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

Abstract

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 structurefunction 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.

The Residues Determining Differences in Ion Affinities among the Alternative Splice Variants F, A, and B of the Mammalian Renal Na-K-Cl Cotransporter (NKCC2)

“…By itself, this degree of conservation suggests a central role in the membrane transport function of the protein, and we have noted that the periodic spacing of these conserved residues suggests a functionally important ␣-helical structure of the first part of ICL1 (14). This hypothesis has been strongly supported by the preliminary evaluation of the work reported here (15), as well as by related studies in shark NKCC2 (16,17).…”

“…The current study and those of others (16,17) have highlighted residues that determine ion affinities in NKCC2 and are presumed to be part of the translocation pocket, complementing related findings in NKCC1 (2).…”

Homooligomeric and Heterooligomeric Associations between K+-Cl– Cotransporter Isoforms and between K+-Cl– and Na+-K+-Cl– Cotransporters

“…In contrast to NKCC1, for instance, the KCCs behave as Na ϩ -independent carriers that are modestly sensitive to bumetanide (4,16,25,26,32,33), and compared with KCC1, KCC2 exhibits higher affinity for Cl Ϫ (16,(22)(23)(24)(25). Heterooligomers that are composed of NKCC1 and a KCC or of different KCCs could thus display characteristics that differ substantially from those of NKCC1 or KCC homooligomers, leading to diversity in the physiological and pharmacologic properties of cation-Cl Ϫ cotransport among various tissues.…”

“…An alternative scenario would be that all of the CCCs can interact with both Na ϩ and K ϩ but that they exhibit a preference for K ϩ when the subunits at work only include KCC isoforms. This scenario would explain why the KCCs behave as Na ϩ -independent systems when expressed heterologously (4 -8, 16, 22-26) and why NKCC1 can mediate some levels of K ϩ -Cl Ϫ cotransport in the absence of extracellular Na ϩ (13,32,33,46). In the current study, we have begun to address the interesting question of whether oligomeric assemblies between certain CCCs could lead to the formation of functionally unique structures by conducting ion transport studies in oocytes injected with NKCC1 with or without KCC4.…”

“…5). As previously described (15,25,26,32,33), this protocol included sequential incubations (at ϳ22°C) in various media (detailed in 86 Rb ϩ was detected with the TopCountNXT counter (Packard). For each condition tested, counts were averaged and converted into flux rates as explained in the legend to Fig.…”