Molecular characteristics and physiological roles of Na+ -K+ -Cl- cotransporter 2

Abstract

Na⁺ -K⁺ -Cl^- cotransporter 2 (NKCC2; SLC12A1) is an integral membrane protein that comes as three splice variants and mediates the cotranslocation of Na⁺ , K⁺ , and Cl^- ions through the apical membrane of the thick ascending loop of Henle (TALH). In doing so, and through the involvement of other ion transport systems, it allows this nephron segment to reclaim a large fraction of the ultrafiltered Na⁺ , Cl^- , Ca²⁺ , Mg²⁺ , and HCO₃^- loads. The functional relevance of NKCC2 in human is illustrated by the many abnormalities that result from the inactivation of this transport system through the use of loop diuretics or in the setting of inherited disorders. The following presentation aims at discussing the physiological roles and molecular characteristics of Na⁺ -K⁺ -Cl^- cotransport in the TALH and those of the individual NKCC2 splice variants more specifically. Many of the historical and recent data that have emerged from the experiments conducted will be outlined and their larger meaning will also be placed into perspective with the aid of various hypotheses.

Keywords: Bartter syndrome; Na+-K+-Cl− cotransporter 2; cation-Cl− cotransporter; high blood pressure; loop of Henle; renal physiology.

Figure 1

Hydropathy plot model of NKCC2 and phylogram of CCC family. (a) Residues are shown as round or square forms (one form per residue) and putative glycosylation sites as branched lines. Residues in yellow are those of the alternatively spliced exon, residue in blue differ among the variants, and residues in green correspond to known phosphoacceptor sites in the N‐terminus. The model was drawn using the program PLOT. (b) The tree was constructed through the programs Clustal Omega and FigTree v1.4.3 (Sievers et al., 2011) using the longest human (hu) residue sequences for each of the CCCs shown. Members of this family belong to four different subclasses. The scale corresponds to a genetic distance. Sequences used: NKCC1, NP_001037.1; NKCC2, NP_000329.2; NCC, NP_000330.2; KCC1, NP_005063.1; KCC2, NP 001128243.1; KCC3, NP_598408.1; KCC4, NP_006589.2; CCC8, NP_064631.2; CCC9, NP_078904.3. CCC, cation‐Cl⁻ cotransporter; KCC1, K⁺–Cl⁻ cotransporter 1; NCC, Na⁺–Cl⁻ cotransporter; NKCC2, Na⁺–K⁺–Cl⁻ cotransporter 2

Figure 2

NKCC2 splice variants. The primary transcript counts 97,776 nucleotides from the start of exon A (before exon 1) to the end of exon 26. It is represented in this figure by boxes (for the exons) and lines (for the untranslated regions) that are drawn to scale on the horizontal axis. Regions in orange correspond to neosequences before a polyadenylation site in intron 16. Because of this polyadenylation site, the NKCC2∆CT transcripts should thus form typical polyA‐tailed mRNA. Variants NKCC2B, NKCC2A, and NKCC2F are found in several species such as hu, rabbit (rb), and mouse (ms) kidney while variants NKCC2B∆CT, NKCC2A∆CT, and NKCC2F∆CT are only found in ms kidney (Mount, Baekgaard et al., 1999). I, residues encoded from intronic sequences; mRNA, messenger RNA; NKCC2, Na⁺–K⁺–Cl⁻ cotransporter 2; ∆CT, variants in which exons 17–26 (corresponding to most of the C‐terminus) are missing; *, stop codon

Figure 3

Residue sequences of exon 4 in Na⁺–K⁺–Cl⁻ cotransporter 2 (NKCC2). Blue is used to indicate that residues differ among variants (NKCC2B, NKCC2A, and NKCC2F) and species (rb, hu, and ms). Residues in the gray box belong to transmembrane domain 2 and residues in the white box to composite site 1

Figure 4

Distribution of Na⁺–K⁺–Cl⁻ cotransporter 2 (NKCC2) splice variants in adult ms kidney by in situ hybridization. Exon 4 was detected for each variant with a specific radioactive riboprobe on a sagittal tissue section, signals were converted to colors (red for NKCC2B, green for NKCC2A, and blue for NKCC2F) and images were superimposed. Adapted with permission from Igarashi et al. (1995). The color mount was generated by Dr Biff Forbush (Yale University, CT)

Figure 5

Apparent affinities of Na⁺–K⁺–Cl⁻ cotransporter 2 (NKCC2) splice variants for Na⁺, Rb⁺, and Cl⁻. The data shown correspond to mean K _m ratios ± standard error among three independent studies (as described in the text). In each study, these ratios were obtained by dividing the mean K _m determined for either variant with the mean K _m determined for NKCC2F. These constants are all seen to be higher for NKCC2F and analogous between NKCC2A and NKCC2B. On the basis of our observations, V _max was also found to be higher for NKCC2F and similar between NKCC2A and NKCC2B (not shown)

Figure 6

Structure of zebrafish Na⁺–K⁺–Cl⁻ cotransporter 1 based on a cryo‐electron microscopy density map. (a) Topology model of a monomer. Are shown: the transmembrane domains, extracellular loops (ELs), intracellular loops (ILs; also called connecting loops [CLs] in the text), linker helix (α0), as well as α‐ and β‐helices in the C‐terminus. (b) Structural model of a dimer. Are seen: the membrane‐associated domain (MAD) and the C‐termini (CTs). Adapted with permission from Xu et al. (1994)

Figure 7

Model of ion transport and causes of NKCC2 dysfunction in the TALH. Ion transport: K⁺ recycling through K⁺ channels (b) in the apical TALH and through K⁺ channels (not shown) and the Na⁺/K⁺ ATPase pump (f) in the basolateral TALH cause both the luminal and serosal fluid to be positively charged (Greger, 1985; Greger & Schlatter, 1981; Hamilton & Devor, 2012; Hebert & Andreoli, 1984; Hebert, Culpepper, & Andreoli, 1981; Hurst, Duplain, & Lapointe, 1992). However, outwardly directed Cl⁻ conductive pathways (c–e) in the basolateral TALH decreases the effect of K⁺ recycling on the serosal side such that active NaCl reabsorption in this nephron segment causes the luminal‐to‐serosal transepithelial potential to increase along with the paracellular reabsorption of certain cations (Gu et al., 2009; Guinamard, Chraibi, & Teulon, 1995; Winters, Zimniak, Mikhailova, Reeves, & Andreoli, 2000; Yoshitomi, Koseki, Taniguchi, & Imai, 1987). NKCC2 dysfunction: Different types of Bartter syndromes (BS) are listed along with an overview of their clinical manifestations (see Cunha and Heilberg (2018) as to why these manifestations vary among the types). The color of the headings “BS” match those of the proteins affected. BSIVB appears in various shades given that it is associated with pathogenic mutations in both CLCNKA and CLCNKB. In this figure, the salt‐losing nephropathy that has been linked to melanoma antigen D2 has term BSVI arbitrarily. The proteins shown are (a) NKCC2 (SLC12A1), (b) ROMK2 or ROMK3 (KCNJ1), (c) CLCNKB, (d) Barttin, (e) CLCNKA, (f) the Na⁺/K⁺ ATPase, (g) Ca²⁺‐sensing receptor, (h) melanoma antigen D2, and (i) claudins (isoforms 14, 16, or 19). N, normal; NKCC2, Na⁺–K⁺–Cl⁻ cotransporter 2; TALH, thick ascending loop of Henle