A Novel Type of ATP Block on a Ca 2+-Activated K + Channel from Bullfrog Erythrocytes

Abstract

Using the patch-clamp technique, we have identified an intermediate conductance Ca 2+-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 Ca 2+ concentration ( K 1/2 = 600 nM) but voltage-independent. These basic characteristics are similar to those of human and frog erythrocyte Ca 2+-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 0 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 Mg 2+, 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.