2.A.30 The Cation-Chloride Cotransporter (CCC) Family

Members of the CCC family, found in animals, plants, fungi, archaea and bacteria, can catalyze NaCl/KCl symport, NaCl symport, or KCl symport depending on the system. The NaCl/KCl symporters are specifically inhibited by bumetanide while the NaCl symporters are specifically inhibited by thiazide. Most characterized CCC family proteins are from higher animals, but several have been identified in greeen algae, mosses, grasses, dicots and bacteria (Henderson et al. 2018). Homologues have been sequenced from Caenorhabditis elegans (worm), Saccharomyces cerevisiae (yeast) and Synechococcus sp. (blue green bacterium).  These proteins show sequence similarity to members of the APC family (TC #2.A.3). CCC family proteins are usually large (between 1000 and 1200 amino acyl residues), and possess 12 putative transmembrane spanners flanked by large N-terminal and C-terminal hydrophilic domains. Henderson et al. 2018 have provided evidence for two phylogenetic clades which they called CCC1 and CCC2.

Two splice variants of NKCC2 are identical except for a 23 aa membrane domain. They have different affinities for Na+, K+ and Cl-. This segment (residues 216-233 in NKCC2) were examined for ion selectivity. Residue 216 affects K+ binding while residue 220 only affects Na+ binding. These two sites are presumed to be adjacent to each other (Gagnon et al., 2005). Cation-chloride cotransporters (CCCs) play roles in setting the Cl- driving force in nerves (Düsterwald et al. 2018).

Each of the major types of CCC family members in mammals exist as paralogous isoforms. These may differ in substrates transported. For example, of the four currently recognized KCl transporters, KCC1 and KCC4 both recognize KCl with similar affinities, but KCC1 exhibits anion selectivity: Cl- > SCN- = Br- > PO4-3 > I-, while KCl4 exhibits anion selectivity: Cl- > Br- > PO4-3 = I- > SCN-. Both are activated by cell swelling under hypotonic conditions (Mercado et al., 2000). These proteins may cotransport water (H2O) (Mollajew et al., 2010).

One member of the CCC family, the thiazide-sensitive NaCl cotransporter (NCC) of man is involved in 5% of the filtered load of NaCl in the kidney. Mutations in NCC cause the recessive Gitelman syndrome. NCC is a dimer in the membrane (de Jong et al., 2003). It is regulated by RasGRP1 which mediates the PE induced suppression of NCC activity through the stimulation of the MAPK pathway (Ko et al., 2007).

CCCs share a conserved structural scaffold that consists of a transmembrane transport domain followed by a cytoplasmic regulatory domain. Warmuth et al. (2009) determined the x-ray structure of the C-terminal domain of a CCC from the archaeon Mehanosarcina acetivorans. It shows a novel fold of a regulatory domain, distantly related to universal stress proteins. The protein forms dimers in solution, consistent with the proposed dimeric organization of eukaryotic CCC transporters.

The generalized transport reaction for CCC family symporters is:

{Na+ + K+ + 2Cl-} (out) ⇌ {Na+ + K+ + 2Cl-} (in).

That for the NaCl and KCl symporters is:

{Na+ or K+ + Cl-} (out) ⇌ {Na+ or K+ + Cl-} (in).

This family belongs to the APC Superfamily.



Adachi, M., Y. Asakura, Y. Sato, T. Tajima, T. Nakajima, T. Yamamoto, and K. Fujieda. (2007). Novel SLC12A1 (NKCC2) mutations in two families with Bartter syndrome type 1. Endocr J 54: 1003-1007.

Amadeo, A., A. Coatti, P. Aracri, M. Ascagni, D. Iannantuoni, D. Modena, L. Carraresi, S. Brusco, S. Meneghini, A. Arcangeli, M.E. Pasini, and A. Becchetti. (2018). Postnatal Changes in K/Cl Cotransporter-2 Expression in the Forebrain of Mice Bearing a Mutant Nicotinic Subunit Linked to Sleep-Related Epilepsy. Neuroscience 386: 91-107.

Berenbrink, M., S. Völkel, P. Koldkjaer, N. Heisler, and M. Nikinmaa. (2006). Two different oxygen sensors regulate oxygen-sensitive K+ transport in crucian carp red blood cells. J. Physiol. 575: 37-48.

Boettger, T., M.B. Rust, H. Maier, T. Seidenbecher, M. Schweizer, D.J. Keating, J. Faulhaber, H. Ehmke, C. Pfeffer, O. Scheel, B. Lemcke, J. Horst, R. Leuwer, H.C. Pape, H. Völkl, C.A. Hübner, and T.J. Jentsch. (2003). Loss of K-Cl co-transporter KCC3 causes deafness, neurodegeneration and reduced seizure threshold. EMBO. J. 22: 5422-5434.

Chamma, I., Q. Chevy, J.C. Poncer, and S. Lévi. (2012). Role of the neuronal K-Cl co-transporter KCC2 in inhibitory and excitatory neurotransmission. Front Cell Neurosci 6: 5.

Döding, A., A.M. Hartmann, T. Beyer, and H.G. Nothwang. (2012). KCC2 transport activity requires the highly conserved L₆₇₅ in the C-terminal β1 strand. Biochem. Biophys. Res. Commun. 420: 492-497.

de Jong, J.C., P.H.G.M. Willems, F.J.M. Mooren, L.P.W.J. van den Heuvel, N.V.A.M. Knoers, and R.J.M. Bindels. (2003). The structural unit of the thiazide-sensitive NaCl cotransporter is a homodimer. J. Biol. Chem. 278: 24302-24307.

Düsterwald, K.M., C.B. Currin, R.J. Burman, C.J. Akerman, A.R. Kay, and J.V. Raimondo. (2018). Biophysical models reveal the relative importance of transporter proteins and impermeant anions in chloride homeostasis. Elife 7:. [Epub: Ahead of Print]

Gagnon, E., Bergeron, M.J., Daigle, N.D., Lefoll, M.H., and Isenring, P. (2005). Molecular mechanisms of cation transport by the renal Na+-K+-Cl- cotransporter: structural insight into the operating characteristics of the ion transport sites. J. Biol. Chem. 280: 32555-32563.

Gagnon, M., M.J. Bergeron, G. Lavertu, A. Castonguay, S. Tripathy, R.P. Bonin, J. Perez-Sanchez, D. Boudreau, B. Wang, L. Dumas, I. Valade, K. Bachand, M. Jacob-Wagner, C. Tardif, I. Kianicka, P. Isenring, G. Attardo, J.A. Coull, and Y. De Koninck. (2013). Chloride extrusion enhancers as novel therapeutics for neurological diseases. Nat. Med. 19: 1524-1528.

Gauvain, G., I. Chamma, Q. Chevy, C. Cabezas, T. Irinopoulou, N. Bodrug, M. Carnaud, S. Lévi, and J.C. Poncer. (2011). The neuronal K-Cl cotransporter KCC2 influences postsynaptic AMPA receptor content and lateral diffusion in dendritic spines. Proc. Natl. Acad. Sci. USA 108: 15474-15479.

Haas, M. and B. Forbush, III. (2000). The Na-K-Cl cotransporter of secretory epithelia. Annu. Rev. Physiol. 62: 515-534.

Hamann, S., J.J. Herrera-Perez, T. Zeuthen, and F.J. Alvarez-Leefmans. (2010). Cotransport of water by the Na+-K+-2Cl- cotransporter NKCC1 in mammalian epithelial cells. J. Physiol. 588: 4089-4101.

Henderson, S.W., S. Wege, and M. Gilliham. (2018). Plant Cation-Chloride Cotransporters (CCC): Evolutionary Origins and Functional Insights. Int J Mol Sci 19:.

Hertz, L., L. Peng, and D. Song. (2015). Ammonia, like K+, stimulates the Na+, K+, 2 Cl- cotransporter NKCC1 and the Na+,K+-ATPase and interacts with endogenous ouabain in astrocytes. Neurochem Res 40: 241-257.

Huang Y., Wang JJ. and Yung WH. (2013). Coupling between GABA-A receptor and chloride transporter underlies ionic plasticity in cerebellar Purkinje neurons. Cerebellum. 12(3):328-30.

Ivakine, E.A., B.A. Acton, V. Mahadevan, J. Ormond, M. Tang, J.C. Pressey, M.Y. Huang, D. Ng, E. Delpire, M.W. Salter, M.A. Woodin, and R.R. McInnes. (2013). Neto2 is a KCC2 interacting protein required for neuronal Cl- regulation in hippocampal neurons. Proc. Natl. Acad. Sci. USA 110: 3561-3566.

Jo, J., G.H. Son, B.L. Winters, M.J. Kim, D.J. Whitcomb, B.A. Dickinson, Y.B. Lee, K. Futai, M. Amici, M. Sheng, G.L. Collingridge, and K. Cho. (2010). Muscarinic receptors induce LTD of NMDAR EPSCs via a mechanism involving hippocalcin, AP2 and PSD-95. Nat Neurosci 13: 1216-1224.

Ko B., L.M. Joshi, L.L. Cooke, N. Vazquez, M.W. Musch, S.C. Hebert, G. Gamba, R.S. Hoover. Phorbol ester stimulation of RasGRP1 regulates the sodium-chloride cotransporter by a PKC-independent pathway. Proc. Natl. Acad. Sci. U.S.A. 104: 20120-20125.

Llano, O., S. Smirnov, S. Soni, A. Golubtsov, I. Guillemin, P. Hotulainen, I. Medina, H.G. Nothwang, C. Rivera, and A. Ludwig. (2015). KCC2 regulates actin dynamics in dendritic spines via interaction with β-PIX. J. Cell Biol. 209: 671-686.

Mercado, A., L. Song, N. Vázquez, D.B. Mount, and G. Gamba. (2000). Functional comparison of the K+-Cl- cotransporters KCC1 and KCC4. J. Biol. Chem. 275: 30326-30334.

Mistry, A.C., B.M. Wynne, L. Yu, V. Tomilin, Q. Yue, Y. Zhou, O. Al-Khalili, R. Mallick, H. Cai, A.A. Alli, B. Ko, A. Mattheyses, H.F. Bao, O. Pochynyuk, F. Theilig, D.C. Eaton, and R.S. Hoover. (2016). The Sodium Chloride Cotransporter (NCC) and Epithelial Sodium Channel (ENaC) Associate. Biochem. J. [Epub: Ahead of Print]

Mollajew, R., F. Zocher, A. Horner, B. Wiesner, E. Klussmann, and P. Pohl. (2010). Routes of epithelial water flow: aquaporins versus cotransporters. Biophys. J. 99: 3647-3656.

Mount, D.B., A. Mercado, L. Song, J. Xu, A.L. George, Jr., E. Delpire, and G. Gamba. (1999). Cloning and characterization of KCC3 and KCC4, new members of the cation-chloride cotransporter gene family. J. Biol. Chem. 274: 16355-16362.

Mount, D.B., R.S. Hoover, and S.C. Hebert. (1997). The molecular physiology of electroneutral cation-chloride cotransport. J. Membr. Biol. 158: 177-186.

Pacheco-Alvarez, D., P.S. Cristóbal, P. Meade, E. Moreno, N. Vazquez, E. Muñoz, A. Díaz, M.E. Juárez, I. Giménez, and G. Gamba. (2006). The Na+:Cl- cotransporter is activated and phosphorylated at the amino-terminal domain upon intracellular chloride depletion. J. Biol. Chem. 281: 28755-28763.

Park, J.H. and M.H. Saier, Jr. (1996). Phylogenetic, structural and functional characteristics of the Na-K-Cl cotransporter family. J. Membr. Biol. 149: 161-168.

Piermarini, P.M., D.C. Akuma, J.C. Crow, T.L. Jamil, W.G. Kerkhoff, K.C.M.F. Viel, and C.M. Gillen. (2017). Differential expression of putative sodium-dependent cation-chloride cotransporters in Aedes aegypti. Comp Biochem Physiol A Mol Integr Physiol 214: 40-49. [Epub: Ahead of Print]

Pressey, J.C., V. Mahadevan, C.S. Khademullah, Z. Dargaei, J. Chevrier, W. Ye, M. Huang, A.K. Chauhan, S.J. Meas, P. Uvarov, M.S. Airaksinen, and M.A. Woodin. (2017). A kainate receptor subunit promotes the recycling of the neuron-specific K+-Cl- co-transporter KCC2 in hippocampal neurons. J. Biol. Chem. 292: 6190-6201.

Puskarjov, M., P. Seja, S.E. Heron, T.C. Williams, F. Ahmad, X. Iona, K.L. Oliver, B.E. Grinton, L. Vutskits, I.E. Scheffer, S. Petrou, P. Blaesse, L.M. Dibbens, S.F. Berkovic, and K. Kaila. (2014). A variant of KCC2 from patients with febrile seizures impairs neuronal Cl- extrusion and dendritic spine formation. EMBO Rep 15: 723-729.

Russell, J.M. (2000). Sodium-potassium-chloride cotransport. Physiol. Rev. 80: 211-276.

Schwale, C., S. Schumacher, C. Bruehl, S. Titz, A. Schlicksupp, M. Kokocinska, J. Kirsch, A. Draguhn, and J. Kuhse. (2016). KCC2 knockdown impairs glycinergic synapse maturation in cultured spinal cord neurons. Histochem Cell Biol 145: 637-646.

Seaayfan, E., N. Defontaine, S. Demaretz, N. Zaarour, and K. Laghmani. (2015). OS9 interacts with NKCC2 and targets its immature form for the endoplasmic-reticulum-associated degradation pathway. J. Biol. Chem. [Epub: Ahead of Print]

Shrestha S., Park J., Ahn SJ. and Kim Y. (2015). PGE2 MEDIATES OENOCYTOID CELL LYSIS VIA A SODIUM-POTASSIUM-CHLORIDE COTRANSPORTER. Arch Insect Biochem Physiol. 89(4):218-29.

Somasekharan, S., J. Tanis, and B. Forbush. (2012). Loop diuretic and ion-binding residues revealed by scanning mutagenesis of transmembrane helix 3 (TM3) of Na-K-Cl cotransporter (NKCC1). J. Biol. Chem. 287: 17308-17317.

Stechman, M.J., N.Y. Loh, and R.V. Thakker. (2007). Genetics of hypercalciuric nephrolithiasis: renal stone disease. Ann. N.Y. Acad. Sci. 1116: 461-484.

Wang, J., C. Sun, N. Gerdes, C. Liu, M. Liao, J. Liu, M.A. Shi, A. He, Y. Zhou, G.K. Sukhova, H. Chen, X.W. Cheng, M. Kuzuya, T. Murohara, J. Zhang, X. Cheng, M. Jiang, G.E. Shull, S. Rogers, C.L. Yang, Q. Ke, S. Jelen, R. Bindels, D.H. Ellison, P. Jarolim, P. Libby, and G.P. Shi. (2015). Interleukin 18 function in atherosclerosis is mediated by the interleukin 18 receptor and the Na-Cl co-transporter. Nat. Med. 21: 820-826.

Warmuth, S., I. Zimmermann, and R. Dutzler. (2009). X-ray structure of the C-terminal domain of a prokaryotic cation-chloride cotransporter. Structure 17: 538-546.

Witte M., Reinert T., Dietz B., Nerlich J., Rubsamen R. and Milenkovic I. (2014). Depolarizing chloride gradient in developing cochlear nucleus neurons: underlying mechanism and implication for calcium signaling. Neuroscience. 261:207-22.

Worrell, R.T., L. Merk, and J.B. Matthews. (2008). Ammonium transport in the colonic crypt cell line, T84: role for Rhesus glycoproteins and NKCC1. Am. J. Physiol. Gastrointest. Liver Physiol. 294: G429-440.

Wu, H., X. Che, J. Tang, F. Ma, K. Pan, M. Zhao, A. Shao, Q. Wu, J. Zhang, and Y. Hong. (2016). The K+-Cl- Cotransporter KCC2 and Chloride Homeostasis: Potential Therapeutic Target in Acute Central Nervous System Injury. Mol Neurobiol 53: 2141-2151.

Zhu, M.H., T.S. Sung, M. Kurahashi, L.E. O''Kane, K. O''Driscoll, S.D. Koh, and K.M. Sanders. (2016). Na+-K+-Cl- co-transporter (NKCC) maintains the chloride gradient to sustain pacemaker activity in interstitial cells of Cajal. Am. J. Physiol. Gastrointest Liver Physiol ajpgi.00277.2016. [Epub: Ahead of Print]


TC#NameOrganismal TypeExample

NaCl/KCl symporter; the orthologue in humans when mutated can be responsible for Bartter syndrome, an autosomal recessive disease (Stechman et al., 2007).


NaCl/KCl cotransporter of Rattus norvegicus


The Na+/K+Cl- cotransporter, NKCC1 of 1036 aas and 11 or 12 TMSs. In several insects, it is involved in prostaglandin E2-promoted immune responses. PGE2 mediates oenocytoid cell lysis (a class of lepidopteran hemocytes: OCL) via a specific membrane receptor to release inactive prophenoloxidase (PPO) into the hemolymph (Shrestha et al. 2015).



NKCC1 of Bombyx mori (Silk moth)

2.A.30.1.11NaCl symporter (activated by phosphorylation of the N-terminal domain upon Cl- depletion (Pacheco-Alvarez et al., 2006))Animals NaCl cotransporter of Rattus norvegicus

Electroneutral NaCl symporter, NCC (Gitelman syndrome transporter). NCC is also an Interleukin-18 (IL18)-binding protein that collaborates with the IL18 receptor in cell signaling, inflammatory molecule expression, and experimental atherogenesis (Wang et al. 2015). NCC and the α- and γ-subunits of the epithelial Na+ channel, which together determine salt balance and blood pressure, directly interact with each other with functional consequences (Mistry et al. 2016).


SLC12A3 (NCC) of Homo sapiens


KCl symporter, KCC1. Water can be cotransported with KCl (Mollajew et al., 2010).


KCl cotransporter KCC1 of Rattus norvegicus (Q63632)


KCl symporter KCC2. It influences postsynaptic AMPA receptor content and lateral diffusion in dendritic spines (Gauvain et al., 2011). It plays a role in inhibitory and excitatory neurotransmission in neurons (Chamma et al., 2012). KCC2 transport activity requires the highly conserved L(675) in the C-terminal β1 strand (Döding et al., 2012). Direct physical coupling between the GABA-A receptor and the KCC2 chloride transporter underlies ionic plasticity in cerebellar purkinje neurons in response to brain-derived neurotrophic factor (BDNF) (Huang et al., 2013).  KCC2 is neuron-specific and is essential for Cl(-) homeostasis and fast inhibitory synaptic transmission in the mature CNS. KCC2 is regulated by the single-pass transmembrane protein neuropilin and tolloid like-2 (Neto2). Neto2 is required to maintain the normal abundance of KCC2 and specifically associates with the active oligomeric form of the transporter (Ivakine et al. 2013). Loss of the Neto2:KCC2 interaction reduced KCC2-mediated Cl- extrusion, resulting in decreased synaptic inhibition in hippocampal neurons.  KCC2 mediates the efflux of Cl-out of neurons and plays a role in inhibitory GABAergic and glycinergic neurotransmission. It also participates in the regulation of various physiological processes of neurons, including cell migration, dendritic outgrowth, spine morphology, and dendritic synaptogenesis (Wu et al. 2016). Down-regulation of KCC2 is associated with multiple neurological diseases and is particularly relevant to acute central nervous system (CNS) injury.


KCl cotransporter KCC2 of Rattus norvegicus

2.A.30.1.15KCl symporter, KCC3 (Andermann Syndrome protein)AnimalsSLC12A6 of Homo sapiens
2.A.30.1.16Solute carrier family 12 member 7 (Electroneutral potassium-chloride cotransporter 4) (K-Cl cotransporter 4)AnimalsSLC12A7 of Homo sapiens
2.A.30.1.17Solute carrier family 12 member 4 (Electroneutral potassium-chloride cotransporter 1) (Erythroid K-Cl cotransporter 1) (hKCC1)AnimalsSLC12A4 of Homo sapiens

Solute carrier family 12 member 5 (Electroneutral potassium-chloride cotransporter 2) (K-Cl cotransporter 2) (hKCC2) (Neuronal K-Cl cotransporter). Direct physical coupling between the GABA-A receptor and the KCC2 chloride transporter underlies ionic plasticity in cerebellar purkinje neurons in response to brain-derived neurotrophic factor (BDNF) (Huang et al., 2013).  KCC2 is responsible for maintaining low Cl- concentrations in neurons of the CNS.  Loss of activity of this transporter provides a mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain (Gagnon et al. 2013).  Mediates chloride extrusion in mature neurons, and it regulates the development and morphology of dendritic spines through structural interactions with the actin cytoskeleton  through interaction with the b isoform of Rac/Cdc42 guanine nucleotide exchange factor, β-PIX (Llano et al. 2015). KCC2 affects the maturation of glycinergic synapses in cultured spinal cord neurons (Schwale et al. 2016). Kainate receptors regulate KCC2 expression in the hippocampus (Pressey et al. 2017).


SLC12A5 of Homo sapiens


K+,Cl--cotransporter, KCC or Slc12a5b of 1117 aas and 12 TMSs. Ion transport via an ortholog is oxygen-sensitive and is regulated by two different oxygen sensors in crucian carp (Carassius carassius) (Berenbrink et al. 2006).

KCC of Danio rerio (Zebrafish) (Brachydanio rerio)


Solute carrier family 12 member 1 (Bumetanide-sensitive sodium-(potassium)-chloride cotransporter 2) (Kidney-specific Na-K-Cl symporter, NKCC2) Mutations cause type I Bartter syndrome (BS), a life threatening kidney disease featuring arterial hypotension along with electrolyte abnormalities (Adachi et al. 2007).  An OS9-mediated ERAD pathway in renal cells degrades immature NKCC2 proteins (Seaayfan et al. 2015). Regulated by AMPK (see 8.A.104.1.1).


SLC12A1 of Homo sapiens

2.A.30.1.20Possible NaCl/KCl or KCl symporter, Axi4 PlantsAxi4 of Nicotiana tabacum

Potassium/chloride cotransporter, KCC2 of 1139 aas and 12 TMSs. It mediates electroneutral potassium-chloride cotransport in mature neurons and is required for neuronal Cl- homeostasis. As a major extruder of intracellular chloride, it establishes the low neuronal Cl- levels required for chloride influx after binding of GABA-A and glycine to their receptors, with subsequent hyperpolarization and neuronal inhibition (Puskarjov et al. 2014). Postnatal changes in expression in the forebrain of mice bearing a mutant nicotinic subunit has been linked to Sleep-Related Epilepsy (Amadeo et al. 2018).


KCC2 of Homo sapiens


Sodium-coupled cation-chloride cotransporter of 859 aas and 11 TMSs.  There are three paralogues (Piermarini et al. 2017).

CCC of Aedes aegypti (Yellowfever mosquito) (Culex aegypti)


NaCl/KCl symporter (basolateral), NKCC1 (may also transport NH4+ and water); (Worrell et al., 2008; Hamann et al., 2010). Loop diuretic and ion binding residues have been identified (Somasekharan et al., 2012).  NKCC1 is the major Cl--loader responsible for the depolarizing action of GABA/glycine receptors at postnatal days 3-5 in cochlear nucleus neurons (Witte et al. 2014). Stimulated by ammonia (Hertz et al. 2015).  NKCC maintains Cl- gradients to sustain pacemaker activity (TC# 1.A.1.5.10) in interstitial cells of Cajal (Zhu et al. 2016).


SLC12A2 of Homo sapiens

2.A.30.1.5NaCl/KCl symporterAnimalsNaCl/KCl symporter of Squalus acanthias (Shark)
2.A.30.1.6 solute carrier family 12 (potassium/chloride transporters), member 9AnimalsSLC12A9 of Homo sapiens
2.A.30.1.7 solute carrier family 12 (potassium/chloride transporters), member 8AnimalsSLC12A8 of Homo sapiens

Na+,K+,Cl--cotransporter, NKCC or Slc12a2, of 1120 aas and 12 TMSs.  Ion transport via an ortholog is oxygen-sensitive and is regulated by two different oxygen sensors in crucian carp (Carassius carassius) (Berenbrink et al. 2006).

NKCC of Danio rerio (Zebrafish) (Brachydanio rerio)

2.A.30.1.9Possible NaCl/KCl symporter Animals NaCl/KCl cotransporter of Manduca sexta

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