3 CATION INFLUX CURRENTS ACTIVATED BY PURINERGIC RECEPTOR STIMULATIONIN RAT MEGAKARYOCYTES

1. Simultaneous patch clamp and fura-2 fluorescence measurements were used to study ATP-evoked membrane currents and intracellular [Ca2+] ([ Ca2+](i)) changes in rat megakaryocytes. 2. At negative potentials, un der conditions that blocked K+ currents, 20 mu M ATP activated a bipha sic inward current and a concurrent biphasic increase in [Ca2+](i). Th e initial [Ca2+](i) increase was due to Ca2+ influx whereas the delaye d (1.70 +/- 0.13 s, mean +/- S.D.) increase was at least partly due to the release ofinternal Ca2+ stores. 3. The initial current was activa ted within 100 ms, inactivated mithin 1-4 s and was carried by both Na + and Ca2+. 4. The delayed current was also transient and carried main ly by Na+ when Ca2+ buffering in the pipette was low. This Na+ conduct ance did not require an increase in [Ca2+](i) for activation, but was triggered by inositol 1,4,5-trisphosphate (IP3), or a metabolite of IP 3. 5. Buffering of [Ca2+](i) changes with BAPTA revealed a third curre nt activated by Ca2+ release from internal stores. This channel was se lective for divalent cations with the permeability sequence Ca2+ great er than or equal to Ba2+ > Mn2+, Mg2+. 6. Adenosine-5'-O-3-thiotriphos phate (ATP gamma S), like ATP, evoked all three influx currents, where as ADP only stimulated Ca2+ release and the two currents associated wi th it. Increasing the external divalent cation concentration abolished the ATP-evoked Ca2+ release and delayed currents but not the initial transient current. 7. We conclude that rat megakaryocytes express two types of purinergic receptor. One type, activated by ATP, is closely c oupled to a non-selective cation channel. The second type, which recog nizes ATP(4-) and ADP(3-), activates Ca2+ release and two types of mem brane conductance, one more selective for monovalent cations, the othe r highly selective for Ca2+.