A role for SNAP-25 but not VAMPs in store-mediated Ca2+ entry in human platelets.
Store-mediated Ca2+ entry (SMCE) is a major mechanism for Ca2+ influx in non-excitable cells. Recently, a conformational coupling mechanism allowing coupling between transient receptor potential channels (TRPCs) and IP3 receptors has been proposed to activate SMCE. Here we have investigated the role of two soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs), which are involved in membrane trafficking and docking, in SMCE in human platelets. We found that the synaptosome-associated protein (SNAP-25) and the vesicle-associated membrane proteins (VAMP) coimmunoprecipitate with hTRPC1 in platelets. Treatment with botulinum toxin (BoNT) E or with tetanus toxin (TeTx), induced cleavage and inactivation of SNAP-25 and VAMPs, respectively. BoNTs significantly reduced thapsigargin- (TG) and agonist-evoked SMCE. Treatment with BoNTs once SMCE had been activated decreased Ca2+ entry, indicating that SNAP-25 is required for the activation and maintenance of SMCE. In contrast, treatment with TeTx had no effect on either the activation or the maintenance of SMCE in platelets. Finally, treatment with BoNT E impaired the coupling between naturally expressed hTRPC1 and IP3 receptor type II in platelets. From these findings we suggest SNAP-25 has a role in SMCE in human platelets.
Redondo
Pedro C
PC
Department of Physiology, University of Extremadura, Cáceres, Spain.
Harper
Alan G S
AG
Salido
Ginés M
GM
Pariente
Jose A
JA
Sage
Stewart O
SO
Rosado
Juan A
JA
D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001905:Botulinum Toxins / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008565:Membrane Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D050683:R-SNARE Proteins; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D050052:TRPC Cation Channels; D013744:Tetanus Toxin / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
D015220:Calcium Channels; D004791:Enzyme Inhibitors; D006490:Hemostatics; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D018160:Receptors, Cytoplasmic and Nuclear; C494208:SNAP25 protein, human; D050825:Synaptosomal-Associated Protein 25; D050052:TRPC Cation Channels; D013744:Tetanus Toxin; C096351:transient receptor potential cation channel, subfamily C, member 1; D019284:Thapsigargin; D013917:Thrombin; D001905:Botulinum Toxins; D002118:Calcium; C096326:botulinum toxin type E