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

Michael J. Mason and Martyn P. Mahaut-Smith

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.

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