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)

Abstract

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.

Differential regulation of NFAT5 by NKCC2 isoforms in medullary thick ascending limb (mTAL) cells

“…induced by high NaCl concentration is consistent with previous reports that this isoform exhibits a high-capacity transport function (16,20,47). The ability of this isoform to regulate NFAT5 expression as well as transcriptional activity is complementary to high-capacity transport as it may provide a sensitive mechanism that protects TAL cells from increases in hypertonicity.…”

Intramolecular and Intermolecular Fluorescence Resonance Energy Transfer in Fluorescent Protein-tagged Na-K-Cl Cotransporter (NKCC1)

“…6 Thus, we attempted insertion of FPs in a region of the C terminus that is locally very poorly conserved among NKCCs, is absent in KCCs, and has a high degree of predicted disorder using PONDR (27) or DisEMBL (28) (Fig. 1A).…”

“…h s lo 6 with the cotransfectant of hslo and NKCC (k). However, the very small magnitude of the latter changes argues that the unusual properties of N-terminal-tagged NKCC1 are in some way responsible for the largest fraction of the effect.…”

“…NKCC1 and NKCC2 are activated by decreases in cell volume and cell [Cl Ϫ ] through phosphorylation of a regulatory domain that is present in the N terminus of both transporters (1,2), probably by SPAK kinase (3,4). Ion transport properties are thought to be determined by 12 transmembrane domains and a proposed re-entrant loop structure between transmembrane domains 1 and 2 (5,6). The mechanism by which the phosphoregulatory domain in the N terminus interacts with the transmembrane domain or the large C terminus to control function is completely unknown.…”

Enhanced insulin secretion and improved glucose tolerance in mice with homozygous inactivation of the Na+K+2Cl− co-transporter 1

“…05-0 . 1 mM; IC 50 NKCC2 Zw2-3 mM; Gamba 2005), and both transporters exhibit different ion affinities and transport rates (Isenring et al 1998, Gimenez & Forbush 2007. Therefore, we examined the impact of a single dose of BTD on glucose tolerance.…”