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Cell Signal. 2011 Nov;23(11):1850-6. doi: 10.1016/j.cellsig.2011.06.022. Epub 2011 Jul 3.

Functional role of the calmodulin- and inositol 1,4,5-trisphosphate receptor-binding (CIRB) site of TRPC6 in human platelet activation.

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  • 1Department of Physiology, Cell Physiology Research Group, University of Extremadura, Cáceres, 10003, Spain.

Abstract

BACKGROUND:

All identified mammalian TRPC channels show a C-terminal calmodulin (CaM)- and inositol 1,4,5-trisphosphate receptors (IP(3)Rs)-binding (CIRB) site involved in the regulation of TRPC channel function.

OBJECTIVES:

To assess the basis of CaM/IP(3)Rs binding to the CIRB site of TRPC6 and its role in platelet physiology.

METHODS:

Protein association was detected by co-immunoprecipitation and Western blotting, Ca(2+) mobilization was measured by fluorimetric techniques and platelet function was analyzed by aggregometry.

RESULTS:

Co-immunoprecipitation of TRPC6 with CaM or the IP(3)Rs at different cytosolic free Ca(2+) concentrations ([Ca(2+)](c)) indicates that the association between these proteins is finely regulated by cytosolic Ca(2+) via association of CaM and displacement of the IP(3)Rs at high [Ca(2+)](c). Thrombin-stimulated association of TRPC6 with CaM or the IP(3)Rs was sensitive to 2-APB and partially inhibited by dimethyl BAPTA loading, thus suggesting that the association between these proteins occurs through both Ca(2+)-dependent and -independent mechanisms. Incorporation of an anti-TRPC6 C-terminal antibody, whose epitope overlaps the CIRB region, impaired the dynamics of the association of TRPC6 with CaM and the IP(3)Rs, which lead to both inhibition and enhancement of thrombin- and thapsigargin-evoked Ca(2+) entry in the presence of low or high, respectively, extracellular Ca(2+) concentrations, as well as altered thrombin-evoked platelet aggregation.

CONCLUSIONS:

Our results indicate that the CIRB site of TRPC6 plays an important functional role in platelets both modulating Ca(2+) entry and aggregation through its interaction with CaM and IP(3)Rs.

Copyright © 2011 Elsevier Inc. All rights reserved.

PMID:
21757000
[PubMed - indexed for MEDLINE]
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