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

Members of the CCC family, found in animals, plants, fungi 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 one has been partially characterized from Nicotiana tabacum (a plant), and homologous ORFs have been sequenced from Caenorhabditis elegans (worm), Saccharomyces cerevisiae (yeast) and Synechococcus sp. (blue green bacterium). The latter proteins are of unknown function. 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.

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).

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.

References associated with 2.A.30 family:

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. 14532115
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. 22363264
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. 12704198
Döding, A., A.M. Hartmann, T. Beyer, and H.G. Nothwang. (2012). KCC2 transport activity requires the highly conserved L(675) in the C-terminal β1 strand. Biochem. Biophys. Res. Commun. [Epub: Ahead of Print] 22414695
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. 16027154
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. 24097188
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. 21878564
Haas, M. and B. Forbush, III. (2000). The Na-K-Cl cotransporter of secretory epithelia. Annu. Rev. Physiol. 62: 515-534. 10845101
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. 20819947
Huang, Y., J.J. Wang, and W.H. Yung. (2013). Coupling Between GABA-A Receptor and Chloride Transporter Underlies Ionic Plasticity in Cerebellar Purkinje Neuron.s. Cerebellum. [Epub: Ahead of Print] 23341142
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. 23401525
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. 20852624
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.
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. 10913127
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. 21112289
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. 10347194
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. 9263880
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. 16887815
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. 8801348
Russell, J.M. (2000). Sodium-potassium-chloride cotransport. Physiol. Rev. 80: 211-276. 10617769
Somasekharan, S., J. Tanis, and B. Forbush. (2012). Loop diuretic and ion binding residues revealed by scanning mutagenesis of transmembrane helix 3 (TM3) of the Na-K-Cl Cotransporter (NKCC1). J. Biol. Chem. [Epub: Ahead of Print] 22437837
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. 17872384
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. 19368887
Witte, M., T. Reinert, B. Dietz, J. Nerlich, R. Rübsamen, and I. Milenkovic. (2014). Depolarizing chloride gradient in developing cochlear nucleus neurons: Underlying mechanism and implication for calcium signaling. Neuroscience. [Epub: Ahead of Print] 24388924
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. 18032481