UNIVERSITY OF CALIFORNIA Los Angeles The subproteome of mitoBKCa channels from cardiomyocytes reveals novel insights into its mitochondrial import mechanism and function A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Molecular and Medical Pharmacology by Jin Zhang 2016 © Copyright by Jin Zhang 2016 ABSTRACT OF DISSERTATION The subproteome of mitoBKCa channels from cardiomyocytes reveals novel insights into its mitochondrial import mechanism and function by Jin Zhang Doctor of Philosophy in Molecular and Medical Pharmacology University of California, Los Angeles, 2016 Professor Ligia G Toro De Stefani, CO-Chair Professor Riccardo Olcese, CO-Chair BKCa channels are widely expressed ion channels and characterized by their large conductance to potassium and sensitivity to calcium and voltage. It is typically observed at the plasma membrane in the majority of cell types, with adult cardiomyocytes being an exception. Previously, both electrophysiological and immunological studies have detected BKCa channels in + cardiomyocytes mitoplasts, confirming the presence of this K channel (mitoBKCa); however, its physiological functions have not yet fully understood. This work is the first one to examine the interactome of mitoBKCa channels in adult heart (isolated cardiomyocytes and whole ventricle). We used a directed proteomic approach aided by co-immunoprecipitation with BKCa antibodies and pull-down with recombinant DEC sequences. ii As a result, we identified an extensive network of the mitoBKCa, channel and overall, a total of 1079 different proteins were identified as partners of the BKCa channel in cardiomyocytes and left ventricle, including 151 mitochondrial proteins. Two putative protein partners were selected to validate and further examine the associations with BKCa channels: i) the Tom22 from the mitochondrial import system, and ii) the adenine nucleotide translocator (ANT), which is linked to oxidative phosphorylation and the regulation of mPTP. We first demonstrated the interactions of mitoBKCa with Tom22 through reciprocal co- immunoprecipitation (CO-IP), and then further confirmed that both Tom22 and BKCa were targeted into mitochondria through cell fractionation. This result raises the possibility of the functional interaction between the two proteins in mitochondria, supporting a potential import mechanism of BKCa channel through Tom22. Additionally, we identified the transmembrane domain of BKCa channel (1-711) as the main interacting regions with Tom22 through CO-IP. Next, we verified and studied the interactions between mitoBKCa and ANT with similar approaches. As a result, mitoBKCa was able to co-immunoprecipitate ANT, and the presence of DEC sequence in BKCa-DEC enhanced the ability of ANT to associate with BKCa by ~30%. More importantly, this interaction has a very high likelihood to happen in the mitochondria, as supported by the cell fractionation experiment that the majority of ANT and a considerate amount of mitoBKCa were targeted into mitochondria. This finding indicates a molecular link between mitoBKCa and mPTP, as ANT acts like an important regulatory component of it. Furthermore, the transmembrane domain of BKCa (1-343) can also contribute to the molecular interaction between BKCa channel and ANT. iii The dissertation of Jin Zhang is approved. Enrico Stefani Karla Marie Koehler Samson A Chow Jing Huang Riccardo Olcese, Committee CO-Chair Ligia G Toro De Stefani, Committee CO-Chair University of California, Los Angeles 2016 iv TABLE OF CONTENTS Abstract of the Dissertation ii List of Figures and tables vi Vita xi Chapter 1: Introduction 1 Chapter 2: Hypothesis 22 Chapter 3: Overview 24 Chapter 4: Material and Method 28 Chapter 5: Cardiac BKCa mitochondrial interactome: physiological relevance 37 Chapter 6: Tom22, a potential mitochondrial import mechanism for BKCa channel 52 Chapter 7: BKCa interaction with ANT 61 Chapter 8: Discussion and Conclusion 69 Chapter 9: Reference 77 v LISTS OF FIGURES AND TABLES Chapter 1 Figure 1. Topology of BKCa channel 4 Figure 2. The TOM complex – Common Entry for Distinct Biogenesis Pathways 13 Figure 3. TIM23/PAM- presequence translocase 15 Figure 4. TIM22- Carrier Protein Translocase 17 Figure 5. The topology of ADP/ATP carrier (ANT) 19 Figure 6. Proposed MPTP complex architecture 21 Chapter 5 Figure 7. MitoBKCa is associated with 13 distinctive mitochondrial functions in the heart 40 Table 1. BKCa channel-interacting mitochondrial proteins identified by LC/MS/MS 41 Table 2. BKCa channel-interacting proteins from Endoplasmic Reticulum, Golgi and Nucleus identified by LC/MS/MS 46 Chapter 6 Figure 8. mitoBKCa interacts with Tom22 in HEK293T cells 54 Figure 9. Both mitoBKCa and Tom22 are imported into mitochondria in HEK293T cells 57 Figure 10. BKCa channel transmembrane region interacts with Tom22 60 vi Chapter 7 Figure 11. BKCa interacts with ANT in HEK293T cells 63 Figure 12. BKCa and ANT are imported into mitochondria of HEK293T cells 65 Figure 13. Molecular determinant of BKCa channel interaction with ANT 68 Chapter 8 Figure 14. Scheme of potential mitoBKCa import route and interactions in mitochondria 76 vii ACKNOWLEDGEMENTS This research was partially supported by grants from the NIH grant HL107418 (LT, ES, RO). Chapters 2-8 are a version of the following manuscript: Jin Zhang, Min Li, Zhu Zhang, Ronhui Zhu, Riccardo Olcese, Enrico Stefani and Ligia Toro. (2016). The mitochondrial BKCa channel cardiac subproteome reveals BKCa association with the mitochondrial import receptor subunit Tom22, and the adenine nucleotide translocator, ANT (Submitted to Mitochondrion) To Dr. Ligia Toro, I thank you from the bottom of my heart for opening your lab doors to me. It is an honor to have you as my advisor. Your unconditional support has been invaluable in shaping my personal and professional development. Your unbridled passion for science, patience, immerse knowledge, and your encouragement for my scientific and creative independence deeply impressed me and have always been paramount in motivating me through my graduate career. I could not have imagined having a better advisor and mentor for my Ph.D. study. To Dr. Enrico Stefani, I thank you for always being there for me as an additional mentor. I always enjoyed our mind stimulating discussions, and your wealth of scientific knowledge and experiences has helped me enormously along my Ph.D. study. Nevertheless, your receptiveness to new ideas and cutting edge technologies and wholehearted passion for life has always been a limitless source of inspiration. viii To Dr. Riccardo Olcese, I give you my sincerest gratitude for welcoming me and supporting me as a member of your lab. You are a mentor and also a friend for me. Thank you for all the understanding and lessons you have taught me, which does not only benefit my professional career but also shape my personal growth. I am very grateful to you for holding me to a high research standard and enforcing strict validations for each research result. To Dr. Carla Koehler, I thank you for opening your lab door to me and skills needed to continue my Ph.D. research. Your insightful comments and constructive criticisms at different stages of my research were thought-provoking and they helped me focus my ideas. To Dr. Sam Chow, I thank you for always be there to listen and give advice during my Ph.D. study. I am deeply grateful to you for the long discussions that helped me sort out my research and personal life as well. Thank you for being my teacher, guide, mentor and friend. To Dr. Jin Huang, I thank you for your encouragement and practical advice. I am also thankful to you for reading my reports; commenting on my views and helping me understand and enrich my ideas. To the past and present members of the Toro Lab and Stefani Lab, your endless support, discussion, and critiques, your unabashed encouragement, friendship, and smiles, I thank you for providing color to what could have been a drab graduate experience. ix To my friends and classmates, I am eternally grateful to you all for adding endless, hysterical laughter through even the most difficult of personal and professional challenges. If the value of a person is defined by the quality of the company that he keeps, then I am incomparably rich beyond my wildest dreams. Last but not least, to my family, I owe you my everything for believing in me and offering your unconditional support, love, and encouragement through this endeavor of graduate school. For without your collective love and trust, I would not be half the woman that I am today. x VITA Education Ph.D. Candidate: Pharmacology (GPA: 3.7/4 --- UCLA expected June 2016 Advisor: Ligia Toro and Enrico Stefani Dissertation: BK Channel Subproteome Reveals its Interaction with ADP/ATP Translocase and Hints on its Mitochondrial Import Mechanism M.S.: Pharmacology (GPA: 3.9/4) --- Rush University, Chicago, IL July 2012 Advisor: Sanda Predescu Dissertation: Downregulation of Intersectin-1s in human lung cancer may contribute to tumorigenesis M.D.: Clinical Medicine --- Nantong Medical School, Nantong, China July 2010 • Achieved merit-based scholarship for 5 consecutive years Peer-Reviewed Publications Zhang J, Li M, Zhang Z, Zhu R, Olcese R, Stefani E, Toro L. “The mitochondrial BKCa channel cardiac subproteome reveals BKCa association with the mitochondrial import receptor subunit Tom22, and the adenine nucleotide translocator, ANT.” Submitted to Mitochondrion. Jeganathan N, Predescu D, Zhang J, Sha F, Bardita C, Patel M, Wood S, Borgia J, Balk R, Predescu S. Rac1-Mediated Cytoskeleton Rearrangements Induced by Intersectin-1s Deficiency 1. Submitted to Molecular Cancer. Wu Y, Wu X, Lu R, Zhang J, Toro L, Stefani E. “Resonant Scanning with Large Field of View Reduces Photo bleaching and Enhances Fluorescence Yield in STED Microscopy.” Sci Rep. 2015 Oct 1;5:14766. Balderas E, Zhang J, Stefani E, Toro L. “Mitochondrial BK channel.” Front Physiol. 2015 Mar 31;6:104. Abstracts and Talks Jin Zhang, Zhu Zhang, Enrico Stefani, and Ligia Toro.