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Assessment of Molecular Action of Direct Gating and Allosteric Modulatory Effects of Carisoprodol (Somartm) on GABA a Receptors

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Assessment of Molecular Action of Direct Gating and Allosteric Modulatory Effects of Carisoprodol (Somartm) on GABA a Receptors Graduate Theses, Dissertations, and Problem Reports 2015 Assessment of molecular action of direct gating and allosteric modulatory effects of carisoprodol (SomaRTM) on GABA A receptors Manoj Kumar Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Kumar, Manoj, "Assessment of molecular action of direct gating and allosteric modulatory effects of carisoprodol (SomaRTM) on GABA A receptors" (2015). Graduate Theses, Dissertations, and Problem Reports. 6022. https://researchrepository.wvu.edu/etd/6022 This Dissertation is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Dissertation has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. ASSESSMENT OF MOLECULAR ACTION OF DIRECT GATING AND ALLOSTERIC MODULATORY EFFECTS OF MEPROBAMATE (MILTOWN®) ON GABAA RECEPTORS Manish Kumar, MD, MS Dissertation submitted to the School of Pharmacy at West Virginia University in partial fulfillment of Requirements for the degree of DOCTOR OF PHILOSOPHY IN PHARMACEUTICAL AND PHARMACOLOGICAL SCIENCES Glenn H. Dillon, PhD., Chair Rae Matsumoto, PhD Patrick S. Callery, PhD Hanting Zhang, MD, PhD Robert K Griffith, PhD Department of Pharmaceutical Sciences Morgantown, West Virginia May 2016 Keywords: GABAA receptors, GABAA receptor isoforms, Carisoprodol, Meprobamate, Muscle relaxant, Drug Abuse Copyright 2016 Manish Kumar ABSTRACT ASSESSMENT OF MOLECULAR ACTION OF DIRECT GATING AND ALLOSTERIC MODULATORY EFFECTS OF MEPROBAMATE (MILTOWN®) ON GABAA RECEPTORS Manish Kumar Meprobamate (tradenames Miltown, Equanil) is a schedule IV anxiolytic and the primary metabolite of the muscle relaxant carisoprodol. Meprobamate modulates GABAA (- aminobutyric acid type A) receptors, and has been described as having barbiturate-like activity. A thorough understanding of its mechanism remains ill-defined, however. To gain insight into its actions, we have conducted a series of studies using recombinant GABAA receptors. In xz GABAA receptors (where x = 1-6 and z = 1-3), the ability to enhance GABA-mediated current was evident for all subunit isoforms, with the largest effect observed in 5-expressing receptors. Direct gating was comparable with all subunits, although the effect was attenuated in α3-expressing receptors. Allosteric potentiating and direct gating effects were comparable in 112 and 122 receptors, whereas allosteric effects were enhanced in 12 compared to 122 receptors. In “extrasynaptic” GABAA receptors (α1β3δ and α4β3δ receptors), meprobamate allosterically enhanced both EC20 and saturating GABA currents, and directly activated these receptors with an efficacy comparable to that of GABA. Bemegride, which antagonizes effects of pentobarbital, attenuated direct gating effects of meprobamate. Whereas pentobarbital directly gated homomeric β3 receptors, meprobamate did not, and instead blocked the spontaneously open current present in these receptors. In wild type homomeric ρ1 receptors, both pentobarbital and meprobamate were ineffective in direct gating; a mutation (W328M) previously shown to confer sensitivity to pentobarbital, did not confer sensitivity to meprobamate. Our results provide additional insight into the actions of meprobamate and parent therapeutic agents such as Carisoprodol as well as possible domains involved in the direct gating actions of the ligands including transmembrane domains TM4. Further, using site- directed-mutagenesis and whole cell patch clamp electrophysiology in transiently transfected HEK293 cells, we examined the role of GABAA receptor α-subunit transmembrane domain 4 (TM4) and transmembrane domain 2 (TM2) amino acids in direct gating, and inhibitory actions of meprobamate. Mutation of α3 valine at position 440 to leucine (present in the equivalent position in the α1 subunit) increased the direct gating potency and efficacy of meprobamate. In R-α1β2 heteromeric GABAA receptor, both pentobarbital and carisoprodol had inhibitory effect for allosteric modulatory action at higher concentrations. Meprobamate did not have an inhibitory effect at the concentrations tested. T6’F mutation of the β2-subunit abolished the inhibition of carisoprodol but not of pentobarbital. Thus, our studies provide further insight into actions of meprobamate, its parent drug carisoprodol and their comparison to barbiturate pentobarbital. DEDICATION I dedicate my dissertation to my mother Mrs. Manju Kumari whose tireless patience and encouragement helped me in toughest of the times. My Grand Father, Late Mr. Awadh Kishore Das. My aunt Bindu Kumari, whose first words when I joined medical school was, “win a Nobel prize in medicine”. My father, Mr. Sachchidanand Roy, who stood behind me through thick and thin and last but not the least, my mentor Dr. Glenn H. Dillon. iv ACKNOWLEDGMENTS I would like to thank these important people who have helped and guided me in achieving my goals. Dr. Glenn H. Dillon: An excellent mentor, excellent scientist, excellent administrator, excellent family man and an amazing person. I am extremely lucky to have you as mentor for not only learning science but every moment spent with you was a learning experience. So, Thank you. Dawn Dillon: An amazing person, who did not let us miss our family away from home. Thank you. Dr. Rae Matsumoto, Dr. Patrick S. Callery, Dr. Hanting Zhang, Dr. Robert K Griffith: Thank you for being part of my committee and enriching my scientific experience with valuable suggestions. My Family members, Siblings, Ashish Kumar, Madhulika Roy, Uncles, Ashok Kumar, Abhay Kumar, Friends, Lokesh Gowda, Ashwini Saxena, Vishal Yadav, Manoj Kumar, Abhishek Kumar, Anuj Kumar. Thank you all for the valuable support. Lab members, Zheng-Ian, Ren-Qi-Huang, Cathy-Bell Horner, Manoj Kumar, John M. Freund for their valuable suggestions during day to day working in lab. v TABLE OF CONTENTS TITLE……………………………………………………………………………………………. i ABSTRACT………………………………………………………………………………………ii DEDICATION…………………………………………………………………………………...iv ACKNOWLEDGEMENTS……………………………………………………………………...v TABLE OF CONTENTS………………………………………………………………………..vii LIST OF FIGURES…………………………………………………………...............................ix LIST OF ABBREVIATIONS………………………………………………………………...... xi CHAPTER – I INTRODUCTION………………………………………….…………………..1 History of Meprobamate…………………………….……………………………….......1 Chemistry and Physiological Effects of Meprobamate…………………………….….....5 Pharmacology and Metabolism of Meprobamate………………………………….…......7 Side Effect, Abuse and Withdrawal of Meprobamate………………………….…...…....8 GABAA Receptors………………………………………………………….…..…….....11 Structure of GABAA receptors and Subunit Classes and Roles…………………….......15 Various Modes of GABAA receptor Subunit Classes and Roles…………...……….......17 Pharmacology of GABA receptors..……………………………………….……...…......23 Aims of the Study…………………………………………………………………..…...24 References………………………………………………….........................…………...25 CHAPTER – II ASSESSMENT OF DIRECT GATING AND ALLOSTERIC MODULATORY EFFECTS OF MEPROBAMATE IN GABAA RECEPTORS……………………………………………………………………...….49 ABSTRACT………………………………………………………………………………....50 INTRODUCTION…………………………………………….……………………..............51 vi MATERIALS AND METHODS………………………………………………….…..…….53 Cell Culture and Transfection ……………………………..…………………………...53 Electrophysiology…………….…………………………...………………………........53 Chemicals and solutions…………………………………………………...…………...54 Experimental protocol ……………………………………….……………...………....55 Data Analysis………………………………………….……………………………….56 RESULTS ……………………………………………………………………….……...…..57 Influence of α-subunit isoform of meprobamate-mediated activity of the GABAA receptor…………………………………………….………………………….57 Influence of β-subunit isoform of meprobamate-mediated activity of the GABAA receptor……………………………………………………………………..…57 Influence of γ-subunit isoform of meprobamate-mediated activity of the GABAA receptor ………………………………………………………….……………58 Effects of meprobamate in “extrasynaptic” receptors……………………….………….58 Further assessment of barbiturate like effects of meprobamate………………………...59 DISCUSSION…………………………………………………………………….………....62 ACKNOWLEDGEMENTS……………………………………………………..…..............68 TABLES…………………………………...………………………………………..…….....75 FIGURES…………………………………….……………………………………………...77 REFERENCES………………………………………………………….……………..….....69 CHAPTER – III A SINGLE AMINO ACID RESIDUE AT TRANSMEMBRANE 4 OF THE ALPHA 1 SUBUNIT INFLUENCES CARISOPRODOL DIRECT GATING EFFICACY AT GABAA RECEPTORS……………………………...…………....92 ABSTRACT………………………………………………………………………..………...94 INTRODUCTION……………………………………………...…………………….…..….95 vii MATERIAL AND METHODS……………………………………………………………97 Cell Culture and Transfection………………………………………..…………….….97 Whole-cell patch clamp electrophysiology……………..……………………….…….97 Chemicals and solutions……………………………………………………………….98 Experimental Protocol…………………………………………….…….………..........98 Data Analysis……………………………………………………………….……….....99 Plasmids and site-directed-mutagenesis……………………………………………….100 RESULTS…………………………………………………………………………..….......100 TM4 amino acid
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