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Phospholipase C epsilon (PLC?) induced TRPC6 activation: a common but redundant mechanism in primary podocytes.

ABSTRACT: In eukaryotic cells, activation of phospholipase C (PLC)-coupled membrane receptors by hormones leads to an increase in the intracellular Ca(2+) concentration [Ca(2+) ]i . Catalytic activity of PLCs results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) which opens DAG-sensitive classical transient receptor channels 3, 6, and 7 (TRPC3/6/7), initiating Ca(2+) influx from the extracellular space. Patients with focal segmental glomerulosclerosis (FSGS) express gain-of-function mutants of TRPC6, while others carry loss-of-function mutants of PLC?, raising the intriguing possibility that both proteins interact and might work in the same signalling pathway. While TRPC6 activation by PLC? and PLC? isozymes was extensively studied, the role of PLC? in TRPC6 activation remains elusive. TRPC6 was co-immunoprecipitated with PLC? in a heterologous overexpression system in HEK293 cells as well as in freshly isolated murine podocytes. Receptor-operated TRPC6 currents in HEK293 cells expressing TRPC6 were reduced by a specific PLC? siRNA and by a PLC? loss-of-function mutant isolated from a patient with FSGS. PLC?-induced TRPC6 activation was also identified in murine embryonic fibroblasts (MEFs) lacking G?q/11 proteins. Further analysis of the signal transduction pathway revealed a G?12/13 Rho-GEF activation which induced Rho-mediated PLC? stimulation. Therefore, we identified a new pathway for TRPC6 activation by PLC?. PLC?-/- podocytes however, were undistinguishable from WT podocytes in their angiotensin II-induced formation of actin stress fibers and their GTP?S-induced TRPC6 activation, pointing to a redundant role of PLC?-mediated TRPC6 activation at least in podocytes.

SUBMITTER: Kalwa H

PROVIDER: S-EPMC4811033 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Phospholipase C epsilon (PLCε) induced TRPC6 activation: a common but redundant mechanism in primary podocytes.

Kalwa Hermann H Storch Ursula U Demleitner Jana J Fiedler Susanne S Mayer Tim T Kannler Martina M Fahlbusch Meike M Barth Holger H Smrcka Alan A Hildebrandt Friedhelm F Gudermann Thomas T Dietrich Alexander A

Journal of cellular physiology 20150601 6

In eukaryotic cells, activation of phospholipase C (PLC)-coupled membrane receptors by hormones leads to an increase in the intracellular Ca(2+) concentration [Ca(2+) ]i . Catalytic activity of PLCs results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) which opens DAG-sensitive classical transient receptor channels 3, 6, and 7 (TRPC3/6/7), initiating Ca(2+) influx from the extracellular space. Patients with foca ...[more]

PMID: 25521631

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Project description:Transient receptor potential classical (or canonical) (TRPC)3, TRPC6, and TRPC7 are a subfamily of TRPC channels activated by diacylglycerol (DAG) produced through the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) by phospholipase C (PLC). PI(4,5)P2 depletion by a heterologously expressed phosphatase inhibits TRPC3, TRPC6, and TRPC7 activity independently of DAG; however, the physiological role of PI(4,5)P2 reduction on channel activity remains unclear. We used Förster resonance energy transfer (FRET) to measure PI(4,5)P2 or DAG dynamics concurrently with TRPC6 or TRPC7 currents after agonist stimulation of receptors that couple to Gq and thereby activate PLC. Measurements made at different levels of receptor activation revealed a correlation between the kinetics of PI(4,5)P2 reduction and those of receptor-operated TRPC6 and TRPC7 current activation and inactivation. In contrast, DAG production correlated with channel activation but not inactivation; moreover, the time course of channel inactivation was unchanged in protein kinase C-insensitive mutants. These results suggest that inactivation of receptor-operated TRPC currents is primarily mediated by the dissociation of PI(4,5)P2. We determined the functional dissociation constant of PI(4,5)P2 to TRPC channels using FRET of the PLC? Pleckstrin homology domain (PHd), which binds PI(4,5)P2, and used this constant to fit our experimental data to a model in which channel gating is controlled by PI(4,5)P2 and DAG. This model predicted similar FRET dynamics of the PHd to measured FRET in either human embryonic kidney cells or smooth muscle cells, whereas a model lacking PI(4,5)P2 regulation failed to reproduce the experimental data, confirming the inhibitory role of PI(4,5)P2 depletion on TRPC currents. Our model also explains various PLC-dependent characteristics of channel activity, including limitation of maximum open probability, shortening of the peak time, and the bell-shaped response of total current. In conclusion, our studies demonstrate a fundamental role for PI(4,5)P2 in regulating TRPC6 and TRPC7 activity triggered by PLC-coupled receptor stimulation.

Dataset Information

Phospholipase C epsilon (PLC?) induced TRPC6 activation: a common but redundant mechanism in primary podocytes.

Publications

Phospholipase C epsilon (PLCε) induced TRPC6 activation: a common but redundant mechanism in primary podocytes.

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