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Papers In Press, published online ahead of print October 14, 2002
Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520-8026
Corresponding Author: steven.hebert{at}yale.edu
Intracellular ATP and membrane associated phosphatidylinositol phospholipids, like PIP2 [PI(4,5)P2], regulate the activity of ATP-sensitive K+ (KATP) 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) adenosine 5¡¦-triphosphate] to the COOH-terminal cytosolic domains of the pore-forming subunits of Kir1.1 and Kir6.x channels (1). In addition, PIP2 competed for TNP-ATP binding on the COOH-termini of Kir1.1 and Kir6.x channels providing a mechanism that can account for PIP2 antagonism of ATP inhibition of these channels (2). To localize the ATP-binding site within the COOH-terminus of Kir1.1, we produced and purified MBP fusion proteins containing truncated and/or mutated Kir1.1 COOH-termini and examined the binding of TNP-ATP and competition by PIP2. A truncated COOH-terminal fusion protein construct, MBP_1.1Cƒ´C170, containing the first 39 amino acid residues distal to the 2nd 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 NH2-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. PIP2 competed TNP-ATP binding to MBP_1.1Cƒ´C170 with an EC50 of 10.9 ƒÝM. Mutation of any one of three arginine residues (R188A/E, R203A and R217A), that are conserved in Kir1.1 and KATP channels and are involved in ATP and/or PIP2 effects on channel activity, dramatically reduced TNP-ATP binding to MBP_1.1ƒ´C170. In contrast, mutation of a 4th conserved residue (R212A) exhibited slightly enhanced TNP-ATP binding and increased affinity for PIP2 competition of TNP-ATP (EC50 = 5.7 ƒÝM). These studies suggest that the first 39 COOH-terminal amino acid residues form an ATP-PIP2 binding domain in Kir1.1 and possibly the Kir6.x ATP-sensitive K+ channels.
J. Biol. Chem, 10.1074/jbc.M208679200
Submitted on August 23, 2002
Revised on October 8, 2002
Accepted on October 14, 2002
Localization of the ATP/PIP2-binding site to a 39 amino acid region of the carboxy-terminus of the ATP-regulated K+ channel Kir1.1
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