HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 51, 49366-49373, December 20, 2002

This Article Full Text Full Text (PDF) All Versions of this Article: 277/51/49366    most recent M208679200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dong, K. Articles by Hebert, S. C. Search for Related Content PubMed PubMed Citation Articles by Dong, K. Articles by Hebert, S. C. Social Bookmarking                      What's this?

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^*

Ke Dong, LieQi Tang, Gordon G. MacGregor, and Steven C. Hebert^§

From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8026

Intracellular ATP and membrane-associated phosphatidylinositol phospholipids, like PIP₂ (PI(4,5)P₂), 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, 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. 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 PIP₂. A truncated COOH-terminal fusion protein construct, MBP_1.1CC170, 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 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_1.1CC170 with an EC₅₀ of 10.9 µM. 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 PIP₂ effects on channel activity, dramatically reduced TNP-ATP binding to MBP_1.1C170. In contrast, mutation of a fourth conserved residue (R212A) exhibited slightly enhanced TNP-ATP binding and increased affinity for PIP₂ competition of TNP-ATP (EC₅₀ = 5.7 µM). 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.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				O. Pochynyuk, Q. Tong, J. Medina, A. Vandewalle, A. Staruschenko, V. Bugaj, and J. D. Stockand Molecular Determinants of PI(4,5)P2 and PI(3,4,5)P3 Regulation of the Epithelial Na+ Channel J. Gen. Physiol., September 24, 2007; 130(4): 399 - 413. [Abstract] [Full Text] [PDF]

				O. Pochynyuk, Q. Tong, A. Staruschenko, and J. D. Stockand Binding and direct activation of the epithelial Na+ channel (ENaC) by phosphatidylinositides J. Physiol., April 15, 2007; 580(2): 365 - 372. [Abstract] [Full Text] [PDF]

				J. D. Brady, E. D. Rich, J. R. Martens, J. W. Karpen, M. D. Varnum, and R. L. Brown Interplay between PIP3 and calmodulin regulation of olfactory cyclic nucleotide-gated channels PNAS, October 17, 2006; 103(42): 15635 - 15640. [Abstract] [Full Text] [PDF]

				O. Pochynyuk, Q. Tong, A. Staruschenko, H.-P. Ma, and J. D. Stockand Regulation of the epithelial Na+ channel (ENaC) by phosphatidylinositides Am J Physiol Renal Physiol, May 1, 2006; 290(5): F949 - F957. [Abstract] [Full Text] [PDF]

				O. Pochynyuk, A. Staruschenko, Q. Tong, J. Medina, and J. D. Stockand Identification of a Functional Phosphatidylinositol 3,4,5-Trisphosphate Binding Site in the Epithelial Na+ Channel J. Biol. Chem., November 11, 2005; 280(45): 37565 - 37571. [Abstract] [Full Text] [PDF]

				S. C. Hebert, G. Desir, G. Giebisch, and W. Wang Molecular Diversity and Regulation of Renal Potassium Channels Physiol Rev, January 1, 2005; 85(1): 319 - 371. [Abstract] [Full Text] [PDF]

				K. V. Quinn, Y. Cui, J. P. Giblin, L. H. Clapp, and A. Tinker Do Anionic Phospholipids Serve as Cofactors or Second Messengers for the Regulation of Activity of Cloned ATP-Sensitive K+ Channels? Circ. Res., October 3, 2003; 93(7): 646 - 655. [Abstract] [Full Text] [PDF]