title: Localization of the ATP/phosphatidylinositol 4,5 diphosphate-binding site to a 39-amino acid region of the carboxyl terminus of the ATP-regulated K+ channel Kir1.1.
abstract: Intracellular ATP and membrane-associated phosphatidylinositol phospholipids, like PIP(2) (PI(4,5)P(2)), regulate the activity of ATP-sensitive K(+) (K(ATP)) and Kir1.1 channels by direct interaction with the pore-forming subunits of these channels. We previously demonstrated direct binding of TNP-ATP (2',3'-O-(2,4,6-trinitrophenylcyclo-hexadienylidene)-ATP) to the COOH-terminal cytosolic domains of the pore-forming subunits of Kir1.1 and Kir6.x channels. In addition, PIP(2) competed for TNP-ATP binding on the COOH termini of Kir1.1 and Kir6.x channels, providing a mechanism that can account for PIP(2) antagonism of ATP inhibition of these channels. To localize the ATP-binding site within the COOH terminus of Kir1.1, we produced and purified maltose-binding protein (MBP) fusion proteins containing truncated and/or mutated Kir1.1 COOH termini and examined the binding of TNP-ATP and competition by PIP(2). A truncated COOH-terminal fusion protein construct, MBP_1.1CDeltaC170, containing the first 39 amino acid residues distal to the second transmembrane domain was sufficient to bind TNP-ATP with high affinity. A construct containing the remaining COOH-terminal segment distal to the first 39 amino acid residues did not bind TNP-ATP. Deletion of 5 or more amino acid residues from the NH(2)-terminal side of the COOH terminus abolished nucleotide binding to the entire COOH terminus or to the first 49 amino acid residues of the COOH terminus. PIP(2) competed TNP-ATP binding to MBP_1.1CDeltaC170 with an EC(50) of 10.9 microm. Mutation of any one of three arginine residues (R188A/E, R203A, and R217A), which are conserved in Kir1.1 and K(ATP) channels and are involved in ATP and/or PIP(2) effects on channel activity, dramatically reduced TNP-ATP binding to MBP_1.1DeltaC170. In contrast, mutation of a fourth conserved residue (R212A) exhibited slightly enhanced TNP-ATP binding and increased affinity for PIP(2) competition of TNP-ATP (EC(50) = 5.7 microm). These studies suggest that the first 39 COOH-terminal amino acid residues form an ATP-PIP(2) binding domain in Kir1.1 and possibly the Kir6.x ATP-sensitive K(+) channels.
authors.affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8026, USA.
authors.forename: Ke
authors.initials: K
authors.lastname: Dong
authors.forename: LieQi
authors.initials: L
authors.lastname: Tang
authors.forename: Gordon G
authors.initials: GG
authors.lastname: MacGregor
authors.forename: Steven C
authors.initials: SC
authors.lastname: Hebert
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D007700:Kinetics; D008027:Light; D058619:Maltose-Binding Proteins; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry* / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry* / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid
chemical_list: D002352:Carrier Proteins; C510692:Kcnj1 protein, rat; D058619:Maltose-Binding Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011993:Recombinant Fusion Proteins; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D001120:Arginine