| 1.A.13 The Epithelial Chloride Channel (E-ClC) Family
Mammals have multiple isoforms (at least 6 different gene products plus splice variants (Evans et al., 2004)) of epithelial chloride channel proteins. The first member of this family to be characterized was a respiratory epithelium, Ca2+-regulated, chloride channel protein isolated from bovine tracheal apical membranes. It was biochemically characterized as a 140 kDa complex. The purified complex when reconstituted in a planar lipid bilayer behaved as an anion-selective channel. It was regulated by Ca2+ via a calmodulin kinase II-dependent mechanism. When the cRNA was injected into Xenopus oocytes, an outward rectifying, DIDS-sensitive anion conductance was measured. A related gene, Lu-ECAM, was cloned from the bovine aortic endothelial cell line, BAEC. It is expressed in the lung and spleen but not in the trachea. Homologues are found in several mammals, and at least four paralogues (hCaCC-1-4) are present in humans, each with different tissue distributions.
The bovine EClC protein has 903 amino acids and four putative TMSs at residue positions 7-27, 331-351, 617-337 and 883-903. Distant (partial) homologues may be present in plants, ciliates and bacteria, Synechocystis (Sll0103; 420 aas) and E. coli (YfbK; 575 aas), so at least some domains within E-ClC family proteins have an ancient origin. E-ClC proteins show significant sequence similarity with CCA-α2δ family members (8.A.18).
Gibson et al. (2005) have shown that the human calcium-activated chloride channel, hCLCA1, is a secreted, non-integral membrane protein, and therefore suggest that this protein and its homologues are not ion channels at all. They also point out that the protein may not be sufficiently hydrophobic to insert into the membrane. Moreover, the proteins in subfamily 1.A.13.2 do not have α-TMSs, but hydrophilic domains in these proteins show sequence similarity to hydrophilic regions in proteins of family 8.A.18. The proteins without α-TMSs may not be channels. Thus, while some members of the E-ClC family appear to be able to form anion channels, others may not have this capacity.
The generalized reaction catalyzed by EClC family members is:
Cl- (in)  Cl- (out)
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| References: |
Agnel, M., T. Vermat, and J. Culouscou. (1999). Identification of three novel members of the calcium-dependent chloride channel (CaCC) family predominantly expressed in the digestive tract and trachea. FEBS Lett. 455: 295-301.
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Antonets, K.S., K.V. Volkov, A.L. Maltseva, L.M. Arshakian, A.P. Galkin, and A.A. Nizhnikov. (2016). Proteomic Analysis of Escherichia coli Protein Fractions Resistant to Solubilization by Ionic Detergents. Biochemistry (Mosc) 81: 34-46.
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Barrett, K.E. and S.J. Keely. (2000). Chloride secretion by the intestinal epithelium: molecular basis and regulatory aspects. Annu. Rev. Physiol. 62: 535-572.
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Bartenschlager, F., N. Klymiuk, C. Weise, B. Kuropka, A.D. Gruber, and L. Mundhenk. (2022). Evolutionarily conserved properties of CLCA proteins 1, 3 and 4, as revealed by phylogenetic and biochemical studies in avian homologues. PLoS One 17: e0266937.
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Elble, R.C., G. Ji, K. Nehrke, J. DeBiasio, P.D. Kingsley, M.I. Kotlikoff, and B.U. Pauli. (2002). Molecular and functional characterization of a murine calcium-activated chloride channel expressed in smooth muscle. J. Biol. Chem. 277: 18586-18591.
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Evans, S.R., W.B. Thoreson, and C.L. Beck. (2004). Molecular and functional analyses of two new calcium-activated chloride channel family members from mouse eye and intestine. J. Biol. Chem. 279: 41792-41800.
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Fuller, C.M., I.I. Ismailov, D.A. Keeton, and D.J. Benos. (1994). Phosphorylation and activation of a bovine tracheal anion channel by Ca2+/calmodulin-dependent protein kinase II. J. Biol. Chem. 269: 26642-26650.
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Gibson, A., A.P. Lewis, K. Affleck, A.J. Aitken, E. Meldrum, and N. Thompson. (2005). hCLCA1 and mCLCA3 are secreted non-integral membrane proteins and therefore are not ion channels. J. Biol. Chem. 280: 27205-27212.
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Lee, R.M. and S.M. Jeong. (2016). [Identification of a Novel Calcium (Ca^(2+))-Activated Chloride Channel Accessory Gene in Xenopus laevis]. Mol Biol (Mosk) 50: 106-114.
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Lee, R.M., R.H. Ryu, S.W. Jeong, S.J. Oh, H. Huang, J.S. Han, C.H. Lee, C.J. Lee, L.Y. Jan, and S.M. Jeong. (2011). Isolation and Expression Profile of the Ca-Activated Chloride Channel-like Membrane Protein 6 Gene in Xenopus laevis. Lab Anim Res 27: 109-116.
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Ran, S. and D.J. Benos. (1992). Immunopurification and structural analysis of a putative epithelial Cl- channel protein isolated from bovine trachea. J. Biol. Chem. 267: 3618-3625.
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Ran, S., C.M. Fuller, M. Pia Arrate, R. Latorre, and D.J. Benos. (1992). Functional reconstitution of a chloride channel protein from bovine trachea. J. Biol. Chem. 267: 20630-20637.
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Rogers, D.F. (2003). Pulmonary mucus: Pediatric perspective. Pediatr Pulmonol 36: 178-188.
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Sala-Rabanal, M., Z. Yurtsever, K.N. Berry, and T.J. Brett. (2015). Novel Roles for Chloride Channels, Exchangers, and Regulators in Chronic Inflammatory Airway Diseases. Mediators Inflamm 2015: 497387.
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Seltmann, K., B. Hettich, S. Abele, S. Gurri, V. Mantella, J.C. Leroux, and S. Werner. (2024). Transport of CLCA2 to the nucleus by extracellular vesicles controls keratinocyte survival and migration. J Extracell Vesicles 13: e12430.
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Xin, W., J. Zhang, H. Zhang, X. Ma, Y. Zhang, Y. Li, and F. Wang. (2022). CLCA2 overexpression suppresses epithelial-to-mesenchymal transition in cervical cancer cells through inactivation of ERK/JNK/p38-MAPK signaling pathways. BMC Mol. Cell Biol. 23: 44.
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Yoon, I.S., S.M. Jeong, S.N. Lee, J.H. Lee, J.H. Kim, M.K. Pyo, J.H. Lee, B.H. Lee, S.H. Choi, H. Rhim, H. Choe, and S.Y. Nah. (2006). Cloning and heterologous expression of a Ca2+-activated chloride channel isoform from rat brain. Biol Pharm Bull 29: 2168-2173.
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| Examples: |
| TC# | Name | Organismal Type | Example |
| 1.A.13.1.1 | Voltage-gated bovine epithelial Cl- channel protein (Ca2+-activated), bEClC. In rats, two possible paralogues (rbCLCA1 and A2) are expressed in the CNS and peripheral organs (Yoon et al., 2006). CLCA1 may play a role in inflammatory airway diseases (Sala-Rabanal et al. 2015). It is called the von Willebrand factor type A, the DUF1973 protein. | Mammals | EClC of Bos taurus (NP_001070824) |
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| 1.A.13.1.10 | Calcium-activated chloride channel, CLCA2 regulator-2 of 943 aas and 2 TMSs, N- and C-terminal. CLCA2 overexpression suppresses
epithelial-to-mesenchymal transition in cervical cancer cells through
inactivation of ERK/JNK/p38-MAPK signaling pathways (Xin et al. 2022). Transport of CLCA2 to the nucleus by extracellular vesicles controls keratinocyte survival and migration (Seltmann et al. 2024). | | CLCA2 of Homo sapiens |
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| 1.A.13.1.2 | Ca2+-activated Cl- channel-2, CaCC-2 | Mammals | CaCC-2 of Homo sapiens |
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| 1.A.13.1.3 | The Ca-activated chloride channel-6 (Lee et al., 2011). | Animals | Ca-CLC-6 of Xenopus laevis (F7IYU6) |
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| 1.A.13.1.4 | Calcium-activated chloride channel regulator family member 3 (Calcium-activated chloride channel family member 3) (hCLCA3) | Animals | CLCA3P of Homo sapiens |
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| 1.A.13.1.5 | Putative lipoprotein of 1054 aas and 1-3 TMSs. | Spirochaetes | Putative lipoprotein of Leptospira biflexa |
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| 1.A.13.1.6 | Ca+2-activated Cl- channel, or Chloride Channel Accessory 1 (CLCA1 or CACC1), of 914 aas. CLCA1 may play a role in inflammatory airway diseases (Sala-Rabanal et al. 2015). It is thought to play a role in Cl- secretion in the intestine (A. Quach, personal communication). Airway mucus hypersecretion is a clinical feature of a number of
childhood diseases, including asthma and bronchitis-associated
conditions (Rogers 2003). | | CLCA1 of Homo sapiens |
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| 1.A.13.1.7 | xCLCA3; xCLCA2 of 942 aas and 7 TMSs. xCLCA3 contains a predicted signal sequence, multiple
sites of N-linked glycosylation, N-myristoylation, PKA, PKC, and casein kinase II
phosphorylation sites, five putative hydrophobic segments, and the HExxH metalloprotease motif.
Additionally, the transmembrane prediction server yielded a preserved N-terminal CLCA domain and a
von Willebrand factor type A domain with one transmembrane domain in the C-terminal region (Lee and Jeong 2016).
xCLCA3 is expressed in a number of tissues, with strong expression
in the brain, colon, small intestine, lung, kidney, and spleen, and poor expression in the heart and
liver. xCLCA3 may be a candidate CLCA family member as well as a
metalloprotease, rather than just an ion channel accessory protein. | | xCLCA3 of Xenopus laevis (African clawed frog) |
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| 1.A.13.1.8 | VWFA domain-containing protein, CLCA1 of 935 aas and 2 TMSs, N- and C-terminal, and possibly one that is centrally located. The galline CLCA1 displays close genetic distances to mammalian clusters 1, 3 and 4 (Bartenschlager et al. 2022). | | CLCA1 of Gallus gallus (Chicken) |
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| 1.A.13.1.9 | VWFA domain-containing protein, CLCA2, chloride-transporting anion channel, regulated by calcium ions, of 1005 aas and 2 TMSs, one N-terminal and one C-terminal (Bartenschlager et al. 2022). | | CLCA2 of Gallus gallus (Chicken) |
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| Examples: |
| TC# | Name | Organismal Type | Example |
| 1.A.13.2.1 | Hypothetical protein, HP | Plants | HP of Oryza sativa (B8AFH9) |
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| 1.A.13.2.2 | Sll0103 | Bacteria | Sll0103 of Synechocystis (Q55874) |
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| 1.A.13.2.3 | The YfbK/CaClC homologue of 575aas and 0 TMSs. YfbK has amyloidogenic regions due to asparagine- and glutamine-rich regions which is a
common feature of many known amyloid proteins. This correlates with detergent-induced denaturation resistance (Antonets et al. 2016). | Bacteria | YfbK of E. coli (P76481) |
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| 1.A.13.2.4 | Von Willebrand factor type A domain protein of 536 aas and 1 N-terminal TM | | Von Willebrand factor of Tuwongella immobilis |
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| Examples: |
| TC# | Name | Organismal Type | Example |
| 1.A.13.3.1 | Von Willebrand factor type A protein, vWFA. (905 aas; 2 N-terminal and 1 C-terminal TMSs) | Bacteria | vWFA of Chloroflexus aurantiacus (A9WIT9) |
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| Examples: |
| TC# | Name | Organismal Type | Example |
| 1.A.13.4.1 | Bacterial homologue, BatB, of mammalian Ca-CLC channels (N- and C-terminal TMSs) | Bacteria | BatB of Myxococcus fulvus (F8CM01) |
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| Examples: |
| TC# | Name | Organismal Type | Example |
| 1.A.13.5.1 | Uncharacterized protein of 252 aas and 2-3 TMSs | Proteobacteria | UP of E. coli |
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| Examples: |
| TC# | Name | Organismal Type | Example |