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

Three alternatively spliced variants of the renal Na-K-Cl cotransporter (NKCC2) are found in distinct regions of the thick ascending limb of the mammalian kidney; these variants mediate Na ؉ K ؉ 2Cl ؊ transport with different ion affinities. Here, we examine the specific residues involved in the variant-specific affinity differences, utilizing a mutagenic approach to change the NKCC2B variant into the A or F variant, with functional expression in Xenopus oocytes. The splice region contains the second transmembrane domain (TM2) and the putative intracellular loop (ICL1) connecting TM2 and TM3. It is found that the B variant is functionally changed to the F variant by replacement of six residues, half of the effect brought about by three TM2 residues and half by three ICL1 residues. The involvement of the ICL1 residues strongly suggests that this region of ICL1 may actually be part of a membrane-embedded domain. Changing six residues is also sufficient to bring about the smaller functional change from the B to the A variant; three residues in TM2 appear to be primarily responsible, two of which correspond to residues involved in the B-to-F changes. A B-variant mutation reported in a mild case of Bartter disease was found to render the cotransporter inactive. These results identify the combination of amino acid variations responsible for the differences among the three splice variants of NKCC2, and they support a model in which a reentrant loop following TM2 contributes to the chloride binding and translocation domains.

supporting

confidence: 74%

Section: Residues Responsible For the Difference In Chloride Affinitycontrasting

confidence: 38%

See 1 more Smart Citation

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)

Gíménez

Forbush

2007

“…In some studies, however, alternate transport modes have been proposed, e.g. 2Na ϩ :1K ϩ :3Cl Ϫ for squid NKCC1 (16), 1Na ϩ :1K ϩ :1Cl Ϫ for NKCC2A (17,18), and K ϩ /K ϩ exchange for both NKCC1 and NKCC2A (17)(18)(19). We and others have hypothesized that large differences in Cl Ϫ affinities between the 2 Cl Ϫ binding sites could account for the apparent 1Na ϩ :1K ϩ :1Cl Ϫ stoichiometry (17,18), and incomplete reactions during the transport cycle, for the Na ϩ -independent exchange mode (18 -19).…”

mentioning

confidence: 99%

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

Gagnon

Bergeron

Daigle

et al. 2005

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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.

“…Immunolocalization of KCCs Expressed in X. laevis Oocytes-These studies were performed as described in previous publications by our group (41)(42)(43)(44). Briefly, oocytes cryosections (10 m) were postfixed for 30 min in paraformaldehyde and incubated sequentially with a primary and secondary antibody (for 1 h each at room temperature).…”

Section: B) Kcc4mentioning

confidence: 99%

Identification of Key Functional Domains in the C Terminus of the K+-Cl– Cotransporters

Bergeron¹,

Gagnon²,

Caron

et al. 2006

Self Cite

The K؉ -Cl ؊ cotransporter (KCC) isoforms constitute a functionally heterogeneous group of ion carriers. Emerging evidence suggests that the C terminus (Ct) of these proteins is important in conveying isoform-specific traits and that it may harbor interacting sites for 4␤-phorbol 12-myristate 13-acetate (PMA)-induced effectors. In this study, we have generated KCC2-KCC4 chimeras to identify key functional domains in the Ct of these carriers and single point mutations to determine whether canonical protein kinase C sites underlie KCC2-specific behaviors. Functional characterization of wild-type (wt) and mutant carriers in Xenopus laevis oocytes showed for the first time that the KCCs do not exhibit similar sensitivities to changes in osmolality and that this distinguishing feature as well as differences in transport activity under both hypotonic and isotonic conditions are in part determined by the residue composition of the distal Ct. At the same time, several mutations in this domain and in the proximal Ct of the KCCs were found to generate allosteric-like effects, suggesting that the regions analyzed are important in defining conformational ensembles and that isoform-specific structural configurations could thus account for variant functional traits as well. Characterization of the other mutants in this work showed that KCC2 is not inhibited by PMA through phosphorylation of its canonical protein kinase C sites. Intriguingly, however, the substitutions N728S and S940A were seen to alter the PMA effect paradoxically, suggesting again that allosteric changes in the Ct are important determinants of transport activity and, furthermore, that the structural configuration of this domain can convey specific functional traits by defining the accessibility of cotransporter sites to regulatory intermediates such as PMAinduced effectors.The cation-Cl Ϫ cotransporter (CCC) 3 family includes Na ϩ -dependent and Na ϩ -independent ion carriers (1-8). The latter group is con- (25,26). The importance of the KCCs in these processes is suggested more specifically by the behavior of a KCC4 Ϫ/Ϫ mouse, which was found to exhibit renal tubular acidosis (24), and a KCC2 Ϫ/Ϫ mouse, which was found to exhibit generalized epilepsy (26). The mechanisms that lead to KCC deactivation or activation during a change in cell volume and Cl i Ϫ are still poorly understood despite a large number of studies on the subject of K ϩ -Cl Ϫ cotransport regulation.Based on the effect of various pharmacological agents, a popular model that has emerged over the years is one in which K ϩ -Cl Ϫ cotransport is reduced through carrier phosphorylation and induced through carrier dephosphorylation, and in which such modifications result from the concerted action of protein kinases and protein phosphatases at relevant regulatory sites (14 -17, 27-36). It should be mentioned, however, that most of these studies have addressed the issue of cell volume-dependent, rather than Cl i Ϫ -dependent regulation.Although different types of protein phosphatase and kinases tha...