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Gating Mechanisms of the Canonical Trp Channel Isoform Trpc4 The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 8-2015 GATING MECHANISMS OF THE CANONICAL TRP CHANNEL ISOFORM TRPC4 Dhananjay P. Thakur Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Biophysics Commons, Cellular and Molecular Physiology Commons, Integrative Biology Commons, and the Medicine and Health Sciences Commons Recommended Citation Thakur, Dhananjay P., "GATING MECHANISMS OF THE CANONICAL TRP CHANNEL ISOFORM TRPC4" (2015). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 621. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/621 This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. GATING MECHANISMS OF THE CANONICAL TRP CHANNEL ISOFORM TRPC4 By Dhananjay Thakur M.Sc., M.S. APPROVED: ______________________________ Michael X. Zhu, Ph.D. Advisory Professor ______________________________ Carmen W. Dessauer, Ph.D. ______________________________ Richard B. Clark, Ph.D. ______________________________ Roger G. O’Neil, Ph.D. ______________________________ Neal M. Waxham, Ph.D. APPROVED: ____________________________ Dean, The University of Texas Graduate School of Biomedical Sciences at Houston GATING MECHANISMS OF THE CANONICAL TRP CHANNEL ISOFORM TRPC4 A DISSERTATION Presented to the Faculty of The University of Texas Health Science Center at Houston and The University of Texas MD Anderson Cancer Center Graduate School of Biomedical Sciences in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY by Dhananjay Thakur M.S., M.Sc. Houston, Texas August, 2015 Dedication This dissertation is dedicated to my family: Juilie Thakur, Arundhati Thakur and Pramod Thakur Their endless love and support have made everything possible iii Acknowledgements This wonderfully convoluted trajectory that my journey in science has followed so far has been perceptibly and imperceptibly guided by several people. Friends, family, mentors and colleagues - and the distinctions blurred very often - have made me who I am. To Dr. Michael X. Zhu, I owe the deepest gratitude, for guiding me with great patience through the strangest and roughest roads that this project took. Dr. Zhu taught me to make that critical distinction between testable and non-testable hypotheses, and the importance of clearly defining both negative and positive controls for all tests, which very often, turned out not to be a trivial exercise. These lessons have shaped the way I think about every scientific problem now. Regularly, the tendency towards reductionism clashed with the demands for a comprehensive hypothesis. Without Dr. Zhu’s guidance, I would not have been able to achieve that dynamic balance between these two states that is necessary for the resolution of complex biological problems. I would also like to especially thank the members of my advisory and candidacy examination committees, Dr. Carmen Dessauer, Dr. Richard Clark, Dr. Roger O’Neil, Dr. Neal M. Waxham, Dr. Hongzhen Hu, Dr. Michael Beirlein, Dr. Alemayehu Gorfe and Dr. Raymond Grill. Their guidance – in both technical aspects and the scientific process – has been invaluable. Their early lessons in maintaining an eye for detail while not losing the forest for the trees, their generous help with insights stemming from their extensive knowledge, as well as with materiel – protocols, reagents and instruments have been invaluable for my work and development as a scientist. Dr. Jinbin Tian and Dr. Jaepyo Jeon were closely involved in several steps in this work. I am deeply thankful for their guidance and collaboration iv and I am looking forward to many more equally enjoyable and fruitful interactions. The American Heart Association made the last two years of research possible through their generous funding. I am very grateful to the reviewers of my grant applications who (anonymously) taught me how to chisel out a good grant application. I also thank the University Of Texas Graduate School Of Biomedical Sciences, and especially - Dr. Priscilla Saunders, who established and supported the Investing in Student Futures Award, which gave strong and timely encouragement. Students, staff and faculty in the Department of Integrative Biology were extremely generous with their reagents, advice and friendship. Without the deepest friendships that evolved over the last several years, life/science would have been impossible. For those invaluable hours filled with laughter, food, bickering and the mutual indulgence in each other’s existential crises, I would like to thank David, Mykola, Renu, Kelsey, Caitlin, Karolina, Jennifer, Tanya, Yukti, Yingmin, Alexis, Nabila, Faiza, Aparna, Radhika, Rasika, Ching-On, Kevin, Sujay, Sourabh, Deepti, Franco, Susanna, Dries, and Joe. The blame for my transition from physics to biology lands squarely on the shoulders of Ambrose Bierce, who with one flick of a pen (“Brain, n. An apparatus with which we think we think.” The Devil's Dictionary, 1911) planted a seed in my head – that any comprehension of physics was meaningless without its application in biology – which set me off on this journey that has taken me to the TRP channel world. I would also like to thank my mentors in Physics, Dr. Raka Dabhade and Dr. Pandit Vidyasagar at the University of Pune, and Mr. Preesty Thomas at Tata Motors Ltd. Their guidance and encouragement helped me through every important step and transition in my early career. Lastly, I would like to v thank my family again for their unrelenting love and support. Their patience and understanding was critical for this work. vi AGATING MECHANISMS OF THE CANONICAL TRP CHANNEL ISOFORM TRPC4 Dhananjay Thakur, M.Sc., M.S. Advisory Professor: Michael X. Zhu, Ph.D. Non-selective cation channels formed by Transient Receptor Potential Canonical (TRPC) proteins play important roles in regulatory and pathophysiological processes. These channels are known to be activated downstream from phospholipase C (PLC) signaling. However, the mechanism by which the PLC pathway activates TRPC4/C5 remains unclear. Uniquely, TRPC4 is maximally activated only when two separate G protein pathways, Gq/11 and Gi/o, are co- stimulated, making it a coincidence detector of Gq/11- and Gi/o -coupled receptor activation. Using HEK293 cells co-expressing mouse TRPC4β and selected G protein-coupled receptors, I observed that coincident stimulation of Gi/o proteins and PLCδ1 (and not Gq/11-PLCβ) is necessary and sufficient for TRPC4 activation. In cells co-expressing TRPC4 and Gi/o -coupled µ opioid receptor, µ agonist DAMGO elicited currents in a biphasic manner, with an initial slow phase preceding a second, rapidly developing phase. While the currents were dependent on intracellular 2+ 2+ Ca and phosphatidylinositol 4,5-bisphosphate (PIP2), both Ca and PIP2 also exhibited inhibitory effects. Depleting PIP2 abolished the biphasic kinetics and facilitated channel activation by weak Gi/o stimulation. TRPC4 activation was inhibited by knocking down PLCδ1 and almost entirely eliminated by a dominant-negative PLCδ1 mutant or a constitutively active RhoA mutant. These results demonstrate an integrative mechanism of TRPC4 for detection of coincident Gi/o, vii Ca2+, and PLC signaling, wherein TRPC4 and PLCδ1 are functionally coupled. This mechanism is not shared with the closely related TRPC5, implicating unique roles of TRPC4 in signal integration. Intracellular acidification further facilitated channel activation in a bimodal manner, with moderate acidification accelerating the Gi/o-TRPC4 response, while strong acidification was inhibitory. This regulation by H+ is functionally and mechanistically distinct from that by Ca2+, which involves not only Ca2+-dependent PLCδ1 activation but also a direct modulation by Ca2+- calmodulin. Thus, our findings indicate that TRPC4 is maximally activated when (A) Gi/o and 2+ + PLCδ1 are stimulated and (B) intracellular concentrations of PIP2, Ca and H fall within specific ranges. These findings indicate that TRPC4 serves as a unique coincidence sensor of intracellular environmental changes that accompany not only Gi/o stimulation and PLC signaling but also, likely, other pathophysiological conditions, such as metabolic changes and hypoxic stress. viii Table Of Contents Approval Sheet………………………………………...………………..………………………...i Title Page……………………………………………………………………………………........ii Dedication ....................................................................................................................... ………..iii Acknowledgements ....................................................................................................................... iv Abstract ...................................................................................................................................... vii Abbreviations .............................................................................................................................
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