Dissecting the Role of Presynaptic GABA a Receptors in Nerve Terminal Function by Philip Long Department of Pharmacology, The School of Pharmacy, 29 - 39 Brunswick Square, London, WCIN lAX A thesis submitted for the degree of Doctor of Philosophy in Pharmacology for the University of London 2007 - 1 - %pOL Of ProQuest Number: 10104232 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10104232 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 This thesis describes research conducted in the School of Pharmacy, University of London between 2003 and 2006 under the supervision of Dr Jasmina Jovanovic. I certify that the research described is original and that any parts of the work that have been conducted by collaboration are clearly indicated. I also certify that I have written all the text herein and have clearly indicated by suitable citation any part of this dissertation that has already appeared in publication. Signature A Date ^ 6 - /o - _ 9 - Abstract Fast synaptic inhibition in the mammalian brain is mediated principally by GABAa receptors, a large and diverse family of Cl permeable ion channels. Emerging evidence points to the presynaptic localisation of these receptors and their role in regulating intraterminal Ca^"^ levels and neurotransmitter release in some areas of the CNS. We used purified nerve terminals (synaptosomes) to establish the presence of functional presynaptic GABAa receptors in the rat neocortex. As a biochemical read-out of presynaptic GABAa receptor activity, we measured Ca^^-dependent changes in the phosphorylation state of synapsin I. A dose-dependent decrease in synapsin I phosphorylation was detected in response to bicuculline, picrotoxin and GABase, while muscimol and isoguvacine caused a dose-dependent increase in synapsin I phosphorylation. Immunohistochemical analysis revealed this phospho-form of synapsin I to be localised to glutamatergic nerve terminals. In functional studies, GABAa receptor activation by muscimol caused a dose-dependent inhibition of glutamate release, which was abolished by picrotoxin. In order to investigate the mechanism(s) by which presynaptic GABAa receptor activity leads to inhibition of glutamate release, we measured release in the presence of the NKCCl antagonist bumetanide, which abolished the muscimol-induced inhibition of glutamate release. When we subsequently investigated the role of voltage-gated Ca^^ channels on muscimol- dependent inhibition of glutamate release, we found that muscimol inhibited glutamate release in the presence of co-conotoxin GVIA, co-agatoxin IVA and NiCb. However, the inhibition of release by muscimol was abolished by SNX-482 and nifedipine. Muscimol- dependent decrease in release was also abolished in the presence of W7. Our results indicate that the activation of presynaptic GABAa receptors in the rat neocortex is coupled to depolarisation of the nerve terminal membrane, leading to inhibition of glutamate release via Ca^'^-dependent Ca^'^ channel inhibition linked to L- and R-type VDCCs. -3 Acknowledgements I would like to begin by thanking my supervisor Dr Jasmina Jovanovic for all her assistance and guidance throughout the course of my PhD, especially for her constructive comments while writing this thesis. I would also like to express my gratitude to Dr Talvinder Sihra for integrating me into his lab and providing creative input and technical help. Thanks to Dr Audrey Mercer and Rahima Begum for their help carrying out the immunohistochemistry. Finally, thanks to my friends and family for their help and support during this time, especially Rosa Sancho for ensuring I didn’t have to live in a box and to Chris Moore and Sabina Muneton for proof-reading my thesis. -4 Contents Abstract .......................................................................................................................................... 3 Acknowledgements .......................................................................................................................4 Abbreviations ...............................................................................................................................11 1. Introduction ......................................................................................................................... 16 1.1 GAB A as a neurotransmitter ..................................................................................... 17 1.2 GAB A Receptors ....................................................................................................... 19 1.2.1 GABAa Receptors ..................................................................................................19 1.2.2 GABAb Receptors ...................................................................................................19 1.2.3 GAB Ac Receptors ...................................................................................................19 1.3 GABAa receptor structure ........................................................................................ 20 1.4 Regulation of GABAa receptor function by allosteric modulators ........................23 1.5 Synthesis, expression and trafficking of GABAa receptors .................................. 27 1.6 Subcellular localisation of GABAa Receptors ........................................................33 1.7 Modulation of GABAa receptors by protein phosphorylation and association with signalling molecules ............................................................................................................... 34 1.8 Presynaptic GABAa Receptors .................................................................................38 1.9 Cation co-transporters determine the functional outcome of GABAa receptor activation ..................................................................................................................................42 1.10 Neurotransmitter release ........................................................................................... 44 1.11 Synapsins ....................................................................................................................48 1.12 Voltage-gated Ca^^ Chaimels ..................................................................................... 51 1.13 Modulation of neurotransmitter release by presynaptic neurotransmitter receptors ......................................................................................................................................54 1.13.1 Presynaptic G-Protein Coupled Receptors ..................................................... 54 1.13.1.1 Metabotropic Glutamate Receptors .................................................................. 54 1.13.1.2 GAB As receptors ...............................................................................................55 1.13.1.3 Histamine receptors ........................................................................................... 55 1.12.1.4 Adenosine receptors .......................................................................................... 56 1.13.1.5 Muscarinic acetylcholine receptors .................................................................. 56 1.13.2 Presynaptic ligand-gated ion channels ................................................................. 57 - 5 - 1.13.2.1 Glycine receptors .............................................................................................. 57 1.13.2.2 Nicotinic acetylcholine receptors ..................................................................... 57 1.13.2.3 5HT) receptors ...................................................................................................58 1.13.2.4 NMDA receptors ................................................................................................59 1.13.2.5 AMPA receptors .................................................................................................59 1.13.2.6 Kainate receptors ................................................................................................60 1.13.2.7 P2X receptors .....................................................................................................61 1.13.2.8 V anilloid receptors ............................................................................................ 61 1.14 Isolated Nerve Terminals (Synaptosomes) ..............................................................61 1.15 Aims............................................................................................................................62 2. Materials and Methods ........................................................................................................65 3. Regulation of presynaptic Ca^^-dependent signalling pathways and synapsin I phosphorylation by GABAa receptors ...................................................................................... 80 4. Modulation of glutamate release by presynaptic GABAa receptors ............................115 5. Modulation of Plasma Membrane Potential and Intraterminal