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. 

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).