ADP and inositol trisphosphate evoke oscillations of a monovalent cation conductance in rat megakaryocytes.
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.
The Physiological Laboratory, Downing Street, Cambridge CB2 3EG, UK.
J F
JF
Hussain
M P
MP
Mahaut-Smith
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002412:Cations / Q000378:metabolism; D002586:Cesium / Q000494:pharmacology; D018999:Charybdotoxin / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D005942:Gluconates / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D008297:Male; D008533:Megakaryocytes / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D008536:Meglumine / Q000031:analogs & derivatives / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010507:Periodicity; D015221:Potassium Channels / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D012965:Sodium Chloride / Q000494:pharmacology
D002412:Cations; D002614:Chelating Agents; D002712:Chlorides; D005456:Fluorescent Dyes; D005942:Gluconates; D015221:Potassium Channels; C051643:N-methylglucamine gluconate; D018999:Charybdotoxin; D002586:Cesium; D012965:Sodium Chloride; D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D008536:Meglumine; D015544:Inositol 1,4,5-Trisphosphate; C028019:cesium chloride; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D016257:Fura-2