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Extrasynaptic GABA-A Receptors

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Extrasynaptic GABA-A Receptors 1 Functional properties of 4-PIOL at synaptic and extrasynaptic GABA-A receptors Bijal Patel A thesis submitted to University College London for the Degree of Doctor of Philosophy October 2014 Department of Neuroscience, Physiology and Pharmacology University College London Gower Street WC1E 6BT 2 Declaration I, Bijal Patel, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I can confirm that this has been indicated in the thesis. 3 Abstract GABAA receptors are the major inhibitory ligand-gated ion channels in the mammalian CNS. They mediate their physiological effects via two temporally and spatially distinct forms of signalling, denoted as phasic and tonic inhibition. These two forms of inhibition are mediated by distinct GABAA receptor subtypes, with phasic inhibition relying on the activation of synaptically-located γ2 subunit-containing receptors, and tonic inhibition requiring the activation of extrasynaptic receptors, predominantly thought to contain δ-subunits. The importance of tonic inhibition in regulating cell and network excitability has become increasingly apparent. Moreover, elevated tonic currents accompany neurological disorders such as stroke and absence epilepsy, suggesting that selectively reducing tonic inhibition might be therapeutically useful. Due to a lack of δ-selective antagonists, the theoretically predominant antagonist profile of the weak partial agonist, 4-PIOL, was studied as a potential mechanism for selectively reducing tonic inhibition. The functional effects of 4-PIOL were investigated firstly on whole-cell GABA- activated currents of several recombinant γ2- and δ-containing receptors expressed in HEK293 cells. As expected for a partial agonist, 4-PIOL exhibited both agonist- (at γ2-subunit GABAA receptors) and antagonist-type (at δ-subunit receptors) behaviours, depending on the GABA concentration. 4-PIOL was then assessed on tonic and phasic currents of cerebellar granule cells (CGCs), hippocampal pyramidal neurons and thalamic relay-neurons. In CGCs, 4-PIOL inhibited tonic currents, without affecting spontaneous inhibitory postsynaptic currents (sIPSCs); whereas in hippocampal and thalamic relay neurons, 4-PIOL enhanced, or reduced, tonic currents depending on the extrasynaptic GABA concentration, consistent with an action at extrasynaptic γ2-containing receptors. Moreover, 4-PIOL antagonised sIPSCs in these two brain regions, in accord with targeting presynaptic and postsynaptic GABAA receptors. In conclusion, the therapeutic potential of GABAA receptor partial agonists, such as 4-PIOL will be critically dependent on not only the ambient GABA concentration, but also on the relative expression of different GABAA receptor subtypes. 4 Acknowledgements Firstly, my heartfelt thanks goes to my supervisor and mentor, Prof. Trevor Smart. I will be forever grateful for his unending support, guidance and enduring positivity, which have been invaluable on both an academic, and personal level. I would also like to thank the MRC, for providing the financial support to complete this project. This project would not have been possible without the unwavering support, and invaluable feedback provided by Damian and Martin. Their excellent teaching and guidance have helped unravel, what was sometimes a very confusing project! I would also like to thank, both past and present members of the Smart lab (especially Phil and Megan), who have been a great source of support and entertainment over the past few years, and have made every day in the lab a fun and enjoyable experience. I am grateful to my amazing friends and family, for all their love and support over the past few years. In particular, a special thanks goes to the Biomeds, the ‘Baywatch babes’, my Ricards ladies and the Lemmons, who have each been an excellent source of encouragement and entertainment. Finally, my biggest thanks goes to my family, especially my mum, dad and brother, whose love and ‘encouragement’ have given me the strength to complete this PhD. 5 Contents Declaration ........................................................................................................................................... 2 Abstract ................................................................................................................................................ 3 Acknowledgements ............................................................................................................................... 4 Contents ............................................................................................................................................... 5 List of figures......................................................................................................................................... 9 List of Tables ........................................................................................................................................11 List of equations ...................................................................................................................................12 List of Abbreviations ............................................................................................................................13 Chapter 1: Introduction ........................................................................................................................17 1.1. GABAA receptors ............................................................................................................................. 17 1.1.1. GABAA receptor structure ....................................................................................................... 18 1.1.2. Assembly of GABAA receptors................................................................................................. 21 1.1.3. Trafficking of GABAA receptors ............................................................................................... 21 1.1.4. GABAA receptor modulation: Phosphorylation ....................................................................... 25 1.1.5. GABAA receptor modulation: Pharmacology .......................................................................... 27 1.2. Tonic and Phasic currents in the CNS .............................................................................................. 30 1.2.1. Subunit composition of synaptic and extrasynaptic GABAA receptors ................................... 32 1.2.2. Sources and estimates of ambient GABA levels ..................................................................... 36 1.2.3. Pharmacology of tonic and phasic currents ........................................................................... 38 1.3. Pathophysiological conditions associated with elevated GABAA receptor tonic conductances ...... 44 1.3.1. Learning and memory/Cognitive impairments ....................................................................... 44 1.3.2. Motor recovery after stroke ................................................................................................... 47 1.3.3. Absence epilepsy .................................................................................................................... 47 1.4. Thesis Aims: .................................................................................................................................... 51 1.4.1. Summary of thesis aims .......................................................................................................... 53 Chapter 2: Materials and Methods .......................................................................................................54 2.1. Site directed mutagenesis ............................................................................................................... 54 2.2. Reagents ......................................................................................................................................... 57 6 2.3. HEK293 cell culture and Electrophysiology ..................................................................................... 58 2.3.1. HEK293 cell culture ................................................................................................................. 58 2.3.2. HEK293 cell transfection ......................................................................................................... 58 2.3.3. HEK293 whole-cell electrophysiology ..................................................................................... 60 2.3.4. Analysis of GABA concentration-response curves .................................................................. 62 2.3.5. Calculating Spontaneous Channel Activity ............................................................................. 63 2.4. Cerebellar granule cell cultures ...................................................................................................... 64 2.5. Hippocampal cultures ..................................................................................................................... 64 2.6. Brain Slice electrophysiology .......................................................................................................... 65 2.6.1. Animals ................................................................................................................................... 65 2.6.2. Preparation of brain slices ...................................................................................................... 65 2.6.3. Whole-cell electrophysiology in slices and neuronal
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