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)
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).
EClC of Bos taurus (NP_001070824)
The Ca-activated chloride channel-6 (Lee et al., 2011).
Ca-CLC-6 of Xenopus laevis (F7IYU6)
Putative lipoprotein of 1054 aas and 1-3 TMSs.
Putative lipoprotein of Leptospira biflexa
Ca+2-activated Cl- channel, CLCA1 or CACC1 or 914 aas. CLCA1 may play a role in inflammatory airway diseases (Sala-Rabanal et al. 2015).
CLCA1 of Homo sapiens
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)
Hypothetical protein, HP
HP of Oryza sativa (B8AFH9)
Sll0103 of Synechocystis (Q55874)
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).
YfbK of E. coli (P76481)
Von Willebrand factor type A protein, vWFA. (905 aas; 2 N-terminal and 1 C-terminal TMSs)
vWFA of Chloroflexus aurantiacus (A9WIT9)
Bacterial homologue, BatB, of mammalian Ca-CLC channels (N- and C-terminal TMSs)
BatB of Myxococcus fulvus (F8CM01)
Uncharacterized protein of 252 aas and 2-3 TMSs
UP of E. coli