Protein kinase C-dependent phosphorylation of transient receptor potential canonical 6 (TRPC6) on serine 448 causes channel inhibition.
TRPC6 is a cation channel in the plasma membrane that plays a role in Ca(2+) entry following the stimulation of a G(q)-protein coupled or tyrosine kinase receptor. A dysregulation of TRPC6 activity causes abnormal proliferation of smooth muscle cells and glomerulosclerosis. In the present study, we investigated the regulation of TRPC6 activity by protein kinase C (PKC). We showed that inhibiting PKC with GF1 or activating it with phorbol 12-myristate 13-acetate potentiated and inhibited agonist-induced Ca(2+) entry, respectively, into cells expressing TRPC6. Similar results were obtained when TRPC6 was directly activated with 1-oleyl-2-acetyl-sn-glycerol. Activation of the cells with carbachol increased the phosphorylation of TRPC6, an effect that was prevented by the inhibition of PKC. The target residue of PKC was identified by an alanine screen of all canonical PKC sites on TRPC6. Unexpectedly, all the mutants, including TRPC6(S768A) (a residue previously proposed to be a target for PKC), displayed PKC-dependent inhibition of channel activity. Phosphorylation prediction software suggested that Ser(448), in a non-canonical PKC consensus sequence, was a potential target for PKCδ. Ba(2+) and Ca(2+) entry experiments revealed that GF1 did not potentiate TRPC6(S448A) activity. Moreover, activation of PKC did not enhance the phosphorylation state of TRPC6(S448A). Using A7r5 vascular smooth muscle cells, which endogenously express TRPC6, we observed that a novel PKC isoform is involved in the inhibition of the vasopressin-induced Ca(2+) entry. Furthermore, knocking down PKCδ in A7r5 cells potentiated vasopressin-induced Ca(2+) entry. In summary, we provide evidence that PKCδ exerts a negative feedback effect on TRPC6 through the phosphorylation of Ser(448).
Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada.
Simon M
SM
Bousquet
Michaël
M
Monet
Guylain
G
Boulay
D019943:Amino Acid Substitution; D002217:Carbachol / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D005923:Glomerulosclerosis, Focal Segmental / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008916:Miotics / Q000494:pharmacology; D020125:Mutation, Missense; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D051745:Protein Kinase C-delta / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics / Q000378:metabolism; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*; D000074287:TRPC6 Cation Channel; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology
D002273:Carcinogens; D007527:Isoenzymes; D008916:Miotics; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; D014662:Vasoconstrictor Agents; D014667:Vasopressins; D012694:Serine; D002217:Carbachol; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate