Potassium channel openers require ATP to bind to and act through sulfonylurea receptors

K-ATP channels are composed of a small inwardly rectifying K+ channel subunit, either K(IR)6.1 or K(IR)6.2, plus a sulfonylurea receptor, SUR1 or SUR2 (A or B), which belong to the ATP-binding cassette superfamily. SUR1/K(IR)6.2 reconstitute the neuronal/pancreatic beta-cell channel, whereas SUR2A/K(IR)6.2 and SUR2B/K(IR)6.1 (or K(IR)6.2) are proposed to reconstitute the cardiac and the vascular-smooth-muscle-type K-ATP channels, respectively. We report that potassium channel openers (KCOs) bind to and act through SURs and that binding to SUR1, SUP2A and SUR2B requires ATP, Non-hydrolysable ATP-analogues do not support binding, and Mg2+ or Mn2+ are required. Point mutations in the Walker A motifs or linker regions of both nucleotide-binding folds (NBFs) abolish or weaken [H-3]P1075 binding to SUR2B, rendering reconstituted SUR2B/K(IR)6.2 channels insensitive towards KCOs, The C-terminus of SUR affects KCO affinity with SUR2B similar to SUR1 > SUR2A. KCOs belonging to different structural classes inhibited specific [H-3]P1075 binding to SUR2B in a monophasic manner, with the exception of minoxidil sulfate, which induced a biphasic displacement. The affinities of KCO binding to SUR2B were 3.5-8-fold higher than their potencies for activation of SUR2B/K(IR)6.2 channels. The results establish that SURs are the KCO receptors of KATP channels and suggest that KCO binding requires a conformational change induced by ATP hydrolysis in both NBFs.