3 CATION INFLUX CURRENTS ACTIVATED BY PURINERGIC RECEPTOR STIMULATION IN 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, under conditions that blocked K+ currents, 20 mu M ATP activated a biphasic inward current and a concurrent biphasic increase in [Ca2+](i). The initial [Ca2+](i) increase was due to Ca2+ influx whereas the delayed (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 activated 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 mainly by Na+ when Ca2+ buffering in the pipette was low. This Na+ conductance did not require an increase in [Ca2+](i) for activation, but was triggered by inositol 1,4,5-trisphosphate (IP3), or a metabolite of IP3. 5. Buffering of [Ca2+](i) changes with BAPTA revealed a third current activated by Ca2+ release from internal stores. This channel was selective for divalent cations with the permeability sequence Ca2+ greater than or equal to Ba2+ > Mn2+, Mg2+. 6. Adenosine-5'-O-3-thiotriphosphate (ATP gamma S), like ATP, evoked all three influx currents, whereas ADP only stimulated Ca2+ release and the two currents associated with 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 coupled to a non-selective cation channel. The second type, which recognizes ATP(4-) and ADP(3-), activates Ca2+ release and two types of membrane conductance, one more selective for monovalent cations, the other highly selective for Ca2+.