Voltage modulation of IP₃-dependent Ca²⁺ release during G-protein-coupled receptor (GPCR) activation has been described in a number of cell types but most extensively studied in the non-excitable megakaryocyte (MK) (Mahaut-Smith et al. 1999). To gain further insight into the underlying mechanism, we have now compared the voltage-dependence to Ca²⁺ signalling during activation of different receptors and downstream signalling events in the MK.

Male Wistar rats or C57BL/6 mice were humanely killed by CO₂ inhalation and cervical dislocation. Whole-cell patch clamp and Ca²⁺_i recordings from marrow MKs, adapted to allow simultaneous flash photolysis, were conducted as described previously (Mahaut-Smith et al. 1999; Mason & Mahaut-Smith, 2001). Depolarisation-dependent Ca²⁺ mobilisation was assessed using 80 mV pulses from -75 mV.

Ca²⁺ increases evoked by several agonists acting via Gα_q-coupled receptors were voltage-dependent with the rank order: ADP>thromboxaneA₂> serotonin (55 ± 5, 33 ± 6 and 11 ± 4 %, respectively (S.E.M.), as a percentage of the response to 1 µM ADP, 300 nM U46619 and 10 µM 5-HT). Activation of IP₃ receptors following release of IP₃ (n = 9) or G-PIP₂ (n = 10) from caged precursors, alone or in combination with stimulation of diacylglycerol-dependent pathways (100 µM OAG, n = 8, or 1 µM PMA, n = 4), failed to promote voltage control of Ca²⁺ release. Similarly, activation of phospholipase-Cλ by platelet-derived growth factor (50 ng/ml) did not lead to voltage-dependent Ca²⁺ release (n = 4). In P2Y₁^-/- MKs, agonist and voltage-dependent Ca²⁺ increases were absent during ADP application (1 µM; n = 43), but still observed during U46619 (300 nM; n = 15) or serotonin (10 µM; n = 19). Ca²⁺ increases induced by intracellular GTPλS (50 µM) were potentiated 5.7 fold by depolarization, however responses to both GTPλS and voltage were not clearly observed in P2Y₁^-/- MKs (n = 5 and 9). Sensitisation of IP₃ receptors with thimerosal (100 µM) induced a small voltage-dependent Ca²⁺ release (n = 7), but this also required P2Y₁ receptors (n = 19).

These data indicate that signalling via a number of Gα_q-coupled receptors in the MK is voltage-dependent and that this response to ADP requires P2Y₁ receptors. Evidence to date (see also Martinez-Pinna, 2003) supports the hypothesis that the voltage sensor lies at the level of the GPCR or its interaction with G-proteins, rather than a downstream signalling event.

This work was funded by the BHF and MRC