Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC323
Functional involvement of the calmodulin/inositol 1,4,5-trisphosphate receptor-binding region of TRPC6 in human platelets
N. Dionisio2, L. Albarrán2, J. J. López1,2, A. Berna-Erro2, G. M. Salido2, J. A. Rosado2
1. INSERM U770, Universit
Canonical transient receptor potential channels (TRPCs) have been reported to play an important role in intracellular Ca2+ homeostasis in a number of non-excitable cells, including human platelets. TRPCs contain a conserved calmodulin (CaM)/inositol 1,4,5-trisphosphate (IP3) receptor-binding (CIRB) site in the cytosolic C-terminal region, which binds both IP3 receptor and CaM. The latter has been suggested to be tethered to the TRPCs in a Ca2+ concentration-dependent manner and to be displaced competitively by the activated IP3 receptor (Zhang et al., 2001). In human plateles, TRPC6 are suggested to participate in receptor-activated, diacylglycerol-mediated Ca2+ influx, as well as in store-operated Ca2+ entry (Authi, 2007; Jardin et al., 2009), although little is known about the functional role of the CIRB region of TRPC6 in platelets. Here we have investigated the functional relevance of the CIRB region of TRPC6 in human platelets. Blood was drawn from healthy volunteers with local ethical committee approval. Cytosolic free Ca2+ concentration ([Ca2+]c) measurement, immunoprecipitation, Western blotting and platelet aggregation were performed as previously described (Redondo et al., 2006; Alexandru et al., 2008). Co-immunoprecipitation studies revealed that treatment of platelets with ionomycin in the presence of increasing concentrations of extracellular Ca2+, leading to a gradual increase in [Ca2+]c, results in Ca2+ concentration-dependent dissociation of TRPC6 from the IP3 receptors and association with CaM. Ca2+ concentration-dependent interaction of TRPC6 with IP3 receptors or CaM was impaired by introduction in the cells of a peptide corresponding to TRPC6825-875, containing the CIRB site. Introduction of TRPC6825-875 into cells did not alter the association of TRPC1 or Orai1 with IP3 receptors or CaM, but significantly modified both thapsigargin- and thrombin-evoked Ca2+ entry in a [Ca2+]c-dependent manner, enhancing Ca2+ entry at high [Ca2+]c while attenuating it at low [Ca2+]c. Furthermore, introduction of the TRPC6825-875 into platelets significantly reduced thrombin-evoked platelet aggregation. These results support a functional role of the CIRB of TRPC6 in human platelets.
Where applicable, experiments conform with Society ethical requirements