Phospholipase C Epsilon (PLCε) 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 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.

PLCε1 regulates SDF-1α–induced lymphocyte adhesion and migration to sites of inflammation

“…PLCe1 was also shown to modulate adrenergic receptor-dependent cardiac contraction and to inhibit cardiac hypertrophy (22,23). Patients with focal segmental glomerulosclerosis have been found to have loss-of-function mutations in PLCe1, and PLCe1 itself interacts with transient receptor potential channel 6 (24)(25)(26)(27). Even more intriguing is the fact that a genome-wide association study identified the susceptibility loci for dengue shock syndrome at PLCe1 (28).…”

Podocyte Purinergic P2X4 Channels Are Mechanotransducers That Mediate Cytoskeletal Disorganization

“…Enrichment of podocytes was .95% as assessed by cell morphology, staining with an antipodocin antibody (Sigma-Aldrich) and by expression of nephrin, synapotopodin, and Wilms tumor protein (WT1), which is described in detail elsewhere. 57 Cell membrane capacity was used as another selection criterion for podocytes. Cell membrane capacity was estimated to be 71.262.3 pF (n=362) for primary podocytes; thus, there was no significant difference to conditionally immortalized murine podocytes, which had cell membrane capacities of 63.463.3 pF (n=52).…”

Pharmacological inhibition of focal segmental glomerulosclerosis‐related, gain of function mutants of TRPC6 channels by semi‐synthetic derivatives of larixol

“…OAG‐induced current densities in PASMCs were about 20‐fold higher than in podocytes, which only reached about 1 pA·pF −1 . Interestingly, few studies utilizing freshly isolated podocytes instead of the immortalized cell lines confirmed these very small current amplitudes upon stimulation with angiotensin II (Eckel et al ., ; Kalwa et al ., ).…”

“…TRPC6 channels are expressed in a variety of tissues and cell types, including vascular smooth muscle cells, mesangial cells and podocytes. Although the presence of TRPC6 in podocytes has been demonstrated by several groups, there is considerable variability concerning the abundance of the channel protein and the size of functional responses to TRPC6 channel activators (Anderson et al ., ; Abkhezr et al ., ; Kalwa et al ., ). As dysregulation of TRPC6 channel expression in podocytes can occur in response to various stimuli, TRPC6 channels may also play a seminal role in secondary forms of FSGS.…”