Date of Graduation

8-2015

Document Type

Dissertation (PhD)

Program Affiliation

Cell and Regulatory Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Michael X. Zhu

Committee Member

Carmen W. Dessauer

Committee Member

Richard B. Clark

Committee Member

Roger G. O’Neil

Committee Member

Neal M. Waxham

Abstract

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 Ca2+ and phosphatidylinositol 4,5-bisphosphate (PIP2), both Ca2+ 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, 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 PLCδ1 are stimulated and (B) intracellular concentrations of PIP2, Ca2+ 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.

Keywords

TRP Channels, G proteins, TRPC4, Phospholipase C, calcium, protons, calmodulin, PIP2

Available for download on Saturday, December 31, 2016

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