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. CCC family members play critical roles in regulating cell volume, controlling ion absorption and secretion across epithelia, and maintaining intracellular chloride homeostasis. These transporters are primary targets for some of the most commonly prescribed drugs (Chew et al. 2019). CCCs may influence the polarity of GABA signalling in mouse hippocampal parvalbumin interneurons (Otsu et al. 2020). Acute intravenous NaCl and volume expansion reduces Sodium-Chloride Cotransporter abundance and phosphorylation in urinary extracellular vesicles (Wu et al. 2022).
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 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 humans 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). Potassium-chloride cotransporters KCC1 to KCC4 mediate the coupled export of potassium and chloride across the plasma membrane and play important roles in cell volume regulation, auditory system function, and gamma-aminobutyric acid (GABA) and glycine-mediated inhibitory neurotransmission. Xie et al. 2020 presented 2.9- to 3.6-Å resolution structures of full-length human KCC2, KCC3, and KCC4. All three KCCs adopt a similar overall architecture, a domain-swap dimeric assembly, and an inward-facing conformation. One unexpected N-terminal peptide binds at the cytosolic facing cavity and locks KCC2 and KCC4 in an autoinhibition state. The C-terminal domain (CTD) directly interacts with the N-terminal inhibitory peptide, and the relative motions between the CTD and the transmembrane domain suggest that CTD regulates KCCs' activities by adjusting the autoinhibitory effect. CCCs share a conserved structural scaffold that consists of a transmembrane transport domain followed by a cytoplasmic regulatory domain (Xie et al. 2020).
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. SLC12 transporter cryo-EM structures confirm featires such as the presence of 12 transmembrane domains and the formation of a functional dimer as well as the presence of strategic salt bridges that explain why transporters are stabilized in specific conformations. Cryo-EM structures show similarities with other transport protein structures, especially regarding the position of the cations (Delpire 2025).
Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations with chloride across the plasma membrane and are vital for cell volume regulation, salt reabsorption in the kidney, and γ-aminobutyric acid (GABA)-mediated modulation in neurons. Liu et al. 2019 presented cryo-EM structures of human potassium-chloride cotransporter KCC1 in potassium chloride or sodium chloride at 2.9- to 3.5-Å resolution. KCC1 exists as a dimer, with both extracellular and transmembrane domains involved in dimerization. The structural and functional analyses, along with computational studies, revealed one potassium site and two chloride sites in KCC1, which are all required for the ion transport activity. KCC1 adopts an inward-facing conformation, with the extracellular gate occluded. The KCC1 structures allowed the authors to model a potential ion transport mechanism in KCCs and provide a blueprint for drug design (Liu et al. 2019).
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).
References:
NaCl/KCl symporter; the orthologue in humans when mutated can be responsible for Bartter syndrome, an autosomal recessive disease (Stechman et al., 2007).
Animals
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).
Animals
NKCC1 of Bombyx mori (Silk moth)
NaCl symporter (activated by phosphorylation of the N-terminal domain upon Cl- depletion (Pacheco-Alvarez et al., 2006)). The thiazide sensitive Na+:Cl- cotransporter (NCC) provides the principal route for salt reabsorption in the apical membrane of the distal convoluted tubule (DCT) in mammals and plays a fundamental role in managing blood pressure. The cotransporter is targeted by thiazide diuretics, a highly prescribed medication that is effective in treating arterial hypertension and edema (Moreno et al. 2023). The structure has been solved by cryoEM, and the structure/function relationships have been reviewed (Moreno et al. 2023).
Animals
NaCl cotransporter of Rattus norvegicus
Electroneutral NaCl symporter, NCC (Gitelman syndrome transporter) (Correia et al. 2022; Bi et al. 2023). 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). The cryo-EM structure of the human sodium-chloride cotransporter, NCC, has been solved (Nan et al. 2022). NCC mediates the coupled import of sodium and chloride across the plasma membrane, playing vital roles in kidney extracellular fluid volume and blood pressure control. The full-length structure of human NCC, with 2.9 Å resolution for the transmembrane domain and 3.8 Å for the carboxyl-terminal domain, has been solved. In this structure, NCC adopts an inward-open conformation and a domain-swap dimeric assembly. Conserved ion binding sites among the cation-chloride cotransporters and the Na2 site are observed in the structure. A unique His residue in the substrate pocket in NCC potentially interacts with Na1 and Cl1 and might also mediate the coordination of Na2 through a Ser residue. Putative observed water molecules may participate in the coordination of ions and TM coupling. Together with transport activity assays, this structure provides the first glimpse of NCC and defines ion binding sites, promoting drug development for hypertension targeting on NCC (Nan et al. 2022). NCC drives salt reabsorption in the kidney and plays a decisive role in balancing electrolytes and blood pressure. Thiazide and thiazide-like diuretics inhibit NCC-mediated renal salt retention and have been cornerstones for treating hypertension and edema since the 1950s. Zhao et al. 2024 determined NCC co-structures individually complexed with the thiazide drug hydrochlorothiazide, and two thiazide-like drugs chlorthalidone and indapamide, revealing that they fit into an orthosteric site and occlude the NCC ion translocation pathway. Aberrant NCC activation by the WNKs-SPAK kinase cascade underlies Familial Hyperkalemic Hypertension. Zhao et al. 2024 showed that an intracellular amino-terminal motif of NCC, once phosphorylated, associates with the carboxyl-terminal domain, and together, they interact with the transmembrane domain. These interactions suggest a phosphorylation-dependent allosteric network that directly influences NCC ion translocation. A missense variant in the SLC12A3 gene enhances aberrant splicing causing Gitelman syndrome (Law et al. 2025). Finerenone provides a novel treatment for Gitelman syndrome (Jiang et al. 2024). Upregulation of SLC12A3 and SLC12A9 confers aggressiveness and unfavorable prognosis in uveal melanoma (Yan et al. 2023). Gitelman syndrome (GS) is one of the most common hereditary renal tubular disorders, characterised by hypokalemia, hypomagnesemia, hypocalciuria, and metabolic alkalosis. The primary cause of this disorder resides in the SLC12A3 gene, which encodes the NaCl cotransporter in the distal convoluted tubule, and for which more than 500 mutations associated with GS have been described (Tomás-Simó et al. 2025).
Animals
SLC12A3 (NCC) of Homo sapiens
KCl symporter, KCC1. Water can be cotransported with KCl (Mollajew et al., 2010). KCCs are regulated through an N-terminal plug of the ion pathway (Kock Flygaard et al. 2021). KCC1 shows anion specificity in the order: Cl- > SCN- = Br- > PO4-3 > I- (Mercado et al. 2000).
Animals
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. Structural changes in the extracellular loop 2 of the murine KCC2 potassium chloride cotransporter modulate ion transport (Hartmann et al. 2021). Gephyrin, the main scaffolding molecule at GABAergic synapses, interacts with KCC2 to regulate its surface expression and function in cortical neurons (Al Awabdh et al. 2021). The expression system influences the stability, maturation efficiency, and oligomeric properties of KCC2 (Kok et al. 2024). VEGF, but not BDNF, prevents the downregulation of KCC2 induced by axotomy in extraocular motoneurons (Capilla-López et al. 2024).
Animals
KCl cotransporter KCC2 of Rattus norvegicus
KCl symporter, KCC3 (Andermann Syndrome protein) of 1150 aas and 12 TMSs. Erythroid-specific inactivation of Slc12a6/Kcc3 by EpoR promoter-driven Cre expression reduces K-Cl cotransport activity in mouse erythrocytes (Shmukler et al. 2022). Both KCC3b and KCC3a seem to be needed for maintaining cell volume during enhanced inward cotransport of Na+-glucose in proximal tubules and Na+-HCO3- in intercalated cells (Ferdaus and Delpire 2023). In addition, apical KCC3a might couple to pendrin function to recycle Cl-, particularly in conditions of low salt diet and therefore low Cl- delivery to the distal tubule. This function is critical in alkalemia, and KCC3a function in the pendrin-expressing cells may contribute to the K+ loss which is observed in alkalemia (Ferdaus and Delpire 2023). Renewable and highly specific binders against KCC3 have been isolated (Gelová et al. 2024).
Animals
SLC12A6 of Homo sapiens
Solute carrier family 12 member 7 (Electroneutral potassium-chloride cotransporter 4) (K-Cl cotransporter 4, KCC4) It has an anion selectivity of Cl- > Br- > PO4-3 = I- > SCN- for KCC4) (Mercado et al. 2000).
Animals
SLC12A7 (KCC4) of Homo sapiens
Solute carrier family 12 member 4 (Electroneutral potassium-chloride cotransporter 1, KCC1) (Erythroid K-Cl cotransporter 1) (hKCC1). It is activated by cell swelling and may contribute to cell volume homeostasis as well as being involved in the regulation of basolateral Cl- exit in NaCl absorbing epithelia. Isoform 4 has no transport activity. The kinase, WNK3, activates NKCC1/2 and NCC but inhibits the KCCs (Cruz-Rangel et al. 2011). Liu et al. 2019 presented cryo-EM structures of human KCC1 in potassium chloride or sodium chloride at 2.9- to 3.5-Å resolution. KCC1 exists as a dimer, with both extracellular and transmembrane domains involved in dimerization. The structural and functional analyses, along with computational studies, reveal one potassium site and two chloride sites in KCC1, which are all required for the ion transport activity. The structure reveals an inward-facing conformation, with the extracellular gate occluded. The KCC1 structures allowed the authors to model a potential ion transport mechanism in KCCs and provide a blueprint for drug design (Liu et al. 2019). KCC1 bound with the VU0463271 inhibitor in an outward-open state has been solved (Zhao et al. 2022). In contrast to many other amino acid-polyamine-organocation transporter cousins, opening the KCC1 extracellular ion permeation path does not involve hinge-bending motions of TMS 1 and TMS6 half-helices. Instead, rocking of TMS3 and TMS8, together with displacements of TMS4, TMS9, and a conserved intracellular loop 1 helix, underlie alternate opening and closing of extracellular and cytoplasmic vestibules. KCC1 exists in one of two distinct dimeric states via different intersubunit interfaces (Zhao et al. 2022).
Animals
SLC12A4 of Homo sapiens
Solute carrier family 12 member 5 (Electroneutral potassium-chloride cotransporter 2) (K-Cl cotransporter 2) (hKCC2) (Neuronal K-Cl cotransporter) of 1139 aas and 12 TMSs. 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). It is 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 regulates neuronal excitability and hippocampal activity via interaction with Task-3 channels (Goutierre et al. 2019). Coordinated downregulation of KCC2 and the GABAA receptor contributes to inhibitory dysfunction during seizure induction (Wan et al. 2020). The expression system influences stability, maturation efficiency, and oligomeric properties of KCC2 (Kok et al. 2024).
Animals
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). It is regulated by AMPK (see 8.A.104.1.1). The WNK kinase-dependent pathway can affect both the trafficking and the intrinsic activity of NKCC2, which therefore plays a doubly important role in carrier regulation (Marcoux et al. 2019). The gene encodes three splice variants. They are identical to each other except for TMS2 and the following connecting segment (CS2). These variants do not share the same localization, transport characteristics and physiological roles along the thick assending loop of Henle (Marcoux et al. 2019). NKCC2 regulation in the thick ascending limb has been updated and involves membrane trafficking, phosphorylation, and protein-protein interactions (Maskey et al. 2024).
Animals
SLC12A1 of Homo sapiens
K+/Cl- Cotransporter, Kcc-2, of 1129 aas and probably 11 TMSs in a 5 + 6 TMS arrangement. The expression of its gene was responsive to the presence of Ivermectin at 10 to 100 nM concentrations (Dube et al. 2023).
Kcc-2 of Caenorhabditis elegans
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)
Bumetanide-sensitive NaCl/KCl symporter (basolateral), NKCC1 or SLC12A2 (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 important for regulating cell volume, hearing, blood pressure, and hyperpolarizing GABAergic and glycinergic signaling in the central nervous system. 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). Its activity is 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). It uses the existing Na+ and/or K+ gradients to move Cl- into or out of cells. NKCC1 plays fundamental roles in regulating trans-epithelial ion movement, cell volume, chloride homeostasis and neuronal excitability. Yang et al. 2020 reported a cryo-EM structure of human NKCC1 captured in a partially loaded, inward-open state. NKCC1 assembles into a dimer, with the first ten TMSs harboring the transport core and TM11-TM12 helices lining the dimer interface. TMSs 1 and 6 break alpha-helical geometry halfway across the lipid bilayer where ion binding sites are organized around these discontinuous regions. NKCC1 may harbor multiple extracellular entryways and intracellular exit sites, raising the possibility that K+, Na+, and Cl- ions may traverse along their own routes for translocation (Yang et al. 2020). It is present in the basolateral membrane of crypt epithelial cells and mediates uptake of these three ions into these cells (A. Quach, personal communication). NKCC1 mediates trans-epithelial Cl- secretion and regulates excitability of some neurons, while NKCC2 is critical to renal salt reabsorption. Bumetanide is a mainstay for treating edema and hypertension. Cryo-EM structures reveal an outward-facing conformation of NKCC1, showing bumetanide wedged into a pocket in the extracellular ion translocation pathway. Zhao et al. 2022 defined the translocation pathway and the conformational changes necessary for ion translocation. An NKCC1 dimer was observed with separated transmembrane domains and extensive transmembrane and C-terminal domain interactions. An N-terminal phosphoregulatory domain that interacts with the C-terminal domain, suggests a mechanism whereby (de)phosphorylation regulates NKCC1 by tuning the strength of this domain association (Zhao et al. 2022). The cryo-EM structure of the human NKCC1 transporter revealed the mechanisms of ion coupling and specificity (Neumann et al. 2022). In human tissue, NKCC1 plays a critical role in regulating cytoplasmic volume, fluid intake, chloride homeostasis, and cell polarity. Moseng et al. 2022 reported four structures of human NKCC1, both in the absence and presence of loop diuretic (bumetanide or furosemide), using single-particle cryoEM. These structures reveal various novel conformations of the hNKCC1 dimer. They also reveal two drug-binding sites located at the transmembrane and cytosolic carboxyl-terminal domains, respectively. This allows delineation of an inhibition mechanism that involves a coupled movement between the cytosolic and transmembrane domains of hNKCC1 (Moseng et al. 2022). Genetic loss of NKCC1 gives rise to Bartter Syndrome (Cunha and Heilberg 2018; see Bartter Syndrome in Wilipedia). The basolateral calciumsensing receptor has the ability to downregulate the activity of this transporter upon activation. Once transported into the tubule cells, sodium ions are actively transported across the basolateral membrane by Na+,K+-ATPases, and chloride ions pass by facilitated diffusion through basolateral chloride channels. Potassium, however, is able to diffuse back into the tubule lumen through apical potassium channels, returning a net positive charge to the lumen and establishing a positive voltage between the lumen and interstitial space. This charge gradient is obligatory for the paracellular reabsorption of both calcium and magnesium ions. Gut microbial nitrogen recycling and cellular uptake of ammoniumallows the improvment of bortezomib resistance in multiple myeloma (Zhu et al. 2024). NKCC1 operates through a rocking-bundle mechanism (Ruiz Munevar et al. 2024). Ammonium enters multiple myeloma (MM) cells via SLC12A2, promoting chromosomal instability and drug resistance by stabilizing the NEK2 ser/thr protein kinase. Furosemide, a loop diuretic, downregulates SLC12A2, thereby inhibiting ammonium uptake by MM cells and improving progression-free survival (Zhu et al. 2024). NKCC1 operates through a rocking-bundle mechanism (Ruiz Munevar et al. 2024). SLC12A2 is a theraputic target for colorectal cancer (Chen et al. 2024). Inhibiting the NKCC1/AQP4 pathway has a beneficial effect on neurological injury in a rat model of high-altitude cerebral edema (Geng et al. 2025).
Animals
SLC12A2 or NKCC1 of Homo sapiens
Solute carrier family 12 (potassium/chloride transporters), member 9. Biallelic loss-of-function variants of SLC12A9 cause lysosome dysfunction and a syndromic neurodevelopmental disorder (Accogli et al. 2024). Upregulation of SLC12A3 and SLC12A9 confers aggressiveness and unfavorable prognosis in uveal melanoma (Yan et al. 2023).
Animals
SLC12A9 of Homo sapiens
solute carrier family 12 (potassium/chloride transporters), member 8 of 714 aas and 13 TMSs in a 6 + 5 + 2 TMS arrangement. This system has been imiplicated in Autism Spectrum Disease (Ben-Mahmoud et al. 2024).
Animals
SLC12A8 of Homo sapiens
Na+,K+,Cl--cotransporter, NKCC1or Slc12a2/a4, of 1136 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). Chew et al. 2019 determined the cryo-EM structure which defined the architecture of this protein family and revealed how cytosolic and transmembrane domains are strategically positioned for communication. Structural analyses, functional characterizations and computational studies revealed the ion-translocation pathway, ion-binding sites and key residues involved in transport activity, thus providing insights into ion selectivity, coupling and translocation, and establishing a framework for understanding the physiological functions of CCCs as well as interpreting disease-related mutations (Chew et al. 2019). The 3-D structures of this protein and the humanKCC! (TC# 2.A.30.1.4) have been compared and reviewed (Delpire and Guo 2020). While cation-Cl- cotransporters share the overall architecture of carriers belonging to the amino acid-polyamine-organocation (APC) superfamily and some of their substrate binding sites, several new features have been revealed. (1) The large extracellular domain between TMSs 5 and 6 stabilizes the dimer and forms a cap that likely participates in extracellular gating. (2) Conservation of the K+ and Cl- binding sites and a second Cl- coordination site were revealed. (3) an extracellular gate is formed by conserved salt bridges between charged residues located toward the ends of TMSs 3 and 4, and there is an additional neighboring bridge in the hKCC1 structure. (4) Multiple points of contacts occur between the monomers forming the cotransporter homodimer units. These involve the TMSs, the COOH-terminal domains, and the large extracellular loop for hKCC1 (Delpire and Guo 2020).
NKCC of Danio rerio (Zebrafish) (Brachydanio rerio)