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Papers In Press, published online ahead of print July 18, 2005
Department of Medicine, Laval University, Québec, Québec G1R2J6
Corresponding Author: paul.isenring{at}crhdq.ulaval.ca
Two variants of the renal Na-K-Cl cotransporter (NKCC2), called NKCC2A and F, 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 an alpha-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 Km(Cl), 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 Km(Rb) while the other (residue 220) only affected Km(Na). We have thus identified 2 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 alpha-helix, our results imply, furthermore, that the ion transport sites in NKCC2 could be physically linked.
J. Biol. Chem, 10.1074/jbc.M505511200
Submitted on May 19, 2005
Accepted on July 18, 2005
Molecular mechanisms of cation transport by the renal Na-K-Cl cotransporter: Structural insight into the operating characteristics of the ion transport sites
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