Western University Scholarship@Western Electronic Thesis and Dissertation Repository 6-17-2013 12:00 AM Regulation of TRPM2 channels by Fyn kinase Matthew L. Johnston The University of Western Ontario Supervisor Dr. John F. MacDonald The University of Western Ontario Graduate Program in Physiology A thesis submitted in partial fulfillment of the equirr ements for the degree in Master of Science © Matthew L. Johnston 2013 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Cellular and Molecular Physiology Commons Recommended Citation Johnston, Matthew L., "Regulation of TRPM2 channels by Fyn kinase" (2013). Electronic Thesis and Dissertation Repository. 1308. https://ir.lib.uwo.ca/etd/1308 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. REGULATION OF TRPM2 CHANNELS BY FYN KINASE (Thesis format: Monograph) by Matthew Lawrence Johnston Graduate Program in Physiology A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science The School of Graduate and Postdoctoral Studies The University of Western Ontario London, Ontario, Canada © Matthew L. Johnston 2013 i Abstract TRPM2 is a calcium-permeable non-selective cation channel that acts as a mediator of cell death in response to oxidative stress. It has been shown that oxidative stress increases TRPM2 tyrosine phosphorylation and activation – an effect that is blocked by PP2, a non-specific inhibitor of Src family kinases. However, the kinase and target TRPM2 tyrosine residue(s) involved have not yet been identified. Here, we investigated the potential regulation of TRPM2 by Fyn. Intracellular application of recombinant Fyn potentiated TRPM2 currents in HEK293 cells expressing inducible TRPM2 (TRPM2- HEK293 cells). Further, a physical interaction between Fyn and TRPM2 was demonstrated by co-immunoprecipitation in TRPM2-HEK293 cells. Additionally, tyrosine phosphorylation of TRPM2 was induced by Fyn in TRPM2-HEK293 cells, and the amount of phosphorylation detected was related to the activation state of Fyn. We propose that by augmenting TRPM2 activity, Fyn kinase potentiates cellular calcium overload and facilitates cell death in response to oxidative stress. Keywords TRPM2, Fyn kinase, tyrosine phosphorylation, protein-protein interaction, calcium ii Co-Authorship Statement Drs. John MacDonald and Michael Jackson supervised and contributed to the design of all experiments discussed in this work. Dr. Jillian Belrose contributed to the design of the biochemistry experiments conducted in this work. Further, Dr. Belrose conducted an experiment assessing the effect of Fyn on TRPM2 currents in cultured hippocampal neurons (Appendix A, Fig. S1). Dr. Fabiana Caetano conducted preliminary electrophysiology experiments assessing the effect of Fyn on TRPM2. These experiments contributed to the design of the electrophysiological experiments described in this work. Additionally, Dr. Hongbin Li performed an experiment assessing the effect of a Fyn-interfering peptide (Fyn(39-57)) on TRPM2 currents in cultured hippocampal neurons (Appendix A, Fig. S2). iii Acknowledgments Firstly, I would like to extend my gratitude to my supervisors, Dr. John MacDonald and Dr. Michael Jackson for providing me with the opportunity to learn and develop under your guidance. Your expertise, feedback and support have been invaluable to the completion of this project. I have greatly enjoyed the time I have spent in your lab. I would also like to thank the members of my graduate advisory committee: Dr. Vania Prado, Dr. Stephen Ferguson, Dr. Wei-Yang Lu and Dr. Timothy Regnault. Your expertise, feedback and advice were highly beneficial to this project. Additionally, the funding provided for this project by the Canadian Institute of Health Research (CIHR), the Ontario Graduate Scholarship (OGS) program and Schulich School of Medicine and Dentistry is gratefully acknowledged. Additionally, I would like to thank the members of the MacDonald/Jackson lab for their continued support. Particularly, I would like to thank Dr. Jillian Belrose for invaluable contributions to these experiments, as well as Dr. Fabiana Caetano for helping to lay the groundwork for this project. Additionally, my gratitude is owed to Natalie Lavine for the technical assistance and time she devoted to this project. Also, I would like to thank Dr. Hongbin Li, Dr. Gang Lei, Brian Lockhart, Oies Hussein, Dr. Catherine Trepanier and Dr. Kai Yang for their support, feedback and various other contributions to this work. Completion of this project would not have been possible without this support. Lastly, I would like to thank my family and friends for the support they have provided throughout my academic career. iv Table of Contents Abstract ............................................................................................................................... ii Co-Authorship Statement................................................................................................... iii Acknowledgments.............................................................................................................. iv Table of Contents ................................................................................................................ v List of Tables .................................................................................................................... vii List of Figures .................................................................................................................. viii List of Appendices ............................................................................................................. ix List of Abbreviations .......................................................................................................... x Section 1: Introduction ........................................................................................................ 1 1.1 General introduction ............................................................................................... 2 1.2 Transient receptor potential melastatin 2 (TRPM2) ............................................... 2 1.3 Structural and biophysical properties of TRPM2 ................................................... 5 1.4 Oxidative stress and TRPM2 activation ................................................................. 9 1.5 Physiological roles of TRPM2 .............................................................................. 11 1.6 TRPM2 channels in CNS disease ......................................................................... 12 1.7 TRPM2 regulation mechanisms ............................................................................ 14 1.8 TRPM2 regulation by tyrosine phosphorylation................................................... 15 1.9 Fyn kinase ............................................................................................................. 16 1.10 TRPM2, Fyn and NMDA receptor signaling ...................................................... 19 1.11 Rationale and hypothesis ..................................................................................... 20 Section 2: Materials and Methods..................................................................................... 22 2.1 Cell culture ............................................................................................................ 23 2.2 Whole-cell voltage-clamp electrophysiology ....................................................... 23 2.3 Generation of TRPM2 constructs ......................................................................... 24 2.4 Transfection .......................................................................................................... 28 2.5 Immunoprecipitation and Western blotting .......................................................... 29 2.6 Drugs and peptides ................................................................................................ 29 2.7 Data analysis and statistics.................................................................................... 30 Section 3: Results .............................................................................................................. 31 3.1 Regulation of TRPM2 currents by Fyn kinase ..................................................... 32 3.2 Expression of Fyn and TRPM2 in TRPM2-HEK293 cells ................................... 34 3.3 Tyrosine phosphorylation of TRPM2 induced by Fyn kinase .............................. 36 3.4 Physical interaction between Fyn kinase and TRPM2 ......................................... 39 3.5 Expression of Fyn and TRPM2 in HEK293T cells .............................................. 40 3.6 Tyrosine phosphorylation of wild-type and mutant TRPM2 by Fyn kinase ........ 44 3.7 Physical interaction between Fyn kinase and wild-type or mutant TRPM2 ......... 45 v Section 4: Discussion ........................................................................................................ 50 4.1 Summary of key findings ...................................................................................... 51 4.2 Regulation of TRPM2 by Fyn kinase ................................................................... 51 4.3 Identification of TRPM2 tyrosine residue(s) responsible for Fyn modulation ..... 53