Expression and role of TRPC proteins in human platelets: evidence that TRPC6 forms the store-independent calcium entry channel.
Store-operated Ca(++) entry (SOCE) is thought to comprise the major pathway for Ca(++) entry in platelets. Recently, a number of transient receptor potential (TRP) proteins, which have been divided into 3 groups (TRPC, TRPM, and TRPV), have been suggested as SOCE channels. We report the expression and function of TRPC proteins in human platelets. TRPC6 is found at high levels and TRPC1 at low levels. Using purified plasma (PM) and intracellular membranes (IM), TRPC6 is found in the PM, but TRPC1 is localized to the IM. Using Fura-2-loaded platelets, we report that, in line with TRPC6 expression, 1-oleoyl-2-acetyl-sn-glycerol (OAG) stimulated the entry of Ca(++) and Ba(2+) independently of protein kinase C. Thrombin also induced the entry of Ca(++) and Ba(2+), but thapsigargin, which depletes the stores, induced the entry of only Ca(++). Thus, thrombin activated TRPC6 via a SOCE-independent mechanism. In phosphorylation studies, we report that neither TRPC6 nor TRPC1 was a substrate for tyrosine kinases. TRPC6 was phosphorylated by cAMP-dependent protein kinase (cAMP-PK) and associated with other cAMP-PK substrates. TRPC1 was not phosphorylated by cAMP-PK but also associated with other substrates. Activation of cAMP-PK inhibited Ca(++) but not Ba(2+) entry induced by thrombin and neither Ca(++) nor Ba(2+) entry stimulated by OAG. These results suggest that TRPC6 is a SOCE-independent, nonselective cation entry channel stimulated by thrombin and OAG. TRPC6 is a substrate for cAMP-PK, although phosphorylation appears to not affect cation permeation. TRPC1 is located in IM, suggesting a role at the level of the stores.
D000595:Amino Acid Sequence; D001692:Biological Transport; D001792:Blood Platelets / Q000502:physiology*; D002118:Calcium / Q000097:blood*; D015220:Calcium Channels / Q000097:blood* / Q000737:chemistry / Q000235:genetics; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000097:blood; D004789:Enzyme Activation; D016257:Fura-2; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010710:Phosphates / Q000097:blood; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel
D015220:Calcium Channels; D010446:Peptide Fragments; D010710:Phosphates; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; C096351:transient receptor potential cation channel, subfamily C, member 1; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium; D016257:Fura-2