ADP and inositol trisphosphate evoke oscillations of a monovalent cation conductance in rat megakaryocytes

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Abstract
A combination of conventional whole‐cell patch clamp recordings and fura‐2 fluorescence photometry was used to study the membrane currents during oscillations of intracellular Ca2+concentration ([Ca2+]i) in single rat megakaryocytes. At a holding potential of ‐60 mV, in NaCl external saline and KCl internal saline with low levels of Ca2+buffering, 10 μmADP evoked [Ca2+]ioscillations and simultaneous Ca2+‐gated K+currents at a frequency of 3–10 spikes min−1. A smaller inward current was also activated, with a time course that identified this component as the inositol 1,4,5‐trisphosphate (IP3)‐activated monovalent cation current previously demonstrated in rat megakaryocytes. Cs+replacement of internal K+combined with 100 nM external charybdotoxin (CTX) abolished the outward currents and revealed that an inward current was also transiently activated during each [Ca2+]ispike. This underlying conductance was permeable to Na+and Cs+, but possessed little or no permeability to Cl−or divalent cations. Intracellular dialysis with IP3(5‐50 μm) activated the monovalent cationic conductance prior to release of Ca2+from intracellular stores. The [Ca2+]iincrease was associated with a second phase of cationic current, implying that both IP3and Ca2+can activate this conductance. Buffering of [Ca2+]iwith BAPTA abolished the second phase of current, leaving monophasic spikes of inward current, often occurring at regular intervals. These data demonstrate that a monovalent cation current, which results in Na+influx under normal ionic conditions, oscillates in response to ADP receptor stimulation due to activation by both IP3and [Ca2+]i. This provides a route for long‐term Na+entry in the megakaryocyte following stimulation of receptors coupled to phospholipase C activation and may play a role in cell shape change.