title: Functional role of the calmodulin- and inositol 1,4,5-trisphosphate receptor-binding (CIRB) site of TRPC6 in human platelet activation.
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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Department of Physiology, Cell Physiology Research Group, University of Extremadura, Cáceres, 10003, Spain.
authors.forename: N
authors.initials: N
authors.lastname: Dionisio
authors.forename: L
authors.initials: L
authors.lastname: Albarran
authors.forename: A
authors.initials: A
authors.lastname: Berna-Erro
authors.forename: J M
authors.initials: JM
authors.lastname: Hernandez-Cruz
authors.forename: G M
authors.initials: GM
authors.lastname: Salido
authors.forename: J A
authors.initials: JA
authors.lastname: Rosado
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D001792:Blood Platelets / Q000502:physiology*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D018274:Electroporation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D015544:Inositol 1,4,5-Trisphosphate / Q000737:chemistry / Q000378:metabolism*; D015539:Platelet Activation / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology*; D050052:TRPC Cation Channels / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D000074287:TRPC6 Cation Channel; D019284:Thapsigargin / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D002147:Calmodulin; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; D004533:Egtazic Acid; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; C058180:5,5'-dimethyl-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate; D013917:Thrombin; D002118:Calcium