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J. Biol. Chem., Vol. 281, Issue 40, 29817-29829, October 6, 2006

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 281/40/29817    most recent M606152200v1 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 Ding, J. Articles by Fliegel, L. Search for Related Content PubMed PubMed Citation Articles by Ding, J. Articles by Fliegel, L. Social Bookmarking                      What's this?

Structural and Functional Characterization of Transmembrane Segment VII of the Na⁺/H⁺ Exchanger Isoform 1^*

Jie Ding¹², Jan K. Rainey¹³, Caroline Xu, Brian D. Sykes⁴, and Larry Fliegel⁵

From the Department of Biochemistry and Protein Engineering Network of Centres of Excellence, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

The Na⁺/H⁺ exchanger isoform 1 is an integral membrane protein that regulates intracellular pH by exchanging one intracellular H⁺ for one extracellular Na⁺. It is composed of an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the structural and functional aspects of the critical transmembrane segment VII (TM VII, residues 251–273) by using alanine scanning mutagenesis and high resolution NMR. Each residue of TM VII was mutated to alanine, the full-length protein expressed, and its activity characterized. TM VII was sensitive to mutation. Mutations at 13 of 22 residues resulted in severely reduced activity, whereas other mutants exhibited varying degrees of decreases in activity. The impaired activities sometimes resulted from low expression and/or low surface targeting. Three of the alanine scanning mutant proteins displayed increased, and two displayed decreased resistance to the Na⁺/H⁺ exchanger isoform 1 inhibitor EMD87580. The structure of a peptide of TM VII was determined by using high resolution NMR in dodecylphosphocholine micelles. TM VII is predominantly -helical, with a break in the helix at the functionally critical residues Gly²⁶¹–Glu²⁶². The relative positions and orientations of the N- and C-terminal helical segments are seen to vary about this extended segment in the ensemble of NMR structures. Our results show that TM VII is a critical transmembrane segment structured as an interrupted helix, with several residues that are essential to both protein function and sensitivity to inhibition.

Received for publication, June 27, 2006 , and in revised form, July 20, 2006.

^* 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 atomic coordinates and structure factors (code 2HTG) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The on-line version of this article (available at http://www.jbc.org) contains supplemental text, Figs. S1 and S2, and supplemental Refs. 1–10.

¹ Both authors contributed equally to this work.

² Supported by Alberta Heritage Foundation for Medical Research and the Canadian Institutes of Health Research Strategic Training Institute in Membrane Proteins and Cardiovascular Disease.

³ Recipient of postdoctoral fellowships from the Natural Sciences and Engineering Research Council of Canada, the Alberta Heritage Foundation for Medical Research, and the Canadian Institutes of Health Research Strategic Training Institute in Membrane Proteins and Cardiovascular Disease.

⁴ Recipient of support as a Canada Research Chair in Structural Biology.

⁵ Supported by a grant from the Canadian Institutes of Health Research and a Scientist Award from Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed: Dept. of Biochemistry, 347 Medical Science Bldg., University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-1848; Fax: 780-492-0886; E-mail: lfliegel{at}ualberta.ca.

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