Voltage-dependent Ca2+ release in rat megakaryocytes requires functional IP3 receptors

Using simultaneous whole-cell patch-clamp and fluorescence measurements of [Ca2+]i in rat megakaryocytes we have investigated the requirement for functional inositol 1,4,5-trisphosphate (IP3) receptors in Ca2+ release induced by membrane depolarization during agonist stimulation. Voltage-dependent Ca2+ release was observed during application of the IP3-generating agonists U46619 (a thromboxane A2 analogue) and ADP. Furthermore, voltage-dependent Ca2+ release was observed in the absence of exogenous agonist following sensitization of IP3 receptors with thimerosal. Depolarization-induced Ca2+ release was not detected during depletion of intracellular Ca2+ stores by thapsigargin. Thus, depletion of stores alone is not sufficient to confer voltage dependence upon the Ca2+ release mechanism. Block of IP3 receptors by carbacyclin-stimulated elevations in cAMP, uncaging of cAMP or exposure to a high concentration of caffeine reversibly abolished Ca2+ increases stimulated by both ADP and depolarization. The cAMP-dependent block was prevented by a peptide inhibitor of protein kinase A, indicating that an alteration of adenylate cyclase activity leading to modulation of protein kinase A activity does not underlie the control of Ca2+ release by voltage. These results are consistent with the requirement for functional IP3 receptors for voltage control of Ca2+ release from intracellular stores during inositol lipid signalling. The data also indicate the involvement of a voltage sensor downstream of surface membrane receptors in the depolarization-evoked Ca2+ response.