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Na–K–Cl cotransporter

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solute carrier family 12 member 1
Identifiers
SymbolSLC12A1
Alt. symbolsNKCC2
NCBI gene6557
HGNC10910
OMIM600839
Orthologs286
RefSeqNM_000338
UniProtQ13621
Other data
LocusChr. 15 q21.1
Search for
StructuresSwiss-model
DomainsInterPro
solute carrier family 12 member 2
Identifiers
SymbolSLC12A2
Alt. symbolsNKCC1
NCBI gene6558
HGNC10911
OMIM600840
Orthologs20283
RefSeqNM_001046
UniProtP55011
Other data
LocusChr. 5 q23.3
Search for
StructuresSwiss-model
DomainsInterPro
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.[1] 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.[2]

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[3].

Function

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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.[3] The rate of transport of these ions are regulated by phosphorylation sites, which present on both NKCC isoforms[4].

NKCC1

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The NKCC1 isoform consists of about 1,200 amino acids, with about 500 amino acids residues giving rise to twelve hydrophobic transmembrane regions[5]. 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[6]. 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[5].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[5]. Additionally, bumetanide, a loop 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 that secrete fluids, called exocrine glands.[7] In cells of these organs, NKCC1 is commonly found in the basolateral membrane,[8] the part of the cell membrane closest to the blood vessels. Exon 21 possesses a translocation 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 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.[8] Specifically in the cochlea, NKCC1 is present in the stria vascularis, spiral ligament, and spiral ganglia[8]. Similarly, NKCC1 expression decreases with aging, resulting in progressive hearing loss[10]. Additionally, NKCC1 is present in the dark cells of the vestibule and contributes to generation of the endolymph of the vestibular system[11].

NKCC1 is also expressed in many regions of the brain during early development, but not in adulthood.[12] 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.[13]

NKCC1 has been identified in Sertoli cells, spermatocytes, and spermatids in the male reproductive system[14]. 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[14]. Additionally, the NKCC1 knockdown mice also exhibit a decreased testicle size compared to wild-type mice[14]. 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[4].

NKCC2

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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[3]. Additionally, the NKCC2 isoform does not contain exon 21, which results in NKCC2 being translocated to the apical membrane[4]. 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[4]. 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[15].

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[16] 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.[17] 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:

  1. Increases the amount of active osmolytes in urine by decreasing absorption of sodium
  2. Erases the papillar gradient
  3. 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.[18][19]

Genetics

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NKCC1 and NKCC2 are encoded by genes on the long arms of chromosomes 5[5] and 15,[20] 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.[20]

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[21]. Additionally, NKCC1 expression is upregulated when the SLC12A2 promoter is hypomethylated due to transcription factor Sp1 binding[22].

Unlike SLC12A2, the promotor for gene SLC12A1, which encodes for NKCC2, does not contain either a TATA box or Sp1 binding sites[4]. 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[4].

Kinetics

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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.[23] In this way, NKCC2 is able to function properly along the range of sodium concentrations found along the thick ascending limb.

See also

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References

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  1. ^ Haas M (October 1994). "The Na-K-Cl cotransporters". Am. J. Physiol. 267 (4 Pt 1): C869–85. doi:10.1152/ajpcell.1994.267.4.C869. PMID 7943281. S2CID 22680398.
  2. ^ Hebert, SC; Mount, DB; Gamba, G (February 2004). "Molecular physiology of cation-coupled Cl− cotransport: the SLC12 family". Pflügers Archiv: European Journal of Physiology. 447 (5): 580–593. doi:10.1007/s00424-003-1066-3. PMID 12739168. S2CID 21998913.
  3. ^ a b c Russell, J. M. (January 2000). "Sodium-potassium-chloride cotransport". Physiological Reviews. 80 (1): 211–276. doi:10.1152/physrev.2000.80.1.211. ISSN 0031-9333. PMID 10617769. S2CID 8909659.
  4. ^ a b c d e f Terjung, Ronald, ed. (2011-01-17). Comprehensive Physiology (1 ed.). Wiley. doi:10.1002/cphy.c170018. ISBN 978-0-470-65071-4. PMID 29687903.
  5. ^ a b c d e Payne, John A; Forbush, Bliss (1995-01-01). "Molecular characterization of the epithelial NaKCl cotransporter isoforms". Current Opinion in Cell Biology. 7 (4): 493–503. doi:10.1016/0955-0674(95)80005-0. ISSN 0955-0674. PMID 7495568.
  6. ^ Payne, John A.; Xu, Jian-Chao; Haas, Melanie; Lytle, Christian Y.; Ward, David; Forbush, Bliss (July 1995). "Primary Structure, Functional Expression, and Chromosomal Localization of the Bumetanide-sensitive Na-K-Cl Cotransporter in Human Colon". Journal of Biological Chemistry. 270 (30): 17977–17985. doi:10.1074/jbc.270.30.17977. ISSN 0021-9258. PMID 7629105.
  7. ^ Haas M, Forbush B (2000). "The Na-K-Cl cotransporter of secretory epithelia". Annu. Rev. Physiol. 62: 515–34. doi:10.1146/annurev.physiol.62.1.515. PMID 10845101.
  8. ^ a b c Delpire E, Lu J, England R, Dull C, Thorne T (June 1999). "Deafness and imbalance associated with inactivation of the secretory Na-K-2Cl co-transporter". Nat. Genet. 22 (2): 192–5. doi:10.1038/9713. PMID 10369265. S2CID 23779936.
  9. ^ Carmosino, Monica; Giménez, Ignacio; Caplan, Michael; Forbush, Biff (October 2008). Mostov, Keith E. (ed.). "Exon Loss Accounts for Differential Sorting of Na-K-Cl Cotransporters in Polarized Epithelial Cells". Molecular Biology of the Cell. 19 (10): 4341–4351. doi:10.1091/mbc.e08-05-0478. ISSN 1059-1524. PMC 2555935. PMID 18667527.
  10. ^ Diaz, Rodney C.; Vazquez, Ana Elena; Dou, Hongwei; Wei, Dongguang; Cardell, Emma Lou; Lingrel, Jerry; Shull, Gary E.; Doyle, Karen Jo; Yamoah, Ebenezer N. (2007-11-02). "Conservation of Hearing by Simultaneous Mutation of Na,K-ATPase and NKCC1". Journal of the Association for Research in Otolaryngology. 8 (4): 422–434. doi:10.1007/s10162-007-0089-4. ISSN 1525-3961. PMC 2538340. PMID 17674100.
  11. ^ Ciuman, R R (February 2009). "Stria vascularis and vestibular dark cells: characterisation of main structures responsible for inner-ear homeostasis, and their pathophysiological relations". The Journal of Laryngology & Otology. 123 (2): 151–162. doi:10.1017/S0022215108002624. ISSN 0022-2151. PMID 18570690.
  12. ^ Dzhala VI, Talos DM, Sdrulla DA, Brumback AC, Mathews GC, Benke TA, Delpire E, Jensen FE, Staley KJ (November 2005). "NKCC1 transporter facilitates seizures in the developing brain". Nat. Med. 11 (11): 1205–13. doi:10.1038/nm1301. PMID 16227993. S2CID 25348736.
  13. ^ Ben-Ari Y, Gaiarsa JL, Tyzio R, Khazipov R (October 2007). "GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations". Physiol. Rev. 87 (4): 1215–84. doi:10.1152/physrev.00017.2006. PMID 17928584.
  14. ^ a b c Pace, Amy J.; Lee, Eddie; Athirakul, Krairek; Coffman, Thomas M.; O’Brien, Deborah A.; Koller, Beverly H. (2000-02-15). "Failure of spermatogenesis in mouse lines deficient in the Na+-K+-2Cl– cotransporter". Journal of Clinical Investigation. 105 (4): 441–450. doi:10.1172/JCI8553. ISSN 0021-9738. PMC 289162. PMID 10683373.
  15. ^ Alshahrani, Saeed; Almutairi, Mohammed Mashari; Kursan, Shams; Dias-Junior, Eduardo; Almiahuob, Mohamed Mahmoud; Aguilar-Bryan, Lydia; Di Fulvio, Mauricio (December 2015). "Increased Slc12a1 expression in β-cells and improved glucose disposal in Slc12a2 heterozygous mice". Journal of Endocrinology. 227 (3): 153–165. doi:10.1530/JOE-15-0327. ISSN 0022-0795. PMC 4623298. PMID 26400961.
  16. ^ Lytle C, Xu JC, Biemesderfer D, Forbush B (December 1995). "Distribution and diversity of Na-K-Cl cotransport proteins: a study with monoclonal antibodies". Am. J. Physiol. 269 (6 Pt 1): C1496–505. doi:10.1152/ajpcell.1995.269.6.C1496. PMID 8572179.
  17. ^ Gamba G, Friedman PA (May 2009). "Thick ascending limb: the Na(+):K (+):2Cl (-) co-transporter, NKCC2, and the calcium-sensing receptor, CaSR". Pflügers Arch. 458 (1): 61–76. doi:10.1007/s00424-008-0607-1. PMC 3584568. PMID 18982348.
  18. ^ Rieg T, Tang T, Uchida S, Hammond HK, Fenton RA, Vallon V (January 2013). "Adenylyl cyclase 6 enhances NKCC2 expression and mediates vasopressin-induced phosphorylation of NKCC2 and NCC". Am. J. Pathol. 182 (1): 96–106. doi:10.1016/j.ajpath.2012.09.014. PMC 3532715. PMID 23123217.
  19. ^ Ares GR, Caceres PS, Ortiz PA (December 2011). "Molecular regulation of NKCC2 in the thick ascending limb". Am. J. Physiol. Renal Physiol. 301 (6): F1143–59. doi:10.1152/ajprenal.00396.2011. PMC 3233874. PMID 21900458.
  20. ^ a b Simon DB, Karet FE, Hamdan JM, DiPietro A, Sanjad SA, Lifton RP (June 1996). "Bartter's syndrome, hypokalaemic alkalosis with hypercalciuria, is caused by mutations in the Na-K-2Cl cotransporter NKCC2". Nat. Genet. 13 (2): 183–8. doi:10.1038/ng0696-183. PMID 8640224. S2CID 42296304.
  21. ^ Ando, Zen-ichi; Sato, Shigeru; Ikeda, Keiko; Kawakami, Kiyoshi (June 2005). "Slc12a2 is a direct target of two closely related homeobox proteins, Six1 and Six4". The FEBS Journal. 272 (12): 3026–3041. doi:10.1111/j.1742-4658.2005.04716.x. ISSN 1742-464X. PMID 15955062.
  22. ^ Cho, Hyun-Min; Lee, Hae-Ahm; Kim, Hye Young; Lee, Dong-Youb; Kim, In Kyeom (July 2013). "Recruitment of Specificity Protein 1 by CpG hypomethylation upregulates Na+-K+-2Cl− cotransporter 1 in hypertensive rats". Journal of Hypertension. 31 (7): 1406–1413. doi:10.1097/HJH.0b013e3283610fed. ISSN 0263-6352. PMID 24006039.
  23. ^ Plata C, Meade P, Vazquez N, Hebert SC, Gamba G (Mar 2002). "Functional properties of the apical Na+-K+-2Cl- cotransporter isoforms". J. Biol. Chem. 277 (13): 11004–12. doi:10.1074/jbc.M110442200. PMID 11790783.
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