The carboxyl termini of KATP channels bind nucleotides

Abstract

ATP-sensitive potassium (KATP) channels are expressed in many excitable, as well as epithelial, cells and couple metabolic changes to modulation of cell activity. ATP regulation of KATP 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 KATP 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 KATP channels but not to the COOH terminus of the ATP-insensitive channel, Kir2.1. TNP-ATP binding was specific for the COOH termini of KATP channels because this nucleotide did not bind to the NH2 termini of Kir1.1 or Kir6.1. The affinities for TNP-ATP binding to KATP 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 KATP 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 KATP tetrameric channels contain the nucleotide-binding pockets of these metabolically regulated channels with four potential nucleotide-binding sites/channel tetramer.