title: 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.
abstract: OBJECTIVE
  The pancreatic beta cell ATP-sensitive potassium (K(ATP)) channel, composed of the pore-forming alpha subunit Kir6.2, a member of the inward rectifier K+channel family, and the regulatory beta subunit sulfonylurea receptor 1 (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 K(ATP) 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.
  
  
  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 Kir6.2DeltaC26 K332A repeatedly generated K(+)currents in inside-out membrane patches that were sensitive to ATP, but were only weakly activated by 1 mumol/l palmitoyl-CoA ester. Compared with the control channel (Kir6.2DeltaC26), Kir6.2DeltaC26 K332A displayed unaltered ATP sensitivity but significantly decreased sensitivity to palmitoyl-CoA esters. Coexpression of Kir6.2DeltaC26 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 Kir6.2DeltaC26+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
  These results verify that LC-CoA 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.
authors.affiliation: Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden. Robert.branstrom@ki.se
authors.forename: R
authors.initials: R
authors.lastname: Bränström
authors.forename: I B
authors.initials: IB
authors.lastname: Leibiger
authors.forename: B
authors.initials: B
authors.lastname: Leibiger
authors.forename: G
authors.initials: G
authors.lastname: Klement
authors.forename: J
authors.initials: J
authors.lastname: Nilsson
authors.forename: P
authors.initials: P
authors.lastname: Arhem
authors.forename: C A
authors.initials: CA
authors.lastname: Aspinwall
authors.forename: B E
authors.initials: BE
authors.lastname: Corkey
authors.forename: O
authors.initials: O
authors.lastname: Larsson
authors.forename: P-O
authors.initials: PO
authors.lastname: Berggren
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism / Q000494:pharmacology*; D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D003981:Diazoxide / Q000494:pharmacology; D005260:Female; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008820:Mice, Obese; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D010171:Palmitoyl Coenzyme A / Q000378:metabolism / Q000494:pharmacology; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014981:Xenopus; D000099161:Kcnj11 Channel
chemical_list: D000214:Acyl Coenzyme A; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003981:Diazoxide; D010171:Palmitoyl Coenzyme A; D024661:Potassium Channels, Inwardly Rectifying; D000099161:Kcnj11 Channel