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pubmed:abstractText |
1. 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. 2. At a holding potential of -60 mV, in NaCl external saline and KCl internal saline with low levels of Ca2+ buffering, 10 microM ADP evoked [Ca2+]i oscillations 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. 3. 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+]i spike. This underlying conductance was permeable to Na+ and Cs+, but possessed little or no permeability to Cl- or divalent cations. 4. Intracellular dialysis with IP3 (5-50 microM) activated the monovalent cationic conductance prior to release of Ca2+ from intracellular stores. The [Ca2+]i increase was associated with a second phase of cationic current, implying that both IP3 and Ca2+ can activate this conductance. Buffering of [Ca2+]i with BAPTA abolished the second phase of current, leaving monophasic spikes of inward current, often occurring at regular intervals. 5. 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 IP3 and [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.
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