Abstract
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(3+). Stretch activation of podocyte TRPC6 persisted in the presence of inhibitors of phospholipase C (U-73122) and phospholipase A2 (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(2+) 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.
Keywords:
TRPC6; glomerular filtration; mechanosensitive; podocyte; slit diaphragm.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Calcium Channel Blockers / pharmacology
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Cell Line
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Cell Membrane / metabolism
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Chlorobenzoates / pharmacology
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Cinnamates / pharmacology
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Cytochalasin D / pharmacology
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Diglycerides / pharmacology
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Estrenes / pharmacology
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Guanosine Diphosphate / analogs & derivatives
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Guanosine Diphosphate / pharmacology
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HEK293 Cells
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Humans
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Imidazoles / pharmacology
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Intercellular Signaling Peptides and Proteins
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Intracellular Signaling Peptides and Proteins / genetics
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Intracellular Signaling Peptides and Proteins / metabolism*
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Ion Channel Gating
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Kidney Glomerulus / metabolism*
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Membrane Proteins / genetics
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Membrane Proteins / metabolism*
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Mice
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Mutation
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Nephrotic Syndrome
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Peptides / pharmacology
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Phosphodiesterase Inhibitors / pharmacology
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Phospholipase A2 Inhibitors
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Podocytes / metabolism*
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Pyrrolidinones / pharmacology
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RNA Interference
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RNA, Small Interfering
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Rats
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Spider Venoms / pharmacology
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TRPC Cation Channels / genetics
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TRPC Cation Channels / metabolism*
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TRPC6 Cation Channel
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Thionucleotides / pharmacology
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Type C Phospholipases / antagonists & inhibitors
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ortho-Aminobenzoates / pharmacology
Substances
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Calcium Channel Blockers
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Chlorobenzoates
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Cinnamates
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Diglycerides
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Estrenes
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Imidazoles
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Intercellular Signaling Peptides and Proteins
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Intracellular Signaling Peptides and Proteins
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MTx4 protein, Grammostola spatulata
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Membrane Proteins
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NPHS2 protein
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Peptides
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Phosphodiesterase Inhibitors
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Phospholipase A2 Inhibitors
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Pyrrolidinones
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RNA, Small Interfering
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Spider Venoms
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TRPC Cation Channels
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TRPC6 Cation Channel
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Thionucleotides
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Trpc6 protein, mouse
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ortho-Aminobenzoates
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1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione
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Guanosine Diphosphate
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Cytochalasin D
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guanosine 5'-O-(2-thiodiphosphate)
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2-(4-amylcinnamoyl)amino-4-chlorobenzoic acid
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Type C Phospholipases
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1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole