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J Biol Chem, Vol. 274, Issue 7, 3931-3933, February 12, 1999

COMMUNICATION
Direct Photoaffinity Labeling of the Kir6.2 Subunit of the ATP-sensitive K⁺ Channel by 8-Azido-ATP

Kouichi Tanabe, Stephen J. Tucker^§, Michinori Matsuo, Peter Proks^§, Frances M. Ashcroft^§, Susumu Seino, Teruo Amachi, and Kazumitsu Ueda

From the  Laboratory of Biochemistry, Division of Applied Life Sciences, Kyoto University Graduate School of Agriculture, Kyoto 606-8502, Japan, the ^§ University Laboratory of Physiology, Oxford OX1 3PT, United Kingdom, and the  Department of Molecular Medicine, Chiba University Graduate School of Medicine, Chuo-ku, Chiba 260-8670, Japan

ATP-sensitive potassium channels are under complex regulation by intracellular ATP and ADP. The potentiating effect of MgADP is conferred by the sulfonylurea receptor subunit of the channel, SUR, whereas the inhibitory effect of ATP appears to be mediated via the pore-forming subunit, Kir6.2. We determined whether ATP directly interacts with a binding site on the Kir6.2 subunit to mediate channel inhibition by analyzing binding of a photoaffinity analog of ATP (8-azido-[-³²P]ATP) to membranes from COS-7 cells transiently expressing Kir6.2. We demonstrate that Kir6.2 can be directly labeled by 8-azido-[-³²P]ATP but that the related subunit Kir4.1, which is not inhibited by ATP, is not labeled. Photoaffinity labeling of Kir6.2 is reduced by approximately 50% with 100 µM ATP. In addition, mutations in the NH₂ terminus (R50G) and the COOH terminus (K185Q) of Kir6.2, which have both been shown to reduce the inhibitory effect of ATP upon Kir6.2 channel activity, reduced photoaffinity labeling by >50%. These results demonstrate that ATP binds directly to Kir6.2 and that both the NH₂- and COOH-terminal intracellular domains may influence ATP binding.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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