Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Oct 1;279(40):41792-800.
doi: 10.1074/jbc.M408354200. Epub 2004 Jul 28.

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

Affiliations
Free PMC article

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

Stella R Evans et al. J Biol Chem. .
Free PMC article

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.

Figures

Fig. 1
Fig. 1. Alignment of all mouse CLCA isoforms
Consensus sites for phosphorylation by Ca2+/calmodulin-dependent protein kinase II, PKA, and PKC are marked for mCLCA5 and mCLCA6 (small open square, mCLCA5 Ca2+/calmodulin-dependent protein kinase II; large open square, PKA; open circle, PKC; small filled square, mCLCA6 Ca2+/calmodulin-dependent protein kinase II; large filled square, PKA; filled circle, PKC (PhosphoBase version 2.0, Center for Biological Sequence Analysis). Sites are marked for all mCLCAs for monobasic proteolytic cleavage (↓), signal sequence (underlined) (SignalPV1.1, Center for Biological Sequence Analysis), and multiple cysteine motif (C). Additionally, consensus sites for N-linked glycosylation are indicated (N with * overhead). The alignment was generated by the Clustal method.
Fig. 1
Fig. 1. Alignment of all mouse CLCA isoforms
Consensus sites for phosphorylation by Ca2+/calmodulin-dependent protein kinase II, PKA, and PKC are marked for mCLCA5 and mCLCA6 (small open square, mCLCA5 Ca2+/calmodulin-dependent protein kinase II; large open square, PKA; open circle, PKC; small filled square, mCLCA6 Ca2+/calmodulin-dependent protein kinase II; large filled square, PKA; filled circle, PKC (PhosphoBase version 2.0, Center for Biological Sequence Analysis). Sites are marked for all mCLCAs for monobasic proteolytic cleavage (↓), signal sequence (underlined) (SignalPV1.1, Center for Biological Sequence Analysis), and multiple cysteine motif (C). Additionally, consensus sites for N-linked glycosylation are indicated (N with * overhead). The alignment was generated by the Clustal method.
Fig. 2
Fig. 2. Phylogenetic tree showing the relation of all CLCAs
The dendogram was generated by Clustal method alignment on the European Bioinformatics Institute website with complete amino acid sequences for all CLCAs.
Fig. 3
Fig. 3. Diagram of the mCLCA6 splice variant
Exon 8 and a portion of exon 10 are removed in the mCLCA6 splice variant (mCLCA6sv). The removal of exon 8 (starts at amino acid 397) causes a shift in the reading frame, thus changing the sequence of exon 9 (which we call exon 9a). The removal of a portion of exon 10 causes a reversion to the normal reading frame in the latter portion of exon 10. Shown below the splice variant schematic is a diagram of the amino acid sequence removed from the splice variant (sv) (as compared with wild type (wt, dark gray)) and the new sequence generated in exon 9a (light gray).
Fig. 4
Fig. 4. Tissue expression of mCLCA5 and mCLCA6 by RT-PCR
mCLCA5 shows the highest levels of expression in eye and spleen, whereas mCLCA6 is expressed predominantly in intestine and stomach. The lower band in the mCLCA6 intestine and stomach lanes represents the splice variant (mCLCA6sv). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) verifies RNA integrity and loading. b, brain; e, eye; h, heart; i, intestine; k, kidney; li, liver, lu, lung, skm, skeletal muscle; sp, spleen; st, stomach; t, testes; (−), negative control.
Fig. 5
Fig. 5. Biochemical characterization of mCLCA5, mCLCA6, and mCLCA6 splice variant proteins
A, immunoblotting analysis of immunoprecipitated EGFP-tagged CLCAs from transfected tsA201 cells. mCLCA5 and mCLCA6 are detected as 65- and 155-kDa bands, whereas the mCLCA6 splice variant is detected as 65 and 145 kDa. The 65-kDa band corresponds to the cleaved carboxyl terminus, and the 155 (or 145)-kDa band corresponds to full-length protein. B, in vitro translation without (−M) and with (+M) microsomal membranes. l-[35S]Methionine-labeled proteins were resolved by SDS-PAGE and detected by phosphorscreen. mCLCA6 and the mCLCA6 splice variant (mCLCA6sv) show an increase in size due to glycosylation in the presence of microsomal membranes from 90 to 110 kDa for mCLCA6 or 80 to 100 kDa for the mCLCA6 splice variant. C, immunoblot analysis of a PGNase digestion of lysate from tsA201 cells transfected with EGFP-tagged mCLCA6. The mCLCA6 carboxyl terminus shows a decrease in size from 65 to 60 kDa due to the removal of N-linked glycosyl groups by PGNase.
Fig. 6
Fig. 6. tsA201 cells expressing EGFP-tagged mCLCA5 (A), mCLCA6 (B), and mCLCA6 splice variant (mCLCA6sv) (C)
Note the apparent membrane localization of all three proteins. mCLCA5 and mCLCA6 show the sharpest membrane staining and are present at cell-cell junctions.
Fig. 7
Fig. 7. Cl currents were evoked in CLCA-expressing cells by ionomycin-stimulated calcium influx
A, currents evoked in mCLCA6-expressing HEK293 cells by a series of voltage steps (−70 to +70 mV, 150 ms). B, currents evoked in the same cell by voltage steps provided after ionomycin (10 μm) application. Ionomycin stimulated an inward current at the holding potential of −50 mV and increased the amplitude of currents evoked by the various voltage steps. C, current/voltage relationship for the steady state difference current. The difference current was obtained by subtracting step-evoked currents obtained before the application of ionomycin from the currents obtained after ionomycin application. The difference current shows a positive slope conductance and reversed around −17 mV. D, mean reversal potentials for currents evoked by ionomycin in untransfected control cells (n = 10) and cells expressing mCLCA5 (n = 7), mCLCA6 (n = 7), and mCLCA6 splice variant (mCLCA6sv) (n = 7). Current/voltage relationships used to calculate reversal potentials were measured at the peak of the ionomycin-evoked current, which was attained 3 min after ionomycin application in transfected cells and 6 min after ionomycin application in untransfected control cells.
Fig. 8
Fig. 8. Niflumic acid inhibits ionomycin-evoked currents in CLCA-expressing cells
Ionomycin-evoked currents (Vh = − 50 mV) were significantly inhibited by niflumic acid (0.1 mm) in HEK293 cells expressing mCLCA5 (n = 7, p = 0.0004, t test), mCLCA6 (n = 7, p < 0.0001), and the mCLCA6 splice variant (mCLCA6sv) (n = 7, p < 0.0001) but not in untransfected (n = 10, p = 0.97) or mock-transfected (n = 4, p = 0.47, not shown) control cells.

Similar articles

See all similar articles

Cited by 18 articles

See all "Cited by" articles

Publication types

Associated data

Feedback