Dual role of tubulin-cytoskeleton in store-operated calcium entry in human platelets

Two mechanisms for store-operated Ca2+ entry (SOCE) regulated by two independent Ca2+ stores, the dense tubular system (DTS) and the acidic stores, have been described in platelets. We have previously suggested that coupling between the type II IP3 receptor (IP3RII) and hTRPC1, involving reorganization of the actin microfilaments, play an important role in SOCE. However, the involvement of the tubulin microtubules, located beneath the plasma membrane, remains unclear. Here we show that the microtubule disrupting agent colchicine reduced Ca2+ entry stimulated by low concentrations (0.1 U/mL) of thrombin, which activates SOCE mostly by depleting acidic Ca2+-store. Consistently, colchicine reduced SOCE activated by 2,5 di-(tertbutyl)-1,4-hydroquinone (TBHQ), which selectively depletes the acidic Ca2+ stores. In contrast, colchicine enhanced SOCE mediated by depletion of the DTS, induced by high concentrations of thapsigargin (TG), which depletes both the acidic Ca2+ stores and the DTS, the major releasable Ca2+ store in platelets. These findings were confirmed by using Sr2+ as a surrogate for Ca2+ entry. Colchicine attenuated the coupling between IP3RII and hTRPC1 stimulated by thrombin while it enhanced that evoked by TG. Paclitaxel, which induces microtubular stabilization and polymerization, exerted the opposite effects on thrombin- and TG-evoked SOCE and coupling between IP3RII and hTRPC1 compared with colchicine. Neither colchicine nor paclitaxel altered the ability of platelets to extrude Ca2+. These findings suggest that tubulin microtubules play a dual role in SOCE, acting as a barrier that prevents constitutive SOCE regulated by DTS, but also supporting SOCE mediated by the acidic Ca2+ stores.