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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Authors: | Dionisio N Albarran L Berna-Erro A Hernandez-Cruz J M Salido G M Rosado J A |
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Affiliation: | a Department of Physiology, Cell Physiology Research Group, University of Extremadura, Cáceres, 10003, Spainb Laboratorio de Análisis Clínicos Hernandez-Cruz, Cáceres, 10003, Spain |
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Abstract: | BackgroundAll identified mammalian TRPC channels show a C-terminal calmodulin (CaM)- and inositol 1,4,5-trisphosphate receptors (IP3Rs)-binding (CIRB) site involved in the regulation of TRPC channel function.ObjectivesTo assess the basis of CaM/IP3Rs binding to the CIRB site of TRPC6 and its role in platelet physiology.MethodsProtein association was detected by co-immunoprecipitation and Western blotting, Ca2+ mobilization was measured by fluorimetric techniques and platelet function was analyzed by aggregometry.ResultsCo-immunoprecipitation of TRPC6 with CaM or the IP3Rs at different cytosolic free Ca2+ concentrations ([Ca2+]c) indicates that the association between these proteins is finely regulated by cytosolic Ca2+ via association of CaM and displacement of the IP3Rs at high [Ca2+]c. Thrombin-stimulated association of TRPC6 with CaM or the IP3Rs was sensitive to 2-APB and partially inhibited by dimethyl BAPTA loading, thus suggesting that the association between these proteins occurs through both Ca2+-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 IP3Rs, which lead to both inhibition and enhancement of thrombin- and thapsigargin-evoked Ca2+ entry in the presence of low or high, respectively, extracellular Ca2+ concentrations, as well as altered thrombin-evoked platelet aggregation.ConclusionsOur results indicate that the CIRB site of TRPC6 plays an important functional role in platelets both modulating Ca2+ entry and aggregation through its interaction with CaM and IP3Rs. |
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Keywords: | CIRB TRPC6 Human platelets Calmodulin IP3 receptors Calcium Aggregation |
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