Direct photoaffinity labeling of Kir6.2 by [gamma-P-32]ATP-[gamma]4-azidoanilide

ATP-sensitive potassium (K-ATP) channels are under complex regulation by in tracellular ATP and ADP. The potentiatory effect of MgADP is conferred by t he sulfonylurea receptor subunit of the channel, SUR, whereas the inhibitor y effect of ATP appears to be mediated via the pore-forming subunit, Kir6.2 . We have previously reported that Kir6.2 can be directly labeled by 8-azid o-[gamma-P-32]ATP. However, the binding affinity of 8-azido-ATP to Kir6.2 w as low probably due to modification at 8' position of adenine. Here we demo nstrate that Kir6.2 can be directly photoaffinity labeled with higher affin ity by [gamma-P-32]ATP-[gamma]4-azidoanilide ([gamma-P-32]ATP-AA), containi ng an unmodified adenine ring. Photoaffinity labeling of Kir6.2 by [gamma-P -32]ATP-AA is not affected by the presence of Mg2+, consistent with Mg2+-in dependent ATP inhibition of K-ATP channels. Interestingly, SUR1, which can be strongly and specifically photoaffinity labeled by 8-azido-ATP, was not photoaffinity labeled by ATP-AA. These results identify key differences in the structure of the nucleotide binding sites on SUR1 and Kir6.2. (C) 2000 Academic Press.