TheNa–K–Clcotransporter (NKCC) is atransport protein that aids in thesecondary active transport ofsodium,potassium, andchloride intocells.^[1] In humans there are two isoforms of thismembrane transport protein,NKCC1 andNKCC2, encoded by two differentgenes (SLC12A2 andSLC12A1 respectively). Two isoforms of the NKCC1/Slc12a2 gene result from keeping (isoform 1) or skipping (isoform 2) exon 21 in the final gene product.^[2]

NKCC1 is widely distributed throughout the human body; it has important functions inorgans thatsecrete fluids. In contrast, NKCC2 is found specifically in thekidney, where it extracts sodium, potassium, and chloride from theurine so they can bereabsorbed into theblood.^[3]

NKCC proteins aremembrane transport proteins that transportsodium (Na),potassium (K), andchloride (Cl) ions across thecell membrane. Because they move each solute in the same direction, they are consideredsymporters. They maintain electroneutrality by moving two positively charged solutes (sodium and potassium) alongside two parts of a negatively charged solute (chloride). Thus thestoichiometry of the transported solutes is 1Na:1K:2Cl. Althoughsquid giant axons are the only notable exception with a stoichiometry of 2Na:1K:3Cl, electroneutrality across the protein transporter is still maintained.^[3] The rate of transport of these ions are regulated byphosphorylation sites, which present on both NKCC isoforms.^[4]

The NKCC1 isoform consists of about 1,200amino acids, with about 500 amino acids residues giving rise to twelve hydrophobictransmembrane regions.^[5] However, evidence of a shorter NKCC1mRNA transcript (6.7 kb to 7-7.5 kb) in skeletal muscle cells gives support that further NKCC1 variants exists in a tissue-specific manner.^[6] Thecarboxy-terminal of the NKCC1 cotransporter contains multiplephosphorylation sites and is highly conserved across species, while in contrast, theamino-terminal contains at least one phosphorylation site and is poorly conserved across species^[5].Focusing on the transmembrane regions,mutagenesis-driven affinity studies have revealed the second transmembrane region as the determinant of cation affinity, whilechloride affinity was determined by transmembrane regions four through seven.^[5] Additionally,bumetanide, aloop diuretic, was found to bind to transmembrane regions 2 through 7, 11, and 12.^[5]

NKCC1 is widely distributed throughout the body, especially in organs thatsecrete fluids, calledexocrine glands.^[7] In cells of these organs, NKCC1 is commonly found in thebasolateral membrane,^[8] the part of thecell membrane closest to theblood vessels. Exon 21 possesses atranslocation sequence that targets NKCC1 to the basolateral membrane.^[9] Thus, NKCC1 cotransporters that have been alternatively spliced to exclude exon 21 will be translocated to theapical membrane rather than the basolateral membrane. Its basolateral location gives NKCC1 the ability to transport sodium, potassium, and chloride from the blood into the cell. Other transporters assist in the movement of these solutes out of the cell through its apical surface. The end result is that solutes from the blood, particularly chloride, are secreted into the lumen of these exocrine glands, increasing the luminalconcentration of solutes and causing water to be secreted byosmosis.

In addition to exocrine glands, NKCC1 is necessary for establishing the potassium-richendolymph that bathes part of thecochlea, an organ necessary for hearing. Inhibition of NKCC1, as withfurosemide or otherloop diuretics, can result indeafness.^[8] Specifically in the cochlea, NKCC1 is present in thestria vascularis,spiral ligament, andspiral ganglia.^[8] Similarly, NKCC1 expression decreases withaging, resulting in progressivehearing loss.^[10] Additionally, NKCC1 is present in thedark cells of thevestibule and contributes to generation of theendolymph of thevestibular system.^[11]

NKCC1 is also expressed in many regions of thebrain during early development, but not in adulthood.^[12] This change in NKCC1 presence seems to be responsible for altering responses to the neurotransmittersGABA andglycine from excitatory to inhibitory, which was suggested to be important for early neuronal development. As long as NKCC1 transporters are predominantly active, internal chloride concentrations in neurons is raised in comparison with mature chloride concentrations, which is important for GABA and glycine responses, as respective ligand-gated anion channels are permeable to chloride. With higher internal chloride concentrations, outward driving force for this ions increases, and thus channel opening leads to chloride leaving the cell, thereby depolarizing it. Put another way, increasing internal chloride concentration increases thereversal potential for chloride, given by theNernst equation. Later in development expression of NKCC1 is reduced, while expression of aKCC2K-Cl cotransporter increased, thus bringing internal chloride concentration in neurons down to adult values.^[13]

Activity-dependent regulation of NKCC1 during early neuronal development has been suggested to contribute, together with the upregulation of KCC2, to the developmental shift of GABAergic signalling from depolarizing to hyperpolarizing responses.^[14][15]

NKCC1 has been identified inSertoli cells,spermatocytes, andspermatids in themale reproductive system.^[16] NKCC1 function appears to be critical forspermatogenesis, asknockdown of NKCC1 in mice results inspermatocytes failing to mature intospermatozoa, resulting ininfertility.^[16] Additionally, the NKCC1knockdownmice also exhibit a decreasedtesticle size compared towild-typemice.^[16] The mechanism behind NKCC1-dependent malefertility is unclear, it is possible that the observed decreasedsperm count could be due to either lack of NKCC1 cotransport in the testis or upstream failure of NKCC1-expressing neurons in thehypothalamus to releasegonadotropin-releasing hormone.^[4]

The NKCC2 isoform is smaller than NKCC1, 121kDa versus 195kDa, respectively, primarily because NKCC2 does not contain an 80amino acid sequence present on theN-terminus of NKCC1.^[3] Additionally, the NKCC2 isoform does not contain exon 21, which results in NKCC2 being translocated to theapical membrane.^[4] Compared to NKCC1, exon 1 is divided into two separate exons in NKCC2 and exon 4 isalternatively spliced into forms A, B, and F, which are all exclusive to NKCC2.^[4] NKCC2 expression is thought to be limited torenal cells, although this has been called into question with possible NKCC2 expression inpancreatic β-cells.^[17]

NKCC2 is specifically found in cells of thethick ascending limb of the loop of Henle and themacula densa innephrons, the basic functional units of thekidney. Within these cells, NKCC2 resides in theapical membrane^[18] abutting the nephron'slumen, which is the hollow space containingurine. It thus serves both in sodium absorption and intubuloglomerular feedback.

The thick ascending limb of the loop of Henle begins at the deeper portion of the renal outer medulla. Here, the urine has a relatively high concentration of sodium. As urine moves towards the more superficial portion of the thick ascending limb, NKCC2 is the major transport protein by which sodium is reabsorbed from the urine. This outward movement of sodium and the lack of water permeability in the thick ascending limb, creates a more diluted urine.^[19] According to the stoichiometry outlined above, each sodium ion reabsorbed brings one potassium ion and two chloride ions. Sodium goes on to be reabsorbed into theblood, where it contributes to the maintenance ofblood pressure.

Furosemide and otherloop diuretics inhibit the activity of NKCC2, thereby impairing sodium reabsorption in the thick ascending limb of the loop of Henle. The action of theseloop diuretics also reduces potassium reabsorption through the NKCC2 cotransporter and consequently increases tubular flow rate which enhances potassium secretion and emphasises the hypokalaemic effect.

Impaired sodium reabsorption increases diuresis by three mechanisms:

1. Increases the amount of activeosmolytes in urine by decreasing absorption of sodium
2. Erases the papillar gradient
3. Inhibitstubuloglomerular feedback

Loop diuretics therefore ultimately result in decreased blood pressure.

The hormonevasopressin also stimulates the activity of NKCC2. Vasopressin stimulates sodium chloride reabsorption in the thick ascending limb of the nephron by activating signaling pathways. Vasopressin increases the traffic of NKCC2 to the membrane and phosphorylates someserine andthreonine sites on the cytoplasmic N-terminal of the NKCC2 located in the membrane, increasing its activity. Increased NKCC2 activity aids in water reabsorption in the collecting duct throughaquaporin 2 channels by creating a hypo-osmotic filtrate.^[20][21]

NKCC1 and NKCC2 are encoded bygenes on thelong arms ofchromosomes 5^[5] and15,^[22] respectively. A loss of function mutation of NKCC2 producesBartter syndrome, an autosomal recessive disorder characterized by hypokalemicmetabolic alkalosis with normal to low blood pressure.^[22]

The promotor for gene SLC12A2, which encodes for NKCC1, contains binding sites forhomeoboxtranscription factorsSIX1 andSIX4, which have been shown to upregulate NKCC1 mRNA expression when bound.^[23] Additionally, NKCC1 expression is upregulated when the SLC12A2 promoter is hypomethylated due totranscription factor Sp1 binding.^[24]

Unlike SLC12A2, the promotor for gene SLC12A1, which encodes for NKCC2, does not contain either aTATA box orSp1 binding sites.^[4] Regulatory binding sites in the NKCC2 promotor include sites forhepatocyte nuclear factor 1,cAMP-response element binding protein,CCAAT-enhancer binding proteins, andbasic helix-loop-helix proteins.^[4]

Theenergy required to move solutes across the cell membrane is provided by theelectrochemical gradient of sodium. Sodium's electrochemical gradient is established by theNa/K-ATPase, which is anATP-dependentenzyme. Since NKCC proteins use sodium's gradient, their activity is indirectly dependent on ATP; for this reason, NKCC proteins are said to move solutes by way ofsecondary active transport.There are three isoforms of NKCC2 created by alternative splicing (NKCC2A, B and F). Each one of these isoforms is expressed at different portions of the thick ascending limb and they have different affinity for sodium that correlates with its localization. The isoform F is more predominant in the deeper portion of the thick ascending limb, where the sodium concentration is very high. NKCC2F is the isoform with the lowest affinity for sodium and this allows the cotransporter to work at this sodium rich environment. Conversely, NKCC2B is expressed at the more superficial portion of the thick ascending limb and the macula densa, and it has the highest affinity for sodium. This permits NKCC2B to function in this sodium-depleted environment without saturating. The NKCC2A isoform shows an intermediate distribution and affinity for sodium.^[25] In this way, NKCC2 is able to function properly along the range of sodium concentrations found along the thick ascending limb.

• Cotransport
• Cotransporter

• Sodium-Potassium-Chloride+Symporters at the U.S. National Library of MedicineMedical Subject Headings (MeSH)

• Genes on human chromosome 15
• Genes on human chromosome 5
• Solute carrier family

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