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The ATP-sensitive potassium (K ATP ) channels in pancreatic ␤ cells are critical in the regulation of glucose-induced insulin secretion. Although electrophysiological studies provide clues to the complex control of K ATP channels by ATP, MgADP, and pharmacological agents, the molecular mechanism of K ATP -channel regulation remains unclear. The K ATP channel is a heterooligomeric complex of SUR1 subunits of the ATP-binding-cassette superfamily with two nucleotide-binding folds (NBF1 and NBF2) and the pore-forming Kir6.2 subunits. Here, we report that -channel openers. Sulfonylureas, commonly used in the treatment of non-insulin-dependent diabetes mellitus, stimulate insulin secretion by closing the K ATP channels, whereas K ϩ -channel openers inhibit insulin secretion by opening the K ATP channels (7).The pancreatic ␤ cell K ATP channel is a complex of four SUR1 subunits of the ATP-binding cassette (ABC) superfamily with two nucleotide-binding folds (NBF1 and NBF2) and four Kir6.2 subunits of the inwardly rectifying K ϩ -channel family (8, 9). SUR1 is thought to mediate the stimulatory effect of MgADP and is the primary target for pharmacological agents, such as the sulfonylurea glibenclamide and the K ϩ -channel opener diazoxide. The primary site of ATP inhibition of K ATP -channel activity seems to be in Kir6.2 (10, 11). However, the regulation of the ␤ cell K ATP channels by adenine nucleotides and pharmacological agents is complex. In addition to the inhibitory effect, MgATP enhances ␤ cell K ATPchannel activity (12, 13). ADP also has both stimulatory and inhibitory effects (14,15). Although the interaction of sulfonylureas with SUR1 abolishes the stimulatory effect of MgADP on K ATP channels (10), the sensitivity of the K ATP channel to sulfonylureas is increased in the presence of .Previously, we found that SUR1 strongly binds 8-azido-ATP at NBF1, whereas MgADP is bound at NBF2, and that preincubation of SUR1 with MgADP efficiently inhibits 8-azido-ATP binding to SUR1 (ref. 19; Fig. 1A, preincubation procedure). Because this inhibitory effect of MgADP was reduced by mutations in NBF2, we thought the MgADP bound at NBF2 might facilitate MgADP binding at NBF1 and thereby prevent 8-azido-ATP binding to NBF1. The strong 8-azido-ATP binding to SUR1 made it possible to investigate the biochemical basis of such cooperative interaction. Here, we provide direct biochemical evidence, obtained by postincubation procedure, of the cooperative interaction in nucleotide binding of the two NBFs of SUR1. In addition, we show that the sulfonylurea glibenclamide modulates this cooperative interaction in nucleotide binding of the two NBFs of SUR1. MATERIALS AND METHODS Materials