Expression and role of TRPC proteins in human platelets: evidence that TRPC6 forms the store-independent calcium entry channel

Sheila R Hassock,Michael X Zhu,Claudia Trost,Veit Flockerzi,Kalwant S Authi

doi:10.1182/blood-2002-03-0723

Sheila R Hassock, Michael X Zhu + Show 3 more

Open Access

https://doi.org/10.1182/blood-2002-03-0723

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Abstract

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.

Highlights

Platelet activation forms an integral component of hemostasis and contributes to the events leading to thrombosis
Ank was effective at immunoprecipitating overexpressed hTRPC1, as determined by probing the immunoprecipitates with the HA-epitope antibody in Western blots
Ank is specific for hTRPC1, as it did not recognize mTRPC2, hTRPC3, mTRPC4␣, mTRPC5, or mTRPC6, all overexpressed in QBI293A cells

Summary

Introduction

Platelet activation forms an integral component of hemostasis and contributes to the events leading to thrombosis. Many models have been proposed, which include a conformational coupling mechanism involving the IP3R at the stores signaling to the PM channel,[4] release of a soluble Caϩϩ influx factor (CIF) from the stores that may trigger the opening of the cation channel in the PM,[5] involvement of tyrosine kinases,[6,7] and modifications of the conformational coupling mechanism that include a secretionlike mechanism involving rearrangements of the cytoskeleton[8] and possible channel insertion.[9] Importantly, there is as yet no consensus regarding the identity of the SOCE channel, and until such identification is established, the mechanisms for its regulation will remain contentious. The identities of the SOCE channels themselves may vary between cells

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