The role of lysine 185 in the kir6.2 subunit of the ATP-sensitive channel in channel inhibition by ATP.
1. ATP-sensitive potassium (KATP) channels are composed of pore-forming Kir6.2 and regulatory SUR subunits. A truncated isoform of Kir6.2, Kir6.2DeltaC26, 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 Kir6.2DeltaC26. 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 Ki for ATP inhibition increased from 85 microM 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.
Reimann
F
F
University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK.
Ryder
T J
TJ
Tucker
S J
SJ
Ashcroft
F M
FM
D000255:Adenosine Triphosphate / Q000494:pharmacology / Q000502:physiology*; D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D008239:Lysine / Q000502:physiology*; D051379:Mice; D009154:Mutation / Q000502:physiology; D009865:Oocytes / Q000378:metabolism; D015221:Potassium Channels / Q000009:adverse effects / Q000235:genetics* / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D014982:Xenopus laevis
D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D000255:Adenosine Triphosphate; D008239:Lysine