Abstract
A major pathway for Ca
2+
entry in non-excitable cells is activated following depletion of intracellular Ca
2+
stores. A
de novo
conformational coupling between elements in the plasma membrane (PM) and Ca
2+
stores has been proposed as the most likely mechanism to activate this capacitative Ca
2+
entry (CCE) in several cell types, including platelets. Here we report that a cytochrome P450 metabolite, 5,6-EET, might be a component of the
de novo
conformational coupling in human platelets. In these cells, 5,6-EET induces divalent cation entry without having any detectable effect on Ca
2+
store depletion. 5,6-EET-induced Ca
2+
entry was sensitive to the CCE blockers 2-APB, lanthanum, SKF-96365 and nickel and impaired by incubation with anti-hTRPC1 antibody. Ca
2+
entry stimulated by low concentrations of thapsigargin, which selectively depletes the dense tubular system and induces EET production, was impaired by the cytochrome P450 inhibitor 17-ODYA, which has no effect on CCE mediated by depletion of the acidic stores using 2,5-di-(tert-butyl)-1,4-hydroquinone. We have found that 5,6-EET-induced Ca
2+
entry requires basal levels of H
2
O
2
, which might maintain a redox state favourable for this event. Finally, our results indicate that 5,6-EET induces the activation of tyrosine kinase proteins and the reorganization of the actin cytoskeleton, which might provide a support for the transport of portions of the Ca
2+
store towards the PM to facilitate
de novo
coupling between IP
3
R type II and hTRPC1 detected by coimmunoprecipitation. We propose that the involvement of 5,6-EET in TG-induced coupling between IP
3
R type II and hTRPC1 and subsequently CCE is compatible with the
de novo
conformational coupling in human platelets.