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NEM modification prevents high‐affinity ATP binding to the first nucleotide binding fold of the sulphonylurea receptor, SUR1
Author(s) -
Matsuo Michinori,
Tucker Stephen J.,
Ashcroft Frances M.,
Amachi Teruo,
Ueda Kazumitsu
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)01170-9
Subject(s) - sulfonylurea receptor , nucleotide , photoaffinity labeling , biochemistry , chemistry , n ethylmaleimide , cysteine , intracellular , transporter , binding site , serine , protein subunit , enzyme , gene
Pancreatic β‐cell ATP‐sensitive potassium channels, composed of SUR1 and Kir6.2 subunits, serve as a sensor for intracellular nucleotides and regulate 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 μM 8‐azido‐[α‐ 32 P]ATP or 8‐azido‐[γ‐ 32 P]ATP was inhibited by NEM with K i of 1.8 μM and 2.4 μM, and Hill coefficients of 0.94 and 1.1, respectively. However, when the cysteine residue in the Walker A motif of the first nucleotide binding fold (NBF1) of SUR1 was replaced with serine (C717S), photoaffinity labeling was not inhibited by 100 μM 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.