The carboxyl termini of K_ATP channels bind nucleotides

ATP-sensitive potassium (K_ATP) channels are expressed in many excitable, as well as epithelial, cells and couple metabolic changes to modulation of cell activity. ATP regulation of K_ATP channel activity may involve direct binding of this nucleotide to the pore-forming inward rectifier (Kir) subunit despite the lack of known nucleotide-binding motifs. To examine this possibility, we assessed the binding of the fluorescent ATP analogue, 2′,3′-O -(2,4,6- trinitrophenylcyclo-hexadienylidene)adenosine 5′- triphosphate (TNP-ATP) to maltose-binding fusion proteins of the NH₂- and COOH-terminal cytosolic regions of the three known K_ATP channels (Kir1.1, Kir6.1, and Kir6.2) as well as to the COOH-terminal region of an ATP-insensitive inward rectifier K⁺ channel (Kir2.1). We show direct binding of TNP-ATP to the COOH termini of all three known K_ATP channels but not to the COOH terminus of the ATP-insensitive channel, Kir2.1. TNP-ATP binding was specific for the COOH termini of K_ATP channels because this nucleotide did not bind to the NH₂ termini of Kir1.1 or Kir6.1. The affinities for TNP-ATP binding to K_ATP COOH termini of Kir1.1, Kir6.1, and Kir6.2 were similar. Binding was abolished by denaturing with 4 M urea or SDS and enhanced by reduction in pH. TNP-ATP to protein stoichiometries were similar for all K_ATP COOH-terminal proteins with 1 mol of TNP-ATP binding/ mole of protein. Competition of TNP-ATP binding to the Kir1.1 COOH terminus by MgATP was complex with both Mg²⁺ and MgATP effects. Glutaraldehyde cross-linking demonstrated the multimerization potential of these COOH termini, suggesting that these cytosolic segments may directly interact in intact tetrameric channels. Thus, the COOH termini of K_ATP tetrameric channels contain the nucleotide-binding pockets of these metabolically regulated channels with four potential nucleotide-binding sites/channel tetramer.