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 ii © Copyright 2011 by Nidaa A. Othman All rights reserved 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 ix 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 x 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.