortex Hippocampus

g 60

20 % control bindin

-20 0.01 0.1 1 10 100 [GABA] (μM)

Figure 29. Quantified binding values of [35S]TBPS autoradiography to sagittal cryostat sections of the adult rodent brain. Data are expressed as % control binding. GABA caused a concentration dependent reduction of [35S]TBPS binding in all brain regions. Of the three brain regions examined, 1 μM GABA significantly reduced binding to the cerebellum compared to other brain regions (one-way ANOVA with Tukey’s post-hoc test. ** p < 0.01 compared to other brain regions). Complete displacement of [35S]TBPS binding occurred in the presence of 30 μM GABA. Data are represented as mean ± S.E.M of n ≥ 3 independent experiments. Non-specific binding was determined in the presence of 100 μM picrotoxin.

149

The wash step in both types of assays was presumably critical for removal of

endogenous GABA that could modulate [35S]TBPS bound to different receptor subtypes.

The homogenization step may facilitate the removal of endogenous GABA from the

brain. Furthermore, analysis of autoradiographic data requires manual selection and

demarcation of brain regions of interest and the amount of tissue is limited by two-

dimensional nature of the assay. In contrast, homogenate assays utilizes material from

entire brain nuclei. These methodological differences could account for the greater

variability in the data shown in Figure 29 compared to Figure 27, however, the trend

across the three brain regions examined was similar for both assays. Taken together, both

assays demonstrated that [35S]TBPS binding can be used to identify receptors with high

affinity for GABA in the brain.

Later in this chapter, I will describe the experiments examining the abilities of

gabazine (GBZ) and bicuculline (BIC) (Chang et al., 1999), two well-known competitive

antagonists of GABAA receptors to compete with endogenous GABA for binding and

establish what effect this has on [35S]TBPS binding. I will also examine the contribution

of different GABAA subunits to high affinity receptors in the brain. In the next section, I

will examine the influence of propofol, an allosteric agonist at GABAA receptors, on

[35S]TBPS binding to different brain regions.

150

Propofol modulation of [35S]TBPS binding to brain membranes

Propofol is a widely used intravenous anesthetic that binds to GABAA receptors at an allosteric site within the TM2 domain (Bali and Akabas, 2004). In the presence of low concentrations of GABA, propofol potentiates GABA-evoked currents by increasing the probability of channel opening (Hales and Lambert, 1991). At higher concentrations propofol also directly activates GABAA receptors in the absence of ortheosteric agonist. I examined the effect of propofol applied in the absence of exogenous GABA on

[35S]TBPS binding to membrane preparations from rodent hippocampi, frontal cortices and cerebella. While some GABAA receptors are less affected by the potentiating actions of propofol (Davies et al., 1997b) direct activation appears to be unaffected by subunit composition. Therefore I anticipated that the potency and efficacy of propofol as a modulator of [35S]TBPS binding would be similar in the three brain regions tested.

Propofol caused a concentration-dependent reduction in [35S]TPBS binding to all three brain regions (Fig. 30). The three concentration-response relationships are effectively identical and the fit to the data points revealed a similar IC50 for the inhibition

35 of [ S]TBPS binding by propofol with overlapping 95% confidence intervals. The IC50 values for inhibition of [35S]TBPS binding by propofol were 23.0 ± 1.9, 26.4 ± 2.2 and

23.9 ± 1.4 μM for the frontal cortex, hippocampus and cerebellum, respectively. In addition to similar IC50 values, the Hill coefficient (nH) values were also indistinguishable across the three brain regions (Table 3). Taken together, these data demonstrate that the populations of receptors that bind [35S]TBPS in the frontal cortex, hippocampus and cerebellum have similar affinities for propofol.

151

Figure 30

A 120

100

Cerebellum 80 Frontal Cortex 60 Hippocampus

% control binding 40

0.3 1 10 100 [Propofol] (μM)

Figure 30. Propofol caused a concentration dependent reduction of [35S]TBPS binding in homogenized brain regions. No significant difference was observed between the three brain regions examined. Data are represented as mean ± S.E.M of n ≥ 4 independent experiments performed in triplicate. Non-specific binding was determined in the presence of 100 μM picrotoxin.

152

Table 3.

GABA Propofol

IC50 (μM) nH IC50 (μM) nH

Frontal Cortex 2.0 ± 0.2 2.6 ± 0.4 23.0 ± 1.9 2.7 ± 0.5

Hippocampus 1.9 ± 0.1 1.4 ± 0.1 26.4 ± 2.2 2.66 ± 0.5

Cerebellum 1.1 ± 0.1 1.0 ± 0.1 24.0 ± 1.4 2.56 ± 0.3

35 Table 3. IC50 values and Hill coefficients (nH) for the reduction of [ S]TBPS binding by GABA or propofol in the homogenized membranes of rodent frontal cortices, hippocampi or cerebella. GABA caused a more potent reduction of [35S]TBPS binding to the cerebellum. The Hill coefficients for each brain region demonstrate the presence of different receptor populations in each brain region: where receptors with higher affinities for GABA were present in the cerebellum and hippocampus. In contrast, no differences in the potency of modulation of [35S]TBPS binding by propofol were observed. The Hill coefficients for these experiments demonstrated that receptors in the frontal cortex, hippocampus and cerebellum shared similar affinities for propofol. (Values for GABA are included from Table 2).

153

Propofol modulation of [35S]TBPS binding to recombinant α1β2/3γ2 receptors

I also examined propofol modulation of [35S]TBPS binding to recombinant

α1β2γ2 or α1β3γ2 receptors transiently expressed in HEK293 cells. Propofol caused a clearly biphasic modulation of [35S]TBPS binding to both α1β2γ2 and α1β3γ2 receptors

in the absence of exogenous GABA (Fig. 30). Low concentrations of propofol (< 10 μM)

caused a concentration dependent enhancement of [35S]TBPS binding, while higher

concentrations (> 10 μM) caused a reduction in binding. Binding of [35S]TBPS to

α1β3γ2 receptors was higher compared to binding α1β2γ2 receptors. These data may

reflect mixed populations of homomeric β3 receptors, αβ, or αβγ receptors. Different

groups have examined [35S]TBPS binding and blockade of homomeric β3 receptors as

well as α1β3 receptors (Davies et al., 1997b; Jursky et al., 2000; Chen et al., 2006). I

will revisit the role of different β subunits in [35S]TBPS binding later in this chapter.

These data suggest that activation of the channel by propofol is responsible for

modulation of [35S]TBPS binding observed in homogenate brain assays (Fig. 28). Unlike

the homogenate binding assay there is no GABA present in the recombinant receptor

experiments. The biphasic nature of the modulation of [35S]TBPS binding to recombinant

receptors precluded fitting the data to obtain IC50 values for propofol. However, visual

inspection of the concentration-response relationships in Figure 30 reveals that the

approximate concentrations for half maximal inhibition of [35S]TBPS binding are 50 and

200 μM for α1β2γ2 and α1β3γ2 receptors, respectively. The potency with which

propofol displaces [35S]TBPS bound to recombinant receptors is noticeably lower than its potency determined in brain homogenates. 154

Figure 31

200

180

160

140 α1β3γ2 120 α1β2γ2 100

60 % control binding

0.1 1 10 100 500 [Propofol] (μM)

Figure 31. Propofol modulation of [35S]TBPS binding to recombinant α1β2γ2 and α1β3γ2 receptors in the absence of GABA. Low concentrations of propofol (< 10 μM) enhanced [35S]TBPS binding, while high concentrations ( > 10 μM) caused a reduction in [35S]TBPS binding. Modulation of binding at high concentrations was significantly higher in α1β3γ2 receptors compared to α1β2γ2 receptors (* p < 0.05, **** p< 0.0001, Student’s unpaired t-test). Data are presented as mean ± SEM of n ≥ 4 independent experiments performed in triplicate.

155

Propofol is known to cause GABAA receptor desensitization and its high potency

displacement of [35S]TBPS binding from brain membranes may reflect a potentiation of desensitization by endogenous GABA (Orser et al., 1994; Hales and Adodra, 1995).

There is no GABA in the recombinant receptor preparation and therefore a reduction of

[35S]TBPS binding by propofol better reflects the potency of the anesthetic as an

allosteric agonist. Taken together, these data demonstrate that GABAA receptors can be

identified based on their relative affinity for an allosteric agonist. Using [35S]TBPS

binding to recombinant receptors, the inhibition of [35S]TBPS binding by propofol suggests a role for desensitization of the channel at high agonist concentrations.

In the next section, I will examine the role of endogenous GABA in [35S]TBPS binding to

receptors in brain homogenates and whole slices, using the competitive GABAA antagonists GBZ and BIC.

Gabazine competes with endogenous GABA to modulate [35S]TBPS binding

35 Using [ S]TBPS autoradiography and the competitive GABAA antagonist, GBZ,

I examined the role of endogenous GABA in basal binding of [35S]TBPS to rodent brain

slices (Fig. 32A). GBZ caused significant enhancement of [35S]TBPS binding to

receptors in the cerebellum in what appeared to be the cerebellar granule layer (CGL)

(Fig. 32C). The enhancement observed correlates with the localization of the δ subunit

using in situ hybridization (Pirker et al., 2000) and in knockout animal studies. Using

autoradiography and radioligands selective for different subunits, e.g. subtype selective

benzodiazepines to localize α subunits or [3H]muscimol to localize high affinity δ

156

subunits, knockout mouse models have been used to determine brain regions expressing

different subunits based on changes in radioligand binding in the absence of the subunit

that has been suppressed. Some studies also demonstrated that [35S]TBPS binding to the

cerebellum was not eliminated in the absence of the δ subunit, suggesting that other

subunits may contribute to the differential binding to cerebellar receptors compared to other brain regions (Sinkkonen et al., 2001; Halonen et al., 2009).

In contrast to binding in the cerebellum, GBZ caused a slight reduction of

[35S]TBPS binding to receptors in the frontal cortex (FC), hippocampus (H) and thalamus

(Th) in a concentration dependent manner. Using the same analysis methods described

earlier, I quantified [35S]TBPS binding (Fig. 33) and focused on comparing the frontal

cortex, which is thought to contain primarily αβγ receptors, the cerebellum, which is

known to have a prominent population of α6βδ receptors, and the hippocampus, which

predominantly contains α1β2γ2 and is also thought to have some α4βδ receptors (Nusser

and Mody, 2002; Glykys and Mody, 2007). The significant enhancement of [35S]TBPS

binding observed in the cerebellum did not occur in other brain regions. In the frontal

cortex, there was a slight concentration-dependent reduction in [35S]TBPS binding that

was not observed for the other brain regions examined (# p < 0.05, ## p < 0.01, Student’s

t-test compared to [35S]TBPS binding in the presence of 100 nM GBZ). GBZ had little

effect on [35S]TBPS binding to receptors in the hippocampus.

157

Figure 32 A Basal binding B 100 μM PIC

FC Th CRB

C CGL

1 μM GBZ

10 μM GBZ CGL

100 μM GBZ

Figure 32. Representative [35S]TBPS autoradiography images of sagittal 14 μM cryostat sections in the presence of GBZ. A. Basal binding of [35S]TBPS occurred in the absence of exogenous ligand. Distribution of binding was region-dependent. B. Non-specific 35 binding of [ S]TBPS to GABAA receptors was determined in the presence of 100 μM picrotoxin, which competitively displaced binding from all brain regions. C. GBZ enhances [35S]TBPS binding to the cerebellum (CRB) in what appears to be the cerebellar granule cell layer (CGL), but not in other brain regions. Enlargements of basal [35S]TBPS binding and [35S]TBPS binding in the presence of 10 μM GBZ. Throughout the brain, GBZ caused slight reduction of [35S]TBPS binding. FC, frontal cortex; H, hippocampus; Th, thalamus.

158

Figure 33

150

Cerebellum

Frontal Cortex

100 Hippocampus % control binding 50 # ## ##

0.1 1 10 100 [Gabazine] (μM)

Figure 33. Quantified binding values of [35S]TBPS autoradiography in the presence of gabazine. Non-specific binding was obtained in the presence of 100 μM PIC. GBZ caused a significant enhancement of [35S]TBPS binding to the cerebellum, but not other brain regions (* p < 0.05 compared to the frontal cortex, one-way ANOVA with Tukey’s post-hoc test.). GBZ caused a concentration dependent reduction in [35S]TBPS binding in the frontal cortex and hippocampus (# p < 0.05, ## p < 0.01 in compared to 100 nM GBZ in frontal cortex, Student’s unpaired t-test). Data are represented as mean ± S.E.M. of n ≥ 6 individual rodent brains.

159

Using homogenized membranes of isolated brain regions, I examined the ability of GBZ to modulate [35S]TBPS binding in the absence of exogenous GABA. GBZ caused a significant, concentration-dependent enhancement of [35S]TPBS binding in the cerebellum, but not hippocampus or frontal cortex (* p < 0.05, ** p < 0.01, one-way

ANOVA with Tukey’s post-hoc test, n ≥ 4 independent experiments). In contrast, GBZ caused a slight inhibition of [35S]TBPS binding in the frontal cortex (Fig. 34A). GBZ is a weak inverse agonist at αβγ receptors, which represent the majority of receptors in the cortex (Chang et al., 1999).

To test a direct inhibitory role for GBZ in the absence of GABA, I expressed

α1β2γ2 and α1β3γ2 receptors in HEK293 membranes and examined the ability of GBZ to modulate GABA-independent [35S]TBPS binding. These two subtypes were chosen because they represent the majority of synaptic GABAA receptors in the CNS. GBZ caused a slight, but significant reduction in [35S]TBPS binding to both α1β2γ2 and

α1β3γ2 receptors (Fig. 34B) (# p < 0.05, one-way ANOVA with Tukey’s post-hoc test, compared to binding in the presence of 300 nM GBZ). GBZ was better able to modulate

[35S]TBPS binding to α1β3γ2 receptors compared to α1β2γ2 receptors (stars * p < 0.05,

** p < 0.01, one-way ANOVA with Tukey’s post-hoc test, n ≥ 3 independent experiments performed in triplicate). These data suggested differences in sensitivity to

GBZ imposed by different β subunits, which should be investigated in future experiments. I will revisit the role of the β subunit in [35S]TBPS binding later in this chapter.

160

35 Modulation of [ S]TBPS binding by bicuculline

BIC is more efficacious than GBZ as an inverse agonist at GABAA receptors

(McCartney et al., 2007). Therefore, I investigated the effects of BIC on [35S]TBPS

binding using autoradiography and homogenate binding. I examined the ability of BIC to

modulate [35S]TBPS binding in the absence of exogenous GABA using autoradiography

to detect [35S]TBPS binding in brain slices (Fig. 35). Like GBZ, BIC caused differential

35 modulation of [ S]TBPS binding to the various GABAA receptors expressed throughout the rodent brain (Fig. 35B). Data were quantified as described earlier and are graphically represented in Figure 35C. BIC caused a concentration-dependent enhancement of

35 [ S]TBPS binding to GABAA receptors in the cerebellum, but caused slight reduction of binding from the other brain regions (* p < 0.05 compared to other brain regions, one-

way ANOVA with Tukey’s post-hoc test, n ≥ 6 independent experiments. The

enhancement is consistent with the idea that BIC prevents endogenous GABA from

binding to high affinity receptors in the cerebellum, which would have otherwise caused

diminished [35S]TBPS binding compared to the frontal cortex and cerebellum (Fig. 27).

161

Figure 34

A 180 160 140 Cerebellum 120 Frontal Cortex 100 Hippocampus 80 60

% control binding 40 20 0 0.1 1 10 100 [Gabazine] (μM)

B 100 α1β2γ2

75 α1β3γ2 # # 50

% control binding 25

0 0.3 1 10 100 [Gabazine] (μM)

Figure 34. GBZ modulation of [35S]TBPS binding to homogenate membranes. A. Homogenate membranes of isolated rodent cerebella, frontal cortices or hippocampi bound [35S]TBPS in the absence of exogenous GABA. GBZ caused a concentration dependent enhancement of binding to cerebellar receptors, but not hippocampal or cortical receptors. (* p < 0.05, ** p < 0.01; n ≥ 4 independent experiments performed triplicate) B. GBZ caused a concentration dependent displacement of GABA-independent [35S]TBPS binding to recombinant α1β2γ2 and α1β3γ2 receptors. (# p < 0.05 compared to α1β2γ2; stars * p < 0.05, ** p < 0.01 compared to 300 nM GBZ n ≥ 3 experiments performed in triplicate). Non-specific binding was determined in the presence of 100 μM PIC and statistics were obtained using one-way ANOVA with Tukey’s post-hoc test.

162

I also used BIC to modulate [35S]TBPS binding to homogenate brain membranes

In this assay, BIC caused a concentration dependent enhancement of [35S]TBPS binding

to receptors in the cerebellum (Fig. 36A, * p < 0.05, one-way ANOVA with Tukey’s

post-hoc test) and a slight reduction in binding to hippocampi and frontal cortices.

In Chapter 3, I demonstrated that BIC inhibited GABA-independent binding of

[35S]TBPS to recombinant α1β2γ2 receptors (Fig. 9). I also demonstrated that BIC

significantly reduces GABA-independent functional blockade of α1β2γ2 receptors by

TBPS (Fig. 10). Here I compared α1β2γ2 and α1β3γ2 receptors transiently expressed in

HEK293 cells and found that BIC caused a concentration dependent reduction in

[35S]TBPS binding to both subtypes (Fig. 36B). This reduction represents the GABA-

independent component of accessibility to the TBPS binding site described in Chapter 3.

Taken together, experiments using GBZ and BIC suggest that endogenous GABA

plays a significant role reducing [35S]TBPS binding to brain regions thought to contain

high affinity α6βδ receptors. My results suggest that it is possible to discriminate between different receptor subtypes based on their differing affinities for GABA. The differences in GABA modulation of [35S]TBPS binding observed between the hippocampus, frontal cortex and cerebellum are likely dependent on differences in receptor affinity for GABA and may reflect higher levels of ambient GABA in the cerebellum. Both BIC and to a lesser extent, GBZ, inhibited [35S]TBPS binding to

GABAA receptors in the frontal cortex, presumably composed of α1β2γ2 or α1β3γ2,

which suggests a role for spontaneous, GABA-independent gating in [35S]TBPS binding

subtypes.

163

Figure 35

Total binding 300 nM BIC A B H CRB FC

100 μM PIC 30 μM BIC

C 200

g 150 Cerebellum

Frontal Cortex 100 Hippocampus % control bindin % control 50

0.1 1 10 100 [Bicuculline] (μM)

Figure 35. BIC modulation of [35S]TBPS binding. A. [35S]TBPS bindount to sagittal 16 μM rodent brain slices in the absence of exogenous GABA (top panel). 100 μM PIC competitively displaced [35S]TBPS binding throughout the brain (bottom panel). B. Representative images of modulation of [35S]TBPS binding by BIC demonstrate that binding is enhanced in the cerebellum (CRB). C. Quantified [35S]TBPS autoradiography to adult rodent brain slices. Bicuculline caused a significant enhancement of [35S]TBPS binding in the cerebellum, but not hippocamous (H) or frontal cortex (FC) (* p < 0.05 compared to 100 nM BIC, one-way ANOVA with Tukey’s post-hoc test, n ≥ 3 independent experiments performed in triplicate). Non-specific binding was determined in the presence of 100 μM PIC and in the absence of exogenous GABA.

164

Figure 36

A 200

150 Cerebellum Frontal Cortex 100 Hippocampus

50 % control binding

0.1 1 10 100 [Bicuculline] (μM) B 100

75 α1β2γ2 α1β3γ2 50

25 % control binding

0 0.03 0.1 1 10 100 [Bicuculline] (μM)

Figure 36. Modulation of [35S]TBPS binding by bicuculline. A. Bicuculline caused a concentration dependent enhancement of [35S]TBPS binding to homogenized cerebellar membrane and reduction of [35S]TBPS binding to homogenized frontal cortices (* p < 0.05 compared to 100 nM BIC in each brain region, n ≥ 3 independent experiments performed in triplicate). B. Bicuculline caused a significant concentration dependent modulation of GABA-independent [35S]TBPS binding to recombinant α1β2γ2 receptors expressed in HEK293 membranes, but α1β3γ2 receptors. α1β2γ2 data are included from Figure 9 in Chapter 3 (* p < 0.05 compared to 30 nM BIC, n ≥ 4. Non-specific binding was determined in the presence of 100 μM PIC. Data are presented as mean ± SEM and statistics were obtained using one-way ANOVA with Tukey’s post-hoc test.

165

Both GABA affinity and spontaneous gating are influenced by the presence of different auxiliary subunits (δ, ε, or γ). In the next section, I will discuss the experiments

I conducted to target receptors containing the δ subunit in the rodent brain.

[35S]TBPS binding to receptors containing the δ subunit

GABAA receptors containing the δ subunit have a higher affinity for GABA than other subtypes (Fisher and MacDonald, 1997). Several groups have used the agonists

THIP and muscimol to examine brain regions thought to contain the δ subunit, mainly the cerebellum, thalamus, and hippocampus (Halonen et al., 2009, Mortenson et al., 2010).

However, these agonists also activate receptors lacking the δ subunit, albeit with different efficacies (Mortenson et al., 2010). Radioligand binding to brains obtained from knockout mouse models have provided insight into the localization of the δ subunit throughout the brain (Sinkkonen et al., 2001; Halonen et al., 2009). I planned to use a ligand that would be selective for receptors containing the δ subunit, much like benzodiazepines are selective for receptors containing a γ2 subunit. Until recently, a compound fitting these parameters did not exist.

In 2009, Wafford et al. demonstrated the effects of two δ subunit-selective compounds, DS1 and DS2. DS2 was a selective positive allosteric modulator at recombinant α4β3δ receptors compared to α4β3γ2 or α1β3γ2 receptors, whereas DS1 caused less selective modulation at these subtypes. Furthermore, DS2 enhanced tonic inhibition recorded from thalamic neurons, which is thought to be mediated by α4β2δ.

Thus, DS2 is a useful tool for selectively modulating receptors containing the δ subunit. I 166

sought to use DS2 to modulate receptors containing the δ subunit using [35S]TBPS

autoradiography in brain slices. I focused on [35S]TBPS autoradiography in order to

identify any brain region that contains αβδ receptors sensitive to DS2. I anticipated that

DS2 would reduce [35S]TBPS binding more potently by potentiating the effects of GABA

in brain regions such as the cerebellum in which the δ subunit is enriched.

Representative images depicting the level of [35S]TBPS binding to sagittal rodent

brain slices are shown in Figure 37. Panel A depicts control binding of [35S]TBPS in the

absence of exogenous GABA (left) that is competitively displaced by 100 μM PIC

(middle) and modulation of [35S]TBPS binding by 10 μM DS2 (right). These data were

quantified as described earlier and depicted graphically in Figure 38. While there was a

trend towards a greater reduction in [35S]TBPS binding to cerebellar receptors compared

to receptors in the hippocampus or frontal cortex, these differences were not significant

(Fig. 37B and Fig. 38).

Since DS2 is most effective in the presence of non-saturating concentrations of

GABA (Wafford et al., 2009), I examined the ability of the drug to modulate [35S]TBPS

binding in the presence of low concentrations of exogenous GABA (1 or 3 μM). DS2 caused a concentration dependent reduction in [35S]TBPS binding in the absence of

exogenous GABA (Fig. 38 B,C and D). However, the combination of DS2 and

exogenous GABA showed no significant effect on [35S]TBPS binding to the frontal

cortex (Fig. 38B), hippocampus (Fig. 38C) or cerebellum (Fig 38D). Wafford et al.

(2009) demonstrated that DS2 exhibited a slight positive modulation at receptors

containing the γ2 subunit. My results examining modulation of [35S]TBPS binding by

167

DS2 are more consistent with those anticipated for receptors containing the γ2 subunit rather than the δ subunit, even in the cerebellum where the latter are thought to contribute to high affinity GABAA receptors. This raises the possibility that δ subunit containing receptors cannot bind [35S]TBPS. Alternatively, modulation of [35S]TBPS binding by

DS2 lacks subunit specificity.

Previous studies suggest that the δ subunit reduces [35S]TBPS binding (Hevers et al., 2000). I examined [35S]TBPS binding to recombinant αβδ receptors expressed in

HEK293 cells. I examined the ability of [35S]TBPS to bind to α6β3δ or α4β2δ receptors in the absence of GABA and found that specific binding (fmol/mg protein) was significantly reduced compared to receptors containing a γ2 subunit (Fig. 39A, * p <

0.05, ** p < 0.01, one-way ANOVA with Tukey’s post-hoc test). Interestingly, specific picrotoxin-displaceable [35S]TBPS binding was also significantly reduced in the hippocampus and cerebellum (Fig. 39B, ** p < 0.01 compared to frontal cortex, one-way

ANOVA with Tukey’s post-hoc test).

168

Figure 37 Total binding 100 μM PIC 10 μM DS2 A

H CRB FC

B 1 μM GABA 10 μM DS2 + 1 μM GABA

3 μM GABA 10 μM DS2 + 3 μM GABA

Figure 37. Representative images of [35S]TBPS autoradiography in the presence of different ligands. A. Left to right: [35S]TBPS bound to rodent brains in the absence of exogenous GABA, 100 μM PIC caused full competitive displacement of binding and 10 μM DS2 reduced [35S]TBPS binding to several brain regions. B. Combined actions of 10 μM DS2 and GABA caused enhanced reduction of [35S]TBPS binding (right images) compared to GABA alone (left images). FC, frontal cortex; H, hippocampus; CRB, cerebellum.

169

Figure 38 10 μM DS2 Frontal Cortex A B 100 100

75 75

50 50

% control binding 25 % control binding

0 0 10 μM DS2 + - + - + - 1 μM GABA + + - -

ebellum - - - + + 3 μM GABA Frontal Cortex Hippocampus Cer

Hippocampus C 100 100 Cerebellum

g 75 75

50 50

% control bindin 25 25 % control binding

0 0 10 μM DS2 - + + - + 10 μM DS2 + - + - + 1 μM GABA - - - + + 1 μM GABA - + + - - 3 μM GABA - - - + + 3 μM GABA - - - + +

Figure 38. Modulation of [35S]TBPS binding using a δ subunit-selective ligand, DS2. A. 10 μM DS2 caused ~ 25% reduction of [35S]TBPS binding to receptors in frontal cortices and hippocampi and ~ 45% reduction of [35S]TBPS binding to receptors in the cerebellum. B-D. Combined actions of DS2 (10 μM) and exogenous GABA (1 or 3 μM) in the frontal cortex (B), hippocampus (C) or cerebellum (D). No significant additive effect of DS2 and GABA modulation to [35S]TBPS binding was observed (compared to modulation in the presence of GABA).Non-specific binding was determined in the presence of 100 μM PIC. Data are presented as mean ± SEM, n = 5 independent experiments.

170

To date, there is little evidence of [35S]TBPS binding to receptors containing the δ

subunit. Some groups have examined [35S]TBPS binding in δ knockout mouse models

(Sinkkonen et al., 2001; Halonen et al., 2009) to examine “GABA-insensitive” binding of

[35S]TBPS binding. This is a phenomenon that occurs in some brain regions, mainly the thalamus and cerebellum, where [35S]TBPS remains bound to receptors in the presence of mM concentrations of GABA. [35S]TBPS autoradiography to δ-/- mouse brains

demonstrated that the δ subunit did not contribute to GABA-insensitive [35S]TBPS

binding. These studies are in agreement with work done by Hevers et al., (2000), which

demonstrated that the incorporation of a δ subunit in transiently expressed αβγ receptors

reduces specific binding of [35S]TBPS (Hevers et al., 2000). Although I do not observe

GABA-insensitive [35S]TBPS binding in the plane of section used in my experiments, my

findings support the emerging evidence that receptors containing the δ subunit do not contribute to [35S]TBPS binding in the brain.

171

Figure 39

A B

12 8

n i

6 8 prote

4 fmol/mg fmol/mg protein 4 2

0 x δ δ 2 2 2 2 3 γ γ γ β β 3 3 β2 4 6 β β 6 α α 1 α1 α α Cerebellum Frontal Corte Hippocampus

35 Figure 39. Specific binding of [ S]TBPS to recombinant receptors is dependent on subunit composition. A. Receptors containing the δ subunit had significantly lower specific [35S]-TBPS binding compared to receptors containing the γ2 subunit. No significant difference was found in specific [35S]TBPS binding to receptors combining different α or β subunits with the γ2 subunit. * p <0.05, ** p <0.01 compared to receptors containing the γ2 subunit, n ≥ 4 independent experiments performed in triplicate (α1β2γ2 was excluded from this test due to low n#). B. Specific binding of [35S]TBPS was significantly lower in the hippocampus and cerebellum compared to the frontal cortex. ** p < 0.01 compared to the frontal cortex, n ≥ 16 independent experiments performed in triplicate. Data are represented as mean ± S.E.M and statistics were obtained using one- way ANOVA and Tukey’s post-hoc test. Specific binding values were calculated by comparing PIC-displaceable (specific) binding of 30 nM [35S]TBPS obtained from competition binding studies.

172

The α subunit contributes to GABA affinity

The data discussed earlier in this chapter demonstrate that GABA modulation of

[35S]TBPS binding is more potent in brain regions thought to contain high affinity

extrasynaptic αβδ receptors. However, my data, in agreement with other studies, suggest

that receptors containing the δ subunit do not bind [35S]TBPS (Hevers et al., 2000;

Sinkkonen et al., 2001; Halonen et al., 2009). Besides the δ subunit, different α subunits

affect GABA affinity. I examined [35S]TBPS binding to recombinant receptors

containing either α1 or α6 subunits, based on previous studies demonstrating that the α6

subunit attributes a higher affinity for GABA than the α1 (Fisher et al., 1997; Fisher,

2004). I hypothesized that receptors containing the α6 subunit were responsible for the significant differences in modulation of [35S]TBPS binding to cerebellar receptors caused

by GABA, GBZ and BIC.

Early studies using immunohistochemistry and in situ hybridization have demonstrated that the majority of brain regions contain the α1 subunit, whereas α2-α6 are more discretely distributed (Mohler et al., 1990; Laurie et al., 1992; Wisden et al.,

1992; Stephensen et al., 1995; Pirker et al., 2000). Furthermore, the α4 and α6 subunits are often thought to coexpress with receptors containing the δ subunit in the hippocampus and thalamus, or cerebellum, respectively (Korpi et al., 2002; Halonen et al., 2009). In my experiments, I examined the differences in affinity for GABA attributed by the α1 and α6 subunits, the most widely examined α subunits in the CNS. Since there is no evidence for α6β receptors in the brain, receptors containing the α6 subunit but lacking the δ subunit are likely of the α6βxγ2 subtype. 173

[35S]TBPS bound to α1β2γ2, α1β3γ2 and α6β3γ2 receptors in a picrotoxin-

displaceable, GABA-independent manner (Fig. 40). Modulation of [35S]TBPS binding by

exogenous GABA was biphasic for receptors containing the α1 subunit. However, for the

α6β3γ2 subtype, I observed solely a concentration dependent reduction of [35S]TBPS

binding by GABA. Within the concentration range examined (0.03 μM to 100 μM), the

α6 subunit caused a sinistral shift in the modulation of [35S]TBPS binding compared to receptors containing the α1 subunit. These data concur well with the idea that GABA is more potent at receptors containing the α6 subunit regardless of the presence of the δ

subunit (Fisher et al., 1997). GABA caused complete inhibition of binding in the α1β2γ2

subtype, but not α1β3γ2 or α6β3γ2 receptors. Later in this chapter, I will examine the

role of different β subunits in [35S]TBPS binding to establish the cause of incomplete

inhibition of binding in the presence of the β3 subunit..

The α1 and α6 subunits have also been widely examined in studies comparing

different classes of benzodiazepines. In the next section, I will describe the effects of

different benzodiazepines with selectivity for the α6 subunit on [35S]TBPS binding to

examine whether high affinity GABAA receptors detected in the cerebellum are

composed of α6βxγ2 subunits.

174

Figure 40

175 α6β3γ2

α1β2γ2 150 α1β3γ2 125

100

75 % control binding binding % control 50

0.01 0.1 1 10 100 [GABA] (μM)

35 Figure 40. [ S]TBPS bound to recombinant GABAA receptors in the absence of GABA. The reduction of [35S]TBPS binding by GABA occurs at lower concentrations in higher affinity α6β3γ2 receptors compared to α1β2γ2 receptors. Full inhibition of [35S]TBPS binding by GABA occurred in α1β2γ2 receptors, but not α1β3γ2 or α6β3γ2 receptors. Data are represented as mean ± S.E.M and statistics were obtained using Student’s unpaired t-test.* p < 0.05, ** p < 0.01, *** p <0.001, **** p < 0.0001, n ≥ 4 compared to α1β2γ2 receptors. Non-specific binding was determined in the presence of 100 μM PIC.

175

Benzodiazepine ligands modulate [35S]TBPS binding in the cerebellum

As mentioned earlier in this chapter and in Chapter 1, benzodiazepine ligands

modulate GABAergic function at a variety of GABAA receptor subtypes throughout the

CNS. They can be positive, neutral, or negative modulators that bind some or all subtypes, requiring the presence of α and γ subunits. Furthermore, some benzodiazepine ligands behave differently depending on the α subunit present in the receptor (Rudolph and Mohler, 2004). I hypothesized that TBPS could be used to detect functionally distinct receptors based on their pharmacology and other unique properties. As described earlier, my data suggest that receptors containing the δ subunit do not bind [35S]TBPS. However,

GABA causes more potent modulation of [35S]TBPS to cerebellar receptors compared to

receptors in the frontal cortex and hippocampus. These data are consistent with the

hypothesis that high affinity receptors are present in the cerebellum. I used flunitrazepam

and Ro 15-4513 to modulate [35S]TBPS binding to receptors containing the α6 subunit.

Benzodiazepine modulation of [35S]TBPS binding to cerebellar receptors would indicate

that these high affinity receptors contain the γ2 subunit, which is required to form the

binding site for benzodiazepine ligands on GABAA receptors.

Flunitrazepam is thought to have low affinity for receptors containing the α4 or

α6 subunit (Hadingham et al., 1996; Wingrove et al., 1997), but causes positive

modulation of GABA-evoked responses at most other receptors. Ro 15-4513 is an inverse

benzodiazepine binding site agonist at recombinant α1β2γ2 receptors and an agonist at

the α6β2γ2 subtype in the presence of GABA (Hauser et al., 1997). The same group also

demonstrated that flunitrazepam behaves as a weak inverse agonist at α6β2γ2. Based on

176

these studies, I hypothesized that flunitrazepam would enhance [35S]TBPS binding to

brain regions containing the α6 subunit (regions in which endogenous GABA causes

inhibition of basal [35S]TBPS binding), while reducing [35S]TBPS binding to other

receptors by potentiating the effects of exogenous GABA at those subtypes. Similarly, I

hypothesized that Ro 15-4513 would enhance [35S]TBPS binding throughout the brain at

receptors containing the α1 subunit, but reduce binding to cerebellar receptors, if the α6

subunit contributes to [35S]TBPS binding there.

I examined the effect of flunitrazepam (1 μM) on [35S]TBPS binding applied in

the presence and absence of exogenous GABA (Fig. 41). As described earlier, [35S]TBPS binds in a PIC-displaceable manner in the absence of exogenous GABA, while addition of exogenous GABA (1-10 μM) primarily causes a reduction of binding throughout the brain (Fig. 41A). Flunitrazepam appeared to cause a concentration-dependent enhancement of the GABA-evoked reduction in [35S]TBPS binding in both the frontal

cortex and hippocampus (Fig. 41B). However, quantification of these data revealed no

significant effect on binding (Fig. 42A and B). I was surprised to see that these effects

were not significant when quantified, despite seeing an effect in the representative images

(Fig. 41B). This may have been due to exposure differences across the film used between

experiments that incorporated greater error into the quantification.

In contrast to other brain regions, flunitrazepam appeared to cause a significant enhancement of [35S]TBPS binding in the cerebellum compared to GABA alone (Fig.

42C). In the absence of exogenous GABA, flunitrazepam (1 μM) caused a slight

enhancement in all three brain regions that was most prominent in the cerebellum (Fig.

177

42D). These data are consistent with flunitrazepam behaving as an inverse agonist at

receptors containing the α6 subunit. Taken together, these data suggest that the high

affinity modulation of [35S]TBPS binding by GABA in the cerebellum described earlier is

likely to be due to receptors containing the α6 subunit in receptors that also contain the

γ2 subunit. The effects of flunitrazepam on receptors containing the α1 subunit may have

been masked by the presence of receptors containing different α subunits, whose

sensitivity to flunitrazepam and ability to bind [35S]TBPS binding is not known.

As mentioned earlier, Hauser et al. (1997) also demonstrated that Ro 15-4513

acted as an inverse agonist at α1 subunit-containing receptors and caused positive

modulation of GABA-evoked currents at α6 subunit-containing receptors. I hypothesized

that Ro 15-4513 would enhance [35S]TBPS binding to receptors containing the α1 subunit throughout the brain by preventing the inhibitory effects of GABA. Furthermore,

I hypothesized that Ro 15-4513 would enhance the effects of GABA and cause a greater reduction in [35S]TBPS binding to receptors containing the α6 subunit.

Figure 43 contains representative [35S]TBPS autoradiographs in the presence of

exogenous GABA alone (panel A) or Ro 15-4513 (1 μM) alongside slices exposed to

either no agonist or exogenous GABA (1 to 10 μM, panel B). These data were quantified as described earlier and shown in graphical form in Figure 44. Ro 15-4513 caused a significant concentration dependent enhancement of [35S]TBPS binding in the presence

of GABA at receptors in the frontal cortex (Fig. 44A, ** p < 0.01, Student’s t-test, n ≥ 5),

hippocampus (Fig. 44B, *** p < 0.001, Student’s t-test, n ≥ 5) and cerebellum (Fig. 44C,

* p < 0.05, Student’s t-test, n ≥ 5). This is consistent with an inverse agonist action of Ro 178

15-4513 preventing GABA (3 and 10 μM)-evoked displacement of [35S]TBPS binding to

receptors containing the α1 subunit or other α subunit that supports conventional benzodiazepine binding.

In the absence of exogenous GABA, Ro 15-4513 caused no significant modulation

of [35S]TBPS binding (Fig. 44D). The enhancement of [35S]TBPS binding across the

three brain regions examined suggests that the α1 subunit (or other α subunit other than

α4 or α6) is present in the cerebellum. These data are consistent with immunohistochemistry studies that have demonstrated an overlap of expression of the α1 and α6 subunits in the cerebellum (Pirker et al., 2000). These data also demonstrate that receptors containing the α1 subunit are localized to the cerebellum, are incorporated into functional receptors with the γ2 subunit, and likely contribute to [35S]TBPS binding.

The reduction of [35S]TBPS binding by GABA is likely to be due to increased

desensitization of the channel at high concentrations of agonist (Chapter 4). In contrast,

in Chapter 3, I demonstrated that activation of the channel is required for maximal levels

of [35S]TBPS binding. These data suggest that in order for benzodiazepines to enhance

the GABA-evoked reduction in [35S]TBPS binding, they must increase desensitization. It is unlikely that Ro 15-4513 or flunitrazepam would have reduced [35S]TBPS binding of

receptors containing α6 and α1 subunits, respectively, unless they enhanced steady-state

desensitization. In the future, it would be useful to test the effects of benzodiazepines on

desensitization and subsequent blockade of GABA-evoked currents by TBPS using the

patch-clamp technique.

179

Figure 41 A B 1 μM Flunitrazepam Total Binding (no exogenous GABA) H CRB FC

Exogenous GABA

1 μM

3 μM

10 μM

100 μM PIC (no exogenous GABA)

Figure 41. Representative autoradiography images of modulation of [35S]TBPS binding to 16 μM thick rodent brain slices in the absence and presence of the benzodiazepine flunitrazepam. A. Exogenous GABA causes a concentration dependent reduction in [35S]TBPS binding compared to total binding (top panel).B. Flunitrazepam (1 μM) had a modest effect on [35S]TBPS binding when applied alone (top panel) and potentiated the GABA (1-10 μM)-evoked reduction in binding. C. Flunitrazepam (10 μM) enhanced the [35S]TBPS binding to cerebellar receptors (top panel) when applied alone and potentiated the reduction of [35S]TBPS binding caused by exogenous GABA. FC, frontal cortex, H, hippocampus, and CRB, cerebellum.

180

Figure 42

Hippocampus C A Frontal B Cerebellum 120 150 200 100 150 80 100 60 100

40 50 % control binding binding control % 50 % control binding binding control %

20 binding control %

0 0 0 0 1 3 10 01 3 10 0 1 3 10 1 μM Flunitrazepam 1 uM Flunitrazepam 1 uM Flunitrazepam + [GABA] (μM) + [GABA] (μM) + [GABA] (μM)

D 200

GABA alone 150 Flunitrazepam + GABA 100

50 % control binding binding control % 0 Cerebellum Frontal Cortex Hippocampus

Figure 42. Quantified [35S]TBPS autoradiography in the presence of flunitrazepam (1 μM) in the presence or absence of exogenous GABA. A, B. Flunitrazepam caused no significant modulation of [35S]TBPS binding to cortical receptors in the absence of exogenous GABA. Modulation in the presence of exogenous GABA (1-10 μM) caused no further reduction compared to GABA alone. C. Compared to GABA alone (black bars), flunitrazepam significantly reduced the modulation of [35S]TBPS in the presence of GABA. D. Flunitrazepam enhanced [35S]TBPS binding to the cerebellum in the absence of exogenous GABA. Data are presented as mean ± SEM and statistics were obtained using Student’s unpaired t-test. * p < 0.05, ** p < 0.01, n = 5 independent experiments.

181

Figure 43

A B 1 μM Ro 15 45-13

Total Binding H (no exogenous GABA) FC CRB

Exogenous GABA

1 μM GABA

3 μM GABA

10 μM GABA

100 μM PIC

Figure 43. Representative images of [35S]TBPS autoradiography in 16 μM thick rodent brain slices in the presence of the benzodiazepine inverse agonist Ro 15 45-13. A. Exogenous GABA causes a concentration dependent reduction in [35S]TBPS binding compared to total binding (top panel) and control binding (100 μM PIC).B. Ro 15 45-13 (1 μM) prevented the reduction of [35S]TBPS binding by GABA (1-10 μM) in multiple brain regions. Images are representative of five independent experiments. FC, frontal cortex; H, hippocampus; CRB; cerebellum.

182

Figure 44 Ro 15-4513 + GABA GABA alone

A B Frontal Cortex Hippocampus C Cerebellum 100 100 100

g g 75 75 75 bindin 50 50 50 trol on

25 c

% control binding 25 25 % control bindin % 0 0 0 0 1 3 10 0 1 3 10 0 1 3 10 1 μM Ro 15-4513 + [GABA] 1 μM Ro 15-4513 + [GABA] (μM) 1 μM Ro 15-4513 + [GABA] (μM)

D 100

25 % control binding 0 Cerebellum Cerebellum Frontal Cortex Hippocampus

Figure 44 Quantification of [35S]TBPS autoradiography in the presence of 1 μM Ro 15- 4513 and presence or absence of exogenous GABA. A-C. Ro 15-4513 caused a significant reduction in the effects of exogenous GABA (1-10 μM) in all three brain regions. The combined effects of Ro 15-4513 and GABA were concentration dependent. * p < 0.05, ** p < 0.01, *** p < 0.001, Student’s unpaired t-test. D. Ro 15 45-13 caused no significant modulation of [35S]TBPS binding in the absence of exogenous GABA (one-way ANOVA with Tukey’s post-hoc test. Data are presented as mean ± SEM of five independent experiments.

183

The β subunit influences GABA-evoked modulation of [35S]TBPS binding

As mentioned earlier in this chapter, GABA was unable to fully inhibit [35S]TBPS binding to receptors containing the β3 subunit regardless of the identity of the α subunit

(Fig. 40). Similar results have been obtained in a previous study examining [35S]TBPS

binding to β3 subunit containing receptors (Davies et al., 1997b). By contrast GABA

fully inhibited [35S]TBPS binding to receptors containing the β2 subunit (Fig. 5 and 40).

This is a surprising finding because there is strong amino acid sequence identity between

β2 and β3 subunits. A key difference between these two subunits is there abilities to form

homomeric receptors. The β3 subunit readily forms homomeric receptors, being able to

assemble in the endoplasmic reticulum and traffic to the cell membrane in the absence of

other subunits (Davies et al., 1997b; Taylor et al., 1999). By contrast, other GABAA

subunits, including the β2 subunit, fail to traffic to the cell membrane. GABAA receptors

formed by β3 subunits alone fail to bind GABA, but can be activated by propofol and

bind [35S]TBPS in the absence of activation (Davies et al., 1997b). Therefore a failure of

GABA to inhibit [35S]TBPS binding to cells transfected with α1/6, β3 and γ2 subunits could be caused by the formation of populations of β3 and/or β3γ2 subunits that cannot

bind GABA.

There are four residues (G171, K173, E179, R180) in the extracellular domain of

β3 subunits that confer the ability to traffic to the membrane in the absence of other

subunits. Accordingly, the equivalent residues in the extracellular domain of β2 subunits

(D171, N173, T179, K180) prevent trafficking to the cell membrane (Taylor et al., 1999).

I used the mutant β2(GKER) and β3(DNTK) subunits to test the hypothesis that the 184

contribution of homomeric β3 receptors prevented full displacement of [35S]TBPS by

high concentrations of GABA ( > 1 μM).

I anticipated that [35S]TBPS binding to mutant α1β2(GKER)γ2 receptor would

resemble binding of [35S]TBPS to α1β3γ2 receptors, while α1β3(DNTK)γ2 receptors

would eliminate the possibility of homomeric β3 receptors, resulting in a binding curve

that resembles wild type α1β2γ2 receptors. Surprisingly, incorporation of the mutant

β3(DNTK) subunit caused no significant effect in modulation of [35S]TPBS binding by

GABA (Fig. 45A). At low concentrations of GABA (< 1 μM), binding at mutant

α1β3(DNTK)γ2 receptors resembled binding at α1β2γ2 receptors. However, at high

concentrations of GABA (> 1 μM), [35S]TBPS remained bound in a manner similar to the

α1β3γ2 subtype. In contrast, [35S]TBPS binding to mutant α1β2(GKER)γ2 receptors

resembled [35S]TBPS binding to homomeric β3 receptors at low concentrations of GABA

(< 3 μM). High concentrations of GABA (> 3 μM) caused a small inhibition of

[35S]TBPS binding in a manner similar to α1β3γ2 receptors, as expected (Fig 45B).

These data provide conflicting results. Binding to the “β3-like” mutant

β2(GKER) subunit demonstrates that homomeric β3 receptors likely play a significant

role in the way [35S]TBPS binds to recombinant receptors containing the wild-type β3

subunit. Binding of [35S]TBPS to the “β2-like” mutant β3(DNTK) subunit showed no

significant effect on modulation [35S]TBPS binding by GABA. One disadvantage to

using these mutant subunits is that they have not been characterized in the presence of an

α subunit. It is possible that in addition to affecting trafficking of homomeric receptors,

185 or receptors containing a γ2 subunit alone, these mutants may also affect GABA binding, affinity or efficacy. Nonetheless, the data described in this section suggest that homomeric β3 subunits may have significantly contributed to the reduction in displacement of [35S]TBPS binding by GABA in receptors containing the β3, but not β2 subunit.

In the future, it would be useful to examine the concentration-response relationships for GABA activation of these mutant receptors. Furthermore, it would be interesting to examine whether the β3 subunit contributes substantially to the differential binding of [35S]TBPS throughout the rodent brain. GABA is fully capable of inhibiting

[35S]TBPS binding in most brain regions. As mentioned earlier, some groups have described “GABA-insensitive” binding in the thalamus and cerebellar granule layers.

Using different planes of sectioning, my data demonstrate that high concentrations of

GABA (100 μM) cause full inhibition of [35S]TBPS binding to these brain regions.

Furthermore, knock out mice lacking the β3 subunit retain this GABA-insensitive binding phenomenon (Sinkkonen et al., 2001b). Taken together, these findings demonstrate that homomeric β3 receptors, which cannot be activated by GABA, are not likely to be present in the rodent brain.

186

Figure 45 α1β3γ2 α1β3(DNTK)γ2 α1β2γ2 α1β2(GKER)γ2

A B

175 175 150 150 125 125 100 100 75 75 50 % control binding

25 % control binding 50 0 25 0 0.01 0.1 1 10 100 1000 [GABA] (μM) 0.01 0.1 1 10 100 1000 [GABA] (μM)

Figure 45. Mutant subunits containing N-terminal residues thought to be involved in trafficking and formation of homomeric receptors bind [35S]TBPS differently than wild- type α1β2γ2 or α1β3γ2 receptors. A. [35S]TBPS binding to mutant α1β3(DNTK)γ2 receptors is shifted to the right compared to wild-type α1β3γ2 or α1β2γ2 receptors. B. Binding of [35S]TBPS to α1β2(GKER)γ2 receptor resembles β3 homomeric receptors at GABA concentrations < 3 μM but and α1β3γ2 receptors at GABA concentrations > 3 μM. Data are represented as mean ± S.E.M and statistics were obtained using one-way ANOVA with Tukey’s post-hoc test * p < 0.05, ** p < 0.01, *** p <0.001.

187

Conclusions

The main focus of this chapter was to use [35S]TBPS in three different assays to

examine the potential of using this radioligand as a tool to detect functionally distinct

GABAA receptors in the CNS. I used recombinant receptors transiently expressed in

HEK293 cells to examine [35S]TBPS binding to different receptor subtypes in the

absence of endogenous GABA. These experiments allowed for the greatest manipulation

of the microenvironment compared to the other assays outlined in this chapter.

Homogenate brain assays examined individual brain regions that were isolated and

washed to remove excess endogenous GABA or other modulators present at in vivo

GABAA receptors. This type of assay allowed me to create membrane fractions of

receptors already expressed in the rodent CNS and manipulate the microenvironment

much like the experiments using recombinant receptors. Finally, I used [35S]TBPS

autoradiography to apply the information gained from recombinant or homogenized brain

samples to intact receptors expressed in a brain slice. This approach enabled the analysis

of several brain regions in any given brain slice.

I examined [35S]TBPS binding to receptors containing the ε subunit, which attributes higher affinity for GABA as well as increased spontaneous, GABA independent gating (Whiting et al., 1997; Maksay et al., 2003; Wagner et al., 2005;

McCartney et al., 2007). I found GABA modulation of [35S]TBPS binding to be useful in

distinguishing between αβε and αβγ receptors (Fig. 24). Furthermore, I found that

pregnenolone sulfate and furosemide, two inhibitors of spontaneous, GABA-independent gating, reduced [35S]TBPS binding to recombinant receptors containing the ε subunit

188

(Fig. 25). These data are consistent with my earlier findings (Chapter 3) demonstrating

that spontaneous gating contributes to [35S]TBPS binding. Since the distribution of

receptors containing the ε subunit is not well established in the rodent brain, I examined

whether [35S]TBPS binding autoradiography with pregnenolone sulfate could be used to

detect functional receptors of this subtype. However, the drug lacked specificity in this

regard (Fig. 25). Pregnenolone sulfate may have acted competitively to prevent

[35S]TBPS binding throughout the rodent brain, thereby preventing localization of αβε

receptors. Interestingly, while furosemide blocks tonic currents recorded from

recombinant receptors containing the ε subunit, tonic currents recorded from hippocampal neurons are resistant to inhibition by furosemide (Caraiscos et al., 2004;

McCartney et al., 2007). While furosemide is known to inhibit potassium chloride

cotransporters (KCC) in the digestive tract, the binding site and mechanism of action at

GABAA receptors is still not known. These data demonstrate that furosemide is not the

best ligand to use to localize receptors containing the ε subunit in the CNS. Based on my

findings and the minimal evidence available in the GABAA receptor field, it would be

useful to further investigate other brain regions thought to contain the ε subunit like the locus coeruleus to determine whether this functionally distinct subunit exhibits any physiological relevance in the control of neuronal excitation in the CNS.

With regard to affinity for GABA, [35S]TBPS binding to recombinant receptors

was useful in detecting the high affinity α1β3ε subtype compared to the lower affinity

α1β3γ2 subtype (Fig. 26). I examined the role of agonist affinity in the modulation of

[35S]TBPS binding by GABA and found that brain regions containing high affinity

189

receptors (e.g. cerebellum) bound [35S]TBPS fully in the absence of exogenous GABA.

Furthermore, GABA caused a more potent modulation of [35S]TBPS binding to cerebellar receptors compared to receptors in the hippocampus or frontal cortex (Fig. 27, Table 2).

In contrast, propofol, an allosteric agonist at GABAA receptors, caused a concentration dependent inhibition of [35S]TBPS from all the brain regions examined, with no

significant difference in potency (Fig. 30, Table 3). These data demonstrated that the

potency of propofol in activating or desensitizing these receptors did not differ across the

frontal cortex, hippocampus and cerebellum. Propofol caused a biphasic, concentration

dependent modulation of [35S]TBPS binding in both α1β2γ2 and α1β3γ2 receptors (Fig.

31). The enhancement of [35S]TBPS binding at low concentrations of propofol

demonstrated that propofol activation of the channel facilitated accessibility to the

35 [ S]TBPS binding site. The IC50 and Hill slopes for these experiments were summarized

in Table 3 and demonstrated that receptors in the cerebellum had a distinctly higher

affinity for GABA than other brain regions, while all brain regions exhibited the same

affinity for propofol. Taken together, these data demonstrated that differences in GABA

affinity could be detected using [35S]TBPS binding to different brain regions, while

affinity for allosteric agonist are more easily detected in a recombinant setting.

Since high affinity GABAA receptors are often thought to contain the δ subunit, I sought to examine the role of the δ subunit in the differential binding of [35S]TBPS

throughout the rodent brain. Using [35S]TBPS autoradiography, I found that the high

potency of GABA in the cerebellum was detectable but less clear when compared to

homogenate brain assays described earlier (Fig. 28 and 29 compared to Fig. 27). I

190

hypothesized that endogenous GABA remained bound to high affinity receptors in the

cerebellum during the pre-incubation steps for autoradiography outlined in Chapter 2. To

examine the role of endogenous GABA in the binding of [35S]TBPS to high affinity

receptors, I employed the competitive antagonist GBZ and competitive inverse agonist

BIC. I hypothesized that GBZ and BIC would enhance [35S]TBPS binding in brain

regions where endogenous GABA remained bound. The rationale behind these

experiments was that by preventing binding of endogenous GABA to high affinity receptors, GBZ and BIC would enhance [35S]TBPS binding by counteracting the

reduction caused by GABA. I found that both antagonists significantly enhanced

[35S]TBPS binding to cerebellar receptors, but not receptors in the other brain regions

examined where there was a modest inhibition (Fig. 32-35). Furthermore, in recombinant

αβγ receptors, GBZ and BIC caused slight reductions in [35S]TBPS binding presumably

by reducing spontaneous channel opening as described in Chapter 3.

These data suggest that endogenous GABA may indeed be bound to high affinity

receptors in the cerebellum, causing altered [35S]TBPS binding patterns compared to

receptors in other brain regions. I examined whether the δ subunit contributed to the high

35 affinity GABAA receptors found to [ S]TBPS by using a δ subunit selective ligand, DS2.

I found that DS2 caused no significant effect on [35S]TBPS binding in brain regions

thought to contain the δ subunit (hippocampus or cerebellum) compared to the frontal

cortex (Fig. 36-38). These data are consistent with the idea that the δ subunit does not

support [35S]TBPS binding (Hevers et al., 2000). To examine this further, I transiently

expressed αβδ receptors in HEK293 cells and found that there was negligible GABA-

191

independent, picrotoxin-displaceable [35S]TBPS binding compared to receptors

containing the γ2 subunit (Fig. 39A). Specific binding of [35S]TBPS was also

significantly lower in the cerebellum and hippocampus compared to the frontal cortex

(Fig. 39B). Taken together, these data suggest that the δ subunit reduces [35S]TBPS to

binding, a concept that is consistent with the results of a prior study (Hevers et al., 2000).

However, subunits other than the δ subunit are thought to contribute to different

affinities for GABA. As such, I sought to examine the role of the α and β subunits, which

form the GABA binding site, and γ2 subunits, which are required for benzodiazepine

binding, in [35S]TBPS binding throughout the brain. I focused on the α6 subunit, which is

thought to predominate in the cerebellum and the α1 subunit, which is present throughout

the brain (Pirker et al., 2000). I hypothesized that α6βxγ2 receptors in the cerebellum

contribute to the high potency of GABA modulation of [35S]TBPS binding in this brain

region. I found that GABA caused significantly more potent reduction in [35S]TBPS

binding to recombinant receptors containing the α6 subunit compared to receptors

containing the α1 subunit (Fig. 40).

Since my data suggest that receptors containing the δ subunit do not bind

[35S]TBPS it is likely that the high affinity receptors detected in the cerebellum using this

method contain the γ2 subunit. I employed flunitrazepam and Ro 15-4513 to determine whether the cerebellar receptors support benzodiazepine binding and would therefore

likely contain the γ2 subunit. My data provided conflicting results. In view of the

reported inverse agonist effects of flunitrazepam on receptors containing the α6 subunit

(Hauser et al., 2002) my data are consistent with the idea that α6βxγ2 receptors bind 192

[35S]TBPS in the cerebellum. In view of the inverse agonist effects of Ro 15-4513 on

receptors containing the α1 subunit, my data are consistent with α1 and γ2 subunit

containing receptors binding [35S]TBPS throughout the rodent brain, including the

cerebellum. The overlap in distribution of α1 and α6 subunits made it difficult to distinguish between these populations in the cerebellum, however, this may be due to the presence of receptors containing both α1 and α6 subunits, that have a high affinity for

GABA but may be insensitive to modulation by benzodiazepines. Based on studies by

Fisher et al. (1997), the α1 subunit attributes a lower affinity for GABA than the α6

subunit. They also described receptors containing both the α1 and α6 subunits transiently

expressed with the β3 and γ2 subunit. The contribution of both α1 and α6 subunits increased the affinity for GABA compared to the presence of either the α1 or α6 subunits independently (Fisher et al., 1997).

The lack of effects on high affinity receptors containing α6 and γ2 subunits by Ro

15-4513 may be due to the presence of receptors that contain both α1 and α6 subunits.

Some studies have demonstrated that both α1 and α6 subunits can incorporate into

functional receptors that respond to GABA. Furthermore, these studies using

immunoprecipitation have demonstrated that cultured neurons taken from adult rodent

cerebella may express both α1 and α6 subunits within the same receptor subtype (Pollard

et al., 1995; Thompson et al., 1996). These α1α6βxγ2 receptors were thought to be

benzodiazepine insensitive. Taken together, these data demonstrate that the cerebellum

may contain a mixed population of receptors containing different stoichiometries of

either the α1 or α6 subunits, β subunits, and either γ2 or δ subunits. My findings suggest 193 that both α1 and α6 subunit-containing receptors contribute to [35S]TBPS binding in the cerebellum and that incorporation of the α6 subunit is required for high affinity modulation of [35S]TBPS binding by GABA.

The fact that both benzodiazepines did not potentiate the effects of GABA in inhibition of [35S]TBPS binding demonstrates that these ligands may not promote desensitization of the channel. Positive modulation of GABA-evoked currents by benzodiazepines is known to increase the current amplitude of responses evoked by non- saturating concentrations of GABA (Korpi and Sinkkonen, 2006; Da Settimo et al.,

2007). However, one would suspect that by increasing the affinity of the channel for

GABA, the likelihood of entering the desensitized state would increase. This phenomenon remains under debate (Yueng et al., 2003). Given the complex relationship between affinity, desensitization, and the location of the TBPS binding site within the channel, it would be useful to examine the combined effects of different benzodiazepines and GABA on the ability of TBPS to block GABA-evoked currents using the patch clamp technique.

I also found that receptors containing the β3 subunit did not exhibit full inhibition of [35S]TBPS binding by GABA. I speculated that a high proportion of β3 homomeric receptors, which are insensitive to GABA, were responsible for this phenomenon. To test this, I employed the mutant subunits β3(DNTK) and β2(GKER), which contain residues thought to prevent or allow the trafficking of β2 or β3 subunits (respectively) to the membrane (Taylor et al., 1999). Using these mutants, I found that [35S]TBPS binding to

35 αxβ3γ2 receptors may be affected by homomeric β3 receptors that bind [ S]TBPS but

194 are insensitive to GABA (Fig. 45). My data also demonstrate that β3 homomeric receptors are not likely to be present in the brain.

Taken together, the data outlined in this chapter demonstrate that the diverse pharmacology and heterogeneity of GABAA receptors contribute to the differential binding of [35S]TBPS throughout the CNS. Based on the properties of agonist affinity, subunit composition, and selectivity of different ligands for certain subunits, I have

35 demonstrated that [ S]TBPS binding can distinguish functionally distinct GABAA receptors. In order to use [35S]TBPS as a tool to detect these receptors, it is important to take into account the role of activation outlined in Chapter 3, desensitization outlined in

Chapter 4, and the contribution of individual subunits to these channel properties. In the next chapter, I will explore the relevance of the data presented in this dissertation and suggest future directions for this research.

195

Chapter 6: Discussion

GABAA receptors are known for their heterogeneity across different vertebrate

and invertebrate species. The diverse distribution of functionally distinct GABAA receptors throughout different brain regions suggests a role for individual subtypes in the regulation of distinct physiological properties of GABAergic neurotransmission.

Furthermore, the pharmacology of GABAA receptors is governed by the presence of different subunits within a functional receptor. Throughout my dissertation, I examined the contribution of different GABAA subunits to distinct channel properties, including spontaneous GABA independent gating, agonist-induced activation, agonist-induced desensitization, and the actions of subunit selective ligands, such as benzodiazepines.

The focus of this dissertation was to develop [35S]TBPS, a radiolabeled non-

competitive channel blocker, as a tool to detect functionally distinct GABAA receptors in

the rodent CNS. To accomplish this goal, I used an arsenal of methodologies including

whole cell electrophysiology, radioligand binding and autoradiography, mutant receptors,

and different pharmacological ligands specific for different GABAA subunits. The

rationale for these studies was based on the recent structural models of Cys-loop and

prokaryotic GLIC and ELIC receptor activation and desensitization gates (Wilson and

Karlin 1998, 2001; Unwin, 2005; Hilf et al., 2008; Corringer et al., 2010), as well as

35 three decades of work using [ S]TBPS as a radioligand specific for GABAA receptors.

The gating of the GABAA receptor and other ligand-gated ion channels has been

described in the context of hydrophobic “gates” that prevent or allow ion conductance

across the ion channel. The exact location of these gates remains under debate. However,

196

several groups (Unwin, 2005; Bocquet et al., 2009; Corringer et al., 2010) have

implicated residues lying in the upper TM2 region (9’ and higher, Fig. 1) in the location

of an activation gate in pentameric ligand gated ion channels pLGIC. In earlier chapters, I

described my findings in the context of three categories. I investigated the role of 1)

35 GABAA receptor activation or 2) GABAA receptor desensitization in [ S]TBPS binding

and blockade by TBPS and PIC, and 3) the role of specific subunits in the differential

35 binding of [ S]TBPS to functionally distinct GABAA receptors throughout the CNS.

TBPS is a pro-convulsant ligand that is structurally related to the more widely used ligand, PIC (Lawrence and Casida, 1983; Chen et al., 2006). The binding site for

PIC is thought to envelop the 2’, 6’ and 9’ residues in the TM2 of GABAA receptors

(Buhr et al., 2001; Sedelnikova et al., 2006; Erkkilla et al., 2008). Some studies have demonstrated that the binding site for TBPS includes the 3’ residue (Jursky et al., 2000)

as well as the 2’ and 6’ (Fig. 1 and Chen et al., 2006). In radioligand binding assays, PIC

fully displaces [35S]TBPS from all brain regions, demonstrating that the two ligands share

a common binding domain (Squires et al., 1983). As such, many of the conclusions drawn regarding TBPS have been made based on the broader literature about picrotoxin.

35 The role of GABAA receptor activation in [ S]TBPS binding

Both TBPS and PIC have been described as open-channel blockers because

GABAA receptor activation by episodic GABA application significantly enhances their

blockade or association to the GABAA receptor channel (Newland and Cull-Candy, 1992;

Dillon et al., 1995). This suggests that activation of α1β2γ2 receptors by GABA

197 increases the accessibility of both drugs to their binding sites within the open channel.

Consistent with this, low concentrations of GABA enhance [35S]TBPS binding to many types of GABAA receptors, including the α1β2γ2, α1β3γ2 (described in Chapters 3 and

5) as well as α2βxγ2, α3βxγ2, α5βxγ2, and α6βxγ2 receptors (Luddens and Korpi, 1995).

However, my findings demonstrated that blockade of different GABAA receptors (wild- type α1β2γ2, α1β3γ2 and mutant α1(K278M)β2γ2) by both PIC and TBPS occurred primarily in the absence of GABA. These data are in agreement with studies that have demonstrated that PIC is able to block GABAA receptors in the absence of episodic

GABA stimulation (Newland and Cull-Candy, 1992; Dillon et al., 1995) and studies demonstrating that the hydrophobic barrier for activation lies below the binding site for

TBPS and PIC (Wilson & Karlin, 2001; Bali and Akabas, 2007)

My findings also agree with earlier reports that have demonstrated [35S]TBPS can bind to recombinant receptors entirely in the absence of GABA (Luddens and Korpi,

1995; Davies et al., 1997b). I used recombinant receptors transiently expressed in

HEK293 cells in order to eliminate the contribution of endogenous GABA and/or

35 neurosteroids in the ability of [ S]TBPS to bind different GABAA receptor subtypes.

This technique allowed me to not only examine the role of activation, if any, by endogenous GABA, but also, the role of GABA-independent gating of GABAA receptors in the accessibility of TBPS or PIC to their binding sites within the ion channel. I was interested in the role of both agonist (GABA)-induced channel activation as well as agonist (GABA)-independent channel activation, which occurs spontaneously. Many

GABAA receptor subtypes exhibit intermittent agonist-independent spontaneous gating

198

which can be inhibited by bicuculline (BIC) and may contribute to tonic inhibition in the

CNS (McCartney et al., 2007).

I hypothesized that spontaneous gating of the α1β2γ2 receptor provided TBPS

and PIC access to the open channel in the absence of GABA. Several lines of evidence

support this idea. I used the competitive GABAA receptor inverse agonist BIC to inhibit

agonist-independent gating in the absence of GABA. Blockade by TBPS and binding of

[35S]TBPS to α1β2γ2 receptors was inhibited by BIC. This observation is in keeping with a previous finding that BIC slows the rate of blockade of GABAA receptors by TBPS

(Behrends, 2000). I observed roughly ~25% reduction of GABA-independent binding of

[35S]TBPS and blockade by TBPS in the presence of BIC. The attenuation by BIC of

GABAA receptor blockade was most effective when TBPS was applied in the absence of

episodic activation by GABA. Furthermore, in the absence of GABA, BIC caused a

concentration-dependent inhibition of [35S]TBPS binding to α1β2γ2 receptors. In brain

regions thought to contain primarily αβγ receptors, such as the frontal cortex, BIC also

caused a slight reduction in [35S]TBPS binding. These findings suggest that BIC holds the channel in a closed conformation which hinders TBPS from accessing its binding site.

These findings are consistent with earlier work by Luddens and Korpi (1995), who

35 examined the effects of competitive GABAA receptor antagonists on [ S]TBPS binding

to recombinant receptors and demonstrated that BIC inhibits [35S]TBPS binding.

While BIC is becoming more widely accepted as an inverse agonist, as opposed to

a neutral competitive antagonist, gabazine (GBZ) is considered to be a neutral

competitive antagonist. I initially used GBZ as a control to demonstrate that the effects of

199

BIC on accessibility to the TBPS and PIC binding site were not due to competitive

displacement of the channel blockers from the TM2 domain. However, I found that to

GBZ also inhibited [35S]TBPS binding to recombinant α1β2γ2 and α1β3γ2 receptors, as

well as from receptors in the frontal cortex of the rodent brain, albeit with less efficacy

than BIC. These findings were initially surprising to me, since GBZ is considered to be a

competitive antagonist. However, they are in agreement with studies from Luddens and

Korpi (1995), who observed similar effects of GBZ on [35S]TBPS binding in different

GABAA receptor subtypes. They are also in agreement with electrophysiological studies

that have demonstrated GBZ causes some reduction in both spontaneous GABAA

receptor current (Chang and Weiss, 1999) and current allosterically activated by

neurosteroids (Ueno et al., 1997).

In addition to using pharmacological means to reduce GABA-independent gating

of GABAA receptors, I also employed the mutant α1(K278M) subunit, which reduces the

efficacy of GABA as an agonist at α1β2γ2 receptors (Hales et al., 2006). The conserved

Lys residue affected by the mutation is located within the TM2-TM3 loop at a position in

the α1 subunit thought to be involved in the transduction of GABAA receptor binding to

channel gating (Kash et al., 2003). However, the K to M substitution also reduces the

efficacy of receptor activation by propofol (Hales et al., 2006). Furthermore, unpublished

studies in the lab (Deeb and Hales) have demonstrated that the K to M mutation causes a

reduction of spontaneous, PIC-sensitive, GABA-independent current. This reduction

suggests that the hydrophobic barrier against ion conductance is enhanced by the

mutation. Consistent with spontaneous gating providing access to the convulsant binding

200

site in the absence of GABA, α1(K278M)β2γ2 receptors exhibited a reduced block by

TBPS. These data agree with other studies that have demonstrated that mutations

reducing the gating efficacy of GABAA receptors also result in reduced efficacy of blockade by PIC (Buhr et al., 2001).

The mutant α1(K278M) subunit had dramatic effects on [35S]TBPS binding that

were consistent with my electrophysiological findings. Specific (PIC-displaceable)

[35S]TBPS binding to α1(K278M)β2γ2 receptors in the absence of GABA was

significantly reduced compared to specific binding to wild type GABAA receptors.

Furthermore, BIC did not cause a concentration-dependent inhibition of [35S]TBPS

binding to α1(K278M)β2γ2 receptors in the absence of GABA. Taken together these

findings suggest that a reduction in spontaneous GABAA receptor gating diminishes

[35S]TBPS binding. In keeping with this, low concentrations of GABA caused a marked

enhancement of [35S]TBPS binding well above that seen with wild type receptors,

revealing a greater proportion of the use-dependent component of binding. Based on

these data, I concluded that the mutant α1(K278M) subunit increased the requirement for

GABA-induced binding by [35S]TBPS, and blockade by TBPS or PIC, by reducing the

spontaneous, GABA-independent components described earlier in Chapter 3.

I also examined the K to M mutation on the γ2 subunit. Incorporation of the

mutant γ2(K289M) subunit in receptors with wild-type α1 and β2 subunits caused a

marked reduction in spontaneous, agonist independent gating of GABAA receptors.

Earlier findings from our lab have demonstrated that like the mutant α1(K278M) subunit,

the γ2(K289M) subunit also reduces the efficacy of GABAA receptor gating by GABA.

201

In contrast to the α1(K278M) subunit, the γ2(K289M) subunit did not reduce the potency

of GABA compared to wild-type α1β2γ2 receptors (Hales et al., 2006). Interestingly,

although the γ2(K289M) subunit reduces spontaneous, agonist-independent gating, it did

not reduce PIC-displaceable GABA-independent binding of [35S]TBPS. Furthermore,

while there was a dextral shift in potency of GABA modulation of [35S]TBPS binding

caused by both mutant subunits, the γ2(K289M) subunit did not cause an increase in

[35S]TBPS binding compared to α1β2γ2 receptors. These findings demonstrate that the

mutant α1(K278M) and γ2(K289M) subunits caused changes in efficacy that are

dependent on whether or not the subunit contributes to the orthosteric binding site (α1 or

β2, not γ2). Furthermore, the typical stoichiometry of synaptic GABAA receptors is

thought to consist of 2α, 2β and γ subunit (Whiting et al., 1999). The increased effects on efficacy caused by two mutant α1(K278M) subunits, compared to a single mutant

γ2(K289M) subunit may be a result of the increased contribution of mutant subunits to

the function of the GABAA receptor. This could be tested in the future by using

concatemers which would force the stoichiometry of mutant subunits to a known

conformation that can then be assessed for changes in GABA efficacy and affinity.

In the future, it would be interesting to examine blockade by TBPS and PIC at

these mutant receptors more thoroughly. My findings demonstrate that the mutant

α1(K278M) subunit causes a dextral shift in potency of blockade by TBPS compared to

α1β2γ2 receptors. Based on my findings, I hypothesize that mutant α1β2γ2(K289M)

receptors would not exhibit this shift, although these experiments have not yet been

performed. The corresponding K to M mutation on the β2 subunit was not discussed in 202

this dissertation. Earlier findings in the lab demonstrated that the mutant β2(K276M)

caused a reduction in potency of GABA as well as reduced surface expression (Hales et

al., 2006). In my experiments, I attempted to examine [35S]TBPS binding to mutant

α1β2(K276M)γ2 receptors and found it difficult to achieve consistent results, presumably due to reduced surface expression. Nonetheless, these findings led me to believe that the contribution of different subunits (α, β, γ, δ or ε) to efficacy and gating of GABAA receptors is described by the subunit-specific binding patterns of [35S]TBPS to functionally distinct GABAA receptors.

The 3’, 6’ and 9’ TM2 residues are above the channel activation gate according to

a model of the nACh receptor based on substituted cysteine accessibility in open and closed states (Wilson and Karlin, 1998, 2001). If this model is applicable to the GABAA receptor, then binding to these residues may be possible in the closed state and this could explain the significant component of activity-independent inhibition by PIC and TBPS that occurs in the presence of either BIC or the α1(K278M) subunit. Binding to the 2’ residue, which is close to the intracellular aspect of TM2 and well below the activation gate according to several pentameric ligand-gated ion channel models (Xu and Akabas,

1996; Wilson and Karlin, 1998; Unwin, 2005; Bocquet et al., 2009; Corringer et al.,

2010), would likely be dependent on channel opening. However, the importance of the 2’ residue to PIC binding is unclear. The Rdl receptor, a pesticide-resistant mutant GABA- gated Cl- channel in Drosophila harboring an Ala to Ser substitution, exhibits reduced

potency of block by PIC and TBPS (Zhang et al., 1994). However, the block by PIC of

the mutant channels still occurs at μM concentrations. The functional state of the receptor

203

appears to be a major contributing factor in the discrepancies between my findings, which

compared blockade by TBPS and PIC. The current data in the field, combined with my

findings, suggest that the TBPS binding site is likely to overlap the PIC binding site, but

residues below the 6’ may be more pertinent to blockade by TBPS than by PIC.

35 The role of GABAA receptor desensitization in [ S]TPBS binding

While the interactions of both TBPS and PIC with the GABAA receptor are

favored by channel opening, desensitization differentially affects their binding. As

described earlier, previous studies using TBPS and PIC have demonstrated that GABA

activation of the channel enhances the rate of blockade by TBPS and PIC (Newland and

Cull-Candy, 1992, Dillon et al., 1995). Furthermore, Bloomquist et al., (1991)

demonstrated that prolonged GABA exposure reduces the ability of TBPS to remain

bound to the channel. Based on these studies and the experiments I described in Chapter

3, which demonstrated that spontaneous channel opening and GABA activation of the channel were required for maximal GABAA receptor blockade by TBPS and PIC, I

examined the role of desensitization in the accessibility of TBPS and PIC to their binding

sites within the TM2 domain of GABAA receptors. The rationale for these experiments was that high concentrations of GABA cause inhibition of [35S]TBPS binding.

Radioligand binding assays, such as those described throughout my dissertation, are often

performed for prolonged incubation periods of over one hour. During this time, it is likely

that receptors either reach or approach steady-state, where the proportions of activated,

closed or desensitized receptors are at equilibrium at any given concentration of GABA.

204

The α1(K278M) subunit increased the GABA EC50 and caused an increase in the

half-maximal concentration required for steady-state desensitization. Interestingly, the

γ2(K289M) subunit also increased the GABA EC50 for desensitization, despite no shift in the potency of activation by GABA. However, both mutations caused a reduction in

GABA-independent and GABA-dependent gating efficacy. In my experiments, I focused on the α1(K278M)β2γ2 receptor because it caused the greatest deficit in channel function. However, in the future, it would be interesting to examine the mutant

α1β2γ2(K289M) receptor using rapid agonist application to determine if the shift in potency of desensitization by GABA is due to altered desensitization kinetics. These experiments would reveal the likelihood of the mutant α1β2γ2(K289M) receptor to

transition to the desensitized state and demonstrate whether in these receptors, the shift

from activation to desensitization is more energetically favorable. If so, these findings

would suggest that the mutant γ2(K289M) destabilizes the open state in the presence of

GABA, and reduces the open state in the absence of GABA.

PIC appears to bind preferentially to desensitized receptors (Newland and Cull-

Candy, 1992; Zhang et al., 1994). Using rapid agonist application of GABA to

recombinant α1β2γ2 and mutant α1(K278M)β2γ2 receptors, I demonstrated that

desensitization had no significant effect on blockade by PIC. In contrast, my findings

with the α1β2γ2 receptors, in agreement with previous reports involving other GABAA receptor subtypes (Davies et al., 1997b; Luddens & Korpi, 1995), demonstrate that desensitizing concentrations of GABA abolish [35S]TBPS binding. In rapid agonist

activation experiments, I demonstrated that desensitization significantly reduced the

205

efficacy of blockade by submaximal concentrations of TBPS (0.1 μM). It is likely that

the reduction in fractional available current that occurs with increasing levels of

desensitization is responsible for the reduction in blockade by TBPS. These data

demonstrate that unlike PIC, the binding site for TBPS is rendered less accessible upon

desensitization. However, in the future, it would be useful to examine submaximal concentrations of PIC (< 100 μM) to accurately establish the effects of desensitization on blockade by PIC, using the same protocol described for three concentrations of TBPS

(0.1, 1 and 10 μM).

If the desensitization gating model proposed by Wilson and Karlin (1998, 2001) is applicable to my findings, these data would demonstrate that the binding site for TBPS lies below the desensitization gate, while the binding site for PIC lies at least partially above the desensitization gate. Alternatively, PIC may become trapped below the desensitization gate during prolonged exposures to GABA. This would likely explain the increased affinity for the desensitized channel described by other groups (Newland and

Cull-candy, 1992). This could be easily tested in the future with an ultra-rapid agonist

application system, which, unlike my experimental system, would not be limited by the

rate of solution exchange. An increased rate of unbinding of PIC would demonstrate that

the desensitized channel has a lower affinity for the channel, while a decreased rate of

unbinding would indicate that PIC binds favorably to the desensitized state.

35 Some GABAA receptors remain bound to [ S]TBPS in the presence of high concentrations of GABA (Luddens and Korpi, 1995, Davies et al., 1997b, Halonen et al.,

2009). I hypothesized that the differences in the ability of GABA to reduce [35S]TBPS

206

binding at different GABAA receptor subtypes corresponded to differences in

desensitization attributed by different GABAA subunits. My findings comparing α1β2γ2

and α1(K278M)β2γ2 receptors are consistent with this hypothesis. High concentrations of GABA that cause desensitization in α1β2γ2 receptors also caused a reduction in

[35S]TBPS binding. Similarly, higher concentrations of GABA were required to cause the

same reduction of [35S]TBPS binding in α1(K278M)β2γ2 receptors compared to α1β2γ2

receptors, suggesting the potency of inhibition of [35S]TBPS by GABA was reduced by

the mutant α1(K278M) subunit. Consistent with these findings is the dextral shift in

potency of GABA for desensitization of mutant α1(K278M)β2γ2 receptors compared to

wild-type α1β2γ2 receptors. The increase in the fraction of current available due to reduced potency of desensitization of α1(K278M)β2γ2 receptors correlates with

increased [35S]TBPS binding at high GABA concentrations. Taken together, these

findings demonstrate that receptors with reduced sensitivity to desensitization by a given

concentration of an agonist are likely to bind [35S]TBPS at higher levels than receptors

that are more sensitive to desensitization.

Although my data follow a trend that suggests desensitization plays a vital role in

35 governing the displacement of [ S]TBPS binding from different GABAA receptor

subtypes, there were some flaws in my experimental design. The steady-state

desensitization experiments demonstrated that the mutant α1(K278M) subunit reduced

the potency of desensitization by GABA. In my experiments using rapid agonist application, I used 100 μM GABA to induce desensitization in mutant α1(K278M)β2γ2

receptors. At the time, this seemed logical, since I was comparing the effects of 100 μM 207

GABA on [35S]TBPS binding. At this concentration, [35S]TBPS was fully diminished in

α1β2γ2 receptors, but not mutant α1(K278M)β2γ2 receptors. However, in the

electrophysiological assay, 100 μM GABA is a non-saturating concentration for

α1(K278M)β2γ2 receptors. It would be useful to repeat these experiments using a

saturating concentration of GABA (1 mM or higher) to more reproducibly induce full

activation and desensitization of mutant α1(K278M)β2γ2 receptors. With these

parameters changed, I suspect the results would mimic those obtained from α1β2γ2

receptors, but with a dextral shift in potency of desensitization by GABA, as expected.

Furthermore, the mutant α1(K278M)β2γ2 receptor reduced the potency of

blockade by TBPS. This may be a direct result of the altered gating properties elicited by

the K to M mutation, which render the TBPS binding site less accessible in the absence

of GABA. This would explain the increased level of GABA-induced [35S]TBPS binding

observed for α1(K278M)β2γ2 receptors, but not α1β2γ2(K289M) receptors. My

suspicion is that if the PIC-displaceable, GABA-independent binding of [35S]TBPS to

different receptor subtypes was examined in parallel to all [35S]TBPS binding assays,

those receptors exhibiting greater enhancement of [35S]TBPS binding by GABA would conversely exhibit a reduced level of GABA-independent [35S]TBPS binding. However,

this marked enhancement in GABA modulation of [35S]TBPS binding only occurred at

receptors containing the mutant α1(K278M) subunit.

208

35 The development of [ S]TBPS as a probe for functionally distinct GABAA receptors

It is rare that a radioligand binding assay be described as a functional method of

examining different channel properties. However, [35S]TBPS is unlike most other

radioligands because its binding is dependent on channel function. The location of the

35 [ S]TBPS binding site within the GABAA receptor channel, as opposed to externally at the extracellular agonist or benzodiazepine binding sites, makes it a good candidate for

35 use as a tool to detect GABAA receptor function. Typically, [ S]TBPS is used to localize

GABAA receptors in the CNS (Squires et al., 1983; Korpi and Luddens, 1993; Sinkkonen

et al., 2001). Furthermore, [35S]TBPS binding assays are usually supplemented with

either other radioligand assays such as [3H]muscimol or various radiolabeled

benzodiazepines (Sinkkonen et al., 2001, Hevers et al., 2000; Halonen et al., 2009).

I proposed to use [35S]TBPS as a functional tool on the basis of the relationship

between channel function (activation and desensitization) and levels of binding to

different GABAA receptor subtypes. Based on my earlier findings, which demonstrated

that reducing spontaneous GABA-independent gating also reduced [35S]TBPS binding, I

hypothesized that the ε subunit, an auxiliary subunit known to cause high levels of

spontaneous gating (Davies et al., 2001; McCartney et al., 2007) could be localized in the

rodent CNS. Little is known about the rodent isoforms of the ε subunit (alternatively

spliced ε-long and ε-short subunits) and to date, they have been difficult to identify using conventional techniques such as brain slice electrophysiology, in situ hybridization and immunohistochemstry (Sinkkonen et al., 2000; Davies et al., 2002). I hypothesized that tonic inhibition caused by spontaneous gating could have a physiological role in the

209

control of neuronal inhibition in the absence of GABA. In the last decade, mRNA for the

ε subunit has been most consistently indentified in the rodent locus coeruleus and thalamus (Sinkkonen et al., 2000). The locus coeruleus has been implicated in noradrenergic systems and the control of cognitive disorders as well as sleep (Ogren et

al., 1980), while the thalamus is known to contain several nuclei that are highly

specialized in the control of neurotransmission across the brain as well as from the spinal

cord to the cortex (Haber and Calzavara, 2009). As such, a receptor that exhibited a level

of control that was independent of neuronal firing seemed like a physiological advantage,

or safety mechanism to protect against overexcitation of different neuronal pathways.

I sought to localize the ε subunit based on the property of spontaneous gating. In

recombinant α1β3ε receptors, pregnenolone sulfate and furosemide, two known

inhibitors of spontaneous gating, caused a reduction in [35S]TBPS binding. I

hypothesized that by inhibiting GABA-induced [35S]TBPS binding using GBZ or BIC,

along with spontaneous gating not mediated by the ε subunit, I could simultaneously use either pregnenolone sulfate or furosemide to localize brain regions high in receptors

containing the ε subunit. Based on preliminary data demonstrating that pregnenolone sulfate was a better inhibitor of [35S]TBPS binding in α1β3ε receptors, I chose to focus

on using it to modulate [35S]TBPS autoradiography in sagittal and coronal rodent brain

slices. Several pitfalls came across and as such, these experiments required further

investigation. I was unable to unambiguously localize the locus coeruleus, a brain region

thought to contain high expression levels of mRNA, using [35S]TBPS binding.

210

I was surprised to see that I could not localize receptors containing the ε subunit

in the rodent brain using [35S]TBPS. The lack of specificity of pregnenolone sulfate for

receptors containing the ε subunit made it difficult to isolate αβε receptors throughout the brain. In situ hybridization studies demonstrate that the ε subunit is not localized in the frontal cortex, cerebellum or hippocampus, yet pregnenolone sulfate modulated

[35S]TBPS binding to these regions. Pregnenolone sulfate also causes inhibition of

GABA-evoked currents at receptors containing the γ2 subunit. These findings

demonstrate that pregnenolone sulfate was not specific enough to modulate receptors

solely containing the ε subunit, and not other auxiliary subunits. The neurosteroid binding

site for GABAA receptors lies within the TM1 and possibly TM2 domain (Hosie et al.,

2006; Baker et al., 2010). Interestingly, while pregnenolone sulfate has been shown to

inhibit tonic current caused by the ε subunit (McCartney et al., 2007), it may prevent accessibility to the TBPS binding site (by direct competition) in different GABAA

receptors, regardless of subunit composition. However, since pregnenolone sulfate did

not cause complete displacement of [35S]TBPS binding, it is unlikely that the two ligands

share a common binding site, nor do their binding sites significantly overlap. In the

future, it would be interesting to examine receptors containing rodent ε subunits to determine whether they exhibit similar sensitivities to pregnenolone sulfate, furosemide or other more specific inhibitors of spontaneous gating compared to the human ε subunit used in my experiments.

211

Using [35S]TBPS binding to detect receptors containing the δ subunit

In addition to the ε subunit, two other auxiliary subunits exist. The δ subunit is thought to be present solely in high affinity extrasynaptic receptors, while the γ2 subunit

is thought to be present in most synaptic receptors throughout the brain (Nusser et al.,

1998; Fritschy and Brunig, 2003). Furthermore, the γ subunit is required for

benzodiazepine binding and the distribution and sensitivity of αβγ receptors to different benzodiazepines is primarily governed by the identity of the α subunit (Korpi and

Luddens, 1993; Rudolph and Mohler, 2004; Da Settimo et al., 2007).

GABA modulation of binding of [35S]TBPS to different homogenized brain

regions demonstrated that reductions in [35S]TBPS binding were dependent on the

affinity of the receptor for GABA. These findings agreed with earlier studies which

demonstrated that in the cerebellum, GABA caused non-competitive displacement of

[35S]TBPS binding at lower concentrations than were required in other brain regions

(Sinkkonen et al., 2001). In contrast, modulation of [35S]TBPS binding by propofol

demonstrated that agonists without subunit-selective efficacies could not be used to

distinguish between different brain regions. I confirmed that propofol did not

competitively displace [35S]TBPS by using recombinant α1β2γ2 and α1β3γ2 receptors

transiently expressed in HEK293 cells. Propofol caused a biphasic modulation of

[35S]TBPS binding to both receptor subtypes, which confirmed that the effects of

propofol on [35S]TBPS binding to homogenized brain regions were based on channel

activation, not competitive displacement. At high concentrations of propofol, I

hypothesized that the reduction in [35S]TBPS binding was due to desensitization of

212

receptors. These data also suggest that α1β2γ2 and α1β3γ2 receptors exhibit different

affinities for propofol. However, this may have been due to the presence of homomeric

β3 receptors that are insensitive to GABA, but contain a binding site for [35S]TBPS. I will address this possibility later in this chapter.

For most of my experiments examining GABAA receptors in vivo, I compared the frontal cortex, hippocampus and cerebellum, since these brain regions are well characterized and the subunit composition of GABAA receptors expressed are well

known compared to other brain regions. In [35S]TBPS autoradiography experiments, the

significant sinistral shift in GABA potency observed for cerebellar receptors was masked.

I hypothesized that endogenous GABA remained bound to high affinity cerebellar receptors and used the competitive antagonists BIC and GBZ to prevent binding of

GABA to high affinity receptors. Both ligands caused an increase in [35S]TBPS binding

to cerebellar receptors, consistent with the hypothesis that endogenous GABA remained

bound to high affinity receptors and modulated [35S]TBPS binding prior to addition of

exogenous GABA. Taken together, these data demonstrated that differences in

methodologies between [35S]TBPS autoradiography binding to homogenized brain

membranes contribute to disparate observations using these two techniques.

To elucidate whether the high affinity receptors detected in the cerebellum contained the δ subunit, I attempted to localize αβδ receptors using DS2, a δ-selective

ligand that has been previously described to cause positive allosteric modulation of

35 thalamic α4βxδ receptors (Wafford et al., 2009). [ S]TBPS autoradiography experiments

demonstrated that DS2 showed no selectivity in modulation of [35S]TBPS binding to

213

receptors in the frontal cortex, hippocampus and cerebellum compared to modulation by

GABA alone. DS2 showed reduced efficacy at receptors containing the γ2 subunit

(Wafford et al., 2009), which suggests that it should only modulate [35S]TBPS binding to

receptors containing the δ subunit. However, the lack of significant effect observed in the

presence of DS2 suggests that [35S]TBPS was not binding to receptors containing the δ subunit.

I transiently expressed recombinant α6β3δ and α4β2δ receptors in HEK293 cells and found that PIC-displaceable GABA-independent [35S]TBPS binding was

significantly reduced compared to receptors containing the γ2 subunit. Furthermore, PIC-

displaceable binding was also reduced in cerebellar and hippocampal membranes

compared to the frontal cortex. The addition of GABA to control experiments caused no

significant increase in PIC-displaceable binding. Taken together, these findings agree

with other studies that have demonstrated the incorporation of the δ subunit with αβγ

subunits caused a significant reduction in specific [35S]TBPS binding (Hevers et al.,

2000). They also demonstrate that the δ subunit does not support [35S]TBPS binding.

Studies using a related Drosophila receptor mutation, Rdl, demonstrated that TM2 residues 1’, 2’ and 3’ are required for optimal blockade of GABA-evoked currents by

TBPS and PIC (Zhang et al., 1994). This led me to wonder whether the δ subunit contained the residues necessary to form the TBPS binding site. Studies by Jursky et al.

(2000) used chimeric β3 subunits containing residues from α1 and β3 TM2 domains, expressed with wild-type α1 subunits to determine the binding site for TBPS on GABAA

β3 subunits. The authors demonstrated that three residues in the β3 subunit were required 214

to form a high affinity TBPS binding site in α1β3 GABAA receptors. The residues were

V251, A252 and L253, corresponding to the 1’, 2’ and 3’ residues. Some mutations of these residues reduced the affinity of [35S]TBPS compared to wild-type receptors.

Interestingly, these residues overlap with the Rdl sequence identified in Drosophila

studies (Ffrench-constant et al., 1993). In the GABAA δ subunit, the 2’ residue is a Ser,

not Ala, which is the same substitution that caused insecticide resistance in the Rdl

mutant described above. Taken together, these findings demonstrate that the δ subunit

may not contain the necessary residues to form a high affinity TBPS binding site.

The role of different α subunits in GABA affinity

Since experiments examining GABA modulation of [35S]TBPS binding

demonstrated that receptors in the cerebellum exhibited a high affinity for GABA

compared to other receptor subtypes, I sought to determine which receptor subtypes were

contributing to this high affinity phenomenon. Several groups have demonstrated that the

α6 subunit increases the affinity of a receptor for GABA when compared to receptors containing the α1 subunit (Fisher et al., 1997; Fisher, 2004). I compared [35S]TBPS

binding to recombinant α1β2γ2, α1β3γ2 and α6β3γ2 receptors in the presence of various

concentrations of GABA. PIC-displaceable GABA-independent binding was unaltered by the presence of different α or β subunits. However, the modulation of [35S]TBPS binding

was different across the three subtypes. GABA caused a biphasic modulation of

[35S]TBPS binding to receptors containing the α1 subunit, consistent with the findings of

other groups (Korpi and Luddens, 1995; Davies et al., 1997b). However, at the same

215

concentrations, GABA caused a significantly more potent reduction in [35S]TBPS

binding in receptors containing the α6 subunit, also consistent with the findings of Korpi

and Luddens (1995). This led me to believe that the high affinity GABAA receptors detected using homogenized brain regions and autoradiography were receptors containing the α6βxγ2 subtype, not receptors containing the δ subunit. To strengthen these results in

35 the future, it would be useful to examine modulation of [ S]TBPS binding to α6βxγ2 in a lower concentration range of GABA (< 30 nM) than was used in my experiments.

However, based on studies by Fisher et al., it is unlikely that much lower concentrations would cause significant effects on [35S]TBPS binding, since they do not evoke currents

recorded from recombinant α6β3γ2 receptors (Fisher et al., 1997).

The role of the γ2 subunit in [35S]TBPS binding to cerebellar receptors

I investigated the contribution of the γ2 subunit to [35S]TBPS autoradiography to

cerebellar receptors two different benzodiazepines, flunitrazepam and Ro 15-4513. Since

my earlier findings demonstrated that the δ subunit did not support [35S]TBPS binding, I

35 hypothesized that high affinity GABAA receptors in the cerebellum bound to [ S]TBPS contained the γ2 subunit, which is required for modulation by benzodiazepines. Hauser et al. demonstrated that flunitrazepam was a weak inverse agonist at receptors containing the α6 subunit, but exhibited positive efficacy at receptors containing the α1 subunit. In contrast, they found that Ro 15-4513 behaved as an inverse agonist at receptors containing the α1 subunit, but not the α6 subunit. I used these experiments as a basis for my own autoradiography experiments to examine whether receptors bound to [35S]TBPS 216

in the cerebellum contained the α6 and γ2 subunits. I found that flunitrazepam and Ro

15-4513 caused an enhancement of [35S]TBPS binding in brain regions containing either

the α6 or α1 subunit, respectively, consistent with appropriate inverse agonist effects. In future experiments, it would be useful to examine the effects of flunitrazepam and Ro 15-

4513 on [35S]TBPS to recombinant α1β2γ2 or α6β2γ2 receptors to thoroughly compare

these results to the studies conducted by Hauser et al. (1997).

However, the effects of these benzodiazepines as positive modulators of

GABAergic activity at either receptors containing the α1 subunit (flunitrazepam) or the

α6 subunit (Ro 15-4513) were not seen using [35S]TBPS binding. As described in

Chapter 4, desensitization of GABAA receptors reduces blockade by TBPS. Based on

these findings, I hypothesized that reduction in [35S]TBPS binding by agonists are also

due to desensitization, as shown by the concentration-dependent reduction in [35S]TBPS binding observed in the presence of GABA or propofol. Positive modulation of GABA- evoked currents by benzodiazepine ligands is known to increase the current amplitude of responses evoked by non-saturating concentrations of GABA (Korpi and Sinkkonen,

2006; Da Settimo et al., 2007). One might suspect that by increasing the affinity of the channel for GABA, the likelihood of entering the desensitized state would increase. It is possible that some benzodiazepines with positive efficacy at different GABAA receptors

increase GABA-activation of the channel, but not desensitization. In future experiments,

it would be worthwhile to examine the combined effects of different benzodiazepines and

GABA on the ability of TBPS to block GABA-evoked currents using the patch clamp

technique.

217

The impact of different β subunits on [35S]TBPS binding

Interestingly, I observed significant differences in the patterns of non-competitive

displacement of [35S]TBPS binding by GABA across receptors containing β2 or β3

subunits. The β3 subunit is unique in its ability to traffic to the cell membrane as part of a

functional homomeric receptor, whereas other GABAA subunits lack this ability. To

determine whether my recombinant assays contained a population of homomeric β3

receptors that are insensitive to GABA activation but retain a binding site for [35S]TBPS

(Davies et al., 1997b; Chen et al., 2006), I employed mutant β2 and β3 subunits. Taylor et al. demonstrated that four residues in the β3 subunit (G171, K173, E179, R180) are

required for trafficking to the cell membrane, whereas the corresponding four residues in

the β2 subunit (D171, N173, T179, K180) prevent trafficking of homomeric receptors

(Taylor et al., 1999). The mutant β3(DNTK) subunit caused no significant effect on

[35S]TBPS binding compared to α1β3γ2 receptors. However, the mutant β2(GKER)

subunit caused a pattern that resembled [35S]TBPS binding to homomeric β3 receptors at

low concentrations of GABA (< 1 μM) and resembled α1β3γ2 receptors at higher

concentrations of GABA. Taken together, these combined results demonstrated that

homomeric receptors do represent a cause for concern in experiments examining

recombinant receptors containing the β3 subunit.

One disadvantage to using these mutant subunits is that their effects on GABA

binding, efficacy and potency are not known. Taylor et al. (1999) described these mutants

in the context of receptors containing β and γ2 subunits. As such, while their findings

demonstrated that mutant β2(GKER) subunits could successfully traffic the γ2 subunit to 218

the cell membrane as part of a functional receptor, they lacked definition of the GABA

binding site formed between α and β subunits (Taylor et al., 1999). To follow up on my

findings, it would be beneficial to examine the concentration-response relationships for

GABA activation and desensitization of α1β2(GKER)γ2, α1β2(GKER), α1β3(DNTK)γ2

and α1β3(DNTK) receptors to determine the role of these residues in GABA binding and

efficacy. I propose examining the mutant β subunits in the presence and absence of γ2

subunits in order to determine potential differences in αβ versus αβγ receptors that are

affected by these mutations. The physiological relevance of homomeric β3 receptors is

still unknown. Recently, extrasynaptic α1β3 receptors have been identified in

hippocampal neurons (Mortensen and Smart, 2006). Some [35S]TBPS binding studies

have demonstrated that in parts of the thalamus, [35S]TBPS remains bound to receptors in

the presence of high concentrations of GABA (Halonen et al., 2009). These properties are

similar to the effects of homomeric receptors on [35S]TBPS binding. However, in my

experiments, GABA caused complete inhibition of [35S]TBPS binding to sagittal and

coronal brain slices, suggesting that the GABA-insensitive homomeric β3 receptors do

not exist in these planes. Furthermore, knockout mouse models eliminating expression of

the β3 subunit do not reveal reductions in GABA-insensitive binding, suggesting that

homomeric β3 receptors are not likely to be expressed in the brain (Sinkkonen et al.,

2001b).

219

35 The future of [ S]TBPS as a tool to detect functionally distinct GABAA receptors

The findings presented in this dissertation draw pharmacological relevance to several different disorders of the nervous system. However, using a convulsant ligand like [35S]TBPS as a probe for these disorders in patients is not possible for obvious

reasons. One of the most interesting recent applications of [35S]TBPS binding has been

the use of this assay on postmortem human brain samples. Atack et al. (2007)

demonstrated that frozen brain samples of postmortem human tissue could be used in

[35S]TBPS binding assays. Their findings are the first of their kind to demonstrate that

[35S]TBPS bound successfully to postmortem human tissue. They compared their

findings to similar assays using rodent brain samples and demonstrated that [35S]TBPS bound similarly to both tissue types. Furthermore, modulation of [35S]TBPS binding by

picrotoxin, loreclezole, pentobarbital, GABA and several benzodiazepines was virtually

indistinguishable from samples obtained from rodent brains. These data demonstrate the

possibility of examining biopsy samples taken from diseased patients using [35S]TBPS

binding to identify differential receptor expression or even verify the localization of

mutated subunits. Brain tissue is sometimes removed from patients suffering intractable

epilepsy.

35 [ S]TBPS binding provides the opportunity to characterize functional GABAA receptors associated with different human disease states post mortem. The techniques outlined throughout this dissertation have established a pivotal role for channel function in [35S]TBPS binding that significantly alters the binding patterns across different

35 GABAA receptor subtypes. I have also established that [ S]TBPS can be used to

distinguish between receptors containing subunits with one or several mutated residues. 220

These data demonstrate that [35S]TBPS can also be used to detect genetic aberrations in

animal models of disease, or perhaps human brain tissue from deceased patients.

Differential [35S]TBPS binding demonstrates that the control of neuronal

excitation by GABAA receptors exhibiting different functional properties is localized to

specific brain regions. Armed with the knowledge that ~25% of accessibility to the TBPS binding site is dependent on channel activation, as shown by an enhancement of

[35S]TBPS binding and blockade by GABA, and the fact that inhibition of [35S]TBPS by

high concentrations of GABA is dependent on desensitization of GABAA receptors, any

35 GABAA receptor subtype with the residues necessary to bind [ S]TBPS can be

characterized using this radioligand as a functional tool.

For basic research, [35S]TBPS can be used in conjunction with different

modulatory ligands, or in parallel with other radioligand assays to draw comparisons

across the distinct subtypes modulated in each type of assay. These experiments have

35 been done for several years, however the role of GABAA receptor function in [ S]TBPS binding is often inferred based on electrophysiological data published examining the more widely used antagonist, PIC, not TBPS. In the future, it would be useful to examine blockade by TBPS more thoroughly in order to accurately describe its binding site within

the GABAA receptor channel. This will help to confirm my findings, which have

demonstrated that activation and desensitization have differential effects on blockade of

GABAA receptors by TBPS or PIC. These differential effects identify a need for further

investigation of TBPS on its own, as opposed to inferences based on the effects of PIC at

different GABAA receptors. The GABAA receptor is a highly complex structure, however

my findings have added to the field by more thoroughly describing the role of different 221

35 GABAA subunits and their functional properties in [ S]TBPS binding, a technique

35 widely used by many groups to localize GABAA receptors. Using [ S]TBPS binding in parallel with electrophysiology helps bridge the gap between binding of the ligand within the channel and the functional state of the receptors bound to [35S]TBPS.

222

References

Akabas MH et al. Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the alpha subunit. Neuron. 13: 919-27. (1994)

Amin J and Weiss DS. GABAA receptor needs two homologous domains of the beta- subunit for activation by GABA but not by pentobarbital. Nature. 366: 565-9. (1993)

Atack JR et al. Characterization of [35S]t-butylbicyclophosphorothionate ([35S]-TBPS) binding to GABAA receptors in postmortem human brain. British Journal of

Pharmacology. 150: 1066-1074. (2007)

Atack JR et al. Anxiogenic properties of an inverse agonist selective for α3 subunit- containing GABAA receptors. British Journal of Pharmacology. 144: 357-366. (2005)

Atack JR. Preclinical and clinical pharmacology of the GABAA receptor α5 subtypeselective inverse agonist α5IA. Pharmacology & Therapeutics. 125: 11-26. (2010)

Auerbach A and Akk G. Desensitization of mouse nicotinic acetylcholine receptors.

Journal of General Physiology. 112: 181-97. (1998)

Bai D et al. Distinct functional properties of tonic and quantal inhibitory postsynaptic currents mediated by γ-aminobutyric acidA receptors in hippocampal neurons. Molecular

Pharmacology. 59: 814-824. (2001)

Baker C et al. Multiple roles for the first transmembrane domain of GABAA receptor subunits in neurosteroid modulation and spontaneous channel activity. Neuroscience

Letters. 473: 242-7. (2010)

Bali M and Akabas MH. Defining the propofol binding site location on the GABAA receptor. Molecular Pharmacology. 65: 68-76. (2004) 223

Bali M and Akabas MH. The location of a closed channel gate in the GABAA receptor channel. Journal of General Physiology. 129: 145-159. (2007)

Barberis A et al. Zinc inhibits miniature GABAergic currents by allosteric modulation of

GABAA receptor gating. Journal of Neuroscience. 20: 8618-27. (2000)

Barnes EM. Intracellular trafficking of GABAA receptors. Life Science. 66: 1063-70.

(2000)

Barnes EM. Use-dependent regulation of GABAA receptors. International Review of

Neurobiology. 39: 53-76. (1996)

Barnes NM et al. The 5-HT3 receptor – the relationship between structure and function.

Neuropharmagology. 56: 273-84. (2009)

Bartos M et al. Structural basis of activation of Cys-Loop receptors: the extracellular– transmembrane interface as a coupling region. Molecular Neurobiology. 40: 236-252.

(2009)

Baumann SW et al. Individual properties of the two functional agonist sites in GABA(A) receptors. Journal of Neuroscience. 23: 11158-66. (2003)

Baur R et al. A GABA(A) receptor of defined subunit composition and positioning: concatenation of five subunits. FEBS Letters. 580: 1616-20. (2006)

Baur R et al. Diversity of structure and function of α1α6β3δ GABAA receptors: comparison with α1β3δ and α6β3δ receptors. 285: 17398-405. (2010)

Behrends JC. Modulation by bicuculline and penicillin of the block by t-butyl-bicyclo- phosphorothionate (TBPS) of GABA(A)-receptor mediated Cl(-)-current responses in rat striatal neurones. British Journal of Pharmacology. 129: 402-8. (2000)

224

Belelli D and Lambert JJ. Neurosteroids: endogenous regulators of the GABA(A) receptor. Nature Reviews: Neuroscience. 6: 565-75. (2005)

Belelli D et al. General anaesthetic action at transmitter-gated inhibitory amino acid receptors. TiPS. 20: 496-502. (1999)

Belelli D et al. The interaction of the general anesthetic etomidate with the γ-aminobutyric acid type A receptor is influenced by a single amino acid. PNAS Pharmacology. 94:

11031-36. (1997)

Belujon P et al. Inhibitory transmission in locus coeruleus neurons expressing GABAA receptor epsilon subunit has a number of unique properties. Journal of Neurophysiology.

102: 2312-25. (2009)

Belzung C. The genetic basis of the pharmacological effects of anxiolytics: a review based on rodent models. Behavioral Pharmacology. 12: 451-460. (2001)

Bettler B et al. Molecular structure and physiological functions of GABA(B) receptors.

Physiological Reviews. 84:837-67. (2004)

Bianch MT et al. Microscopic kinetic determinants of macroscopic currents: insights from coupling and uncoupling of GABAA receptor desensitization and deactivation. Journal of

Physiology. 584: 769-87. (2007)

Bianchi MT and Macdonald RL. Neurosteroids shift partial agonist activation of GABAA receptor channels from low- to high-efficacy gating patterns. Journal of Neuroscience. 23:

10934-43. (2003)

225

Bianchi MT and Macdonald RL. Slow phases of GABA(A) receptor desensitization: structural determinants and possible relevance for synaptic function. Journal of

Physiology. 544: 3-18. (2002)

Bieda MC and MacIver MB. Major role for tonic GABAA conductances in anesthetic suppression of intrinsic neuronal excitability. Journal of Neurophysiology. 92: 1658-67.

(2004).

Birnir B et al. Spontaneously opening GABAA in CA1 pyramidal neurons of the rat hippocampus. Journal of Membrane Biology. 174: 21-29. (2000)

Blednov YA et al. Loss of ethanol conditioned taste aversion and motor stimulation in knockin mice with ethanol-insensitive α2-containing GABAA receptors. Journal of

Pharmacology & Experimental Therapeutics. 366: 145-154. (2011)

Bloomquist JR et al. Prolonged exposure to GABA activates GABA-gated chloride channels in the presence of channel-blocking convulsants. Comparative Biochemistry and

Physiology, C. Comparative Pharmacology & Toxicology. 99: 397-402. (1991)

Bocquet N et al. X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation. Nature Letters. 457: 111-4. (2009)

Bogdanov Y et al. Synaptic GABAA receptors are directly recruited from their extrasynaptic counterparts. EMBO Journal. 25: 4381-9. (2006)

Borghese CM. An isoflurane- and alcohol-insensitive mutant GABAA receptor α1 subunit with near-normal apparent affinity for GABA: characterization in heterologous systems and production of knockin mice. Journal of Pharmacology and Experimental

Therapeutics. 319: 208-18. (2006)

226

Botta P et al. Modulation of GABAA receptors in cerebellar granule neurons by ethanol: a review of genetic and electrophysiological studies. Alcohol. 41: 187-199. (2007)

Brejc K et al. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature. 411: 269-76. (2001)

Brickley SG et al. Single-channel properties of synaptic and extrasynaptic GABAA receptors suggest differential targeting of receptor subtypes. Journal of Neuroscience. 19:

2960-2973. (1999).

Bright DP et al. Profound desensitization by ambient GABA limits activation of δ- containing GABAA receptors during spillover. Journal of Neuroscience. 31: 753-63.

(2011)

Buhr A et al. Two novel residues in M2 of the γ-aminobutyric acid type A receptor affecting gating by GABA and picrotoxin affinity. Journal of Biological Chemistry. 276:

7775-7781. (2001)

Calkin PA and Barnes EM. γ-Aminobutyric acid-A (GABAA) agonists down regulate

GABAA/Benzodiazepine receptor polypeptides from the surface of chick cortical neurons.

Journal of Biological Chemistry. 269: 1548-53. (1994)

Caraiscos VB et al. Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by alpha5 subunit-containing gamma-aminobutyric acid type A receptors.

PNAS. 101: 3662-7. (2004)

Carland JE et al. Characterization of the effects of charged residues in the intracellular loop on ion permeation in α1 Glycine receptor channels. Journal of Biological Chemistry.

284: 2023-30. (2009)

227

Carta M et al. Alcohol enhances GABAergic transmission to cerebellar cranule cells via an increase in golgi cell excitability. Journal of Neuroscience. 24: 3746-3751. (2004)

Casida JE and Lawrence LJ. Structure-activity correlations for interactions of bicyclophosphorus esters and some polychlorocycloalkane and pyrethroid insecticides with the brain-specific t-butylbicyclophosphorothionate receptor. Environmental health perspectives. 61: 123-132. (1985)

Chang Y and Weiss DS. Allosteric activation mechanism of the α1β2γ2 γ-Aminobutyric acid type A receptor revealed by mutation of the conserved M2 residue. Biophysical

Journal. 77: 2542-51. (1999)

Chang Y et al. Stoichiometry of a recombinant GABAA receptor. Journal of

Neuroscience. 16:5415-5424. (1996)

Chapell R et al. Activation of protein kinase C induces γ-aminobutyric acid type A receptor internalization in Xenopus Oocytes. Journal of Biological Chemistry. 273:

32595-601. (1998)

Chen C and Okayama H. High-efficiency transformation of mammalian cells by plasmid

DNA. Molecular and Cellular Biology. 7: 2745-52. (1987)

Chen L et al. Structural model for γ-aminobutyric acid receptor noncompetitive antagonist binding: Widely diverse structures fit the same site. PNAS. 103: 5185-90. (2006)

Chen L et al. The gamma-aminobutyric acid type A (GABAA) receptor-associated protein

(GABARAP) promotes GABAA receptor clustering and modulates the channel kinetics.

PNAS. 97: 11557-62. (2000)

228

Chen ZW and Olsen RW. GABAA receptor associated proteins: a key factor regulating

GABAA receptor function. Journal of Neurochemistry. 100: 279-94. (2007)

Collingridge GL et al. A nomenclature for ligand gated ion channels.

Neuropharmacology. 56: 2-5. (2009)

Connolly CN and Wafford KA. The Cys-loop superfamily of ligand-gated ion channels: the impact of receptor structure on function. Biological Society Transactions. 32: 529-

534. (2004)

Connolly CN et al. Subcellular localization of gamma-aminobutyric acid type A receptors is determined by receptor beta subunits. PNAS. 93: 9899-904. (1996)

Corringer PJ et al. Atomic structure and dynamics of pentameric ligand-gated ion channels: new insight from bacterial homologues. Journal of Physiology. 588: 565-72.

(2008)

Corringer PJ et al. Nicotinic receptors at the amino acid level. Annual Review of

Pharmacology & Toxicology. 40: 431-58. (2000)

Crabbe JC et al. Alcohol-related genes: contributions from studies with genetically engineered mice. Addiction Biology. 11: 195-269. (2006)

Da Settimo F et al. GABAA/Bz receptor subtypes as targets for selective drugs. Current

Medicinal Chemistry. 14: 2680-701. (2007)

2+ Davies PA et al. A novel class of ligand-gated ion channel is activated by Zn . Journal of

Biological Chemistry. 278: 712-17. (2003)

Davies PA et al. Alternative transcripts of the GABAA receptor ε subunit in human and rat. Neuropharmacology. 43: 467-475. (2002)

229

Davies PA et al. Evidence for the formation of functionally distinct αβγε GABAA receptors. Journal of Physiology. 537.1: 101-113. (2001)

Davies PA et al. Insensitivity to anaesthetic agents conferred by a class of GABAA receptor subunit. Nature. 385: 820-3. (1997a)

Davies PA et al. Modulation by general anaesthetics of rat GABAA receptors comprised of α1β3 and β3 subunits expressed in human embryonic kidney 293 cells. British Journal of Pharmacology. 120:899-909. (1997b).

Dawson GR et al. An inverse agonist selective for α5 subunit-containing GABAA receptors enhances cognition. Journal of Pharmacology & Experimental Therapeutics.

316: 1335-45. (2006)

Deeb TZ et al. Dynamic modification of a mutant cytoplasmic cysteine residue modulates the conductance of the human 5-HT3A receptor. Journal of Biological Chemistry. 282:

6172-6182. (2007)

Del Castillo J and Katz B. Biophysical aspects of neuro-muscular transmission. Progress in biophysics and biophysical chemistry. 6: 121-70. (1956)

Dibas MI et al. Identification of a novel residue within the second transmembrane domain that confers use-facilitated block by picrotoxin in glycine α1 receptors. Journal of

Biological Chemistry. 277: 9112-7. (2002)

Dibbens LM et al. GABRD encoding a protein for extra- or peri-synaptic GABAA receptors is a susceptibility locus for generalized epilepsies. Human Molecular Genetics.

13: 1315-9. (2004)

230

Dillon GH et al. Enhancement by GABA of the association rate of picrotoxin and tert- butylbicyclophosphorothionate to the rat cloned alpha 1 beta 2 gamma 2 GABAA receptor subtype. British Journal of Pharmacology. 115: 539-45. (1995)

Dixon CI et al. Targeted deletion of the GABRA2 gene encoding alpha2-subunits of

GABA(A) receptors facilitates performance of a conditioned emotional response, and abolishes anxiolytic effects of benzodiazepines and barbiturates. Pharmacology,

Biochemistry & Behavior. 90: 1-8. (2008)

Dopico AM and Lovinger DM. Acute alcohol action and desensitization of ligand-gated ion channels. Pharmacological Reviews. 61: 98-114. (2009)

Downing SS et al. Benzodiazepine modulation of partial agonist efficacy and spontaneously active GABA(A) receptors supports an allosteric model of modulation.

British Journal of Pharmacology. 145: 894-906. (2005)

Ebert B et al. Treating insomnia: Current and investigational pharmacological approaches.

Pharmacology & Therapeutics. 112: 612-29. (2006)

Edgar PP and Schwartz RD. Localization and characterization of 35S-t- butylbicyclophsphorothionate binding in rat brain: an autoradiographic study. Journal of

Neuroscience. 10: 603-612. (1990)

Erkkila B et al. Stoichiometric pore mutations of the GABAAR reveal a pattern of hydrogen bonding with picrotoxin. Biophysical Journal. 74: 4299-4306 (2008).

Erlander MG et al. Two genes encode distinct glutamate decarboxylases. Neuron. 7: 91-

100. (1991)

Essrich C et al. Postsynaptic clustering of major GABAA receptor subtypes requires the gamma 2 subunit and gephyrin. Nature Neuroscience. 1: 563-71. (1998) 231

Farrant M and Nusser Z. Variations on an inhibitory theme: phasic and tonic activation of

GABAA receptors. Nature reviews: Neuroscience. 6: 215-229. (2005)

Feng HJ and Macdonald RL. Proton modulation of α1β3δ GABAA receptor channel gating and desensitization. Journal of Physiology. 92: 1577-1585. (2004)

Feng HJ et al. Context-dependent modulation of αβγ and αβδ GABAA receptors by penicillin: Implications for phasic and tonic inhibition. Neuropharmacology. 56: 161-173.

(2009)

Feng HJ et al. Pentobarbital differentially modulates α1β3δ and α1β3γ2L GABAA receptor currents. Molecular Pharmacology. 66: 988-1003. (2004)

Feng HJ et al. δ subunit susceptibility variants E177A and R220H associated with complex epilepsy alter channel gating and surface expression of α4β2δ GABAA receptors. Journal of Neuroscience. 26: 1499-1506. (2006) ffrench-Constant RF et al. A point mutation in Drosophila GABA receptor confers insecticide resistance. Nature Letters. 363: 449-51. (1993a) ffrench-Constant RF et al. A single-amino acid substitution in a y-aminobutyric acid subtype A receptor locus is associated with cyclodiene insecticide resistance in

Drosophila populations. PNAS. 90: 1957-61. (1993b)

Fisher JL and Macdonald RL. Single channel properties of recombinant GABAA receptors containing γ2 or δ subtypes expressed with α1 and β3 subtypes in mouse L929 cells.

Journal of Physiology. 505: 283-97. (1997)

232

Fisher JL et al. The role of α1 and α6 subtype amino-terminal domains in allosteric regulation of γ-aminobutyric acidA receptors. Molecular Pharmacology. 52: 714-24.

(1997)

Fisher JL. The actions of ether, alcohol and alkane general anaesthetics on GABAA and glycine receptors and the effects of TM2 and TM3 mutations. Journal of Physiology. 561:

433-48. (2004)

Franks NP. General anaesthesia: from molecular targets to neuronal pathways of sleep and arousal. Nature Reviews Neuroscience. 9: 370-386. (2008)

Fritschy JM and Brunig I. Formation and plasticity of GABAergic synapses: physiological mechanisms and pathophysiological implications. Pharmacology &

Therapeutics. 98: 299-323. (2003)

Gallagher MJ et al. The juvenile myoclonic epilepsy GABAA receptor α1 subunit mutation A322D produces asymmetrical, subunit position-dependent reduction of heterozygous receptor currents and α1 subunit protein expression. Journal of

Neuroscience. 24: 5570-8. (2004)

Gallo V et al. γ-aminobutyric acid and benzodiazepine-induced modulation of [35S]-t- butylbicyclophosphorothionate binding to cerebellar granule cells. Journal of

Neuroscience. 5: 2432-2438. (1985)

Galzi JL et al. Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic. Nature. 359: 500-5. (1992)

233

Gay EA and Yakel JL. Gating of nicotinic ACh receptors; new insights into structural transitions triggered by agonist binding that induce channel opening. Journal of

Physiology. 584: 727-33. (2007)

Glykys J and Mody I. Activation of GABAA receptors: views from outside the synaptic cleft. Neuron. 56: 763-70. (2007)

Haas KF and Macdonald RL. GABAA receptor subunit γ2 and δ subtypes confer unique kinetic properties on recombinant GABAA receptor currents in mouse fibroblasts. Journal of Physiology. 514: 27-45. (1999)

Haber SN and Calzavara R. The cortico-basal ganglia integrative network: The role of the thalamus. Brain Research Bulletin. 78: 69-74. (2009)

Hadingham KL et al. Cloning of cDNA sequences encoding human α2 and α3 γ- aminobutyric acidA receptor subunits and characterization of the benzodiazepine pharmacology of recombinant α1-, α2-, α3- and α5-containing human γ-aminobutyric acidA receptors. Molecular Pharmacology. 43: 970-5. (1993)

Hales TG and Lambert JJ. The actions of propofol on inhibitory amino acid receptors of bovine adrenomedullary chromaffin cells and rodent central neurones. British Journal of

Pharmacology. 104: 619-628. (1991)

Hales TG et al. An asymmetric contribution to γ-aminobutyric type A receptor function of a conserved lysine within TM2-3 of α1, β2 and γ2 subunits. Journal of Biological

Chemistry. 281: 17034-17032. (2006)

234

Hales TG et al. The epilepsy mutation, gamma2(R43Q) disrupts a highly conserved inter- subunit contact site, perturbing the biogenesis of GABAA receptors. Molecular and

Cellular Neurosciences. 29: 120-7. (2005)

Halonen LM et al. Brain regional distribution of GABAA receptors exhibiting atypical

GABA agonism: Roles of receptor subunits. Neurochemistry International. 55: 389-96.

(2009)

Hamann M et al. Tonic and spillover inhibition of granule cells control information flow through cerebellar cortex. Neuron. 33: 625-633. (2002)

Hauser CAE et al. Flunitrazepam has an inverse agonist effect on recombinant α6β2γ2-

GABAA receptors via a flunitrazepam binding site. Journal of Biological Chemistry. 272:

11723-7. (1997)

Havoundjian H et al. The permeability of y-aminobutyric acid-gated chloride channels is described by the binding of a "cage" convulsant, t-butylbicyclophosphoro[35S]thionate.

PNAS. 83: 9241-44. (1986)

Herring D et al. Constitutive GABAA receptor endocytosis is dynamin mediated and dependent on a dileucine AP2 adaptin-binding motif within the β2 subunit of the receptor.

Journal of Biological Chemistry. 278: 24046-52. (2003)

Hevers W et al. Assembly of function α6β3γ2δ GABAA receptors in vitro.

Neurochemistry. 11: 4103-6. (2000)

Hilf RJC and Dutzler R. X-ray structure of a prokaryotic pentameric ligand-gated ion channel. Nature Letters. 452: 375-80. (2008)

235

Hilf RJC et al. Structural basis in open channel block in a prokaryotic pentameric ligand- gated ion channel. Nature Structural and Molecular Biology. 17. 1330-7. (2010)

Hodgkin AL and Huxley AF. A quantitative description of membrane current and its application to conduction and excitation in nerve. Journal of Physiology. 117: 500-44.

(1952)

Hog S et al. Structure-activity relationships of selective GABA uptake inhibitors. Current

Topics in Medicinal Chemistry. 6: 1861-82. (2006)

Homanics GE et al. Normal electrophysiological and behavioral responses to ethanol in mice lacking the long splice variant of the γ2 subunit of the γ-aminobutyrate type A receptor. Neuropharmacology. 38: 253:265. (1999)

Hosie AM et al. Conserved site for neurosteroid modulation of GABAA receptors.

Neuropharmacology. 56: 149-54. (2009)

Hosie AM et al. Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites. Nature Letters. 444: 486-489. (2006a)

Hosie AM et al. GABAA receptor needs two homologous domains of the beta-subunit for activation by GABA but not by pentobarbital. Nature Neuroscience. 6: 362-9. (2003)

Hosie AM et al. Neurosteroid binding sites on GABAA receptors. Pharmacology &

Therapeutics. 116: 7-19. (2007)

Hosie AM et al. Replacement of asparagine with arginine at the extracellular end of the second transmembrane (M2) region of insect GABA receptors increases sensitivity to penicillin G. Invertebrate Neuroscience. 6: 75-9. (2006b)

236

Huang RQ et al. Molecular basis for modulation of recombinant α1β2γ2 GABAA receptors by protons. Journal of Neurophysiology. 883-94. (2004)

Im WB et al. Effects of GABA and various allosteric ligands on TBPS binding to cloned rat GABAA receptor subtypes. British Journal of Pharmacology. 112: 1025-1030. (1994)

Imoto K et al. Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance. Nature. 335: 645-8. (1988)

Iwakiri M et al. An immunohistochemical study of GABAA receptor gamma subunits in

Alzheimer’s disease hippocampus: Relationship to neurofibrillary tangle progression.

Neuropathology. 29: 263-9. (2009)

Jarboe CH et al. Structural aspects of picrotoxinin action. Journal of Medicinal chemistry.

11: 729-31. (1968)

Jensen ML et al. Charge selectivity of the Cys-loop receptor family of ligand-gated ion channels. Journal of Neurochemistry. 92: 217-225 (2005).

Jones BL and Henderson LP. Trafficking and potential assembly patterns of ε-containing

GABAA receptors. Journal of Neurochemistry. 103: 1258-71. (2007)

Jones MV and Westbrook GL. Desensitized States Prolong GABAA Channel Responses to Brief Agonist Pulses. Neuron. 15: 181-191. (1995)

Jones MV and Westbrook GL. The impact of receptor desensitization on fast synaptic transmission. Trends in Neuroscience. 19: 96-101. (1996)

Jurd R et al. General anesthetic actions in vivo strongly attenuated by a point mutation in the GABAA receptor β3 subunit. The FASEB Journal. 17: 250-2. (2002)

237

Jursky F et al. Identification of amino acid residues of GABAA receptor subunits contributing to the formation and affinity of the tert-butylbicyclophosphorothionate binding site. Journal of Neurochemistry. 74:1310-1315. (2000)

Kalueff AV. Mapping convulsants’ binding to the GABA-A receptor chloride ionophore:

A proposed model for channel binding sites. Neurochemistry International. 50: 61-8.

(2007)

Kang JQ et al. Why does fever trigger febrile seizures? GABAA receptor gamma2 subunit mutations associated with idiopathic generalized epilepsies have temperature-dependent trafficking deficiencies. Journal of Neuroscience. 26: 2590-7. (2006)

Karlin A. Emerging structure of the nicotinic acetylcholine receptor. Nature Reviews

Neuroscience. 3: 102-14. (2002)

Kash TL et al. Coupling of agonist binding to channel gating in the GABAA receptor.

Nature Letters. 421: 272-5. (2003)

Kasparov S et al. GABA(A) receptor epsilon-subunit may confer benzodiazepine insensitivity to the caudal aspect of the nucleus tractus solitarii of the rat. Journal of

Physiology. 536: 785-96. (2001)

Keller CA et al. The gamma2 subunit of GABA(A) receptors is a substrate for palmitoylation by GODZ. Journal of Neuroscience. 24: 5881-91. (2004)

Kittler JT et al. Constitutive endocytosis of GABAA receptors by an association with the

Adaptin AP2 complex modulates inhibitory synaptic currents in hippocampal neurons.

Journal of Neuroscience. 20: 7972-7. (2000)

238

Kittler JT et al. Huntingtin-associated protein 1 regulates inhibitory synaptic transmission by modulating γ-aminobutyric acid type A receptor membrane trafficking. PNAS. 101:

12736-41. (2004)

Kittler JT et al. Phospho-dependent binding of the clathrin AP2 adaptor complex to

GABAA receptors regulates the efficacy of inhibitory synaptic transmission. PNAS. 102:

14871-6. (2005)

Klein RL and Harris RA. Regulation of GABAA receptor structure and function by chronic drug treatments in vivo and with stably transfected cells. Japanese Journal of

Pharmacology. 70: 1-15. (1996)

Kloda JH and Czajkowski C. Agonist-, antagonist-, and benzodiazepine-induced structural changes in the α1Met113-Leu132 region of the GABAA receptor. Molecular

Pharmacology. 71: 483-493. (2007)

Knabl J et al. Genuine antihyperalgesia by systemic diazepam revealed by experiments in

GABAA receptor point-mutated mice. PAIN. 141: 233-8. (2009)

Knabl J et al. Reversal of pathological pain through specific spinal GABAA receptor subtypes. Nature Letters. 451: 330-5. (2008)

Kneussal M. Dynamic regulation of GABA receptors at synaptic sites. Brain Research

Reviews. 39: 74-83. (2002)

Korpi ER and Luddens H. Regional γ-aminobutyric acid sensitivity of t-

35 Butylbicyclophosphoro[ S]thionate binding depends on γ-aminobutyric acidA receptor α subunit. Molecular Pharmacology. 44: 87-92. (1993)

239

Korpi ER and Sinkkonen ST. GABAA receptor subtypes as targets for neuropsychiatric drug development. Pharmacology & Therapeutics. 109: 12-32. (2006)

Krasowski MD and Harrison NL. The actions of ether, alcohol and alkane general anaesthetics on GABAA and glycine receptors and the effects of TM2 and TM3 mutations. British Journal of Pharmacology. 129: 731-43. (2000)

Kumar S et al. The role of GABAA receptors in the acute and chronic effects of ethanol: a decade of progress. Psychopharmacology. 205: 529-564. (2009)

Lagrange AH et al. Enhanced macroscopic desensitization shapes the response of alpha4 subtype-containing GABAA receptors to synaptic and extrasynaptic GABA. Journal of

Physiology. 578: 655-76. (2007)

Lambert JJ et al. Neurosteroid modulation of GABAA receptors. Progress in

Neurobiology. 71:67-80. (2003)

Laurie DJ et al. The distribution of thirteen GABAA receptor subunit mRNAs in the rat brain. III. Embryonic and postnatal development. Journal of Neuroscience. 12: 4151-72.

(1992)

Law RJ and Lightstone FC. Gaba receptor insecticide non-competitive antagonists may bind at allosteric modulator sites. International Journal of Neuroscience. 118: 705-34.

(2008)

Lee FJS et al. Direct receptor cross-talk can mediate the modulation of excitatory and inhibitory neurotransmission by dopamine. Journal of Molecular Neuroscience. 26: 245-

251. (2005)

240

Lees G and Edwards MD. Modulation of recombinant human γ-aminobutyric acidA receptors by isoflurane. Anesthesiology. 88: 206-17. (1998)

Leil TA et al. GABAA receptor-associated protein traffics GABAA receptors to the plasma membrane in neurons. Journal of Neuroscience. 24: 11429-38. (2004)

Li XJ and Li SH. HAP1 and intracellular trafficking. Trends in Pharmacological Science.

26: 1-3. (2005).

Licatta SC and Rowlett JK. Abuse and dependence liability of benzodiazepine-type drugs:

GABAA receptor modulation and beyond. Pharmacology, Biochemistry & Behavior. 90:

74-89. (2008)

Lindquist CEL et al. Penicillin blocks human α1β1 and α1β1γ2S GABAA channels that open spontaneously. European Journal of Pharmacology. 496: 23-32. (2004)

Liu F et al. Direct protein±protein coupling enables cross-talk between dopamine D5 and

γ-aminobutyric acid A receptors. Nature. 403: 274-280. (2000)

Liu M and Glowa JR. Alterations of GABAA receptor subunit mRNA levels associated with increases in punished responding induced by acute alprazolam administration: an in situ hybridization study. Brain Research. 822: 8-16. (1999)

Livesey MR et al. Structural Determinants of Ca2+ Permeability and Conduction in the

Human 5-Hydroxytryptamine Type 3A Receptor. Journal of Biological Chemistry. 283:

19301-13. (2008)

Lloyd KG et al. The potential use of GABA agonists in psychiatric disorders: evidence from studies with progabide in animal models and clinical trials. Pharmacology,

Biochemistry & Behavior. 18: 957-66. (1983)

241

Lo WY et al. A Conserved Cys-loop Receptor Aspartate Residue in the M3-M4

Cytoplasmic Loop Is Required for GABAA Receptor Assembly. Journal of Biological

Chemistry. 283: 29740-52. (2008)

Loup F et al. Altered expression of α3-containing GABA receptors in the neocortex of patients with focal epilepsy. Brain. 129: 3277-89. (2006)

Luddens H and Korpi ER. GABA antagonists differentiate between recombinant

GABA/Benzodiazepine receptor subtypes. Journal of Neuroscience. 15: 6957-62. (1995)

Luntz-Leybman V et al. Uncoupling of GABA/Benzodiazepine receptor α1, β2 and γ2 subunit mRNA expression in cerebellar purkinje cells of staggerer mutant mice. Journal of Neuroscience. 15: 8121-8130. (1995)

Luscher B and Keller CA. Regulation of GABAA receptor trafficking, channel activity and functional plasticity of inhibitory synapses. Pharmacology & Therapeutics. 102: 195-

221. (2004)

Luu T et al. GABA increases both the conductance and mean open time of recombinant

GABAA channels co-expressed with GABARAP. Journal of Biological Chemistry. 281:

35699-35708. (2007)

Lynch JW. Native glycine receptor subtypes and their physiological roles.

Neuropharmacology. 56: 303-9. (2009)

Macdonald RL and Kang JQ. Molecular pathology of genetic epilepsies associated with

GABAA receptor subunit mutations. Epilepsy Currents: American Epilepsy Society. 9: 18-

23. (2009)

242

Macdonald RL et al. GABAA receptor epilepsy mutations. Biochemical Pharmacology.

68: 1497-1506. (2004)

Macdonald RL et al. Mutations linked to generalized epilepsy in humans reduce GABAA receptor current. Experimental Neurology. 184: S57-S68. (2003)

Maconochie DJ et al. How quickly can GABAA receptors open? Neuron. 12: 61-71.

(1994)

Makela R et al. Cerebellar γ-aminobutyric acid type A receptors: pharmacological subtypes revealed by mutant mouse lines. Molecular Pharmacology. 52: 380-88. (1997)

Maksay G and Simonyi M. Kinetic regulation of convulsant (TBPS) binding by

GABAergic agents. Molecular Pharmacology. 30: 321-328. (1986)

Maksay G et al. The pharmacology of spontaneously open α1β3ε GABAA receptor ionophores. Neuropharmacology. 44: 994-1002. (2003)

Marban E et al. Structure and function of voltage-gated sodium channels. Journal of

Physiology. 508: 647-57. (1998)

Matute C et al. Excitotoxicity in glial cells. European Journal of Pharmacology. 447:

239-246. (2002).

McCartney MR et al. Tonically active GABAA receptors in hippocampal neurons exhibit constitutive GABA-independent gating. Molecular Pharmacology. 71: 539-548. (2007)

McCormick DA and Contreras D. On the cellular and network bases of epileptic seizures.

Annual Review of Physiology. 63: 815-46. (2001)

243

McCracken M et al. A transmembrane amino acid in the GABAA receptor β2 subunit critical for the actions of alcohols and anesthetics. Journal of Pharmacology &

Experimental Therapeutics. 335: 600-6. (2010)

McKernan RM et al. Photoaffinity labeling of the benzodiazepine binding site of α1β3γ2

γ-aminobutyric acidA receptors with flunitrazepam identifies a subset of ligands that interact directly with his102 of the α subunit and predicts orientation of these within the benzodiazepine pharmacophore. Molecular Pharmacology. 54: 33-43. (1998)

Meier J and Grantyn R. Preferential accumulation of GABAA receptor γ2L, not γ2S cytoplasmic loops at rat spinal cord inhibitory synapses. Journal of Physiology. 559: 355-

365. (2004)

Mihic SJ et al. Sites of alcohol and volatile anaesthetic action on GABAA and glycine receptors. Nature Letters. 389: 385-9. (1997)

Miller C. Genetic manipulation of ion channels: a new approach to structure and mechanism. Neuron. 2: 1l95-1205. (1989)

Miller PS and Smart TG. Binding, activation and modulation of Cys-loop receptors.

Trends in Pharmacological Sciences. 31: 161-174. (2010)

Miralles CP et al. Immunocytochemical localization of the β3 subunit of the γ- aminobutyric acidA receptor in the rat brain. Journal of Comparitive Neurology. 413: 535-

548. (1999)

Miyazawa A et al. Structure and gating mechanism of the nicotinic acetylcholine receptor pore. Nature. 423: 949-55. (2003)

244

Mody I. Aspects of homeostatic plasticity of GABAA receptor mediated inhibition.

Journal of Physiology. 562.1: 37-46. (2005)

Mohler H et al. A new benzodiazepine pharmacology. A new benzodiazepine pharmacology. 300: 2-8. (2002)

Mohler H et al. GABAA-receptors: structural requirements and sites of gene expression in mammalian brain. Neurochemical Research. 15: 199-207. (1990)

Monod J et al. On the nature of allosteric transitions: a plausible model. Journal of

Molecular Biology. 12: 88-118. (1965)

Moorhouse AJ et al. Single channel analysis of conductance and rectification in cation- selective, mutant glycine receptor channels. Journal of General Physiology. 119: 411-25.

(2002)

Moragues N et al. GABAA receptor epsilon subunit expression in identified peptidergic neurons of the rat hypothalamus. Brain Research. 967: 285-9. (2003)

Moragues N et al. Localisation of GABA(A) receptor epsilon-subunit in cholinergic and aminergic neurones and evidence for co-distribution with the theta-subunit in rat brain.

Neuroscience. 111: 657-69. (2002)

Morris HV et al. Both α2 and α3 GABAA receptor subtypes mediate the anxiolytic properties of benzodiazepine site ligands in the conditioned emotional response paradigm.

European Journal of Neuroscience. 23: 2493-2504. (2006)

Mortensen M and Smart TG. Extrasynaptic αβ subunit GABAA receptors on rat hippocampal pyramidal neurons. Journal of Physiology. 577: 841-856. (2006)

245

Mortensen M et al. Distinct activities of GABA agonists at synaptic- and extrasynaptic- type GABAA receptors. Journal of Physiology. 588: 1251-68. (2010)

Mortensen M et al. Pharmacology of GABA(A) receptors exhibiting different levels of spontaneous activity. European Journal of Pharmacology. 476: 17-24. (2003)

Mortensen M et al. Subcellular localization of gamma-aminobutyric acid type A receptors is determined by receptor beta subunits. Journal of Physiology. 557: 393-417. (2004)

Mukhtasimova N et al. Detection and trapping of intermediate states priming nicotinic receptor channel opening. Nature Letters. 459: 451-455. (2009)

Munro G et al. Developing analgesics by enhancing spinal inhibition after injury: GABAA receptor subtypes as novel targets. Trends in Pharmacological Sciences. 30: 453-459.

(2009)

Neelands TR et al. Spontaneous and γ-Aminobutyric Acid (GABA)-activated GABAA receptor channels formed by ε subunit-containing isoforms. Molecular Pharmacology. 1:

168-78. (1999)

Newland CF and Cull-Candy SG. On the mechanism of action of picrotoxin on GABA receptor channels in dissociated sympathetic neurones of the rat. Journal of Physiology.

447: 191-213. (1992)

Nobrega JN et al. Alterations in the brain GABAA/benzodiazepine receptor chloride ionophore complex in a genetic model of paroxysmal dystonia: a quantitative autoradigraphic analysis. Neuroscience. 64: 229-39. (1995)

Noebels JL. The biology of epilepsy genes. Annual Review of Neuroscience. 26: 599-625.

(2003)

246

Nury H et al. X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel. Nature. 469: 428-31. (2011)

Nusser Z and Mody I. Selective modulation of tonic and phasic inhibitions in dentate gyrus granule cells. Journal of Neurophysiology. 87: 2624-8. (2002)

Nusser Z et al. Segregation of different GABAA receptors to synaptic and extrasynaptic membranes of cerebellar granule cells. Journal of Neuroscience. 18: 1693-1703. (1998)

Nusser Z et al. The alpha 6 subunit of the GABAA receptor is concentrated in both inhibitory and excitatory synapses on cerebellar granule cells. Journal of Neuroscience.

16: 103-14. (1996)

O’Shea SM and Harrison NL. Arg-274 and Leu-277 of the γ-Aminobutyric Acid Type A

Receptor α2 Subunit Define Agonist Efficacy and Potency. Journal of Biological

Chemistry. 275: 22764-8. (2000)

O’Sullivan GA et al. GABARAP is not essential for GABA receptor targeting to the synapse. European Journal of Neuroscience. 22: 2644-8. (2005)

Ogren SO et al. Evidence for a role of the locus coeruleus noradrenaline system in learning. Neuroscience Letters. 20: 351-6. (1980)

Ogris W et al. Affinity of various benzodiazepine site ligands in mice with a point mutation in the GABAA receptor γ2 subunit. Biochemical Pharmacology. 68: 1621-9.

(2004)

Olsen RW and Sieghart W. GABAA receptors: Subtypes provide diversity of function and pharmacology. Neuropharmacology. 56: 141-148. (2009)

247

Olsen RW et al. GABAA receptor subtypes: the "one glass of wine" receptors. Alcohol.

41: 201-9. (2007)

Olsen RW. Picrotoxin-like channel blockers of GABAA receptors. PNAS. 103: 6081-2.

(2006)

Ortells MO and Lunt GG. Evolutionary history of the ligand-gated ion channel superfamily of receptors. Trends in Neuroscience. 18: 121-27. (1995)

Overstreet LS et al. Slow desensitization regulates the availability of synaptic GABAA receptors. Journal of Neuroscience. 20: 7914-21. (2000)

Park-Chung M et al. Sulfated and unsulfated steroids modulate gamma-aminobutyric acidA receptor function through distinct sites. Brain Research. 830: 720-87. (1999)

Peters JA et al. Novel structural determinants of single channel conductance and ion selectivity in 5-hydroxytryptamine type 3 and nicotinic acetylcholine receptors. Journal of

Physiology. 588: 587-595. (2010)

Peters JA et al. Novel structural determinants of single-channel conductance in nicotinic acetylcholine and 5-hydroxytryptamine type-3 receptors. Biochemical Society

Transactions. 34: 882-6. (2006)

Peters JA et al. The 5-hydroxytryptamine type 3 (5-HT3) receptor reveals a novel determinant of single-channel conductance. Biochemical Society Transactions. 32: 547-

552. (2004)

Petri S et al. The cellular mRNA expression of GABA and glutamate receptors in spinal motor neurons of SOD1 mice. Journal of The Neurological Sciences. 238: 25-30. (2005)

Pirker S et al. GABAA receptors: Immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience. 101: 815-50. (2000) 248

Pollard S et al. Quantitative characterization of α6 and α1α6 subunit-containing native γ- aminobutyric acidA receptors of adult rat cerebellum demonstrates two a subunits per receptor oligomer. Journal of Biological Chemistry. 270: 21285-90. (1995)

Qi ZH et al. Protein kinase C epsilon regulates gamma-aminobutyrate type A receptor sensitivity to ethanol and benzodiazepines through phosphorylation of gamma2 subunits.

Journal of Biological Chemistry. 282: 33052-63. (2007)

Renard S et al. Structural elements of the gamma-aminobutyric acid type A receptor conferring subtype selectivity for benzodiazepine site ligands. Journal of Biological

Chemistry. 274: 13370-4. (1999)

Reynolds DS et al. Sedation and anesthesia mediated by distinct GABAA receptor isoforms. Journal of Neuroscience. 23: 8608-17. (2003)

Rogers CJ et al. Benzodiazepine and beta-carboline regulation of single GABAA receptor channels of mouse spinal neurones in culture. Journal of Physiology. 475: 69-82. (1994)

Rudolph U and Mohler H. Analysis of GABAA receptor function and dissection of the pharmacology of benzodiazepines and general anesthetics through mouse genetics.

Annual Review of Pharmacology and Toxicology. 44: 475-98. (2004)

Rudolph U et al. Benzodiazepine actions mediated by specific gamma-aminobutyric acid(A) receptor subtypes. Nature. 401: 796-800. (1999)

Sanna E et al. Direct activation of GABAA receptors by loreclezole, an anticonvulsant drug with selectivity for the beta-subunit. Neuropharmacology. 35: 1753-60. (1996)

Sattler R and Tymianski M. Molecular mechanisms of glutamate receptor-mediated excitotoxic neuronal cell death. Molecular Neurobiology. 24: 107-29. (2001)

249

Saxena NC and Macdonald RL. Assembly of GABAA Receptor Subunits: Role of the δ

Subunit. Journal of Neuroscience. 14: 7077-86. (1994)

Scheller M and Forman SA. Coupled and uncoupled gating and desensitization effects by pore domain mutations in GABAA receptors. Journal of Neuroscience. 22: 8411-22.

(2002)

Seagar M and Takahashi M. Interactions between presynaptic calcium channels and proteins implicated in synaptic vesicle trafficking and exocytosis. Journal of

Bioenergetics and Biomembranes. 30: 347-56. (1998)

Sedelnikova A et al. Stoichometry of a pore mutation that abolishes picrotoxin-mediated antagonism of the GABAA receptor. Journal of Physiology. 577.2: 569-577. (2006)

Seger DL. Flumazenil – treatment or toxin. Journal of Clinical Toxicology. 42: 209-216.

(2004)

Semyanov A et al. Tonically active GABAA receptors: modulating gain and maintaining the tone. Trends in Neuroscience. 27: 262-9. (2004)

Sergeeva OA et al. Pharmacological properties of GABAA receptors in rat hypothalamic neurons expressing the epsilon-subunit. Journal of Neuroscience. 25: 88-95. (2005)

Sigel E et al. The rat β1-subunit of the GABAA receptor forms a picrotoxin-sensitive anion channel open in the absence of GABA. FEBS letters. 257: 377-379. (1989).

Silivotti LG. What single-channel analysis tells us of the activation mechanism of ligand- gated channels: the case of the glycine receptor. Journal of Physiology. 588: 45-58.

(2010)

250

Sine SM and Engel AG. Recent advances in Cys-loop receptor structure and function.

Nature. 440: 448-55. (2006)

Sinkkonen ST et al. Altered atypical coupling of γ-aminobutyrate type A receptor agonist and convulsant binding sites in subunit-deficient mouse lines. Molecular Brain Research.

86: 179-83. (2001b)

Sinkkonen ST et al. Autoradiographic imaging of altered synaptic alphabetagamma2 and extrasynaptic alphabeta GABAA receptors in a genetic mouse model of anxiety.

Neurochemistry International. 44: 539-47. (2004)

Sinkkonen ST et al. Characterization of γ-aminobutyrate type A receptors with atypical coupling between agonist and convulsant binding sites in discrete brain regions.

Molecular brain research. 86: 168-178. (2001)

Sinkkonen ST et al. GABAA receptor ε and θ subunits display unusual structural variation between species and are enriched in the rat locus ceruleus. Journal of Neuroscience. 20:

3588-95. (2000)

Smith AJ et al. Compounds Exhibiting Selective Efficacy for Different β Subunits of

Human Recombinant γ-Aminobutyric AcidA Receptors. Journal of Pharmacology and

Experimental Therapeutics. 311: 601-9. (2004)

Smith KR et al. Regulation of inhibitory synaptic transmission by a conserved atypical interaction of GABAA receptor β- and γ-subunits with the clathrin AP2 adaptor.

Neuropharmacology. 55: 844-50. (2008)

Song M and Messing RO. Protein kinase C regulation of GABAA receptors. Cell and

Molecular Life Sciences. 62: 119-127. (2005)

251

Sousa A and Ticku MK. Interactions of the neurosteroid dehydroepiandrosterone sulfate with the GABA(A) receptor complex reveals that it may act via the picrotoxin site.

Journal of Pharmacology & Experimental Therapeutics. 282: 827-33. (1997)

Squires RF et al. [35S]t-Butylbicyclophosphorothionate binds with high affinity to brain- specific sites coupled to γ-aminobutyric acid-A and ion recognition sites. Molecular

Pharmacology. 23:326-336. (1983)

Srinivasan S et al. Biphasic modulation of GABAA receptor binding by steroids suggests functional correlates. Neurochemical research. 24: 1363-1372. (1999)

Stephens GJ. G-protein-coupled-receptor-mediated presynaptic inhibition in the cerebellum. Trends in Pharmacological Science. 30:421-30. (2009)

Stephenson FA. The GABAA receptors. Biochemical Journal. 310: 1-9. (1995)

Strecker GJ et al. Zinc and flunitrazepam modulation of GABA-mediated currents in rat suprachiasmatic neurons. Journal of Neurophysiology. 81: 184-191. (1999)

Sun C et al. Diminished neurosteroid sensitivity of synaptic inhibition and altered location of the α4 subunit of GABAA receptors in an animal model of epilepsy. Journal of

Neuroscience. 27: 12641-50. (2007)

Takazawa T and MacDermott AB. Glycinergic and GABAergic tonic inhibition fine tune inhibitory control in regionally distinct subpopulations of dorsal horn neurons. Journal of

Physiology. 588: 2571-87. (2010)

Tallman JF and Gallager DW. Modulation of benzodiazepine site sensitivity.

Pharmacology, Biochemistry & Behavior. 10: 809-13. (1979)

252

Tan KR et al. Neural bases for addictive properties of benzodiazepines. Nature. 463: 769-

775. (2010)

Taylor PM et al. Identification of amino acid residues within GABAA receptor β subunits that mediate both homomeric and heteromeric receptor expression. Journal of

Neuroscience. 19: 6360-71. (1999)

Terai K et al. Immunohistochemical localization of GABAA receptors in comparison with

GABA-immunoreactive structures of the nucleus tractus solitarii of the rat. Neuroscience.

82: 843-52. (1998)

Thompson AJ and Lummis SC. A single ring of charged amino acids at one end of the pore can control ion selectivity in the 5-HT3 receptor. British Journal of Pharmacology.

140: 359-65. (2003)

Thompson CL et al. Developmental regulation of expression of GABAA receptor alpha 1 and alpha 6 subunits in cultured rat cerebellar granule cells. Neuropharmacology. 35:

1337-46. (1996)

Thompson SA et al. Overexpression of the GABA(A) receptor epsilon subunit results in insensitivity to anaesthetics. Neuropharmacology. 43: 662-8. (2002)

Ticku MK et al. Binding of [3H]α-dihydroxypicrotoxinin, a γ-aminobutyric acid synaptic antagonist, to rat brain membranes. Molecular Pharmacology. 14: 391-402. (1978)

Tselin V et al. Assembly of nicotinic and other Cys-loop receptors. Journal of

Neurochemistry. 116: 734-41. (2011)

Twelvetrees AE et al. Delivery of GABAARs to synapses is mediated by HAP1-KIF5 and disrupted by mutant huntingtin. Neuron. 65: 53-65. (2010).

253

Twyman RE et al. Kinetics of open channel block by penicillin of single GABAA receptor channels from mouse spinal cord neurones in culture. Journal of Physiology. 445: 97-127.

(1992)

Ueno S et al. Biciculline and gabazine are allosteric inhibitors of channel opening of the

GABAA receptor. Journal of Neuroscience. 17: 625-634. (1997)

Unwin N. Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution.

Journal of Molecular Biology. 346: 967-989. (2005)

Unwin N. Structure of the acetylcholine gated channel. Novartis Foundation Symposia.

245: 5-15. (2002)

Unwin N. The structure of ion channels in membranes of excitable cells. Neuron. 3: 665-

76. (1989)

Uusi-Oukari M et al. Brain regional heterogeneity of pH effects on GABA(A) receptor- associated [35S]TBPS binding. Neurochemical Research. 29: 771-80. (2004)

Wafford KA and Whiting PJ. Ethanol potentiation of GABAA receptors requires phosphorylation of the alternatively spliced variant of the gamma 2 subunit. FEBS Letters.

313: 113-7. (1992)

Wafford KA et al. Differentiating the role of γ -aminobutyric acid type A (GABAA) receptor subtypes. Biochemical Society Transactions. 32: 553-6. (2004)

Wafford KA et al. Ethanol sensitivity of the GABAA receptor expressed in Xenopus oocytes requires 8 amino acids contained in the gamma 2L subunit. Neuron. 7: 27-33.

(1991)

254

Wafford KA et al. Novel compounds selectively enhance δ subunit containing GABAA receptors and increase tonic currents in thalamus. Neuropharmacology. 56: 182-9. (2009)

Wagner DA et al. Kinetics and Spontaneous Open Probability Conferred by the ε Subunit of the GABAA Receptor. Journal of Neuroscience. 25: 10462-8. (2005)

Waldvogel HJ et al. Differential localization of γ-aminobutyric acid type A and glycine receptor subunits and gephyrin in the human pons, medulla oblongata and uppermost cervical segment of the spinal cord: an immunohistochemical study. Journal of

Comparative Neurology. 518: 305-28. (2010)

Wallner M et al. Ethanol enhances α4β3δ and α6β3δ γ-aminobutyric acid type A receptors at low concentrations known to affect humans. PNAS. 100: 15218-223. (2003)

Wang CT et al. Cation permeability and cation-anion interactions in a mutant GABA- gated chloride channel from Drosophila. Biophysical Journal. 77: 691-700. (1999)

Wang Q et al. Ligand- and Subunit-specific Conformational Changes in the Ligand- binding Domain and the TM2-TM3 Linker of α1β2γ2 GABAA Receptors. Journal of

Biological Chemistry. 285: 40373-86. (2010)

Webb TI and Lynch JW. Molecular pharmacology of the glycine receptor chloride channel. Current Pharmaceutical Design. 13: 2350-67. (2007)

Weinbroum A et al. Use of flumazenil in the treatment of drug overdose: a double-blind and open clinical study in 110 patients. Critical Care Medicine. 24: 199-206. (1996)

Werner DF et al. Inhaled anesthetic responses of recombinant receptors and knockin mice harboring α2(S270H/L277A) GABAA receptor subunits that are resistant to isoflurane.

Journal of Pharmacology & Experimental Therapeutics. 366: 134-144. (2011)

255

Werner DF et al. Knockin mice with ethanol-insensitive alpha1-containing gamma- aminobutyric acid type A receptors display selective alterations in behavioral responses to ethanol. Journal of Pharmacology & Experimental Therapeutics. 319: 219-27. (2006)

Whiting PJ et al. Molecular and functional diversity of the expanding GABA-A receptor gene family. Annals of the New York Academy of Sciences. 868: 645-53. (1999)

Whiting PJ et al. Neuronally restricted RNA splicing regulates the expression of a novel

GABAA receptor subunit conferring atypical functional properties. Journal of

Neuroscience. 17: 5027-37. (1997)

Wickman K and Clapham DE. Ion channel regulation by G proteins. Physiological

Reviews. 75: 865-885 (1995)

Wieland HA et al. A single histidine in GABAA receptors is essential for benzodiazepine agonist binding. Journal of Biological Chemistry. 267: 1426-29. (1992)

Wilkins ME et al. Identification of a β subunit TM2 residue mediating proton modulation of GABA type A receptors. Journal of Neuroscience. 22: 5328-5333. (2002)

Wilkins ME et al. Proton modulation of recombinant GABAA receptors: influence of

GABA concentration and the β subunit TM2–TM3 domain. Journal of Physiology. 567:

365-377. (2005)

Wilson GG and Karlin A. Acetylcholine receptor channel structure in the resting, open and desensitized states probed with the substituted-cysteine-accessibility method. PNAS.

98: 1241-1248. (2001)

Wilson GG and Karlin A. The location of the gate in the acetylcholine receptor channel.

Neuron. 20: 1269-81. (1998)

256

Wingrove PB et al. Key amino acids in the γ subunit of the γ-aminobutyric AcidA receptor that determine ligand binding and modulation at the benzodiazepine site.

Molecular Pharmacology. 52: 874-881. (1997)

Wisden W et al. The distribution of 13 GABAA receptor subunit mRNAs in the rat brain.

I. Telencephalon, diencephalon, mesencephalon. Journal of Neuroscience. 12: 1040-62.

(1992)

2+ Wooltorton JRA et al. Identification of a Zn binding site on the murine GABAA receptor complex: dependence on the second transmembrane domain of β subunits. Journal of

Physiology. 505: 633-640. (1997)

Wu DF et al. A conserved cysteine residue in the third transmembrane domain is essential for homomeric 5-HT3 receptor function. Journal of Physiology. 588: 603-15. (2010)

Xu M and Akabas MH. Identification of channel-lining residues in the M2 Membrane- spanning segment of the GABAA receptor α1 subunit. Journal of General Physiology.

107: 195-205 (1996).

Xu M et al. Interaction of picrotoxin with GABAA receptor channel-lining residues - probed in cysteine mutants. Biophysical Journal. 69: 1858-67. (1995)

Yakel JL. Gating of nicotinic ACh receptors: latest insights into ligand binding and function. Journal of Physiology. 588: 597-602. (2010).

Yamodo IH et al. Conformational changes in the nicotinic acetylcholine receptor during gating and desensitization. Biochemistry. 49: 156-65. (2010)

Yang Z et al. A proposed structural basis for picrotoxinin and picrotin binding in the glycine receptor pore. Journal of Neurochemistry. 103: 580-9. (2007)

257

Yueng J et al. Tonically activated GABAA receptors in hippocampal neurons are high- affinity, low-conductance sensors for extracellular GABA. Molecular Pharmacology.

63:2-8. (2003)

Zeilhofer HU et al. GABAergic analgesia: new insights from mutant mice and subtypeselective agonists. Trends in Pharmacological Sciences. 30: 397-02. (2009)

Zeller A et al. Mapping the contribution of β3-containing GABAA receptors to volatile and intravenous general anesthetic actions. BMC Pharmacology. 7. (2007)

Zhang HG et al. A unique amino acid of the Drosophila GABA receptor with influence of drug sensitivity by two mechanisms. Journal of Physiology. 479:65-75. (1994)

Zhang J et al. Localization of GABAA receptor subunits α1, α3, β1, β2/3, γ1, and γ2 in the salamander retina. Journal of Comparative Neurology. 459: 440-453. (2003)

Zhang N et al. Altered localization of GABA(A) receptor subunits on dentate granule cell dendrites influences tonic and phasic inhibition in a mouse model of epilepsy. Journal of

Neuroscience. 27: 7520-31. (2007)

Zhu WJ et al. Delta subunit inhibits neurosteroid modulation of GABAA receptors.

Journal of Neuroscience. 16: 6648-56. (1996)

258