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This Article Full Text Full Text (PDF) All Versions of this Article: 568/3/931    most recent jphysiol.2005.095638v1 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 John, S. A Articles by Ribalet, B. Search for Related Content PubMed PubMed Citation Articles by John, S. A Articles by Ribalet, B.

¹ UCLA Cardiovascular Research Laboratory and Departments of
² Physiology
³ Medicine (Cardiology), UCLA School of Medicine, Los Angeles, CA 90095, USA

ATP-sensitive K (K_ATP) channels are composed of Kir6, the pore-forming protein, and the sulphonylurea receptor SUR, a regulatory protein. We and others have previously shown that positively charged residues in the C terminus of Kir6.2, including R201 and K185, interact with the and ß phosphate groups of ATP, respectively, to induce channel closure. A positively charged residue in the N terminus, R50, is also important, and has been proposed to interact with either the or ß phosphate group of ATP. To examine this issue, we systematically mutated R50 to residues of different size, charge and hydropathy, and examined the effects on adenine nucleotide sensitivity in the absence and presence of SUR1. In the absence of SUR1, only the size of residue 50 significantly altered ATP sensitivity, with smaller side chains decreasing ATP sensitivity. In the presence of SUR1, however, hydrophathy and charge also played a role. Hydrophilic residues decreased ATP sensitivity more than hydrophobic residues for small size residues, and, surprisingly, negatively charged residues E and D preserved ATP sensitivity and increased ADP sensitivity relative to the wild-type residue R. These observations suggest that a negative charge near position 50, due to either mutation of R50 or the interaction of the phosphate group of ATP with R50, facilitates closure of the ATP-dependent gate. Mutation of the nearby positively charged residue R54, known to be involved in stabilizing channel opening via electrostatic interactions with phosphatidylinositol 4,5-bisphosphate (PIP₂), also caused increased ADP sensitivity as compared with ATP, suggesting a loss of function of ATP's phosphate. Based on these results, we propose that a phosphate group or a negative charge at position 50 initiates channel closure by destabilizing the electrostatic interactions between negative phosphate groups of PIP₂ and residues such as R54.

(Received 29 July 2005; accepted after revision 8 September 2005; first published online 15 September 2005)
Corresponding author B. Ribalet: Department of Physiology, School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA. Email: bribalet{at}mednet.ucla.edu

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