Gating Mechanisms of The Canonical Trp Channel Isoform Trpc4

<p>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, G_q/11 and G_i/o, are co-stimulated, making it a coincidence detector of G_q/11- and G_i/o-coupled receptor activation. Using HEK293 cells co-expressing mouse TRPC4β and selected G protein-coupled receptors, I observed that coincident stimulation of G_i/oproteins and PLCδ1 (and not G_q/11-PLCβ) is necessary and sufficient for TRPC4 activation. In cells co-expressing TRPC4 and G_i/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 G_i/ostimulation. 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 G_i/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 G_i/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) G_i/oand 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 G_i/ostimulation and PLC signaling but also, likely, other pathophysiological conditions, such as metabolic changes and hypoxic stress.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation (PhD)

Year dc:date.available

2015

Author dc:creator

• Thakur, Dhananjay P

Contributors dc:contributor

• Michael X. Zhu
• Carmen W. Dessauer
• Richard B. Clark

• TRP Channels
• G proteins
• TRPC4
• Phospholipase C
• calcium
• protons
• calmodulin
• PIP2
• Biophysics
• Cellular and Molecular Physiology
• Integrative Biology
• Medicine and Health Sciences

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/621

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-1656