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Exploring the Activity of an Inhibitory Neurosteroid at GABAA Receptors

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Exploring the Activity of an Inhibitory Neurosteroid at GABAA Receptors 1 Exploring the activity of an inhibitory neurosteroid at GABAA receptors Sandra Seljeset A thesis submitted to University College London for the Degree of Doctor of Philosophy November 2016 Department of Neuroscience, Physiology and Pharmacology University College London Gower Street WC1E 6BT 2 Declaration I, Sandra Seljeset, 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 The GABAA receptor is the main mediator of inhibitory neurotransmission in the central nervous system. Its activity is regulated by various endogenous molecules that act either by directly modulating the receptor or by affecting the presynaptic release of GABA. Neurosteroids are an important class of endogenous modulators, and can either potentiate or inhibit GABAA receptor function. Whereas the binding site and physiological roles of the potentiating neurosteroids are well characterised, less is known about the role of inhibitory neurosteroids in modulating GABAA receptors. Using hippocampal cultures and recombinant GABAA receptors expressed in HEK cells, the binding and functional profile of the inhibitory neurosteroid pregnenolone sulphate (PS) were studied using whole-cell patch-clamp recordings. In HEK cells, PS inhibited steady-state GABA currents more than peak currents. Receptor subtype selectivity was minimal, except that the ρ1 receptor was largely insensitive. PS showed state-dependence but little voltage-sensitivity and did not compete with the open-channel blocker picrotoxinin for binding, suggesting that the channel pore is an unlikely binding site. By using ρ1-α1/β2/γ2L receptor chimeras and point mutations, the binding site for PS was probed. All chimeras and mutants remained sensitive to PS, raising the question as to whether modulation could be due to indirect interactions between PS and the cell membrane. In hippocampal neurones, the major postsynaptic effect of PS was to increase the IPSC decay rate. However, PS also increased GABA release by activating presynaptic TRPM3 receptors. Upon block of TRPM3, GABA release was reduced by PS due to potentiation of presynaptic Kir2 channel activity. In conclusion, PS directly modulates GABAA receptor kinetics by speeding up current decay at both neuronal and recombinant receptors. At inhibitory synapses, PS can enhance or inhibit GABA release by acting at TRPM3 or Kir2 channels respectively. 4 Acknowledgements Firstly, I would like to thank my supervisor and mentor, Prof. Trevor Smart, whose enduring support and positivity have helped me complete this PhD. I would also like to thank the Wellcome Trust for providing the financial support to complete this project. Many thanks goes to all the members of the Smart lab, past and present, for being a great source of support and entertainment for which I will always be grateful. I would especially like to thank Mike and Duncan for their help with the molecular biology work, and Phil for always being of assistance when my rig or U-tube misbehaved! I am also grateful to Laura for all her help and assistance with the hippocampal cultures. A special thanks goes to Damian for invaluable feedback, guidance and support, and for joining me in those daily tea breaks! I would also like to thank my lovely friends and family. I will forever be grateful to my trumpet teacher Matt for turning my postgraduate student years into a musical adventure that I will never forget, and the members of UCLU Concert Band and SWON who have been like a family to me, providing a great source of support and fun over the past few years. Finally, the biggest of thanks are owed to my best friends Adam, Hazel, Liam and Sam for their love, encouragement and support, and for always being there for me. 5 Contents Declaration ........................................................................................................ 2 Abstract ............................................................................................................. 3 Acknowledgements .......................................................................................... 4 Contents ............................................................................................................ 5 List of figures .................................................................................................... 9 List of tables .................................................................................................... 13 List of equations ............................................................................................. 13 List of abbreviations ....................................................................................... 14 Chapter 1: Introduction .................................................................................. 18 1.1. GABAA receptors .................................................................................... 18 1.1.1. GABAA receptor structure and distribution ....................................... 18 1.1.2. Assembly and trafficking of GABAA receptors .................................. 25 1.1.3. Tonic and phasic inhibition mediated by GABAA receptors .............. 27 1.1.4. GABAA receptor pharmacology and modulation by endogenous ligands ....................................................................................................... 32 1.2. Neurosteroids ......................................................................................... 39 1.2.1. Neurosteroid synthesis, metabolism and expression in the brain .... 40 1.2.2. Molecular targets of inhibitory neurosteroids ................................... 45 1.2.3. Inhibitory neurosteroids and the GABAA receptors .......................... 47 1.2.4. Inhibitory neurosteroids in health and disease ................................. 50 1.3. Project aims ............................................................................................ 53 Chapter 2: Methods ........................................................................................ 56 2.1. HEK293 cell culture ................................................................................ 56 2.2. HEK cell transfection .............................................................................. 56 2.3. Reagents ................................................................................................ 58 2.4. Site-directed mutagenesis and DNA ...................................................... 58 2.5. Hippocampal cell culture ........................................................................ 60 6 2.6. Patch-clamp electrophysiology ............................................................... 61 2.7. Homology modelling ............................................................................... 64 2.8. Data analysis and statistics .................................................................... 64 2.8.1. Analysis of HEK cell recordings ....................................................... 64 2.8.2. Analysis of hippocampal neurone recordings .................................. 66 2.8.3. Statistics .......................................................................................... 67 Chapter 3: Modulation by pregnenolone sulphate of recombinant GABAA receptors expressed in HEK cells ................................................................. 68 3.1. Introduction ............................................................................................. 68 3.2. Results ................................................................................................... 71 3.2.1. PS is a negative allosteric modulator of the α1β2γ2L receptor ........ 71 3.2.2. Pre-applying PS does not increase inhibition ................................... 75 3.2.3. Antagonism by PS at α1β2γ2L is activation-dependent but only weakly voltage-sensitive ............................................................................ 77 3.2.4. PS and PTX do not compete for binding at α1β2γ2L ....................... 80 3.2.5. PS shows no antagonist activity when applied internally via the patch pipette ........................................................................................................ 82 3.2.6. Does PS show any receptor subtype selectivity? ............................ 84 3.2.7. Does PS act as a negative allosteric modulator at receptors that typically exist outside the synapse? ........................................................... 90 3.2.8. PS acts as an antagonist at β3 homomers ...................................... 93 3.2.9. Inhibition by PS at slower- and faster-desensitising GABAA receptor mutants ...................................................................................................... 95 3.3. Discussion .............................................................................................. 98 3.3.1. The mode of inhibition by PS of GABAA receptors ........................... 98 3.3.2. PS exhibits little receptor subtype selectivity at recombinant GABAA receptors expressed in HEK cells ............................................................ 101 3.3.3. PS can antagonise a ‘tonic’ GABA current at the α4β2δ receptor in HEK cells ................................................................................................. 104 3.3.4. The potency of PS may be affected by the kinetics of the GABAA receptor.................................................................................................... 104 3.4. Conclusion ...........................................................................................
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