Single residue (K332A) substitution in Kir6.2 abolishes the stimulatory effect of long-chain acyl-CoA esters:: indications for a long-chain acyl-CoA ester binding motif

Aims/hypothesis The pancreatic beta cell ATP-sensitive potassium (K-ATP) channel, composed of the pore-forming a subunit Kir6.2, a member of the inward rectifier K+ channel family, and the regulatory 0 subunit sulfonylurea receptor I (SUR1), a member of the ATP-binding cassette superfamily, couples the metabolic state of the cell to electrical activity. Several endogenous compounds are known to modulate KATp channel activity, including ATP, ADP, phosphatidylinositol diphosphates and long-chain acyl coenzyme A (LC-CoA) esters. LC-CoA esters have been shown to interact with Kir6.2, but the mechanism and binding site(s) have yet to be identified. Materials and methods Using multiple sequence alignment of known acyl-CoA ester interacting proteins, we were able to identify four conserved amino acid residues that could potentially serve as an acyl-CoA ester-binding motif The motif was also recognised in the C-terminal region of Kir6.2 (R311-332) but not in SUR1. Results Oocytes expressing Kir6.2 Delta C26 K332A repeatedly generated K currents in inside-out membrane patches that were sensitive to ATP, but were only weakly activated by 1 mu mol/l palmitoyl-CoA ester. Compared with the control channel (Kir6.2 Delta C26), Kir6.2 Delta C26 K332A displayed unaltered ATP sensitivity but significantly decreased sensitivity to palmitoyl-CoA esters. Coexpression of Kir6.2 Delta C26 K332A and SUR1 revealed slightly increased activation by palmitoyl-CoA ester but significantly decreased activation by the acyl-CoA esters compared with the wild-type K-ATP channel and Kir6.2 Delta C26+SUR1. Computational modelling, using the crystal structure of KirBac1.1, suggested that K332 is located on the intracellular domain of Kir6.2 and is accessible to intracellular modulators such as LC-CoA esters. Conclusions/interpretation These results verify that LCCoA esters interact at the pore-forming subunit Kir6.2, and on the basis of these data we propose an acyl-CoA ester binding motif located in the C-terminal region.