NOX2 interacts with podocyte TRPC6 channels and contributes to their activation by diacylglycerol: essential role of podocin in formation of this complex

Abstract

Canonical transient receptor potential-6 (TRPC6) channels have been implicated in the pathophysiology of glomerular diseases. TRPC6 channels are typically activated by diacylglycerol (DAG) during PLC-dependent transduction cascades. TRPC6 channels can also be activated by reactive oxygen species (ROS). We previously showed that podocin is required for DAG analogs to produce robust activation of TRPC6 channels in podocytes. Here we show that endogenous TRPC6 channels in immortalized podocytes reciprocally coimmunoprecipitate with the catalytic subunit of the NADPH oxidase NOX2 (gp91(phox)). The NOX2-TRPC6 interaction was not detected in cells stably expressing a short hairpin RNA targeting podocin, although NOX2 and TRPC6 were present at normal levels. Application of a membrane-permeable DAG analog [1-oleoyl-2-acetyl-sn-glycerol (OAG)] increased generation of ROS in podocytes, but this effect was not detected in podocin knockdown cells. OAG also increased steady-state surface expression of the NOX2 regulatory subunit p47(phox). In whole cell recordings, TRPC6 activation by OAG was reduced in podocytes pretreated with the NOX2 inhibitor apocynin, by the pan-NOX inhibitor diphenylene iodonium, and by tempol, a ROS quencher. Cholesterol depletion and disruption of lipid rafts by methyl-β-cyclodextrin reduced activation of podocyte TRPC6 channels by OAG and also eliminated the NOX2-TRPC6 interaction as assessed by coimmunoprecipitation. These data suggest that active NOX2 assembles with TRPC6 at podocin-organized sterol-rich raft domains and becomes catalytically active in response to DAG. The localized production of ROS contributes to TRPC6 activation by chemical stimuli such as DAG. Podocin appears to be necessary for assembly of the NOX2-TRPC6 complex in lipid rafts.

Calcium-Sensing Receptor Stimulation in Cultured Glomerular Podocytes Induces TRPC6-Dependent Calcium Entry and RhoA Activation

“…It remains unclear how the TRPC6 signal and the CaSR signal of podocytes are integrated for the CaSR stimulation. It has been demonstrated that, in podocytes, TRPC6 is localized in the lipid raft domain at plasma membranes [40] and TRPC6 is in a molecular complex with RhoA [16]. In some cell types, CaSR is distributed in a caveolin-rich plasma membrane domain [1]; however, this distribution does not seem to occur in podocytes [9].…”

Phosphoproteomic Analysis Reveals Regulatory Mechanisms at the Kidney Filtration Barrier

“…The PHB domain phosphorylation site discovered in this study may prove to be a modulator of the oligomerization status of podocin at the slit diaphragm in vivo, thereby altering resistance and mechanics in the podocyte in response to various intracellular and extracellular stimuli affecting AGC kinases. 56,57 In fact, aPKC, a kinase with relevance for podocyte biology, 11,40 can induce phosphorylation of this site when coexpressed with podocin in human embryonic kidney (HEK293T) cells (Supplemental Figure 5).…”

Canonical Transient Receptor Potential 6 Channel: A New Target of Reactive Oxygen Species in Renal Physiology and Pathology

“…However, Nox4 might not be the only NADPH oxidase to regulate TRPC6 in podocytes. In a recent study, Nox2 was found to physically interact with TRPC6 channels in podocytes, and the complexes contributed to TRPC6 activation by diacylglycerol, a product generated in G protein-coupled and receptor tyrosine kinase receptor signaling cascades (107).…”