Functional comparison of renal Na-K-Cl cotransporters  between distant species

Gagnon, Édith; Forbush, Bliss; Caron, Luc; Isenring, Paul

doi:10.1152/ajpcell.00262.2002

Cited by 50 publications

(72 citation statements)

References 28 publications

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“…To some extent, these studies are complementary to observations in NKCC2BϪ/Ϫ mice in which TGF function curves were right-shifted, suggesting that NKCC2B is required to maintain a normal TGF responsiveness to subnormal flow. Overall, our data suggest a successive engagement of NKCC2B and NKCC2A because NaCl concentrations at the MD are altered over the TGF-relevant Cl concentration range between 0 and 60 mM (26), Cl concentrations at which changes of NKCC2 transport activity would be expected on the basis of a K m of approximately 9 to 12 mM for NKCC2B and 22 to 45 mM for NKCC2A (6,10,14).…”

Section: Discussionmentioning

confidence: 84%

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Renal Function in Mice with Targeted Disruption of the A Isoform of the Na-K-2Cl Co-Transporter

Oppermann

Mizel

Kim

et al. 2007

Journal of the American Society of Nephrology

106

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Three different full-length splice isoforms of the Na-K-2Cl co-transporter (NKCC2/BSC1) are expressed along the thick ascending limb of Henle (TAL), designated NKCC2A, NKCC2B, and NKCC2F. NKCC2F is expressed in the medullary, NKCC2B mainly in the cortical, and NKCC2A in medullary and cortical portions of the TAL. NKCC2B and NKCC2A were shown to be coexpressed in the macula densa (MD) segment of the mouse TAL. The functional consequences of the existence of three different isoforms of NKCC2 are unclear. For studying the specific role of NKCC2A in kidney function, NKCC2A؊/؊ mice were generated by homologous recombination. NKCC2A؊/؊ mice were viable and showed no gross abnormalities. Ambient urine osmolarity was reduced significantly in NKCC2A؊/؊ compared with wild-type mice, but water deprivation elevated urine osmolarity to similar levels in both genotypes. Baseline plasma renin concentration and the effects of a high-and a low-salt diet on plasma renin concentration were similar in NKCC2A؉/؉ and ؊/؊ mice. However, suppression of renin secretion by acute intravenous saline loading (5% of body weight), a measure of MD-dependent inhibition of renin secretion, was reduced markedly in NKCC2A؊/؊ mice compared with wild-type mice. Cl and water absorption along microperfused loops of Henle of NKCC2A؊/؊ mice were unchanged at normal flow rates but significantly reduced at supranormal flow. Tubuloglomerular feedback function curve as determined by stop flow pressure measurements was left-shifted in NKCC2A؊/؊ compared with wild-type mice, with maximum responses being significantly diminished. In summary, NKCC2A activity seems to be required for MD salt sensing in the high Cl concentration range. Coexpression of both high-and low-affinity isoforms of NKCC2 may permit transport and Cl-dependent tubuloglomerular feedback regulation to occur over a wider Cl concentration range. T he Na-K-2Cl co-transporter (NKCC2), located in the apical membrane of the epithelial cells of the thick ascending limb of Henle (TAL), constitutes the major cellular uptake pathway in this portion of the nephron. In addition, NKCC2 transport activity in macula densa (MD) cells is considered to be the initial step in the signaling chain that links tubular epithelial cells of the TAL with vascular cells of the afferent arteriole (1-5). At least four different full-length splice isoforms of NKCC2 are expressed along the TAL, designated NKCC2A, NKCC2B, NKCC2F, and NKCC2AF (6 -10). These isoforms of the transporter are derived from differential splicing of the variable exon 4 of the NKCC2 gene (7,8,11). Exon 4 encodes for amino acids of the second transmembrane domain and the adjacent intracellular loop of NKCC2, a region that has been shown to be involved in the ion-binding characteristics of the co-transporter (11). Cell-specific splicing results in heterogeneous expression of the various NKCC2 isoforms along the TAL. NKCC2F, the most abundant NKCC2 isoform, is expressed in the medullary TAL, NKCC2B mainly in the cortical TAL, and NKCC2A both in medullar...

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Section: Discussionmentioning

confidence: 84%

“…to 45, and 110 mM for NKCC2B, NKCC2A, and NKCC2F, respectively (6,10,14). It is conceivable, therefore, that expression of isoforms with different ion affinities allows transport activity in a region where ion concentrations vary over a wide range.…”

mentioning

confidence: 99%

Renal Function in Mice with Targeted Disruption of the A Isoform of the Na-K-2Cl Co-Transporter

Oppermann

Mizel

Kim

et al. 2007

Journal of the American Society of Nephrology

106

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“…The three splice variants of NKCC2 differ in their affinities for the three transported ions, in a way that effectively matches the transport K m values to the concentration of ions in the renal tubule (11)(12)(13). Thus, as concentrated NaCl enters the outer medullary portion of the TAL, it is handled by low affinity NKCC2F, and as the fluid becomes diluted in more distal segments of the TAL, intermediate affinity NKCC2A is appropriately matched for effective transport.…”

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confidence: 99%

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

Journal of Biological Chemistry

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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.

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“…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

Journal of Biological Chemistry

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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...

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Functional comparison of renal Na-K-Cl cotransporters between distant species

Cited by 50 publications

References 28 publications

Renal Function in Mice with Targeted Disruption of the A Isoform of the Na-K-2Cl Co-Transporter

Renal Function in Mice with Targeted Disruption of the A Isoform of the Na-K-2Cl Co-Transporter

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)

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

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