Capacitative and non-capacitative signaling complexes in human platelets

Discharge of the intracellular Ca2+ stores activates Ca2+ entry through store-operated channels (SOCs). Since the recent identification of STIM1 and STIM2, as well as the Orai1 homologs, Orai2 and Orai3, the protein complexes involved in Ca2+ signaling needs re-evaluation in native cells. Using real time PCR combined with Western blotting we have found the expression of the three Orai isoforms, STIM1, STIM2 and different TRPCs in human platelets. Depletion of the intracellular Ca2+ stores with thapsigargin, independently of changes in cytosolic Ca2+ concentration, enhanced the formation of a signaling complex involving STIM1, STIM2, Orai1, Orai2 and TRPC1. Furthermore, platelet treatment with the dyacylglicerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) resulted in specific association of Orai3 with TRPC3. Treatment of platelets with arachidonic acid enhanced the association between Orai1 and Orai3 in human platelets and overexpression of Orai1 and Orai3 in HEK293 cells increased arachidonic acid-induced Ca2+ entry. These results indicate that Ca2+ store depletion results in the formation of exclusive signaling complexes involving STIM proteins, as well as Orai1, Orai2 and TRPC1, but not Orai3, which seems to be involved in non-capacitative Ca2+ influx in human platelets.

► Ca2+ store depletion results in a complex, probably regulated by STIMs, including Orai1 and Orai2. ► Orai3 does not associate with STIM1 or STIM2 upon Ca2+ store depletion. ► The association of Orai3 with Orai1 or Orai2 is reduced after Ca2+ store discharge. ► The diacylglycerol analog OAG stimulates specific association of Orai3 with TRPC3. ► Activation of Orai3 leads to non-capacitative Ca2+ entry in human platelets.