Physiology and pathophysiology of SLC12A1/2 transporters

Abstract

The electroneutral Na(+)-K(+)-Cl(-) cotransporters NKCC1 (encoded by the SLC12A2 gene) and NKCC2 (SLC12A1 gene) belong to the Na(+)-dependent subgroup of solute carrier 12 (SLC12) family of transporters. They mediate the electroneutral movement of Na(+) and K(+), tightly coupled to the movement of Cl(-) across cell membranes. As they use the energy of the ion gradients generated by the Na(+)/K(+)-ATPase to transport Na(+), K(+), and Cl(-) from the outside to the inside of a cell, they are considered secondary active transport mechanisms. NKCC-mediated transport occurs in a 1Na(+), 1K(+), and 2Cl(-) ratio, although NKCC1 has been shown to sometimes mediate partial reactions. Both transporters are blocked by bumetanide and furosemide, drugs which are commonly used in clinical medicine. NKCC2 is the molecular target of loop diuretics as it is expressed on the apical membrane of thick ascending limb of Henle epithelial cells, where it mediates NaCl reabsorption. NKCC1, in contrast, is found on the basolateral membrane of Cl(-) secretory epithelial cells, as well as in a variety of non-epithelial cells, where it mediates cell volume regulation and participates in Cl(-) homeostasis. Following their molecular identification two decades ago, much has been learned about their biophysical properties, their mode of operation, their regulation by kinases and phosphatases, and their physiological relevance. However, despite this tremendous amount of new information, there are still so many gaps in our knowledge. This review summarizes information that constitutes consensus in the field, but it also discusses current points of controversy and highlights many unanswered questions.

Figure 1. Sequence homology and domains of the NH₂-terminal tails of NKCC1 & NKCC2

A. Schematic comparison of NKCC1 and NKCC2 amino terminal tails from vertebrates: mammal (Homo sapiens), bird (Gallus gallus), reptile (Anolis carolinensis), amphibian (Xenopus laevis), bone fish (Oreochromis niloticus), and cartilage fish (Squalus acanthias). Identical residues are highlighted in red, conserved residues are highlighted in green, blocks of identical residues are shown in blue, and non-conserved residues are indicated in white (gaps). Note the presence of 2-3 regions of high degree of conservation, corresponding to the SPAK/OSR1 binding motifs, and the phospho-threonines and -serines. Bars indicate the RFxV motifs or SPAK/OSR1 binding site (filled bar), and the VxFxD motif or PP1 binding site (open bar). B. Amino acid alignment of a portion of the N-terminal tail of human and mouse NCC, NKCC1, and NKCC2. Protein sequences were aligned using VectorNti (Invitrogen) and a portion of the alignment is displayed. Threonine and serine residues that are targets of phosphorylation are highlighted with references. The position of the R5 peptide used to generate the R5 antibody [38] is indicated. Residues highlighted in yellow are identical residues, whereas amino acids highlighted in green/blue represent conserved residues.

Figure 2. Structure of the core of human NKCC1

A) Structure of the transmembrane domains and other alpha helices was modeled using the Phyre² protein fold recognition server [72]. The PDB file was viewed using the Visual Molecular Dynamics software (University of Illinois) and the image was rendered after highlighting the alpha helices. The first 5 TMs are colored in blue, whereas the next five TMs are colored in red. Note the proximity and parallelism of TM1-6, TM2-7, TM3-8, and TM4-9. B) Predicted topology of the first 10 transmembrane domains based on similarity to crystal structure of amino acid-polyamine-organocation transporters. The last 2 transmembrane domains are not drawn. C. Alignment of two segments of the human NKCC1 core showing conservation of transmembrane domains. The SV and MM/VV amino acids in human NKCC1 TMD2-7 are replaced by AL and GT in shark NKCC1. The alignment was done with VectorNti 6 (Invitrogen).

Figure 3. Two kinetic models of Na-K-2Cl cotransport

A. Glide symmetry model where a Na⁺ ion binding first on the cotransporter in the outside facing configuration is the first ion coming off in the inward facing configuration. B. Alternative model with more conventional kinetics where Cl⁻ binds first followed by Na⁺, the second Cl⁻, and K⁺. Partial reactions or slippages which are electroneutral in nature (e.g. NaCl movement) are allowed. See text for details and references.

Figure 4. NKCC1 transport of Cl⁻, Br⁻ and I⁻

K⁺ influx was measured in NKCC1-injected Xenopus laevis oocytes in a hyperosmotic solution containing 96 mM NaCl, 4 mM KNO₃, 2 mM Ca(NO₃)₂, 1 mM MgSO4, 60 mM sucrose and 1 mM ouabain in the presence or absence of 20 μM bumetanide (pH 7.4, osmolality 260 mOsM). For halide substitution, NaCl was replaced with NaBr, or NaI. Fluxes are expressed in nanomoles K⁺ oocyte⁻¹ hr⁻¹. Bars represent means ± S.E.M (n= 20-25 oocytes). Values in inset represent sizes (radius) of atoms and their ions in pm.

Figure 5. Na-K-2Cl cotransport in two types of epithelial cells

A. Thick ascending limb epithelial cell model showing apical NKCC2 localization. The driving force for NaCl reabsorption is provided by the basolateral Na⁺/K⁺ pump. Apical K⁺ channel (ROMK) delivers K⁺ in the lumen for NKCC2 function and creates an electropositive lumen. ClCK(_{A and B)} on the basolateral membrane creates a path for Cl⁻ movement and participate in the electronegative blood side. The electrical field generated by the epithelial cells favors the paracellular reabsorption of divalent cations. B. Model of a Cl⁻ secreting epithelial cell with localization of NKCC1 on the basolateral membrane. The cotransporter replenishes Cl⁻ as the anion is transported across the apical through CFTR or other Cl⁻ channels. K⁺, which enters through the pump and NKCC1, leaves the cell through apical and basolateral K⁺ channels (not shown). Note that this model can be used for stria vascularis marginal cells (K⁺ secreting cell) with CFTR substituted for KCNQ1.

Figure 6. Diagram of the mouse medullary and cortical thick ascending limb of henle expressing NKCC2

The low affinity isoform, NKCC2-F, is expressed in the lower portion of the outer medullary TAL where the urine Na⁺ concentration is high. The high affinity isoform, NKCC2-B, is expressed in cortical TAL where the urine Na⁺ concentration is low. The intermediate affinity isoform, NKCC2-A is expressed along the entire TAL. In the outer medulla, the major role of NKCC2 is to transport Na⁺ without water, thus raising the osmolarity of the interstitium. Osmolarity of the urine and interstitium, as well as the urea and Na⁺ concentrations in the urine are provided. Glom = Glomerulus.