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J. Biol. Chem., Vol. 283, Issue 32, 22018-22030, August 8, 2008

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Structural and Functional Characterization of Transmembrane Segment IX of the NHE1 Isoform of the Na⁺/H⁺ Exchanger^*

Tyler Reddy¹², Jie Ding¹³, Xiuju Li, Brian D. Sykes⁴, Jan K. Rainey^¶, and Larry Fliegel, Recipient of an Alberta Heritage Foundation for Medical Research Scientist award⁵

From the Department of Biochemistry and Molecular Biology and ^¶Department of Chemistry, Dalhousie University, Halifax, Nova Scotia B3H 1X5 and the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

The Na⁺/H⁺ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by removing one intracellular H⁺ in exchange for one extracellular Na⁺. It has a large N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the putative transmembrane segment IX (residues 339–363). Each residue was mutated to cysteine in a functional cysteineless NHE1 protein. Of 25 amino acids mutated, 5 were inactive or nearly so after mutation to cysteine. Several of these showed aberrant targeting to the plasma membrane and reduced expression of the intact protein, whereas others were expressed and targeted correctly but had defective NHE1 function. Of the active mutants, Glu³⁴⁶ and Ser³⁵¹ were inhibited >70% by positively charged [2-(trimethylammonium)-ethyl]methanethiosulfonate but not by anionic [2-sulfonatoethyl]methanethiosulfonate, suggesting that they are pore lining and make up part of the cation conduction pathway. Both mutants also had decreased affinity for Na⁺ and decreased activation by intracellular protons. The structure of a peptide representing amino acids 338–365 was determined by using high resolution NMR in dodecylphosphocholine micelles. The structure contained two helical regions (amino acids Met³⁴⁰–Ser³⁴⁴ and Ile³⁵³–Ser³⁵⁹) kinked with a large bend angle around a pivot point at amino acid Ser³⁵¹. The results suggest that transmembrane IX is critical with pore-lining residues and a kink at the functionally important residue Ser³⁵¹.

Received for publication, May 6, 2008 , and in revised form, May 21, 2008.

The atomic coordinates and structure factors (code 2k3c) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). Resonance assignments have been deposited in the BioMagResBank entry 15747.

^* This work was supported in part by grants from Canadian Institutes of Health Research (to L. F. and B. D. S.), from Dalhousie University, the E. Gordon Young Endowment Fund, and the Natural Sciences and Engineering Research Council of Canada (to J. K. R.). 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.

¹ Both authors contributed equally to this work.

² Supported by a Natural Sciences and Engineering Research Council Canada Graduate Scholarship (CGS-M) and recipient of critical professional development funding from the Nova Scotia Health Research Foundation.

³ Recipient of funding from the Alberta Heritage Foundation for Medical Research and from the Canadian Institutes of Health Research Strategic Training Initiative in Membrane Proteins and Cardiovascular Disease.

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

⁵ 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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This article has been cited by other articles:

				B. L. Lee, X. Li, Y. Liu, B. D. Sykes, and L. Fliegel Structural and Functional Analysis of Transmembrane XI of the NHE1 Isoform of the Na+/H+ Exchanger J. Biol. Chem., April 24, 2009; 284(17): 11546 - 11556. [Abstract] [Full Text] [PDF]