The effect of ATP on Ca(2+)-dependent K+ channels of human red cells

The action of ATP on Ca(2+)-dependent K+ channels was studied in fresh human erythrocytes using patch-clamp techniques. Single-channel current was recorded at pH 6.5 from inside-out patches in the presence of symmetrical K+ gluconate solutions, containing both 1 microM free Ca2+ in the bath and 0.5 mM LaCl3 on the pipette side. With no ATP, the electrical activity revealed low-conductance K+ channels (25 pS), which showed inward rectification and an opening kinetics dependent on membrane potential. When ATP (1 mM) and Mg2+ (2 mM) were added together and a depolarizing potential was simultaneously applied, only a high-conductance channel (about 75 pS) was observed. This channel showed no rectifying properties and it was not found if ATP was added in the absence of Mg2+. Channel activity was enhanced by adding fluoride (10 mM) or trifluoperazine (50 microM) whilst it was reduced after incubating with dibutyryl cAMP (50 microM) or alkaline phosphatase (250 U/ml). On the other hand, when fragmented membranes from inside-out vesicles were incubated with gamma-32 P-ATP and 1 microM free Ca2+ under above conditions, only two high-molecular weight polypeptides (235 and 320 kDa) were labelled with 32P. The results suggest that ATP-mediated phosphorylation of Ca(2+)-dependent K+ channels leads to a high-conductance state.