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 μM) 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 μM 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 mM BAPTA) nor hydrolysis-resistant guanine nucleotides (2 mM GDP-β-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.