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J. Biol. Chem., Vol. 280, Issue 37, 32555-32563, September 16, 2005

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Molecular Mechanisms of Cation Transport by the Renal Na⁺-K⁺-Cl^- Cotransporter

STRUCTURAL INSIGHT INTO THE OPERATING CHARACTERISTICS OF THE ION TRANSPORT SITES^*

From the Nephrology Research Group, Department of Medicine, Faculty of Medicine, Laval University, Québec G1R 2J6, Canada

Two variants of the renal Na⁺-K⁺-Cl^- cotransporter (NKCC2), called NKCC2A and NKCC2F, display marked differences in Na⁺, Rb⁺, and Cl^- affinities, yet are identical to one another except for a 23-residue membrane-associated domain that is derived from alternatively spliced exons. The proximal portion of these exons is predicted to encode the second transmembrane domain (tm2) in the form of an -helix, and the distal portion, part of the following connecting segment (cs1a). In recent studies, we have taken advantage of the A–F differences in kinetic behavior to determine which regions in tm2-cs1a are involved in ion transport. Functional characterizations of chimeras in which tm2 or cs1a were interchanged between the variants showed that both regions are important in specifying ion affinities, but did not allow delineating the contribution of individual residues. Here, we have extended these structure-function analyses by studying additional mutants in which variant residues between A and F were interchanged individually in the tm2-cs1a region (amino acid number 216, 220, 223, 229, or 233 in NKCC2). None of the substitutions were found to affect K_{m (C1-)}, suggesting that the affinity difference for anion transport is conveyed by a combination of variant residues in this domain. However, 2 substitutions in the tm2 of F were found to affect cation constants specifically; interestingly, one of these mutations (residue 216) only affected K_{m (Rb+)} while the other (residue 220) only affected K_{m (Na+)}. We have thus identified two novel residues in NKCC2 that play a key role in cation transport. Because such residues should be adjacent to one another on the vertical axis of the tm2 -helix, our results imply, furthermore, that the ion transport sites in NKCC2 could be physically linked.

Received for publication, May 19, 2005 , and in revised form, June 29, 2005.

^* This work was supported by Grants MOP-68949 and MOP-15405 from the Canadian Institute of Health and Research and the Kidney Foundation of Canada. 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 A Fonds de Recherche en Santé du Québec (FRSQ) scholar.

² FRSQ Junior II and Associate Professor of Medicine at Laval University. To whom correspondence should be addressed: L'Hôtel-Dieu de Québec Research Center, 10 Rue McMahon (Rm. 3852), Québec (QC) G1R 2J6, Canada. Tel.: 418-691-5151 (15477); Fax: 418-691-5787; E-mail: paul.isenring{at}crhdq.ulaval.ca.

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