Na–K–Cl cotransporter

{{Short description|Group of transport proteins}} {{infobox protein | Name = solute carrier family 12 member 1 | caption = | image = | width = | HGNCid = 10910 | Symbol = SLC12A1 | AltSymbols = NKCC2 | EntrezGene = 6557 | OMIM = 600839 | HomoloGene = 286 | RefSeq = NM_000338 | UniProt = Q13621 | PDB = | ECnumber = | Chromosome = 15 | Arm = q | Band = 21.1 | LocusSupplementaryData = }} {{infobox protein | Name = solute carrier family 12 member 2 | caption = | image = | width = | HGNCid = 10911 | Symbol = SLC12A2 | AltSymbols = NKCC1 | EntrezGene = 6558 | OMIM = 600840 | HomoloGene = 20283 | RefSeq = NM_001046 | UniProt = P55011 | PDB = | ECnumber = | Chromosome = 5 | Arm = q | Band = 23.3 | LocusSupplementaryData = }} thumb|The basic function of the Na-K-Cl cotransporter (NKCC). The '''Na–K–Cl cotransporter''' ('''NKCC''') is a transport protein that aids in the secondary active transport of sodium, potassium, and chloride into cells.<ref name="pmid7943281">{{cite journal | vauthors = Haas M | title = The Na-K-Cl cotransporters | journal = Am. J. Physiol. | volume = 267 | issue = 4 Pt 1 | pages = C869–85 | date=October 1994 | pmid = 7943281 | doi = 10.1152/ajpcell.1994.267.4.C869 | s2cid = 22680398 }}</ref> In humans there are two isoforms of this membrane transport protein, '''NKCC1''' and '''NKCC2''', encoded by two different genes (''SLC12A2'' and ''SLC12A1'' respectively). Two isoforms of the NKCC1/Slc12a2 gene result from keeping (isoform 1) or skipping (isoform 2) exon 21 in the final gene product.<ref>{{cite journal|last=Hebert|first=SC|author2=Mount, DB|author3= Gamba, G|title=Molecular physiology of cation-coupled Cl<sup>−</sup> cotransport: the SLC12 family.|journal=Pflügers Archiv: European Journal of Physiology|date=February 2004 |volume=447|issue=5|pages=580–593|doi=10.1007/s00424-003-1066-3|pmid=12739168|s2cid=21998913}}</ref>

NKCC1 is widely distributed throughout the human body; it has important functions in organs that secrete fluids. In contrast, NKCC2 is found specifically in the kidney, where it extracts sodium, potassium, and chloride from the urine so they can be reabsorbed into the blood.<ref name=":1" />

==Function== NKCC proteins are membrane transport proteins that transport sodium (Na), potassium (K), and chloride (Cl) ions across the cell membrane. Because they move each solute in the same direction, they are considered symporters. They maintain electroneutrality by moving two positively charged solutes (sodium and potassium) alongside two parts of a negatively charged solute (chloride). Thus the stoichiometry of the transported solutes is 1Na:1K:2Cl. Although squid giant axons are the only notable exception with a stoichiometry of 2Na:1K:3Cl, electroneutrality across the protein transporter is still maintained.<ref name=":1">{{Cite journal|last=Russell|first=J. M.|date=January 2000|title=Sodium-potassium-chloride cotransport|journal=Physiological Reviews|volume=80|issue=1|pages=211–276|doi=10.1152/physrev.2000.80.1.211|issn=0031-9333|pmid=10617769|s2cid=8909659}}</ref> The rate of transport of these ions are regulated by phosphorylation sites, which present on both NKCC isoforms.<ref name=":2">{{Cite book |url=https://onlinelibrary.wiley.com/doi/book/10.1002/cphy |title=Comprehensive Physiology |date=2011-01-17 |publisher=Wiley |isbn=978-0-470-65071-4 |editor-last=Terjung |editor-first=Ronald |edition=1 |language=en |doi=10.1002/cphy.c170018|pmid=29687903 }}</ref>

===NKCC1=== The NKCC1 isoform consists of about 1,200 amino acids, with about 500 amino acids residues giving rise to twelve hydrophobic transmembrane regions.<ref name=":0">{{Cite journal |last1=Payne |first1=John A |last2=Forbush |first2=Bliss |date=1995-01-01 |title=Molecular characterization of the epithelial NaKCl cotransporter isoforms |url=https://linkinghub.elsevier.com/retrieve/pii/0955067495800050 |journal=Current Opinion in Cell Biology |volume=7 |issue=4 |pages=493–503 |doi=10.1016/0955-0674(95)80005-0 |pmid=7495568 |issn=0955-0674|url-access=subscription }}</ref> However, evidence of a shorter NKCC1 mRNA 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.<ref>{{Cite journal |last1=Payne |first1=John A. |last2=Xu |first2=Jian-Chao |last3=Haas |first3=Melanie |last4=Lytle |first4=Christian Y. |last5=Ward |first5=David |last6=Forbush |first6=Bliss |date=July 1995 |title=Primary Structure, Functional Expression, and Chromosomal Localization of the Bumetanide-sensitive Na-K-Cl Cotransporter in Human Colon |journal=Journal of Biological Chemistry |volume=270 |issue=30 |pages=17977–17985 |doi=10.1074/jbc.270.30.17977 |doi-access=free |pmid=7629105 |issn=0021-9258}}</ref> The carboxy-terminal of the NKCC1 cotransporter contains multiple phosphorylation sites and is highly conserved across species, while in contrast, the amino-terminal contains at least one phosphorylation site and is poorly conserved across species<ref name=":0" />.Focusing on the transmembrane regions, mutagenesis-driven affinity studies have revealed the second transmembrane region as the determinant of cation affinity, while chloride affinity was determined by transmembrane regions four through seven.<ref name=":0" /> Additionally, bumetanide, a loop diuretic, was found to bind to transmembrane regions 2 through 7, 11, and 12.<ref name=":0" />

NKCC1 is widely distributed throughout the body, especially in organs that secrete fluids, called exocrine glands.<ref name="pmid10845101">{{cite journal |vauthors=Haas M, Forbush B |title=The Na-K-Cl cotransporter of secretory epithelia |journal=Annu. Rev. Physiol. |volume=62 |pages=515–34 |year=2000 |pmid=10845101 |doi=10.1146/annurev.physiol.62.1.515}}</ref> In cells of these organs, NKCC1 is commonly found in the basolateral membrane,<ref name="pmid10369265" /> the part of the cell membrane closest to the blood vessels. Exon 21 possesses a translocation sequence that targets NKCC1 to the basolateral membrane.<ref>{{Cite journal |last1=Carmosino |first1=Monica |last2=Giménez |first2=Ignacio |last3=Caplan |first3=Michael |last4=Forbush |first4=Biff |date=October 2008 |editor-last=Mostov |editor-first=Keith E. |title=Exon Loss Accounts for Differential Sorting of Na-K-Cl Cotransporters in Polarized Epithelial Cells |journal=Molecular Biology of the Cell |language=en |volume=19 |issue=10 |pages=4341–4351 |doi=10.1091/mbc.e08-05-0478 |issn=1059-1524 |pmc=2555935 |pmid=18667527}}</ref> Thus, NKCC1 cotransporters that have been alternatively spliced to exclude exon 21 will be translocated to the apical 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 luminal concentration of solutes and causing water to be secreted by osmosis.

In addition to exocrine glands, NKCC1 is necessary for establishing the potassium-rich endolymph that bathes part of the cochlea, an organ necessary for hearing. Inhibition of NKCC1, as with furosemide or other loop diuretics, can result in deafness.<ref name="pmid10369265">{{cite journal |vauthors=Delpire E, Lu J, England R, Dull C, Thorne T |title=Deafness and imbalance associated with inactivation of the secretory Na-K-2Cl co-transporter |journal=Nat. Genet. |volume=22 |issue=2 |pages=192–5 |date=June 1999 |pmid=10369265 |doi=10.1038/9713 |s2cid=23779936}}</ref> Specifically in the cochlea, NKCC1 is present in the stria vascularis, spiral ligament, and spiral ganglia.<ref name="pmid10369265" /> Similarly, NKCC1 expression decreases with aging, resulting in progressive hearing loss.<ref>{{Cite journal |last1=Diaz |first1=Rodney C. |last2=Vazquez |first2=Ana Elena |last3=Dou |first3=Hongwei |last4=Wei |first4=Dongguang |last5=Cardell |first5=Emma Lou |last6=Lingrel |first6=Jerry |last7=Shull |first7=Gary E. |last8=Doyle |first8=Karen Jo |last9=Yamoah |first9=Ebenezer N. |date=2007-11-02 |title=Conservation of Hearing by Simultaneous Mutation of Na,K-ATPase and NKCC1 |journal=Journal of the Association for Research in Otolaryngology |language=en |volume=8 |issue=4 |pages=422–434 |doi=10.1007/s10162-007-0089-4 |issn=1525-3961 |pmc=2538340 |pmid=17674100}}</ref> Additionally, NKCC1 is present in the dark cells of the vestibule and contributes to generation of the endolymph of the vestibular system.<ref>{{Cite journal |last=Ciuman |first=R R |date=February 2009 |title=Stria vascularis and vestibular dark cells: characterisation of main structures responsible for inner-ear homeostasis, and their pathophysiological relations |url=https://www.cambridge.org/core/product/identifier/S0022215108002624/type/journal_article |journal=The Journal of Laryngology & Otology |language=en |volume=123 |issue=2 |pages=151–162 |doi=10.1017/S0022215108002624 |pmid=18570690 |issn=0022-2151|url-access=subscription }}</ref>

NKCC1 is also expressed in many regions of the brain during early development, but not in adulthood.<ref name="pmid16227993">{{cite journal |vauthors=Dzhala VI, Talos DM, Sdrulla DA, Brumback AC, Mathews GC, Benke TA, Delpire E, Jensen FE, Staley KJ | title = NKCC1 transporter facilitates seizures in the developing brain | journal = Nat. Med. | volume = 11 | issue = 11 | pages = 1205–13 | date=November 2005 | pmid = 16227993 | doi = 10.1038/nm1301 | s2cid = 25348736 }}</ref> This change in NKCC1 presence seems to be responsible for altering responses to the neurotransmitters GABA and glycine 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 the reversal potential for chloride, given by the Nernst equation. Later in development expression of NKCC1 is reduced, while expression of a KCC2 K-Cl cotransporter increased, thus bringing internal chloride concentration in neurons down to adult values.<ref name="pmid17928584">{{cite journal | vauthors = Ben-Ari Y, Gaiarsa JL, Tyzio R, Khazipov R | title = GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations | journal = Physiol. Rev. | volume = 87 | issue = 4 | pages = 1215–84 | date = October 2007 | pmid = 17928584 | doi = 10.1152/physrev.00017.2006 }}</ref>

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.<ref>Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa K, Pirvola U, Saarma M, Kaila K (1999). "The K+/Cl− co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation". Journal of Neuroscience. 19 (12): 4695–4704. doi:10.1038/16697.</ref><ref>Ben-Ari Y (2002). "Excitatory actions of GABA during development: the nature of the nurture". Nature Reviews Neuroscience. 3 (9): 728–739. doi:10.1038/nrn920.</ref>

NKCC1 has been identified in Sertoli cells, spermatocytes, and spermatids in the male reproductive system.<ref name=":3">{{Cite journal |last1=Pace |first1=Amy J. |last2=Lee |first2=Eddie |last3=Athirakul |first3=Krairek |last4=Coffman |first4=Thomas M. |last5=O’Brien |first5=Deborah A. |last6=Koller |first6=Beverly H. |date=2000-02-15 |title=Failure of spermatogenesis in mouse lines deficient in the Na+-K+-2Cl– cotransporter |journal=Journal of Clinical Investigation |language=en |volume=105 |issue=4 |pages=441–450 |doi=10.1172/JCI8553 |issn=0021-9738 |pmc=289162 |pmid=10683373}}</ref> NKCC1 function appears to be critical for spermatogenesis, as knockdown of NKCC1 in mice results in spermatocytes failing to mature into spermatozoa, resulting in infertility.<ref name=":3" /> Additionally, the NKCC1 knockdown mice also exhibit a decreased testicle size compared to wild-type mice.<ref name=":3" /> The mechanism behind NKCC1-dependent male fertility is unclear, it is possible that the observed decreased sperm count could be due to either lack of NKCC1 cotransport in the testis or upstream failure of NKCC1-expressing neurons in the hypothalamus to release gonadotropin-releasing hormone.<ref name=":2" />

===NKCC2=== The NKCC2 isoform is smaller than NKCC1, 121 kDa versus 195 kDa, respectively, primarily because NKCC2 does not contain an 80 amino acid sequence present on the N-terminus of NKCC1.<ref name=":1" /> Additionally, the NKCC2 isoform does not contain exon 21, which results in NKCC2 being translocated to the apical membrane.<ref name=":2" /> Compared to NKCC1, exon 1 is divided into two separate exons in NKCC2 and exon 4 is alternatively spliced into forms A, B, and F, which are all exclusive to NKCC2.<ref name=":2" /> NKCC2 expression is thought to be limited to renal cells, although this has been called into question with possible NKCC2 expression in pancreatic β-cells.<ref>{{Cite journal |last1=Alshahrani |first1=Saeed |last2=Almutairi |first2=Mohammed Mashari |last3=Kursan |first3=Shams |last4=Dias-Junior |first4=Eduardo |last5=Almiahuob |first5=Mohamed Mahmoud |last6=Aguilar-Bryan |first6=Lydia |last7=Di Fulvio |first7=Mauricio |date=December 2015 |title=Increased Slc12a1 expression in β-cells and improved glucose disposal in Slc12a2 heterozygous mice |url=https://joe.bioscientifica.com/view/journals/joe/227/3/153.xml |journal=Journal of Endocrinology |volume=227 |issue=3 |pages=153–165 |doi=10.1530/JOE-15-0327 |issn=0022-0795 |pmc=4623298 |pmid=26400961}}</ref>

NKCC2 is specifically found in cells of the thick ascending limb of the loop of Henle and the macula densa in nephrons, the basic functional units of the kidney. Within these cells, NKCC2 resides in the apical membrane<ref name="pmid8572179">{{cite journal |vauthors=Lytle C, Xu JC, Biemesderfer D, Forbush B | title = Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies | journal = Am. J. Physiol. | volume = 269 | issue = 6 Pt 1 | pages = C1496–505 | date=December 1995 | pmid = 8572179 | doi = 10.1152/ajpcell.1995.269.6.C1496 }}</ref> abutting the nephron's lumen, which is the hollow space containing urine. It thus serves both in sodium absorption and in tubuloglomerular 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.<ref name="pmid18982348">{{cite journal |vauthors=Gamba G, Friedman PA | title = Thick ascending limb: the Na(+):K (+):2Cl (-) co-transporter, NKCC2, and the calcium-sensing receptor, CaSR | journal = Pflügers Arch | volume = 458 | issue = 1 | pages = 61–76 | date=May 2009 | pmid = 18982348 | doi = 10.1007/s00424-008-0607-1| pmc=3584568}}</ref> 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 the blood, where it contributes to the maintenance of blood pressure.

Furosemide and other loop diuretics inhibit the activity of NKCC2, thereby impairing sodium reabsorption in the thick ascending limb of the loop of Henle. The action of these loop 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: # Increases the amount of active osmolytes in urine by decreasing absorption of sodium # Erases the papillar gradient # Inhibits tubuloglomerular feedback Loop diuretics therefore ultimately result in decreased blood pressure.

The hormone vasopressin 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 some serine and threonine 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 through aquaporin 2 channels by creating a hypo-osmotic filtrate.<ref name="pmid23123217">{{cite journal | vauthors = Rieg T, Tang T, Uchida S, Hammond HK, Fenton RA, Vallon V | title = Adenylyl cyclase 6 enhances NKCC2 expression and mediates vasopressin-induced phosphorylation of NKCC2 and NCC | journal = Am. J. Pathol. | volume = 182 | issue = 1 | pages = 96–106 | date = January 2013 | pmid = 23123217 | pmc = 3532715 | doi = 10.1016/j.ajpath.2012.09.014 }}</ref><ref name="pmid21900458">{{cite journal | vauthors = Ares GR, Caceres PS, Ortiz PA | title = Molecular regulation of NKCC2 in the thick ascending limb | journal = Am. J. Physiol. Renal Physiol. | volume = 301 | issue = 6 | pages = F1143–59 | date = December 2011 | pmid = 21900458 | pmc = 3233874 | doi = 10.1152/ajprenal.00396.2011 }}</ref>

==Genetics== NKCC1 and NKCC2 are encoded by genes on the long arms of chromosomes 5<ref name=":0" /> and 15,<ref name="pmid8640224">{{cite journal |vauthors = Simon DB, Karet FE, Hamdan JM, DiPietro A, Sanjad SA, Lifton RP | title = Bartter's syndrome, hypokalaemic alkalosis with hypercalciuria, is caused by mutations in the Na-K-2Cl cotransporter NKCC2 | journal = Nat. Genet. | volume = 13 | issue = 2 | pages = 183–8 | date=June 1996 | pmid = 8640224 | doi = 10.1038/ng0696-183 | s2cid = 42296304 }}</ref> respectively. A loss of function mutation of NKCC2 produces Bartter syndrome, an autosomal recessive disorder characterized by hypokalemic metabolic alkalosis with normal to low blood pressure.<ref name="pmid8640224"/>

The promotor for gene SLC12A2, which encodes for NKCC1, contains binding sites for homeobox transcription factors SIX1 and SIX4, which have been shown to upregulate NKCC1 mRNA expression when bound.<ref>{{Cite journal |last1=Ando |first1=Zen-ichi |last2=Sato |first2=Shigeru |last3=Ikeda |first3=Keiko |last4=Kawakami |first4=Kiyoshi |date=June 2005 |title=Slc12a2 is a direct target of two closely related homeobox proteins, Six1 and Six4 |url=https://febs.onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2005.04716.x |journal=The FEBS Journal |language=en |volume=272 |issue=12 |pages=3026–3041 |doi=10.1111/j.1742-4658.2005.04716.x |pmid=15955062 |issn=1742-464X|url-access=subscription }}</ref> Additionally, NKCC1 expression is upregulated when the SLC12A2 promoter is hypomethylated due to transcription factor Sp1 binding.<ref>{{Cite journal |last1=Cho |first1=Hyun-Min |last2=Lee |first2=Hae-Ahm |last3=Kim |first3=Hye Young |last4=Lee |first4=Dong-Youb |last5=Kim |first5=In Kyeom |date=July 2013 |title=Recruitment of Specificity Protein 1 by CpG hypomethylation upregulates Na+-K+-2Cl− cotransporter 1 in hypertensive rats |url=https://journals.lww.com/00004872-201307000-00010 |journal=Journal of Hypertension |language=en |volume=31 |issue=7 |pages=1406–1413 |doi=10.1097/HJH.0b013e3283610fed |pmid=24006039 |issn=0263-6352|url-access=subscription }}</ref>

Unlike SLC12A2, the promotor for gene SLC12A1, which encodes for NKCC2, does not contain either a TATA box or Sp1 binding sites.<ref name=":2" /> Regulatory binding sites in the NKCC2 promotor include sites for hepatocyte nuclear factor 1, cAMP-response element binding protein, CCAAT-enhancer binding proteins, and basic helix-loop-helix proteins.<ref name=":2" />

==Kinetics== The energy required to move solutes across the cell membrane is provided by the electrochemical gradient of sodium. Sodium's electrochemical gradient is established by the Na/K-ATPase, which is an ATP-dependent enzyme. 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 of secondary 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.<ref name="pmid11790783">{{cite journal |vauthors=Plata C, Meade P, Vazquez N, Hebert SC, Gamba G | title = Functional properties of the apical Na+-K+-2Cl- cotransporter isoforms | journal = J. Biol. Chem. | volume = 277 | issue = 13 | pages = 11004–12 | date=Mar 2002 | pmid = 11790783 | doi = 10.1074/jbc.M110442200 | doi-access = free }}</ref> In this way, NKCC2 is able to function properly along the range of sodium concentrations found along the thick ascending limb.

==See also== *Cotransport *Cotransporter

==References== {{Reflist|2}}

==External links== * {{MeshName|Sodium-Potassium-Chloride+Symporters}}

Category:Solute carrier family