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 β‐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, SUR2A and SUR2B requires ATP. Non‐hydrolysable ATP‐analogues do not support binding, and Mg 2+ or Mn 2+ are required. Point mutations in the Walker A motifs or linker regions of both nucleotide‐binding folds (NBFs) abolish or weaken [ 3 H]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 ∼ SUR1 > SUR2A. KCOs belonging to different structural classes inhibited specific [ 3 H]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 K ATP channels and suggest that KCO binding requires a conformational change induced by ATP hydrolysis in both NBFs.