TRPC6 mutations associated with focal segmental glomerulosclerosis cause constitutive activation of NFAT-dependent transcription.
Mutations in the canonical transient receptor potential channel TRPC6 lead to an autosomal dominant form of human kidney disease characterized histologically by focal and segmental glomerulosclerosis. Several of these mutations enhance the amplitude and duration of the channel current. However, the effect of these mutations on the downstream target of TRPC6, the nuclear factor of activated T cell (NFAT) transcription factors, has not been previously examined. Here we demonstrate that all three TRPC6 mutations previously shown to enhance channel activity lead to enhanced basal NFAT-mediated transcription in several cell lines, including cultured podocytes. These effects are dependent on channel activity and are dominant when mutants are coexpressed with wild-type TRPC6. While TRPC6 mutants do not demonstrate an increase in basal channel currents, a subset of cells expressing the R895C and E897K mutants have elevated basal calcium levels as measured by Fura-2 imaging. Activation of NFAT by TRPC6 mutants is blocked by inhibitors of calcineurin, calmodulin-dependent kinase II, and phosphatidylinositol 3-kinase. PP2 partially inhibits NFAT activation by mutant TRPC6 independently of Src, Yes, or Fyn. Differences in channel glycosylation and surface expression do not explain the ability of mutants to enhance NFAT activation. Taken together, these results identify the activation of the calcineurin-NFAT pathway as a potential mediator of focal segmental glomerulosclerosis.
Schlöndorff
Johannes
J
Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
Del Camino
Donato
D
Carrasquillo
Robert
R
Lacey
Vanessa
V
Pollak
Martin R
MR
D001596:Benzylamines / Q000494:pharmacology; D019703:Calcineurin / Q000378:metabolism; D065095:Calcineurin Inhibitors; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D016572:Cyclosporine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005923:Glomerulosclerosis, Focal Segmental / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008564:Membrane Potentials; D009025:Morpholines / Q000494:pharmacology; D009154:Mutation; D050778:NFATC Transcription Factors / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D050199:Podocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D043643:Receptor, Muscarinic M1 / Q000378:metabolism; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D000074287:TRPC6 Cation Channel; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D000096926:Benzenesulfonamides
D001596:Benzylamines; D019703:Calcineurin; D065095:Calcineurin Inhibitors; D002118:Calcium; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002867:Chromones; D016572:Cyclosporine; D004791:Enzyme Inhibitors; D009025:Morpholines; D050778:NFATC Transcription Factors; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger; D043643:Receptor, Muscarinic M1; D013449:Sulfonamides; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; C072105:KN 93; D000096926:Benzenesulfonamides; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one