Navigating the multifaceted intricacies of the Na+-Cl- cotransporter, a highly regulated key effector in the control of hydromineral homeostasis

Abstract

The Na⁺-Cl^- cotransporter (NCC; SLC12A3) is a highly regulated integral membrane protein that is known to exist as three splice variants in primates. Its primary role in the kidney is to mediate the cosymport of Na⁺ and Cl^- across the apical membrane of the distal convoluted tubule. Through this role and the involvement of other ion transport systems, NCC allows the systemic circulation to reclaim a fraction of the ultrafiltered Na⁺, K⁺, Cl^-, and Mg⁺ loads in exchange for Ca²⁺ and [Formula: see text]. The physiological relevance of the Na⁺-Cl^- cotransport mechanism in humans is illustrated by several abnormalities that result from NCC inactivation through the administration of thiazides or in the setting of hereditary disorders. The purpose of the present review is to discuss the molecular mechanisms and overall roles of Na⁺-Cl^- cotransport as the main topics of interest. On reading the narrative proposed, one will realize that the knowledge gained in regard to these themes will continue to progress unrelentingly no matter how refined it has now become.

Keywords: Gitelman syndrome; Na+-Cl− cotransporter; SLC12A3; cation-Cl− cotransporter; distal tubule.

Graphical abstract

FIGURE 1.

Functional characterization of the Na⁺-Cl⁻ cotransporter (NCC) during the premolecular era. A: autoradiogram of a slide-mounted rat kidney section prelabeled with [³H]metolazone (MTZ). Signal intensity was considered a marker of NCC abundance and/or Cl⁻ binding by the cotransporter. B: same as A except that the coverslip was examined at higher resolution under oil immersion. C: effects of various conditions, states, factors, or interventions on Na⁺-Cl⁻ cotransport or on NCC abundance. The data shown were mostly from micropuncture/microperfusion studies and [³H]MTZ binding assays. The images displayed in A and B are from Beaumont et al. in Journal of Pharmacology and Experimental Therapeutics (35). They are reproduced in this figure with permission by the copyright holders.

FIGURE 2.

Structural feature of the Na⁺-Cl⁻ cotransporter (NCC). A: hydropathy plot model of rat (rt)NCC3/rtNCC1A. Residues are shown as round or square forms (1 form per residue) and the glycosylation site of rtNCC3/rtNCC1A as a branched line. x and y show how rtNCC3/rtNCC1A differ from the human variants (huNCC1/huNCC1B, huNCC2/huNCC3C, and huNCC3/huNCC1C) in residue composition. Color code used is indicated at right in D. The model was drawn using the program PLOT. B–D: hydropathy plot model, structure, and ion binding sites of huNCC based on cryo-EM determinations. Color code used is indicated at right in D. Ct, COOH terminus; EL, extracellular loop; IL, intracellular loop; Nt, NH₂ terminus; TMD, transmembrane domain. See glossary for additional abbreviations.

FIGURE 3.

Phylogenetic analyses. A: phylogram of functionally active human cation-Cl⁻ cotransporter (huCCC) isoforms. Na⁺-Cl⁻ cotransporter (NCC)β (also called SLC12A10, NCC2, or CCC10) is thus excluded. Sequences used: NKCC1, NP_001037.1; NKCC2, NP_000329.2; NCC, NP_000330.2; K⁺-Cl⁻ cotransporter (KCC)1, NP_005063.1; KCC2, NP_001128243.1; KCC3, NP_598408.1; KCC4, NP_006589.2; CCC8, NP_064631.2; and CCC9, NP_078904.3. B: phylogram of functionally active Danio rerio (dr)CCC, Equus caballus (ec)CCC, and Equus quagga (eq)CCC isoforms. NCCβ is thus included. Sequences used: drNKCC1, NP_001002080.1; drNKCC2, XP_021323409.1; drNCC, NP_001038545.1; drKCC1, XP_691291.2; drKCC4, XP_696060.6; drCCC8, NP_001122020.1; drCCC9, XP_005167859.1; drNCCβ, NP_001154850.2; ecNKCC1, XP_005599514.2; ecNKCC2, XP_003363557.1; ecNCC, XP_014593920.2; ecKCC1, XP_001498498.1; ecKCC4, XP_023481655.1; ecCCC8, XP_001505118.1; ecCCC9, XP_023479371.1; ecNCCβ, LC727702.1; eqNKCC1, XP_046522358.1; eqNKCC2, XP_046510184.1; eqNCC, XP_046540395.1; eqKCC1, XP_046538230.1; eqKCC4, XP_046527564.1; eqCCC8, XP_046521243.1; eqCCC9, XP_046514771.1; and eqNCCβ, XM_046655358.1. The trees were constructed with the programs Clustal Omega and FigTree, using the longest residue sequences for each of the CCCs. In each panel, the scale corresponds to a genetic distance.

FIGURE 4.

Proposed model of ion binding by the Na⁺-Cl⁻ cotransporter (NCC). In the scheme presented, the transport cycle involves 4 ion binding sites (2 for Na⁺ and 2 for Cl⁻) and is based on ordered binding and glide symmetry where first on is first off. This scheme is supported by indirect evidence through [³H]metolazone (MTZ) binding assays and the characterization of NKCC1 (34, 41, 125). The model depicted can be summarized as follows. When the carrier is empty, it adopts an outward-open configuration (1) and undergoes disocclusion (2) to be loaded from the extracellular side by 4 ions (Na1, Cl1, Na2, and Cl2) in successive steps (3–6). Once it is loaded, the carrier becomes occluded momentarily (7), adopts an inward-open configuration (8), and undergoes disocclusion once again (9) to release all 4 ions internally in the same order (10–13). When the carrier is no longer occupied by ions (14), it undergoes another episode of occlusion (15) and is translocated back to the outward-open configuration (1).

FIGURE 5.

Domains of proteins that are involved in Na⁺-Cl⁻ cotransporter (NCC) regulation. Color code used is indicated at right. A: SPAK. B: WNK1. C: kidney-specific (KS)-WNK1. D: WNK4. E: KLHL3. See glossary for additional abbreviations.

FIGURE 6.

Models of Na⁺-Cl⁻ cotransporter (NCC) regulation. A: previous model. B: new model. Circled Ps correspond to $\mathrm{P}{\mathrm{O}}_{\mathrm{4}}^{\mathrm{2-}}$ groups. KS, kidney specific. See glossary for additional abbreviations.

FIGURE 7.

Signaling cascades involved in Na⁺-Cl⁻ cotransporter (NCC) regulation. Color code used is indicated at bottom. CA, catecholamines; CNI, calcineurin inhibitor; Ub, ubiquitin. See glossary for additional abbreviations.

FIGURE 8.

Regulation of the Na⁺-Cl⁻ cotransporter (NCC) by KLHL3 and CUL3. A: regulation of KLHL3 by PKC and PP3. KLHL3 is allowed to interact with WNK1/WNK4 when it is dephosphorylated by PP3 but not when it is phosphorylated by PKC (277, 278). B: regulation of WNK1/WNK4 by the E3 ubiquitin ligase multimeric complex. E3 ligase is comprised more specifically of 1) CUL3 that binds to the Bric-a-brac, tramtrack, broad-complex domain (BTB domain) of KLHL3, 2) ring box protein (RBX) that places KLHL3-bound WNK1/WNK4 close to E2 ubiquitin (Ub) conjugating enzyme (CE) by interacting with both CUL3 and E2 Ub CE, 3) E1 Ub activating enzyme (AE) that prompts E2 Ub CE to be active, 4) E2 Ub CE that sustains ubiquitination of Lys residues within physically recruited WNK1/WNK4, and 5) NEDD8 and COP9 signalosome (CSN) that both allow CUL3 to be active (249, 254, 268, 279,280). When WNK1/WNK4 are thus allowed to interact with KLHL3, they undergo ubiquitination and subsequent degradation by the proteasome. C: regulation of SPAK/OSR1 (and NCC as a result) by WNK1/WNK4. P, phosphorylation. See glossary for additional abbreviations.

FIGURE 9.

Distribution of ion transport systems and membrane receptors in the distal convoluted tubule (DCT)1, DCT2, and connecting tubule (CNT) nephron subsegments. Expression sites are indicated by horizontal lines for each of the proteins listed. They are based on multiple studies but mostly in rodents (see references in text). Note that several of these proteins have been found to be present outside of the segments encompassed by the horizontal lines but at much lower levels. TAL, thick ascending loop. See glossary for additional abbreviations.

FIGURE 10.

Ion transport by distal convoluted tubule (DCT) cells. A: ion transport systems. When the transport systems shown are activated by a decrease in membrane potential (E_m), they are accompanied by + signs in green and when they are activated by an increase in E_m, they are accompanied by – signs also in green. B: effect of changes in Na⁺-Cl⁻ cotransport on E_m. C: effect of changes in Na⁺-Cl⁻ cotransport on electrogenic K⁺ secretion. BL, basolateral membrane; CCD, cortical collecting duct; CNT, connecting tubule. See glossary for additional abbreviations.

FIGURE 11.

Homeostatic roles of the Na⁺-Cl⁻ cotransporter (NCC) in the distal convoluted tubule (DCT) based on 3 examples. A: hyperkalemia. NCC responds to a rise in serum [K⁺] by decreasing its activity to enhance Na⁺ delivery at the ENaC- and ROMK-expressing sites (252, 260, 412, 448). Na⁺ reabsorption should be minimally affected under such circumstances as it is assumed by another ion transport system. It is of note that a rise in serum [K⁺] should theoretically lead to activation of the WNK1/WNK4-SPAK/OSR-NCC pathway in DCT cells by increasing aldosterone production (46, 101, 339, 340). However, it leads in the end to inactivation of this pathway, as it also causes [Cl⁻]_i to rise (252, 260, 412, 448). B: Na⁺ restriction. NCC responds to a decrease in dietary Na⁺ by increasing its activity to enhance Na⁺ reabsorption (46, 101, 339, 340). K⁺ secretion should increase minimally under such circumstances, as Na⁺ delivery at the ENaC- and ROMK-expressing sites decreases. C: hypercalcemia. NCC could respond to a rise in serum [Ca²⁺] by increasing its activity to promote urinary Ca²⁺ loss. The CaSR would be likely involved in this process even if it has been shown to cause NCC activity to decrease when activated on the basolateral membrane of DCT cells (381). It is indeed also expressed on the apical membrane of DCT cells, and its activation at this other location has been shown to increase NCC activity (328, 382). Hypercalcemia would then trigger the apical response by causing Ca²⁺ reabsorption by the thick ascending loop (TAL) to decrease (303, 385). Downward arrow, minimized; upward arrow, maximized. MR, mineralocorticoid receptor. See glossary for additional abbreviations.