The ability of membrane potential to modulate IP₃-dependent Ca²⁺ release independently of Ca²⁺ influx has been described in a number of excitable cell types but has been most extensively studied in the non-excitable rat megakaryocyte (MK) (Mahaut-Smith et al. 1999). We have now examined the limits of sensitivity of this phenomenon to step depolarisations and activation by action potential waveforms (APW) in the MK.

Male Wistar rats were humanely killed by CO₂ inhalation and cervical dislocation. Whole-cell patch clamp and [Ca²⁺]_i recordings from marrow MKs were conducted as described previously (Mahaut-Smith et al. 1999). Depolarising steps of increasing magnitude or duration were applied during activation of P2Y receptors (1 µM ADP). The peak [Ca²⁺]_i response showed marked heterogeneity between cells: range 23-560 nM (n = 17) for a 55 mV, 5 s step from -75 mV. A positive correlation was observed between the average [Ca²⁺]_i increase and amplitude of the voltage step (range 3 to 55 mV) from -75 mV. The responses for 55, 20 and 10 mV steps were 151 ± 34, 84 ± 13 and 45 ± 6 nM, respectively (mean ± S.E.M. n = 17 or 18). The most sensitive cells lacked a threshold potential and showed [Ca²⁺]_i increases with depolarisations of only 3 mV (n = 5). We also examined the dependence of the depolarisation-evoked [Ca²⁺]_i increase upon pulse duration using a step from -85 to +50 mV. The [Ca²⁺]_i response increased with pulse duration up to 700 ms; the average was 48 ± 15 nM (n = 9) for 250 ms compared to 25 ± 4 nM, n = 17 for 100 ms. Again, the most sensitive cells responded with a [Ca²⁺]_i increase to the shortest (25 ms) pulse.

These data suggest that depolarisations of only a few millivolts or tens of milliseconds are sufficient to induce a [Ca²⁺]_i increase via modulation of P2Y receptor-induced Ca²⁺ release. This implies that a variety of voltage waveforms including action potentials can cause Ca²⁺ release via this mechanism. To further test this concept, atrial and ventricular cardiac APWs were applied during stimulation of MKs with 1 µM ADP. These APWs were both capable of inducing a [Ca²⁺]_i increase, but the ventricular waveform generated a larger peak response (182 ± 12 nM, n = 6 vs. 47 ± 6 nM, n = 10).

Our data indicate that voltage control of Ca²⁺ release via a G-protein-coupled receptor is sufficiently sensitive to detect small voltage changes and APW. The role of this novel signalling mechanism in excitable and non-excitable tissues should therefore be considered.

This work was funded by the British Heart Foundation and Medical Research Council.