Abstract
Depletion of intracellular Ca2+ stores results in the activation of SMCE (store-mediated Ca2+ entry) in many cells. The mechanism of activation of SMCE is poorly understood. In human platelets, a secretion-like coupling model may be involved. This proposes that store depletion results in trafficking of portions of the endoplasmic reticulum to the plasma membrane, enabling coupling between proteins in the two membranes. In support of this, we have shown that, in human platelets, agonist-evoked Ca2+ store depletion results in de novo and reversible coupling of the Ins P3RII [type II inositol (1,4,5)trisphosphate receptor] with the putative Ca2+ entry channel hTRPC1 [human canonical transient receptor potential 1 (protein); Rosado, Brownlow and Sage (2002) J. Biol. Chem. 277, 42157-42163]. A crucial test of the hypothesis that this coupling activates SMCE is that it should occur rapidly enough to account for agonist-evoked Ca2+ entry. In the present study, we have used quenched- and stopped-flow approaches to determine the latencies of thrombin-evoked coupling of Ins P3RII with hTRPC1 and of thrombin-evoked bivalent cation entry using Mn2+ quenching of fura 2 fluorescence. Thrombin-evoked Mn2+ entry was detected with a latency of 0.81+/-0.07 s (S.E.M., n =7) or 1.36+/-0.09 s (S.E.M., n =7) at a concentration of 1.0 or 0.1 unit/ml respectively. Coupling between Ins P3RII and hTRPC1, assessed at 100 ms intervals, was first detected with a latency of 0.9 or 1.4 s after stimulation with thrombin at a concentration of 1.0 or 0.1 unit/ml respectively. These results support the hypothesis that de novo coupling of Ins P3RII with hTRPC1 could activate SMCE in human platelets.