ADP-induced rapid inward currents through Ca2+-permeable cation channels in mouse, rat and guinea-pig megakaryocytes: A patch-clamp study

1. The rapid inward currents in mouse megakaryocytes evoked by adenosine diphosphate (ADP), a ubiquitous platelet-activating substance,were studied. Time and current-resolution were improved by using patch-clamp recording and an extracellular fast-perfusion ('Y tube') technique. 2. Application of ADP (40 mu M) to megakaryocytes immersed in physiological saline evoked rapid inward currents(80-340 pA at -42 mV). The cellular responses to a second ADP application were markedly reduced, but in the absence of external Ca2+, responses to repeated ADP application were maintained and did not deteriorate. 3. The ADP-induced current recorded in Ca2+-free external media showed short latency (less than 20 ms) and approximately exponential decay (time constant, 300-550 ms), which was independent of the holding potential and seemed to be caused mainly by receptor desensitization; it took over 5 . 5 min for complete recovery. 4. The ADP concentration-response relationship of the megakaryocytes revealed that the half-maximal concentration and the Hill coefficient-were 12 . 6 mu M and 1 . 4, respectively. 5. An ion replacement experiment showed that the ADP-induced currents could be carried by Na+, Cs+ and K+, but not Cl-, and the cation channels were permeable to Ca2+, Ba2+ and Mg2+. 6. Neither Ca2+ chelators (10 mM EGTA and 10 mat BAPTA) nor hydrolysis-resistant guanine nucleotides (2 mM GDP-beta-S and 0 . 4 mM 5'-guanylylimidodiphosphate) in the internal saline affected the rapid responses to ADP, and ADP-induced currents were recorded in excised membrane patches, suggesting that-the ADP receptor site and the molecular structure forming the cation channel are tightly coupled and/or parts of the same molecule. 7. In rat and guinea-pig megakaryocytes, ADP-induced rapid inward currents showed the same properties as in mouse megakaryocytes.