The role of lysine 185 in the Kir6.2 subunit of the ATP-sensitive channel in channel inhibition by ATP

1. ATP-sensitive potassium (K-ATP) channels are composed of pore-forming Ki r6.2 and regulatory SUR subunits. A truncated isoform of Kir6.2, Kir6.2 Del ta C26, forms ATP-sensitive channels in the absence of SUR1, suggesting the ATP-inhibitory site lies on Kir6.2. 2. Previous studies have shown that mutation of the lysine residue at posit ion 185 (K185) in the C-terminus of Kir6.2 to glutamine, decreased the chan nel sensitivity to ATP without affecting the single-channel conductance or the intrinsic channel kinetics. This mutation also impaired 8-azido[P-32]-A TP binding to Kir6.2. 3. To determine if K185 interacts directly with ATP, we made a range of mut ations at this position, and examined the effect on the channel ATP sensiti vity by recording macroscopic currents in membrane patches excised from Xen opus oocytes expressing wild-type or mutant Kir6.2 Delta C26. 4. Substitution of K185 by a positively charged amino acid (arginine) had n o substantial effect on the sensitivity of the channel to ATP. Mutation to a negatively charged residue markedly decreased the channel ATP sensitivity : the K-i for ATP inhibition increased from 85 mu M to >30 mM when arginine was replaced with aspartic acid. Substitution of neutral residues had inte rmediate effects. 5. The inhibitory effects of ADP, ITP and GTP were also reduced when K185 w as mutated to glutamine or glutamate. 6. The results indicate that a positively charged amino acid at position 18 5 is required for high-affinity ATP binding to Kir6.2. Our results demonstr ate that ATP does not interact with the side-chain of K185. It remains uncl ear whether ATP interacts with the backbone of this residue, or whether its mutation influences ATP binding allosterically.