Opposing effects of podocin on the gating of podocyte TRPC6 channels evoked by membrane stretch or diacylglycerol

Abstract

Anderson M, Kim EY, Hagmann H, Benzing T, Dryer SE. Opposing effects of podocin on the gating of podocyte TRPC6 channels evoked by membrane stretch or diacylglycerol. Am J Physiol Cell Physiol 305: C276 -C289, 2013. First published May 8, 2013 doi:10.1152/ajpcell.00095.2013.-Gain-of-function mutations in the transient receptor potential (TRP) cation channel subfamily C member 6 (TRPC6) gene and mutations in the NPHS2 gene encoding podocin result in nephrotic syndromes. The purpose of this study was to determine the functional significance of biochemical interactions between these proteins. We observed that gating of TRPC6 channels in podocytes is markedly mechanosensitive and can be activated by hyposmotic stretch or indentation of the plasma membrane. Stretch activation of cationic currents was blocked by small interfering RNA knockdown of TRPC6, as well as by SKF-96365 or micromolar La 3ϩ . Stretch activation of podocyte TRPC6 persisted in the presence of inhibitors of phospholipase C (U-73122) and phospholipase A 2 (ONO-RS-082). Robust stretch responses also persisted when recording electrodes contained guanosine 5=-O-(2-thiodiphosphate) at concentrations that completely suppressed responses to ANG II. Stretch responses were enhanced by cytochalasin D but were abolished by the peptide GsMTx4, suggesting that forces are transmitted to the channels through the plasma membrane. Podocin and TRPC6 interact at their respective COOH termini. Knockdown of podocin markedly increased stretch-evoked activation of TRPC6 but nearly abolished TRPC6 activation evoked by a diacylglycerol analog. These data suggest that podocin acts as a switch to determine the preferred mode of TRPC6 activation. They also suggest that podocin deficiencies will result in Ca 2ϩ overload in foot processes, as with gain-of-function mutations in the TRPC6 gene. Finally, they suggest that mechanical activation of TRP family channels and the preferred mode of TRP channel activation may depend on whether members of the stomatin/ prohibitin family of hairpin loop proteins are present.podocyte; glomerular filtration; TRPC6; slit diaphragm; podocyte; mechanosensitive PODOCYTES ARE HIGHLY DIFFERENTIATED multipolar cells that cover the external surface of glomerular capillaries and form an essential component of the kidney ultrafiltration apparatus (46). Primary processes extend from the podocyte cell body and ramify extensively as they wrap around the glomerular capillary. Specialized structures known as foot processes normally emanate at regular spatial intervals from the major processes of podocytes and attach to the surface of the glomerular basement membrane. Connections between interdigitating foot processes, known as slit diaphragms (SDs), are formed by trans-interactions of specialized transmembrane adhesion molecules (26). The interacting extracellular domains of SD proteins form a porous matrix (65) that provides a pathway for convective flow and diffusion of water and small solutes into Bowman's space (7). SDs may have multiple roles, as the...

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).…”

A Disease-causing Mutation Illuminates the Protein Membrane Topology of the Kidney-expressed Prohibitin Homology (PHB) Domain Protein Podocin

“…Podocin P3 L did interact with members of this complex, like nephrin, CD2AP, and TRPC6, indicating that the P3 L mutation per se does not interfere with the interaction. The interaction with nephrin, CD2AP, and TRPC6 has been mapped previously to the C terminus of podocin (10,37). It is likely that the interaction with the glycosylated form is occurring only in the cell lysate.…”

RETRACTION: Angiotensin II and Canonical Transient Receptor Potential-6 Activation Stimulate Release of a Signal Transducer and Activator of Transcription 3–Activating Factor from Mouse Podocytes

“…In some experiments, this was done in the presence of inhibitors or after small-interfering RNA (siRNA) knockdown. For this, both a specific siRNA directed against TRPC6 and nontargeted siRNAs for use in control experiments were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and transfected into podocytes using Lipofectamine (Invitrogen, Grand Island, NY) in serum-reduced medium according to the manufacturer's directions and as previously described elsewhere (Kim et al, 2011;Anderson et al, 2013;Roshanravan and Dryer, 2014). Analysis was performed 48 hours after transfection with siRNA targeting TRPC6 or control siRNA.…”

“…TRPC6 channels expressed in podocyte foot processes occur in a complex with nephrin, podocin, and other slit diaphragm molecules (Reiser et al, 2005;Dryer and Reiser, 2010). However, TRPC6 channels are also expressed in other parts of the cell, including major processes and the cell body, and the gating of TRPC6 in those locations may be regulated somewhat differently (Dryer and Reiser, 2010;Anderson et al, 2013).…”