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).

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. 

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).



This family belongs to the APC Superfamily.

 

References:

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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.

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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.

Marcoux, A.A., S. Slimani, L.E. Tremblay, R. Frenette-Cotton, A.P. Garneau, and P. Isenring. (2019). Endocytic recycling of Na -K -Cl cotransporter type 2: importance of exon 4. J. Physiol. 597: 4263-4276.

Marcoux, A.A., S. Slimani, L.E. Tremblay, R. Frenette-Cotton, A.P. Garneau, and P. Isenring. (2019). Regulation of Na-K-Cl cotransporter type 2 by the with no lysine kinase-dependent signaling pathway. Am. J. Physiol. Cell Physiol. 317: C20-C30.

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.

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Examples:

TC#NameOrganismal TypeExample
2.A.30.1.1

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

 
2.A.30.1.10

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)

 
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
 
2.A.30.1.12

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).

Animals

SLC12A3 (NCC) of Homo sapiens

 
2.A.30.1.13

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).

Animals

KCl cotransporter KCC1 of Rattus norvegicus (Q63632)

 
2.A.30.1.14

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).

Animals

KCl cotransporter KCC2 of Rattus norvegicus

 
2.A.30.1.15

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).

Animals

SLC12A6 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.30.1.17

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

 
2.A.30.1.18

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).

Animals

SLC12A5 of Homo sapiens

 
2.A.30.1.19

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)

 
2.A.30.1.2

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).

Animals

SLC12A1 of Homo sapiens

 
2.A.30.1.20Possible NaCl/KCl or KCl symporter, Axi4 PlantsAxi4 of Nicotiana tabacum
 
2.A.30.1.3

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)

 
2.A.30.1.4

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 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).


Animals

SLC12A2 or NKCC1 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
 
2.A.30.1.8

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)

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