A novel type of ATP block on a Ca²⁺-activated K⁺ channel from bullfrog erythrocytes

Using the patch-clamp technique, we have identified an intermediate conductance Ca²⁺ -activated K⁺ channel from bullfrog (Rana catesbeiana) erythrocytes and have investigated the regulation of channel activity by cytosolic ATP. The channel was highly selective for K⁺ over Na⁺, gave a linear I-V relationship with symmetrical 117.5 mM K⁺ solutions and had a single-channel conductance of 60 pS. Channel activity was dependent on Ca²⁺ concentration (K(1/2) = 600 nM) but voltage-independent. These basic characteristics are similar to those of human and frog erythrocyte Ca²⁺ - activated K⁺ (Gardos) channels previously reported. However, cytoplasmic application of ATP reduced channel activity with block exhibiting a novel bell-shaped concentration dependence. The channel was inhibited most by ~10 μM ATP (P(o) reduced to 5% of control) but less blocked by lower and higher concentrations of ATP. Moreover, the novel type of ATP block did not require Mg²⁺, was independent of PKA or PKC, and was mimicked by a nonhydrolyzable ATP analog, AMP-PNP. This suggests that ATP exerts its effect by direct binding to sites on the channel or associated regulatory proteins, but not by phosphorylation of either of these components.