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

Abstract

1 ATP-sensitive potassium (K_ATP) channels are composed of pore-forming Kir6.2 and regulatory SUR subunits. A truncated isoform of Kir6.2, Kir6.2ΔC26, 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 position 185 (K185) in the C-terminus of Kir6.2 to glutamine, decreased the channel sensitivity to ATP without affecting the single-channel conductance or the intrinsic channel kinetics. This mutation also impaired 8-azido[³²P]-ATP binding to Kir6.2. 3 To determine if K185 interacts directly with ATP, we made a range of mutations at this position, and examined the effect on the channel ATP sensitivity by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing wild-type or mutant Kir6.2ΔC26. 4 Substitution of K185 by a positively charged amino acid (arginine) had no 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 μM to >30 mM when arginine was replaced with aspartic acid. Substitution of neutral residues had intermediate effects. 5 The inhibitory effects of ADP, ITP and GTP were also reduced when K185 was mutated to glutamine or glutamate. 6 The results indicate that a positively charged amino acid at position 185 is required for high-affinity ATP binding to Kir6.2. Our results demonstrate that ATP does not interact with the side-chain of K185. It remains unclear whether ATP interacts with the backbone of this residue, or whether its mutation influences ATP binding allosterically.