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Understanding the Molecular Mechanism of Trp Channel

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Understanding the Molecular Mechanism of Trp Channel UNDERSTANDING THE MOLECULAR MECHANISM OF TRP CHANNEL ACTIVATION/INHIBITION BY STRUCTURAL ANALYSIS by AMRITA SAMANTA Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Advisor: Vera Moiseenkova-Bell Department of Physiology and Biophysics CASE WESTERN RESERVE UNIVERSITY August 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of AMRITA SAMANTA Candidate for the degree of Physiology and Biophysics* Witold Surewicz (Committee Chair) Sudha Chakrapani Vera Moiseenkova-Bell Phoebe Stewart Derek Taylor Isabelle Deschenes May 29, 2018 *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents List of Tables vii List of Figures viii Acknowledgments xi List of Abbreviations xiii Abstract xv Chapter 1: Introduction 1.1 The Transient Receptor Potential (TRP) family of ion channels 2 1.2 TRPC subfamily 2 1.2.1 TRPC1 3 1.2.2 TRPC4 & TRPC5 5 1.2.3 TRPC2, TRPC3, TRPC6 & TRPC7 6 1.3 TRPV Subfamily 7 1.3.1 TRPV1 7 1.3.2 TRPV2 10 1.3.3 TRPV3 & TRPV4 11 i 1.3.4 TRPV5 & TRPV6 12 1.4 TRPM Subfamily 13 1.4.1 TRPM1 & TRPM3 14 1.4.2 TRPM4 & TRPM5 15 1.4.3 TRPM2, TRPM6 & TRPM7 16 1.4.4 TRPM8 17 1.5 TRPA Subfamily 18 1.6 TRPML Subfamily 20 1.6.1 TRPML1 21 1.6.2 TRPML2 & TRPML3 22 1.7 TRPP Subfamily 23 1.7.1 Polycystin family 25 1.7.2 TRPP2, TRPP3 & TRPP5 27 1.8 Purpose of this study 29 1.9 Cryo Electron Microscopy 30 Figures 32 ii Chapter 2: Understanding the molecular mechanism of the TRPV2 channel gating during ligand activation by cryo-EM 2.1 Introduction 37 2.2 Materials and Methods 2.2.1 Stable cell line generation and flux assay 38 2.2.2 Protein expression and purification 40 2.2.3 Cryo-EM data collection 40 2.2.4 Image processing 41 2.2.5 Model building 42 2.3 Results 2.3.1 Role of the pore turret domain in TRPV2 activation and large 43 organic molecules uptake 2.3.2 Architecture of the TRPV2 in apo- and CBD-activated states 44 2.3.3 Conformational differences between apo- and CBD-activated 47 TRPV2 2.4 Discussion 49 Figures 52 iii Chapter 3: Understanding the ligand induced conformational changes of mouse TRPA1 channel during activation and inhibition 3.1 Introduction 71 3.2 Materials and Methods 3.2.1 Expression and purification of TRPA1 74 3.2.2 Limited proteolysis of TRPA1 75 3.2.3 LC-MS analysis 75 3.3 Results 3.3.1 Limited proteolysis and in-solution mass spectrometry to 77 determine ligand induced conformational changes in TRPA1 3.3.2 Analysis of NMM (electrophilic agonist) induced 79 conformational change in TRPA1 3.3.3 Analysis of PF-4840154 (non-electrophilic agonist) induced 80 conformational change in TRPA1 3.3.4 Analysis of menthol (non-electrophilic modulator) induced 82 conformational change in TRPA1 3.3.5 Analysis of A-967079 (non-electrophilic antagonist) induced 83 conformational change in TRPA1 3.4 Discussion 84 iv Table 89 Figures 90 Chapter 4: Discussion and future directions 4.1 Summary 103 4.2 Impact of this study 4.2.1 Application for structural studies of other memorable proteins 105 4.2.2 Application on health and disease 106 4.3 Future directions 4.3.1 Permeation of large organic cations through TRP channels 107 4.3.2 Charecterization of TRPV2 specific blocker and 108 understanding the mechanism of TRPV2 inhibition 4.3.3 Resolving structure of full length TRPA1 reconstituted into 109 nanodisc by using cryo EM 4.3.4 Regions of TRPA1 molecule affected by other electrophilic 110 and non-electrophilic ligands 4.4 Concluding remarks 110 Figures 112 Appendix 117 v Reference 119 vi List of Tables Table 3.1. Modulators of TRPA1 used in this study 89 vii List of Figures A schematic representation of the TRP superfamily of ion Figure 1.1 32 channels High resolution structures for TRP channels initially Figure 1.2 33 resolved A schematic representation of the TRP channel pore Figure 1.3 34 showing the two regions of constriction (gates) A schematic representation of the TRPP1 and TRPP2 Figure 1.4 interacting with each other through their C-terminal region and 35 forming the receptor-channel complex Functional characterization of wild type and truncated Figure 2.1 TRPV2 indicates the pore turret region is essential for 52 passage of both Ca2+ and large cations Figure 2.2 Summary flowchart of TRPV2 data processing 53 Figure 2.3 Resolution data for TRPV2 refinement 54 Cryo-EM analysis of apo and CBD-activated TRPV2 Figure 2.4 55 channel Figure 2.5 Structural details of apo and CBD-activated TRPV2 56 Structural comparison of 4.6Å apo TRPV2 and previously Figure 2.6 58 published ~5Å apo TRPV2 channel Figure 2.7 Putative CBD binding site 59 viii Cryo-EM density map comparison of apo and CBD- Figure 2.8 activated TRPV2 channel indicates large conformational 60 change in the TMD but not in the ARD Comparison of apo and CBD-activated TRPV2 models and Figure 2.9 62 TMD Figure 2.10 Architecture of the apo and CBD-activated TRPV2 pores 64 Structural comparison of full length apo TRPV2, truncated Figure 2.11 65 apo TRPV2 and apo TRPV1 pores Figure 2.12 Comparison of TRPV pore architectures 67 Figure 2.13 Comparison of the pore region of the TRPV channels 68 Electron density corresponding to regions of interest in the Figure 2.14 69 TRPV2 structures validates model assignment Figure 3.1 TRPA1 purification and limited proteolysis of pure TRPA1 90 Construction of a representation of the full-length TRPA1 Figure 3.2 91 channel Figure 3.3 Effect of NMM on TRPA1 conformation 92 Nano- LC-MS/MS spectrum of the modified peptides after Figure 3.4 94 Asp-N digestion of NMM-activated TRPA1 Effect of non-electrophilic modulators on TRPA1 Figure 3.5 96 conformation Figure 3.6 Effect of A-967079 on TRPA1 conformation 97 Dimer representations of TRPA1 showing additional Figure 3.7 99 regions of miscleavage upon ligand interaction ix Cartoon representation of TRPA1 showing the regions Figure 3.8 101 involved in channel gating Cartoon representation of gating mechanism of TRPV2 in Figure 4.1 112 presence of CBD A tree representing the mammalian TRP superfamily of ion Figure 4.2 113 channels with most of the high resolution structures Schematic representation of the principle of limited Figure 4.3 114 proteolysis followed by mass spectrometry Inhibition of TRPV2 activity by PL and preliminary cryo-EM Figure 4.4 115 data of PL-inhibited TRPV2 Cryo-EM analysis of CBD-activated and PL-inhibited Figure 4.5 116 TRPV2 channel x ACKNOWLEDGEMENTS I am especially thankful to my PhD advisor, Dr. Vera Moiseenkova-Bell, for her professional and personal support and guidance during my training as a graduate student in her lab. I would like to thank the previous and present lab members of the Moiseenkova-Bell lab: Dr. Matthew Cohen, who taught me some of the molecular biology techniques and helped me to think critically about projects during the initial few years of my journey; Dr. Kevin Huynh, who taught me a lot of the molecular biology techniques that I used to complete this dissertation; Monica Kane, who was an undergraduate in the lab and brought a lot of fresh energy to the lab; Taylor Hughes, who is a great lab mate and colleague I enjoyed working and learning new techniques together with in the lab; Dr. Ievgen Ignatenko, a great lab mate who is always ready to help. I was also fortunate to have the opportunity to train an undergraduate student, Connor Dawedeit and a high school student, Fatema Uddin, both of whom brought a lot of young energy to the lab. I would like to thank my dissertation committee members (Dr. Witold Surewicz, Dr. Sudha Chakrapani, Dr. Phoebe Stewart, Dr. Derek Taylor and Dr. Isabelle Deschenes) for guiding me to think critically, forcing me to stay focused and supporting me in completion of my dissertation. Additionally, I would like to thank Heather Holdaway and Dr. Sudheer Molugu, who trained me in cryo-EM microscopy; Dr. Janna Kiselar who helped me with mass spectrometry; Dr. George Dubyak who trained me in cell culture and Ca2+ flux assay measurements; Dr. David Lodowski for his collaboration and support, Dr. Yuhang Liu and Dr. Seungil Han for their collaboration and allowing to use their electron microscope. Moreover, I would like xi to acknowledge Dr. Sudha Chakrapani who taught me the technique of electrophysiology and gave me space in her lab during the last few months of my graduation when my lab relocated to UPenn. In addition, I would also like to thank Chakrapani lab members for being great colleagues to work with and good friends. Thank you to my family for believing in me; Diptendu konar – I could not have completed this journey without you and your constant support and Adhrit for being such a patient and understanding child. Finally, I would like to thank all the professors, friends and colleagues whom I did not mention here but who were essential for my success in graduate school. xii List of Abbreviations ADPKD Autosomal Dominant Polycystic Kidney Disease AITC Allyl Isothiocyanate ARD Ankyrin Repeat Domain CBD Cannabidiol Cryo-EM Cryo Electron Microscopy DADS Diallyl Disulphide GBM Glioblastoma Multiforme ICRAC Calcium Release-Activated Calcium Current IP3 Inositol Triphosphate ISOC Store Operated Calcium Current LEL Late Endosome and Lysosome LC-MS Liquid Chromatography Mass Spectrometry NaV channels Voltage gated Sodium channels NHERF Na+/H+ exchanger regulatory factor NOMPC No Mechanoreceptor Potential C PKD Polycystic Kidney Disease PL Piperlongumine xiii REJ Receptor for Egg Jelly RTX/DkTx Resiniferatoxin/Double-knot Toxin S1-S6 Transmembrane helices 1 through
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