In eukaryotic cells, activation of phospholipase C (PLC)-coupled membrane receptors by hormones leads to an increase in the intracellular Ca2+ concentration [Ca2+]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 Ca2+ 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 PLCε, raising the intriguing possibility that both proteins interact and might work in the same signalling pathway. While TRPC6 activation by PLCβ and PLCγ isozymes was extensively studied, the role of PLCε in TRPC6 activation remains elusive. TRPC6 was co-immunoprecipitated with PLCε 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 PLCε siRNA and by a PLCε loss-of-function mutant isolated from a patient with FSGS. PLCε-induced TRPC6 activation was also identified in murine embryonic fibroblasts (MEFs) lacking Gαq/11 proteins. Further analysis of the signal transduction pathway revealed a Gα12/13 Rho-GEF activation which induced Rho-mediated PLCε stimulation. Therefore, we identified a new pathway for TRPC6 activation by PLCε. PLCε-/- podocytes however, were undistinguishable from WT podocytes in their angiotensin II-induced formation of actin stress fibers and their GTPγS-induced TRPC6 activation, pointing to a redundant role of PLCε-mediated TRPC6 activation at least in podocytes.

Gain of function mutations of TRPC6 as well as loss of function mutations in PLC ε were identified in patients suffering from focal segmental glomerular sclerosis (FSGS), a disease displaying increasing proteinuria due to a defect in the glomerular filtration process of the kidney. Along these lines, we have previously shown that PLC ε physically interacts and activates TRPC6 via production of diacylglycerol (DAG) in kidney podocytes to increase the barrier function of the glomerular slit diaphragm. Moreover, down‐regulation of G‐protein α13‐subunits significantly reduced ATII‐mediated TRPC6 activation, whereas siRNAs directed against G protein αq‐subunits did not. By employing Gαq−/− murine embryonic fibroblasts, we were able to dissect Gα12/13‐PLC ε‐mediated from Gαq‐PLCβ‐induced TRPC6 activation using lysophosphatidic acid (LPA) receptor stimulation. Most interestingly, application of LPA resulted in a significantly larger increase in the intracellular Ca2+ concentration ([Ca2+]i) in Gαq−/− cells expressing TRPC6 in comparison to Gαq−/− control cells. Therefore, Gα12/13‐mediated activation of PLC ε via RhoA leading to TRPC6‐induced cation influx might be a general, but commonly‐ignored signal transduction pathway in many cell types.