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	Figure 1. Visual representation of the relationship between the nine human SLC12A cotransporters. The cluster dendrogram highlights two distinct subfamilies: the Na+‐dependent branch on the upper left from the Na+‐independent branch of the bottom right. There are also two orphan members, CCC8 and CCC9 that as yet do not have specific functions or substrates assigned to them. The two Na+‐K+‐2Cl− cotransporters are inhibited by the loop diuretic bumetanide, whereas, the Na+‐Cl− cotransporter is inhibited by thiazides diuretics, such as metolazone and hydrochlorothiazide. The four K+‐Cl− cotransporters are inhibited by furosemide, another loop diuretic, and ML077 a novel more potent inhibitor. The length of dendrogram branches represents the number of amino acid substitutions per site (bar = 0.1 amino acid substitutions per site).
	Figure 2. Model of transmembrane core of NKCC1. Model of NKCC1 core obtained by I‐TASSER based on the crystal structure of several related transporters such as ApcT K158A transporter (PDB: 3gi8C), L‐arginine/agmatine antiporter (PDB: 5j4iA), glutamate‐GABA antiporter GadC (PDB: 4djiA), LeuT (PDB: 2g6HA). (A). The long alpha‐helices representing the transmembrane (TM) domains are displayed as solid straight tubes. Highlighted are TM2 and TM7, which run parallel to each other. (B). Note that the valine (Val) and serine (Ser) residues on TM2 that affect the Na+ affinity are close to the extracellular domain, whereas the two methionine (Met) residues that affect the K+ affinity are located deeper in the topology of the cotransporter. Alanine (Ala), isoleucine (Ile), and leucine (Leu) residues with similar properties are identified on TM7.
	Figure 3. Exon structure of human SLC12A2 (NKCC1) and SLC12A1 (NKCC2). The 27 exons of both the NKCC1 and NKCC2 genes have a high degree of conservation that diverged at the beginning of vertebrate evolution. Exon 1 in the NKCC1 gene (orange) is split into two exons in the NKCC2 gene (blue). As a result, exons 2 thru 20 in NKCC1 align with exons 3 thru 21 in NKCC2. Highlighted in light blue is exon 21 in NKCC1 that is missing in NKCC2, resulting in a realigning of exons 22 thru 27 for both genes. Also highlighted in light blue is exon 4 in the NKCC2 gene that is alternatively spliced giving three mutually exclusive variants (A, B, and F) which provides distinct affinities for Cl−. We have also highlighted the shorter isoform of NKCC2 found in mouse kidney formed by extension of exon 17 (C4 3′ end). In NKCC1, a small 16 amino acid residue exon (exon 21) is alternatively spliced. This exon is absent in the NKCC2 gene.
	Figure 4. Role of NKCC1 in colonic epithelium. The figure separates the surface cells from the crypt cells. (A) In the surface cells, apical Na+ channels and basolateral Na+/K+‐ATPase (pump) participate in Na+ reabsorption. (B) In cells from the crypts, K+ and Cl− channels are expressed on both the apical and basolateral membrane and cells can mediate preferentially Cl− secretion or K+ secretion, depending on stimuli. Many basolateral signals (e.g., VIP, ATP, AngII, and Epinephrine) lead to activation of adenylate cyclase (AC) and production of cAMP, which stimulates CFTR Cl− channels on the apical membrane through protein kinase A (PKA). Cl− is replenished at the serosal (basolateral) side by NKCC1. Part of the Cl− that is secreted can be exchanged for HCO3−, which enters the basolateral membrane through the Na+/HCO3− cotransporter (NBC1). In this Cl− secretory mode, a basolateral K+ conductance is needed to recycle the K+ that enters through the Na+/K+‐ATPase (Pump) and NKCC1. Note that the crypt cells are also able to secrete K+ when Cl− secretion is “silent.” In this case, a Cl− conductance is needed on the basolateral membrane to recycle Cl−.
	Figure 5. NKCC1 in choroid plexus. (A) Intense apical staining of NKCC1 in mouse choroid plexus is evidenced by expression at the surface of finger‐like structures. The rabbit polyclonal antibody was raised against a 74 amino acid portion of the COOH‐terminal tail of the cotransporter (142). Bar = 50 μm. (B) Two possible models of NKCC1 function in choroid plexus (CP) epithelial cells are illustrated. When carbonic anhydrase (CA) activity is high, exchange of Cl− for HCO3− accumulates Cl− into the CP epithelial cell leading to outward NKCC1 transport. In this situation, NKCC1 participates in Na+ secretion and CSF production. Intracerebral application of bumetanide slows down the rate of CSF production. In contrast, if CA activity is low, intracellular Cl− might be lower and the gradients now favor typical inward NKCC1‐mediated transport. The cotransporter in this case might participate in the reabsorption of K+.
	Figure 6. Two cavities in the inner ear (cochlear and endolymphatic sac) with very different ionic compositions. The two compartments have distinct K+ concentrations: 140 mmol/L in the cochlear endolymph and 13 mmol/L in the endolymphatic sac. The stria vascularis produces the K+‐rich endolymphatic fluid. It is a multilayered epithelium composed of basal fibrocytes (Fibrocytes), basal cells (BC), intermediate cells (IC), and marginal cells (MC). The epithelium expresses NKCC1 in abundance as well as Na+/K+‐ATPase (pump) and K+ channels (Kir4.1 and Kv7.1). Gap junctions connect fibrocytes to BC and IC. The driving force for K+ secretion, like in many other epithelia, is the Na+/K+ pump. The cochlea connects to the endolymphatic sac via a narrow canal.
	Figure 7. K+ leak generates varying membrane potentials in different cell types and the endolymphatic potential of the inner ear. (A) The leak of K+ ions from cytosol to the extracellular environment creates a membrane potential (Vm) that varies from ‐8 mV in chondrocytes to ‐70 mV in neurons to ‐95 mV in skeletal muscle. The minus sign indicates that the inside of the cell is negative with respect to the extracellular environment. (B) Similarly, the leak of K+ from the K+‐rich endolymphatic cavity to the interstitium creates the endolymphatic potential (EP). Note the reversed sign (+80‐90 mV), which indicates that the interstitial environment is positive with respect to the endolymphatic cavity. (C) Anatomy of a turn of the cochlea from the inner ear of a wild‐type mouse: SM: scala media, SV: Scala Vestibuli, St.v. stria vascularis, sl: spiral ligament, tm: tectorial membrane, oc: outer hair cells, Rm: Reissner's membrane. (D) The scala media and Reissner's membrane are intact in the inner ear of heterozygous NKCC1 knockout mice. (E) However, in homozygous NKCC1 knockout mice, Reissner's membrane is collapsed and there is no sign of the scala media. Bar = 50 μm.
	Figure 8. Evidence of apoptosis in the seminiferous tubule (gonads) of NKCC1 knockout mice. Panels A and B show thionin‐stained sections treated with DIG‐dUTP, followed by a peroxidase‐conjugated anti‐DIG antibody. Note the presence of only one cell undergoing apoptosis at the base of a seminiferous tubule in a wild‐type control mouse (box in A), whereas multiple apoptotic cells are present in the seminiferous tubule from the homozygous NKCC1 knockout mouse (black arrows in B). Spermatids (sp) are numerous in wild‐type (A), but absent in the knockout mouse (B). Black arrowheads point to spermatogonia. Panels C and D show fluorescein‐based in situ cell death‐detection method. Sections were fixed with paraformaldehyde and exposed to fluorescein‐dUTP. Note the background fluorescence given by spermatids in the seminiferous tubules from control mice (white arrowheads), but absence of TUNEL staining at the base of the seminiferous tubules (C). In the knockout (D), many apoptotic cells are labeled (white arrows). Scale bars = 50 μmol/L.
	Figure 9. Plot of testis versus body weights in heterozygous versus homozygous NKCC1 knockout mice. Adult wild‐type (not shown) and heterozygous NKCC1 knockout mice weighing around 30 g had testis weighing around 200 mg. Severe deficits in both body (60%) and testis (25%) weights were noted in homozygous NKCC1 knockout mice.
	Figure 10. Model of ion transport in Thick Ascending Limb of Henle (TAL). (A) Multiple transport mechanisms act in concert to mediate Na+ reabsorption in the TAL. Na+ enters the apical membrane through NKCC2 and leaves the basolateral membrane through the Na+/K+‐ATPase (pump). Because K+ is limiting in the urine, NKCC2 works in concert with an apical K+ channel: ROMK, which provides K+ for K+‐coupled Na+ transport via NKCC2. The Cl− that enters the cells via NKCC2 on the apical side is extruded by CLCNK Cl− channels. The combined effects of K+ leak through ROMK on the apical membrane and Cl− leak through CLCNKA/B on the basolateral membrane creates an electropositive lumen that facilitates the movement of divalent cations (Ca2+ and Mg2+) through paracellular pathways. NKCC2 is inhibited by the “loop” diuretic furosemide (LASIX). (B) Absence of Na+ reabsorption (or Na+ wasting) occurs in conditions of loss‐of‐function mutations in these transport pathways. Mutations in NKCC2 lead to Bartter's syndrome type I (BARTS1), mutations in ROMK cause Bartter's syndrome type II (BARTS2), and mutations in CLCNK channels or in Barttin (an associated protein) lead to Bartter's syndrome types III and IV (BARTS3 and 4).
	Figure 11. Depiction of cAMP as a “node” in NKCC2 regulation. Activation of adenylate cyclase by G‐coupled protein αs (stimulated by vasopressin receptors) and inhibition by Gαi (activated by prostaglandin receptor) regulates the synthesis of cAMP from ATP. cAMP is degraded into AMP by phosphodiesterases, one of which PDE2, is under the control of the PI3K‐Akt‐eNOS, nitric oxide, cGMP signaling pathway initiated by the endothelin receptor. cAMP has genomic effects, binding to CRE element in the NKCC2 promoter, leading to increased transcription. cAMP also stimulates PKA which plays a role in the forward trafficking of NKCC2 to the plasma membrane. Synaptic protein, VAMP2, is involved in the PKA‐mediated trafficking process.

Further Reading

Russell JM. Sodium-Potassium-Chloride Cotransport. Physiol Rev 80: 211-276, 2000. (261) This is the most comprehensive review ever written on Na+-K+-2Cl- cotransport. It contains a trove of information on the biophysical properties of the cotransporter.

Gagnon KB, Delpire E. Molecular physiology of SPAK and OSR1: Two Ste20-related protein kinases regulating ion transport. Physiol Rev 92: 1577-1617, 2012. (87)

McCormick JA, Ellison DH. The WNKs: atypical protein kinases with pleiotropic actions. Physiol Rev 91: 177-219, 2011. (190) These two reviews summarize most of what is known on the regulatory kinases that act upstream of the Na+-K+-2Cl- cotransporter.

Hoffmann EK, Lambert IH, Pedersen SF. Physiology of cell volume regulation in vertebrates. Physiol Rev 89: 193-277, 2009. (122) This review article is written by the most proficient scientist in the field of cell volume regulation.

Ares GR, Caceres PS, Ortiz PA. Molecular regulation of NKCC2 in the thick ascending limb. Am J Physiol Renal Physiol 301: F1143-1159, 2011 (14) This is a recent review article summarizing most of what is known about the regulation of NKCC2.

Alvarez-Leefmans FJ, Delpire E. Physiology and Pathology of Chloride Transporters and Channels in the Nervous System: From Molecules to Diseases: Academic Press, 2009. (8) This book was contributed by many leaders in the field of Cl- transport and homeostasis. While covering on all mechanisms transporting Cl- and mostly focusing on the nervous system, the book contains several chapters covering the function of the Na+-K+-2Cl- cotransporter, including its function in many epithelial cells.

Teaching Material

E. Delpire, K. B. Gagnon. Na⁺-K⁺-2Cl⁻ Cotransporter (NKCC) Physiological Function in Nonpolarized Cells and Transporting Epithelia. Compr Physiol. 8: 2018, 871-901.

Didactic Synopsis

Major Teaching Points:

• Na⁺-K⁺-2Cl⁻ cotransporters are secondary active transport mechanisms.
• NKCC1 and NKCC2 are phosphoproteins, active when phosphorylated and inactive when dephosphorylated.
• Transport through NKCC is most active when intracellular Cl⁻ is low.
• NKCC1 is a mechanism of cell volume regulation that facilitates regulatory volume increase after cell shrinkage.
• NKCC1 is a basolateral cotransporter in most epithelial cells. It participates in Cl⁻ based fluid secretion in airway, gastric and intestinal epithelium, as well as sweat, lacrimal, and salivary glands.
• NKCC1 also participates in the formation of the K⁺-rich endolymph in inner ear.
• NKCC1 is highly expressed on the apical membrane of choroid plexus epithelial cells. Its direction of transport might be reversed due to high intracellular Cl⁻ concentration maintained by carbonic anhydrase and a Cl⁻/HCO₃⁻ exchanger.
• In nonepithelial cells, the cotransporter participates in Cl⁻ homeostasis impacting for instance smooth muscle cell contraction and synaptic transmission.
• NKCC2 is an apical cotransporter. It lacks a COOH-terminal motif that sends NKCC1 to the basolateral membrane of epithelial cells.
• NKCC2 participates in NaCl reabsorption in the thick ascending limb of Henle and fluid and Cl⁻ sensing in the macula densa.

Didactic Legends

The figures—in a freely downloadable PowerPoint format—can be found on the Images tab along with the formal legends published in the article. The following legends to the same figures are written to be useful for teaching.

Figure 1. Teaching points: The SCL12A family of transporters is relatively small as it only comprises of nine genes. Two genes (CCC8 and CCC9) encode orphan proteins or proteins to which function has yet to be described. Based on the length of the lines separating the members of a subfamily, there is less genetic diversity in the four K⁺-Cl⁻ cotransporters than the three Na⁺-(K⁺)-Cl⁻ cotransporters. Based on the chemical gradients of transporter ions, the Na⁺-dependent cotransporters carry ions into the cell (out to in), whereas the Na⁺-independent cotransporters carrion ions out of the cell. Note that transport direction reflects net transport only, as transporters are able to move ions in both directions.

Figure 2. Teaching points:The Figure is a simulated representation of the transmembrane domains of NKCC1. It is modeled based on known crystal structure of distantly related transporters, as there is no crystal structure of NKCC1 available. Hydrophobic alpha-helices are represented as tubes whereas hydrophilic linkers are represented as threads. Note the compactness of the structure and the parallel orientation of several pairs of transmembrane domains. Panel B adds the volume occupied by specific amino acids involved in ion affinity. Notice that the amino acid residues turn around the axis of the transmembrane domain—which reflects the structure of the alpha helix with a 3.6 residues per turn.

Figure 3. Teaching points:The two genes (SLC12A1 and SLC12A2) have a very similar exon/intron structure, which is expected as the SLC12A1 gene arose from gene duplication. Overtime during evolution some changes occurred, but they are minimal compared to the overall gene structure. Most striking differences are the size of the introns, the sizes of the first and last exons, and the presence of some gene-specific exons (light blue). Alternatively spliced exons are the three exons 4 in SLC12A1—labeled A, B, and F, and exon 21 in SLC12A2.

Figure 4. Teaching points: A variety of transporters are expressed on the apical and basolateral membranes of epithelial cells. The figure presents three models. In (A), we represent an intestinal surface cell, which function is to reabsorb Na⁺. The transcellular pathway for Na⁺ movement consists of apical Na⁺ channels and the basolateral Na⁺/K⁺ pump. Note the direction of the arrows, pointing to the movement of Na⁺ from the luminal side to the blood side (i.e., reabsorption). In (B), we represent a crypt cell, or a cell that lives deeper in an intestinal crypt, in Cl⁻ secreting mode (larger cell on left) or K⁺ secreting mode (narrower cell on the right). Note that NKCC1 and the Na⁺/K⁺ pump on the basolateral side are active in the two models. Difference between the two models include activation of apical Cl⁻ channels and basolateral K⁺ conductance under Cl⁻secretion mode; while activation of apical K⁺ channels and basolateral Cl⁻ conductance under K⁺ secretion mode. Bicarbonate can be secreted along with Cl⁻ through a basolateral cotransport mechanism and an apical exchanger mechanism. Also note that several receptors are located on the basolateral membrane (in red). Through activation of adenylcyclase (AC) and the production of cAMP, they stimulate apical Cl⁻ channels.

Figure 5. Teaching points:The choroid plexus constitutes a unique epithelium as both the Na⁺/K⁺ pump and NKCC1 are located on the apical (CSF facing) membrane. This raises an interesting conundrum as to the function of NKCC1 and the direction of transport. In (A), we propose that the epithelium can switch from a Na⁺ secretion mode (top model) to K⁺ reabsorption mode (bottom model). To make this work, one assumes high carbonic anhydrase (CA) activity leading to production of large amounts of bicarbonate. As HCO₃⁻ is exchanged with Cl⁻ at the basolateral membrane, cytosolic Cl⁻ raises enough to reverse the direction of NKCC1 transport. In this model, both NKCC1 and the pump participate to CSF Na⁺ secretion, while K⁺ can cycle at the apical membrane. In (B), we propose an alternative model where the direction of NKCC1 transport remains inward. K⁺ can be reabsorbed through the combined action of NKCC1 and the pump. If the pump was alone in mediating Na⁺ secretion and K⁺ reabsorption, these two processes would always be linked. NKCC1 on the same membrane then the pump unlinks K⁺ reabsorption from Na⁺ secretion.

Figure 6. Teaching points: The stria vascularis is a stratified epithelium in the inner ear. As seen in the Figure it is composed of multiple cell layers. Cells from the three basal layers are connected by gap junctions or large structures that connect the cytoplasm of cells and allow the movement of small substances. Basolateral NKCC1 together with apical K⁺ channels in both fibrocytes and marginal cells (MC) provide a pathway for K⁺ secretion from blood to the endolymphatic cavity. Note the unusually high concentration of K⁺in the endolymphatic cavity. The endolymph is a K⁺ rich medium, unlike all other environments external to cells. In this figure, we also drew the epithelium of the endolymphatic sac, which is thought to be reabsorptive. Studies have shown that NKCC2 might be expressed on the apical membrane providing a pathway for Na⁺ and fluid reabsorption. Note that in contrast to our model, many models still consider the endolymphatic sac to contain a K⁺-rich endolymph. Notice the small passage that connects the endolymphatic sac from the main cavity. It is meant to represent the long and narrow tube that connects the sac to the saccule. It does not provide an accurate representation of the anatomy.

Figure 7. Teaching points: K⁺ is the major ion involved in creating the electrical potential across the plasma membrane of a cell (A) or across the wall of the inner ear (B). Because the reference electrode is outside and the interior of the cell is negative, the membrane potential has a negative value (from -8 mV in chondrocytes to -95 mV in skeletal muscle). Similarly, because the reference electrode is outside and the endolymphatic cavity is positive, due to the marginal cells leak of K⁺, the endolymphatic potential has a positive value. In C to E, the structure of the inner ear is shown. The focus is on the Reissner's membrane (Rm) which separates the scala media (SM) from the scala vestibule (SV). The K⁺-rich endolymph that is secreted by the stria vascularis (St.v.) epithelium fills the scala media. In the absence of fluid secretion, the scala media is empty and the Reissner's membrane sits on top of the stria vascularis. Thus, striking morphological or anatomical changes signal functional changes.

Figure 8. Teaching points:Size of the testes in NKCC1 knockout mice can be explained by absence of spermatid maturation. Notice the mass of spermatozoa filling the lumen of the seminiferous tubule in heterozygous mice (A) versus complete absence in homozygous knockouts (B). The Figure also reports Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) for detection of apoptotic cell death. As cells undergo apoptosis, the cell's DNA is broken, exposing blunt-ends that can be labeled with HRP-conjugated (A and B) or FITC-conjugated dUTPs (C and D). As shown in the panels, an increased number of positive (red for A and B, and fluorescent green for C and D) cells is detected in the tubules from homozygote mice.

Figure 9. Teaching points: Homozygote NKCC1 mice are smaller and have smaller testis sizes than their heterozygote counterparts. While this information could be visualized with bar graphs, by plotting the size of the testes on one axis against/versus the overall body weight on the other axis, once can see (i) a correlation between testis weight and body weight (almost parallel red and blue lines), and (ii) a clear drop in testis weights in the homozygote mice, indicating a testis-specific phenotype.

Figure 10. Teaching points: The absorptive function of the Thick Ascending Limb is explained in (A). Transporters on apical and basolateral sides work in concert to move Na⁺ and Cl⁻ from lumen to blood. As in most epithelia, the transport is driven via ATP consumption by the Na⁺/K⁺ pump. Na⁺ and Cl⁻ enter the apical membrane through NKCC2, while Cl⁻ is released at the serosal side by CLC-Ka/b Cl⁻ channels. It is believed that the K⁺ concentration in the urine is limiting to the movement of Na+ through NKCC2, which is mitigated by (ROMK) K⁺ channels, which recycle the K⁺ that enters the cell through NKCC2. The leak of K⁺ at the apical membrane in combination with the leak of Cl⁻ at the basolateral membrane creates an electropositive lumen that helps the movement of divalent cations through paracellular pathways. The system is so well designed that mutations in either component of the Na⁺ absorptive pathway gives rise to variants of Bartter syndrome (BARTS 1-4).

Figure 11. Teaching points: NKCC2 function is regulated at many levels: transcription, trafficking, endocytosis. One pathway not displayed here is phosphorylation by WNK/SPAK-OSR1 kinases, which likely affects transport cycle turnover of transporters already existing at the plasma membrane. The Figure focuses on a major signaling molecule, cAMP, which leads to NKCC2 activation. Vasopressin activates, whereas prostaglandin inhibits, adenylate cyclase, the enzyme that produces cAMP. Endothelin, through production of nitrous oxide, activate guanylyl cyclase, which produces cGMP, which in turn activates the enzyme that degrades cAMP. Thus, activation of endothelin receptor inhibits NKCC2 function. cAMP not only affects NKCC2 function at the genomic level (through a cAMP-responsive element, CRE) in the promoter of NKCC2, but also facilitates the forward trafficking of the transporter and its insertion in the plasma membrane through PKA and VAMP2.