Alternative splicing in sodium channels: Biophysical and functional effects in Nav1.1, Nav1.2 & Nav1.7. Andrianos Liavas A thesis submitted to University College London for the degree of Doctor of Philosophy Department of Clinical and Experimental Epilepsy Institute of Neurology Queen Square London WC1N 3BG 1 Declaration I, Andrianos Liavas, confirm that the work presented in this thesis is my original research work. Where contributions of others are involved, this has been clearly indicated in the thesis. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. 2 Acknowledgements I would like to thank first of all my supervisor, Dr Stephanie Schorge, for her continuous care and support that went far beyond the duties of an ordinary supervisor and for which I'm truly greatful. I would also like to thank my professors and colleagues, in my lab as well as in previous labs I've worked in, for sharing their time, knowledge, expertise as well as their everyday life with me, which has ended up in great partnerships and true friendships. A special thanks to Gabriele, my lab brother, (I'm gonna miss working together my friend!), the whole 808 gang, with all of which we came really close together (and not just because of lack of office space!), the Friday night pub crew (and Monday and Thursday sometimes...!) and especially Elodie, for being truly special to me. A very big thank you also to my family, my brother Mathios and all my friends and beloved ones for always being there for me. It's their constant love and support through which I found the strength and courage to persist through the difficulties and finally reach the end of this big journey today. Finally, I would also like to thank my examiners, Dr Martin Stocker and Prof Richard Baines, for finding the time to read and correct my thesis. 3 Abstract Alternative splicing in voltage-gated sodium channels can affect pathophysiological conditions, including epilepsy and pain. A conserved alternative splicing event in sodium channel genes, including SCN1A, SCN2A and SCN9A, gives rise to the neonatal (5N) and adult (5A) isoforms. Differences in the ratio of 5A/5N in Nav1.1 (encoded by SCN1A) in patients may lead to different predisposition to epilepsy or response to antiepileptic drugs (AED). Previous HEK293T whole-cell voltage-clamp recordings showed that Nav1.1-5N channels recover more quickly from fast inactivation than 5A. However it was unknown whether this effect is conserved in Nav1.2 (encoded by SCN2A) and Nav1.7 (SCN9A) channels, or what the functional consequences of this splicing event are for neurons. This project used whole-cell voltage-clamp recordings on heterologously expressed neonatal and adult channels to compare the biophysical properties of the splice isoforms for all three channel types and their modulation by AEDs. It also used current-clamp and dynamic-clamp recordings on transfected hippocampal cultured neurons to assess the effect of splicing on neuronal properties during epileptiform activity. Biophysical analysis in HEK293T cells revealed that splicing profoundly regulates fast inactivation and channel availability during fast, repetitive stimulation, with neonatal channels showing higher availability compared to adult channels and this difference was conserved among Nav1.1, Nav1.2 and Nav1.7. The change in inactivation imposed by splicing can be modeled as a modification of the stability of the inactivation statein resting channels. This change can be eradicated by administration of the AEDs phenytoin and carbamazepine. Current-clamp recordings in transfected neurons showed that the alternatively spliced variantmodifies the rising phase of action potentials for Nav1.1 and Nav1.2 at high firing frequencies, implying a consistent splice- dependent modulation of channel availability. For Nav1.1 in interneurons, this translated to higher firing frequency for the neonatal isoform, which also conferred a higher maximal firing rate during epileptiform events imposed under dynamic-clamp recordings. 4 Table of Contents Chapter 1: Introduction ........................................................................................ 18 1.1 Voltage-gated sodium channel structure and function .......................................................... 18 1.1.1 Discovery and purification of the sodium channel protein ........................................... 18 1.1.2 Na+ channel structure: α-subunits ................................................................................. 19 1.1.3 Sodium channel α-subunit genes: evolution and chromosomal location ..................... 22 1.1.4 β-subunits ..................................................................................................................... 24 1.1.5a Sodium channels subtypes have been appropriated to different locations and functions in neurons. ............................................................................................................................. 27 1.1.5b Non-canonical distribution of sodium channels ......................................................... 31 1.1.6 Molecular properties of VGSCs underlying function: Overview ................................. 31 1.1.7 Voltage Dependence of activation and gating .............................................................. 32 1.1.8 Ion selectivity ............................................................................................................... 35 1.1.9 Inactivation ................................................................................................................... 36 1.1.10 Persistent current ........................................................................................................ 38 1.1.11 VGSC Pharmacology ................................................................................................. 40 1.2 VGSCs and Disease ............................................................................................................... 44 1.2.1 Different Na+ channels are associated with distinct diseases ....................................... 44 1.2.2 SCN9A – Peripheral nerve sodium channelopathies ..................................................... 45 1.2.3 SCN1A & SCN2A – Brain sodium channelopathies ..................................................... 46 1.2.4 Epilepsy ........................................................................................................................ 46 1.2.5 Epilepsy and VGSCs .................................................................................................... 47 1.2.6 Dravet Syndrome .......................................................................................................... 48 1.2.7 GEFS+ .......................................................................................................................... 49 1.2.8 Epilepsy and SCN1A ..................................................................................................... 49 1.2.9 SCN2A and Benign Familial Neonatal Infantile Seizures (BFNIS) ............................. 56 1.2.10 Epilepsy and SCN3A and SCN8A ............................................................................... 57 1.2.11 Pain & Epilepsy and SCN9A ...................................................................................... 58 1.3 VGSCs and use-dependent blockers ...................................................................................... 61 1.3.1 Use-dependent blockers – AEDs .................................................................................. 61 1.3.2 Phenytoin ...................................................................................................................... 61 1.3.3 The modulated receptor model ..................................................................................... 63 5 1.3.4 Molecular basis of phenytoin’s action .......................................................................... 64 1.3.5 Carbamazepine ............................................................................................................. 66 1.4 VGSCs and splicing ............................................................................................................... 68 1.4.1 Splicing ......................................................................................................................... 68 1.4.2 Alternative splicing in the SCN1A gene ....................................................................... 69 1.4.3 SCN1A splicing and epilepsy ........................................................................................ 73 1.4.4 Possible reasons for differences between studies ......................................................... 76 1.4.5 5N overexpression in epilepsy ...................................................................................... 79 1.4.6 Functional studies for Nav transcript variants ............................................................... 80 1.4.7 Dynamic-Clamp as a direct link to epileptiform bursts ................................................ 85 1.5 Experimental aims ................................................................................................................. 87 Chapter 2: General Materials and Methods ....................................................... 89 2.1 Molecular Biology ................................................................................................................. 89