This Article Full Text Full Text (PDF) All Versions of this Article: biophysj.104.051474v1 88/4/2597    most recent Alert me when this article is cited Alert me if a correction is posted Services 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 Sackin, H. Articles by Walters, D. E. Search for Related Content PubMed PubMed Citation Articles by Sackin, H. Articles by Walters, D. E.

Structural Locus of the pH Gate in the Kir1.1 Inward Rectifier Channel

Henry Sackin ^*, Mikheil Nanazashvili ^*, Lawrence G. Palmer , M. Krambis ^* and D. E. Walters ^*

^* Department of Physiology and Biophysics, The Chicago Medical School, North Chicago, Illinois; and Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York

Correspondence: Address reprint requests to Dr. Henry Sackin, Dept. of Physiology and Biophysics, The Chicago Medical School, 3333 Green Bay Rd., North Chicago, IL 60064. Tel.: 847-578-8329; Fax: 847-578-3265; E-mail: henry.sackin{at}rosalindfranklin.edu.

The closed-state crystal structure of prokaryotic inward rectifier, KirBac1.1, has implicated four inner helical phenylalanines near the cytoplasmic side as a possible locus of the channel gate. In the present study, we investigate whether this structural feature corresponds to the physiological pH gate of the renal inward rectifier, Kir1.1 (ROMK, KCNJ1). Kir1.1 is endogenous to the mammalian renal collecting duct and the thick ascending limb of Henle and is strongly gated by internal pH in the physiological range. It has four leucines (L160-Kir1.1b), homologous to the phenylalanines of KirBac1.1, which could function as steric gates near the convergence of the inner (M2) helices. Replacing these Leu-160 residues of Kir1.1b by smaller glycines abolished pH gating; however, replacement with alanines, whose side chains are intermediate in size between leucine and glycine, did not eliminate normal pH gating. Furthermore, a double mutant, constructed by adding the I163M-Kir1.1b mutation to the L160G mutation, also lacked normal pH gating, although the I163M mutation by itself enhanced the pH sensitivity of the channel. In addition to size, side-chain hydrophobicity at 160-Kir1.1b was also important for normal pH gating. Mutants with polar side chains (L160S, L160T) did not gate normally and were as insensitive to internal pH as the L160G mutant. Hence, either small or highly polar side chains at 160-Kir1.1b stabilize the open state of the channel. A homology model of the Kir1.1 closed state, based on the crystal structure of KirBac1.1, was consistent with our electrophysiological data and implies that closure of the Kir1.1 pH gate results from steric occlusion of the permeation path by the convergence of four leucines at the cytoplasmic apex of the inner transmembrane helices. In the open state, K crosses the pH gate together with its hydration shell.

This article has been cited by other articles:

				Z. T. Schug, P. C. A. da Fonseca, C. D. Bhanumathy, L. Wagner II, X. Zhang, B. Bailey, E. P. Morris, D. I. Yule, and S. K. Joseph Molecular Characterization of the Inositol 1,4,5-Trisphosphate Receptor Pore-forming Segment J. Biol. Chem., February 1, 2008; 283(5): 2939 - 2948. [Abstract] [Full Text] [PDF]

				S. M. Dravid, K. Erreger, H. Yuan, K. Nicholson, P. Le, P. Lyuboslavsky, A. Almonte, E. Murray, C. Mosley, J. Barber, et al. Subunit-specific mechanisms and proton sensitivity of NMDA receptor channel block J. Physiol., May 15, 2007; 581(1): 107 - 128. [Abstract] [Full Text] [PDF]

				Y.-Y. Zhang, H. Sackin, and L. G. Palmer Localization of the pH Gate in Kir1.1 Channels Biophys. J., October 15, 2006; 91(8): 2901 - 2909. [Abstract] [Full Text] [PDF]

				H. Sackin, M. Nanazashvili, L. G. Palmer, and H. Li Role of Conserved Glycines in pH Gating of Kir1.1 (ROMK) Biophys. J., May 15, 2006; 90(10): 3582 - 3589. [Abstract] [Full Text] [PDF]