Nucleotide modulation of pinacidil stimulation of the cloned K-ATP channelKir6.2/SUR2A

ATP-sensitive K+ channels are the target for K+ channel openers such as pin acidil. These channels are formed from pore-forming Kir6.2 and regulatory s ulfonylurea receptor (SUR) subunits. Pinacidil activates channels containin g SUR2A (heart, skeletal muscle), but not those containing SUR1 (beta cells ). Surprisingly, binding of the pinacidil analog [H-3]P1075 is dependent on added nucleotides, yet in electrophysiological studies, pinacidil is effec tive in the absence of intracellular nucleotides. To determine the reason f or this anomaly, we examined the functional interactions between pinacidil (or P1075) and nucleotides by expressing cloned Kir6.2/SUR2A channels in Xe nopus laevis oocytes. Both pinacidil and P1075 activated macroscopic Kir6.2 /SUR2A currents in the absence of added nucleotide, but the presence of int racellular ATP or ADP slowed the off-rate of the response. Mutation of the Walker A lysine in a single nucleotide binding domain (NBD) of SUR2A (K707A in NBD1, K1348A in NBD2), abolished this action of nucleotide. The K1348A mutation prevented stimulation by MgADP but had little effect on the amplit ude of the pinacidil response. In contrast, Kir6.2/SUR2A-K707A currents wer e activated by MgADP, but only responded to pinacidil in the presence of Mg -nucleotide. Off-rates in the absence (or presence) of nucleotide were slow er for the pinacidil analog P1075 than for pinacidil, consistent with the h igher affinity of P1075. We suggest that slowing of P1075 dissociation by n ucleotide enables binding to be detected.