The ATP-sensitive potassium (K-ATP) channels in pancreatic beta cells are c
ritical in the regulation of glucose-induced insulin secretion. Although el
ectrophysiological studies provide clues to the complex control of K-ATP ch
annels by ATP, MgADP, and pharmacological agents, the molecular mechanism o
f K-ATP-channel regulation remains unclear. The K-ATP channel is a heterool
igomeric complex of SUR1 subunits of the ATP-binding-cassette superfamily w
ith two nucleotide-binding folds (NBF1 and NBF2) and the pore-forming Kir6.
2 subunits. Here, we report that MgATP and MgADP, but not the Mg salt of ga
mma-thio-ATP, stabilize the binding of prebound 8-azido-[alpha-P-32]ATP to
SUR1. Mutation in the Walker A and B motifs of NBF2 of SUR1. abolished this
stabilizing effect of MgADP. These results suggest that SUR1 binds 8-azido
-ATP strongly at NBF1 and that MgADP, either by direct binding to NBF2 or b
y hydrolysis of bound MgATP at NBF2, stabilizes prebound 8-azido-ATP bindin
g at NBF1. The sulfonylurea glibenclamide caused release of prebound 8-azid
o-[alpha-P-32]ATP from SUR1 in the presence of MgADP or MgATP in a concentr
ation-dependent manner. This direct biochemical evidence of cooperative int
eraction in nucleotide binding of the two NBFs of SUR1 suggests that gliben
clamide both blocks this cooperative binding of ATP and MgADP and, in coope
ration with the MgADP bound at NBF2, causes ATP to be released from NBF1.