Molecular and functional analyses of two new calcium-activated chloride channel family members from mouse eye and intestine.
Two new calcium-activated chloride channel (CLCA) family members, mCLCA5 and mCLCA6, have been cloned from mouse eye and intestine, respectively. mCLCA5 is highly homologous to hCLCA2, and mCLCA6 is highly homologous to hCLCA4. mCLCA5 is widely expressed with strong expression in eye and spleen, whereas mCLCA6 is primarily expressed in intestine and stomach. mCLCA6 is also expressed as a splice variant lacking exon 8 and part of exon 10 in intestine and stomach. Transfection of tsA201 cells with enhanced green fluorescent protein-tagged versions of the three cDNAs reveals protein products of 155 and 65 kDa for mCLCA5 and mCLCA6 and 145 and 65 kDa for the mCLCA6 splice variant. In vitro translation of mCLCA5 generates a 90-kDa protein that does not appear to be glycosylated. mCLCA6 also generates a 90-kDa protein that is glycosylated to a 110-kDa product, whereas the mCLCA6 splice variant generates an 80-kDa product that is 100 kDa after glycosylation. Treatment of enhanced green fluorescent protein-tagged mCLCA6 with PNGase F (peptide: N-glycosidase F) to remove N-linked glycosyl groups shows a reduction in size of the 65 kDa product to 60 kDa. Consistent with the hypothesis that mCLCA5, mCLCA6, and its splice variant encode calcium-activated chloride channels, in HEK293 cells expressing CLCAs ionomycin-evoked increases in intracellular calcium stimulated a current that reversed near Cl(-) equilibrium potential, E(Cl). Furthermore, these currents were inhibited by the chloride channel blocker niflumic acid. Given the prominent role of hCLCA2 in cancer cell adhesion and the unique high level of expression of hCLCA4 in brain, the identification of their murine counterparts presents the opportunity to clarify the role of CLCAs in disease and normal cell physiology.
Evans
Stella R
SR
Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Thoreson
Wallace B
WB
Beck
Carol L
CL
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D003001:Cloning, Molecular; D004594:Electrophysiology; D005123:Eye / Q000737:chemistry*; D006031:Glycosylation; D007422:Intestines / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020033:Protein Isoforms; D016415:Sequence Alignment; D014162:Transfection
D018118:Chloride Channels; C492299:Clca2 protein, mouse; C492979:Clca4a protein, mouse; D020033:Protein Isoforms