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

J. Biol. Chem., Vol. 280, Issue 47, 39637-39643, November 25, 2005

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 280/47/39637    most recent M509328200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted 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 Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Habibian, R. Articles by Dibrov, P. Search for Related Content PubMed PubMed Citation Articles by Habibian, R. Articles by Dibrov, P. Social Bookmarking                      What's this?

Functional Analysis of Conserved Polar Residues in Vc-NhaD, Na⁺/H⁺ Antiporter of Vibrio cholerae^*

Rahim Habibian, Judith Dzioba, Jeannie Barrett, Michael Y. Galperin1, Peter C. Loewen, and Pavel Dibrov2

From the Department of Microbiology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada and National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894

Vc-NhaD is a Na⁺/H⁺ antiporter from Vibrio cholerae with a sharp maximum of activity at pH 8.0. NhaD homologues are present in many bacteria as well as in higher plants. However, very little is known about structure-function relations in NhaD-type antiporters. In this work 14 conserved polar residues associated with putative transmembrane segments of Vc-NhaD have been screened for their possible role in the ion translocation and pH regulation of Vc-NhaD. Substitutions S150A, D154G, N155A, N189A, D199A, T201A, T202A, S389A, N394G, S428A, and S431A completely abolished the Vc-NhaD-mediated Na⁺-dependent H⁺ transfer in inside-out membrane vesicles. Substitutions T157A and S428A caused a significant increase of apparent K_m values for alkali cations, with the K_m for Li⁺ elevated more than that for Na⁺, indicating that Thr-157 and Ser-428 are involved in alkali cation binding/translocation. Of six conserved His residues, mutation of only His-93 and His-210 affected the Na⁺(Li⁺)/H⁺ antiport, resulting in an acidic shift of its pH profile, whereas H93A also caused a 7-fold increase of apparent K_m for Na⁺ without affecting the K_m for Li⁺. These data suggest that side chains of His-93 and His-210 are involved in proton binding and that His-93 also contributes to the binding of Na ions during the catalytic cycle. These 15 residues are clustered in three distinct groups, two located at opposite sides of the membrane, presumably facilitating the access of substrate ions to the third group, a putative catalytic site in the middle of lipid bilayer. The distribution of these key residues in Vc-NhaD molecule also suggests that transmembrane segments IV, V, VI, X, XI, and XII are situated close to one another, creating a transmembrane relay of charged/polar residues involved in the attraction, coordination, and translocation of transported cations.

Received for publication, August 24, 2005 , and in revised form, September 23, 2005.

^* This research was supported by grants from Natural Sciences and Engineering Research Council of Canada (to P. C. L., P. D., R. H., and J. D.) and the Canada Research Chair Program (to P. C. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental table.

¹ Supported by the Intramural Research Program at the National Library of Medicine, National Institutes of Health.

² To whom correspondence should be addressed: Dept. of Microbiology, University of Manitoba, Fort Garry campus, Rm. 425 Buller Bldg., Winnipeg, Manitoba R3T 2N2 Canada. Tel.: 204-474-8059; Fax: 204-474-7603; E-mail: dibrovp{at}ms.umanitoba.ca.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. Barrero-Gil, A. Rodriguez-Navarro, and B. Benito Cloning of the PpNHAD1 transporter of Physcomitrella patens, a chloroplast transporter highly conserved in photosynthetic eukaryotic organisms J. Exp. Bot., August 1, 2007; 58(11): 2839 - 2849. [Abstract] [Full Text] [PDF]

				E. Olkhova, C. Hunte, E. Screpanti, E. Padan, and H. Michel Multiconformation continuum electrostatics analysis of the NhaA Na+/H+ antiporter of Escherichia coli with functional implications PNAS, February 21, 2006; 103(8): 2629 - 2634. [Abstract] [Full Text] [PDF]