Regulation of Ca2+-activated Cl- channel ANO1 (TMEM16A) by different Ca2+ sources in sensory neurons Xin Jin Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Biomedical Sciences March 2015 1 The candidate confirms that the work submitted is his/her own, except where work which has formed part of jointly authored publications has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated below. The candidate confirms that appropriate credit has been given within the thesis where reference has been made to the work of others. Chapter 4, 5, 6, 7 are based on work from jointly authored publication: Activation of the Cl- channel ANO1 by localized calcium signals in nociceptive sensory neurons requires coupling with the IP3 receptor. Jin X, Shah S, Liu Y, Zhang H, Lees M, Fu Z, Lippiat JD, Beech DJ, Sivaprasadarao A, Baldwin SA, Zhang H, Gamper N. Sci Signal. 2013 Aug 27; 6 (290):ra73. Xin Jin et al planned experiments; Xin Jin et al performed experiments; Xin Jin et al analyzed data; Figure 4.5 in panels A-C was contributed by Yani Liu, Figure 5.2 was contributed by Huiran Zhang, Figure 6.1 was contributed by Shihab Shah and Nikita Gamper. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement © 2015 The University of Leeds and Xin Jin 2 Acknowledgements I would like to thank my supervisor, Dr Nikita Gamper for giving me the opportunity to work on such an exciting project, for his advice, encouragement, critical inputs and continuous guidance throughout this project. I am thankful to past and present members of the Gamper group for their valuable discussions and practical help. I would also like to sincerely thank Prof. Hailin Zhang and his group for continuous generous help and practical support. I am extremely grateful to Dr Rao Sivaprasadarao and Dr Stephen Baldwin for their excellent advice, and general help. As it is impossible to mention everyone who has helped me during the course of this project, I would like to say a big thanks to the people in neuroscience and ion channels groups at Leeds. I would like to acknowledge the Faculty of Biological Sciences and University of Leeds for funding. I am grateful to my family, for their encouragement, advice and support throughout my life. III Abstract Proteins of anoctamin (TMEM16) family are the candidate subunits for Ca2+- activated Cl- channels (CaCC). In recent years, studies have shown that anoctamin-1 (TMEM16A or ANO1) plays important physiological roles in processes including epithelial fluid secretion, muscle contraction and olfactory transduction. How the Ca2+ regulates the activity of ANO1 in different tissue is still not clear. In this study, I showed that the excitatory CaCC in nociceptors (small-diameter sensory neurons that are responsible for transmission of painful stimuli) was activated by 2+ the release of Ca from the 1, 4, 5-trisphosphate (IP3)-sensitive intracellular stores in response to bradykinin (BK) or proteases (through protease activated receptor 2). Interestingly, while in the majority of nociceptors, CaCC was induced by Ca2+ release from the stores, only in a few neurons CaCC was activated by the Ca2+ influx through the voltage-gated Ca2+ channels (VGCC). Chelating intracellular Ca2+ with the slow Ca2+ buffer EGTA did not affect CaCC activation by protease activated receptor 2 (PAR2), while BAPTA abolished such activation, suggesting a close proximity of the Ca2+ release sites and CaCC. Membrane fractionation demonstrated that in the dorsal root ganglion (DRG), ANO1, bradykinin receptor 2 (B2R) or PAR2, were co-purified with lipid raft marker caveolin-1. Using various biochemical approaches I further demonstrated that ANO1 physically interacted with the IP3 receptor 1 (IP3R1) in DRG. Moreover, IP3R1, ANO1, B2R, and/or PAR2 were all assembled into functional signalling complexes and the plasma membrane components of the complex which contained ANO1 and GPCRs were tethered to the juxtamembrane regions of the endoplasmic reticulum. Disruption of the membrane microdomains by methyl-beta-cyclodextrin (MβCD) or competitive peptides partially restored coupling of CaCC to VGCC but disrupted coupling between B2R or PAR2 signaling and ANO1. Thus, such molecular complexes dichotomize different Ca2+ sources to provide ANO1-mediated excitation in response to specific ambient signals but protect the channels from global changes in intracellular Ca2+ and prevent sensory neurons from overexcitability. IV Table of contents Acknowledgements ........................................................................................................... III Abstract ............................................................................................................................. IV List of figures .................................................................................................................... IX List of tables ..................................................................................................................... XII Abbreviations ................................................................................................................. XIII Chapter 1 General introduction ........................................................................................... 1 1.1 Nociceptors and pain transmission ........................................................................... 1 1.2. Inflammatory pain .................................................................................................... 3 1.3 Ion channel diversity ................................................................................................. 5 1.3.1 Ligand-gated channels ....................................................................................... 6 1.3.2 Voltage-gated channels ...................................................................................... 6 1.3.3 Cyclic nucleotide-gated (CNG) channels .......................................................... 7 1.3.4 Mechano-gated channels .................................................................................... 8 1.3.5 Water channels ................................................................................................... 9 1.3.6 Cl- channels ........................................................................................................ 9 1.4 Anoctamins ............................................................................................................. 26 1.4.1 ANO1 ............................................................................................................... 26 1.4.2 Other anoctamins ............................................................................................. 39 1.5 Working hypothesis and aims of the study ............................................................. 43 Chapter 2 Materials and methods ..................................................................................... 45 2.1 Materials ................................................................................................................. 45 2.2 DRG culture ............................................................................................................ 45 2.3 Generation DNA constructs .................................................................................... 47 2.4 Electrophysiology ................................................................................................... 49 V 2.5 Immunocytochemistry ............................................................................................ 51 2.6 Immunoprecipitation and Western blotting ............................................................ 51 2.7 Lipid Raft Isolation ................................................................................................. 54 2.8 GST Pulldown Assays ............................................................................................ 54 2.9 Fluorescent Imaging................................................................................................ 55 2.10 In Situ Proximity Ligation Assay (PLA) .............................................................. 55 2.11 Data Analysis ........................................................................................................ 58 Chapter 3. Activation of ANO1 by Ca2+ ........................................................................... 59 3.1 Introduction ............................................................................................................. 59 3.1.1 Ca2+ sensing and gating mechanisms of ion channels ...................................... 59 3.1.2 Voltage gating mechanisms of ANO1 ............................................................. 62 3.1.3 ANO1 gating by heat ....................................................................................... 63 3.1.4 Interaction of ANO1 with other proteins ......................................................... 63 3.1.5 Regulation of ANO1 properties by alternative splicing .................................. 64 3.2 Aims ........................................................................................................................ 65 3.3 Results ..................................................................................................................... 65 3.3.1 Ca2+-activated Cl- currents recorded in HEK293