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J. Biol. Chem., Vol. 283, Issue 39, 26782-26794, September 26, 2008

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Oligomeric Structure and Minimal Functional Unit of the Electrogenic Sodium Bicarbonate Cotransporter NBCe1-A^*

Liyo Kao, Pakan Sassani, Rustam Azimov, Alexander Pushkin, Natalia Abuladze, Janos Peti-Peterdi, Weixin Liu, Debra Newman, and Ira Kurtz¹

From the Division of Nephrology, David Geffen School Medicine, UCLA, Los Angeles, California 90095-1689 and the Departments of Physiology and Biophysics and Medicine, Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, California 90033

The electrogenic sodium bicarbonate cotransporter NBCe1-A mediates the basolateral absorption of sodium and bicarbonate in the proximal tubule. In this study the oligomeric state and minimal functional unit of NBCe1-A were investigated. Wild-type (wt) NBCe1-A isolated from mouse kidney or heterologously expressed in HEK293 cells was predominantly in a dimeric state as was shown using fluorescence energy transfer, pulldown, immunoprecipitation, cross-linking experiments, and nondenaturing perfluorooctanoate-PAGE. NBCe1-A monomers were found to be covalently linked by S-S bonds. When each of the 15 native cysteine residues were individually removed on a wt-NBCe1-A backbone, dimerization of the cotransporter was not affected. In experiments involving multiple native cysteine residue removal, both Cys⁶³⁰ and Cys⁶⁴² in extracellular loop 3 were shown to mediate S-S bond formation between NBCe1-A monomers. When native NBCe1-A cysteine residues were individually reintroduced into a cysteineless NBCe1-A mutant backbone, the finding that a Cys⁹⁹² construct that lacked S-S bonds functioned normally indicated that stable covalent linkage of NBCe1-A monomers was not a necessary requirement for functional activity of the cotransporter. Studies using concatameric constructs of wt-NBCe1-A, whose activity is resistant to methanesulfonate reagents, and an NBCe1-A^T442C mutant, whose activity is completely inhibited by methanesulfonate reagents, confirmed that NBCe1-A monomers are functional. Our results demonstrate that wt-NBCe1-A is predominantly a homodimer, dependent on S-S bond formation that is composed of functionally active monomers.

Received for publication, May 26, 2008 , and in revised form, July 22, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants DK077162, DK058563, DK063125, ES12935, DK64324, and DK74754. 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 To whom correspondence should be addressed: UCLA Division of Nephrology, 10833 Le Conte Ave., Rm. 7-155 Factor Bldg., Los Angeles, CA 90095-1689. E-mail: ikurtz{at}mednet.ucla.edu.

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