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J. Biol. Chem., Vol. 281, Issue 43, 32417-32427, October 27, 2006

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A Cysteine-scanning Mutagenesis Study of Transmembrane Domain 8 of the Electrogenic Sodium/Bicarbonate Cotransporter NBCe1^*

From the Department of Physiology and Biophysics, University of Alabama, Birmingham, Alabama 35294

Na/HCO₃ cotransporters (NBCs) such as NBCe1 are members of a superfamily of bicarbonate transporters that includes anion exchangers. Residues within putative transmembrane domain 8 (TMD8) of anion exchanger 1 are involved in ion translocation (Tang, X. B., Kovacs, M., Sterling, D., and Casey, J. R. (1999) J. Biol. Chem. 274, 3557–3564), and the corresponding domain in NBCe1 variants is highly homologous. We performed cysteine-scanning mutagenesis to examine the role of TMD8 residues in ion translocation by rat NBCe1-A. We accessed function and/or sulfhydryl sensitivity and p-chloromercuribenzene sulfonate (pCMBS) accessibility of 21 cysteine-substituted NBC mutants expressed in Xenopus oocytes using the two-electrode, voltage clamp technique. Five NBC mutants displayed <10% wild-type activity: P743C, A744C, L746C, D754C, and T758C. For the remaining 16 mutants, we compared transporter-mediated inward currents elicited by removing external Na⁺ before and after exposing oocytes to either 2-aminoethylmethane thiosulfonate (MTSEA) or pCMBS. MTSEA inhibited NBC mutants T748C, I749C, I751C, F752C, M753C, and Q756C by 9–19% and stimulated mutants A739C, A741C, L745C, V747C, Q755C, and I757C by 11–21%. pCMBS mildly inhibited mutants A739C, A740, V747C, and Q756C by 5 or 8%, and stimulated I749C by 10%. However, both sulfhydryl reagents strongly inhibited the L750C mutant by 85%. Using the substituted cysteine accessibility method, we examined the accessibility of the NBC mutant L750C under different transporter conditions. pCMBS accessibility is (i) reduced when the transporter is active in the presence of both Na⁺ and , likely due to substrate competition with pCMBS; (ii) reduced in the presence of a stilbene inhibitor; and (iii) stimulated at more positive membrane potentials. In summary, TMD8 residues of NBCe1, particularly L750, are involved in ion translocation, and accessibility is influenced by the state of transporter activity.

Received for publication, July 31, 2006 , and in revised form, August 24, 2006.

^* This work was supported by National Institutes of Health Grant NS 046653 (to M. O. B.) and the American Heart Association, Southeast Affiliate Pre-doctoral Fellowship 0515186B (to S. D. M.). 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 supplemental Fig. S1.

¹ To whom correspondence should be addressed: Dept. of Physiology & Biophysics, University of Alabama at Birmingham, 1918 University Blvd., MCLM 812, Birmingham, AL 35294. Tel.: 205-975-9084; Fax. 205-975-7679; E-mail: bevensee{at}physiology.uab.edu.

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