NEM modification prevents high-affinity ATP binding to the first nucleotide binding fold of the sulphonylurea receptor, SUR1

Pancreatic beta-cell ATP-sensitive potassium channels, composed of SUR1 and Kir6.2 subunits, serve as a sensor for intracellular nucleotides and regul ate glucose-induced insulin secretion. To learn more about the interaction of SUR1 with nucleotides, we examined the effect of N-ethylmaleimide (NEM) modification. Photoaffinity labeling of SUR1 with 5 mu M 8-azido-[a-P-32]AT P or 8-azido-[g-P-32]ATP was inhibited by NEM with K-i of 1.8 mu M and 2.4 mu M, and Hill coefficients of 0.91 and 1.1, respectively. However, when th e cysteine residue in the Walker A motif of the first nucleotide binding fo ld (NBF1) of SUR1 was replaced with serine (C717S), photoaffinity labeling was not inhibited by 100 mu M NEM. These results suggest that NBF1 of SUR1 has a NEM-sensitive structure similar to that of NBF1 of MDR1, a multidrug transporter, and confirm NBF1 as the high-affinity ATP binding site on SUR1 , (C) 1999 Federation of European Biochemical Societies.