Molecular and Functional Analyses of Two New Calcium-activated Chloride Channel Family Members from Mouse Eye and Intestine

Abstract

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.Calcium-activated chloride currents have been characterized physiologically in many cell types including epithelium (1-4), smooth muscle (5-7), skeletal muscle (8), and neurons (9, 10). However, the molecular identities of channels responsible for calcium-activated chloride currents have not been determined.Proteins of the bestrophin and CLCA 1 families have been proposed to function as calcium-activated chloride channels (11)(12)(13)(14)(15)(16)(17)(18)(19).The CLCA gene family encodes proteins with four or five putative transmembrane domains, and many family members appear to function as calcium-activated chloride channels (bCLCA1, mCLCA1, mCLCA4, hCLCA1, and hCLCA2) (15)(16)(17)(18)(19). Other studies suggest that CLCAs may not be chloride channels themselves but may instead modulate chloride channels (20,21) In addition to their potential roles as ion channels or regulators of ion channels, some CLCA family members function as adhesion molecules. For example, bCLCA2 (Lu-ECAM-1), mCLCA1, and hCLCA2 all appear to be cell-cell adhesion molecules that help mediate the colonization of lung by lung metastatic murine B16-F10 melanoma cells (22-24). ␤ 4 integrin expressed on the surface of these melanoma cells can adhere to mCLCA1 in lung endothelium. This lea...

The Putative Chloride Channel hCLCA2 Has a Single C-terminal Transmembrane Segment

“…Homologs were subsequently identified in other species, with at least six in mouse and four in human (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). With the sequencing of numerous genomes, it has become apparent that CLCA proteins are found throughout the chordates and possibly in some invertebrates (13).…”

Murine mCLCA6 Is an Integral Apical Membrane Protein of Non-goblet Cell Enterocytes and Co-localizes With the Cystic Fibrosis Transmembrane Conductance Regulator

“…Both proteins are thought to be expressed in the intestine (Agnel et al 1999;Evans et al 2004), and the hCLCA1/hCLCA4 gene locus has been shown to play a role as modulator of the basic intestinal chloride secretory defect in CF (Ritzka et al 2004). However, the cell type expressing these proteins and their subcellular location are as yet unknown.…”

“…Murine mCLCA6 has recently been discovered as the murine ortholog of human hCLCA4 (Evans et al 2004). Both proteins are thought to be expressed in the intestine (Agnel et al 1999;Evans et al 2004), and the hCLCA1/hCLCA4 gene locus has been shown to play a role as modulator of the basic intestinal chloride secretory defect in CF (Ritzka et al 2004).…”

“…However, several other functions have been proposed and include adhesion molecule function (Abdel-Ghany et al 2003), bicarbonate channel activity (Thevenod et al 2003), modulation of other channel proteins (Greenwood et al 2002;Loewen et al 2002), inhibition of tumor growth by promoting apoptosis , and protease activity (Pawlowski et al 2006). To date, the CLCA gene family comprises 18 members known in eight mammalian species with six murine and four human homologs reported (Cunningham et al 1995;Gandhi et al 1998;Gruber et al 1998a;Gruber and Pauli 1999;Komiya et al 1999;Lee et al 1999;Romio et al 1999;Elble et al 2002;Leverkoehne and Gruber 2002;Evans et al 2004;Anton et al 2005;Al-Jumaily et al 2007). The human and murine CLCA proteins are of considerable interest based on their role in diseases with secretory dysfunction including asthma (Nakanishi et al 2001;Hoshino et al 2002;Range et al 2007).…”

Steroid hormone regulation of Clca3 expression in the murine uterus

“…Six members of this family have been identified, cloned and partially characterized in the mouse: Clca1 (Gandhi et al 1998, Romio et al 1999, Clca2 (Lee et al 1999), Clca3 (Komiya et al 1999), Clca4 (Elble et al 2002), Clca5 and Clca6 (Abdel-Ghany et al 2003, Beckley et al 2004, Evans et al 2004. Structural analysis indicates significant similarities among different CLCA family members, including protein sizes of 902-943 amino acids and four or five transmembrane regions (Pauli et al 2000, Jentsch et al 2002.…”