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

FEBS Letters

Matsuo, Michinori¹  Amachi, Teruo¹  Tucker, Stephen J.²  Ueda, Kazumitsu¹  Ashcroft, Frances M.² 

[1] Laboratory of Biochemistry, Division of Applied Life Sciences, Kyoto University Graduate School of Agriculture, Kyoto 606-8502, Japan;University Laboratory of Physiology, Oxford OX1 3PT, UK

关键词: N-Ethylmaleimide;    ABC transporter;    ATP binding;    Potassium channel;   

DOI  :  10.1016/S0014-5793(99)01170-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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-³²P]ATP or 8-azido-[γ-³²P]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.

【 授权许可】

Unknown