Elsevier

Gene

Volume 228, Issues 1–2, 4 March 1999, Pages 181-188
Gene

Characterization of a murine gene homologous to the bovine CaCC chloride channel

https://doi.org/10.1016/S0378-1119(98)00620-9Get rights and content

Abstract

The bovine CaCC protein is a putative Ca2+-dependent Cl channel of airway epithelial cells. Therefore, CaCC proteins could contribute to transepithelial Cl transport and accordingly modify the phenotype of cystic fibrosis (CF) patients. We have identified a murine EST containing a full-length cDNA coding for a 902-amino-acid protein highly homologous to bovine CaCC. The murine gene (mCaCC) maps to chromosome 3 at the H2–H3 band and is expressed, as indicated by Northern blot analysis, in mouse skin and kidney but not in brain, heart, lung or testis. RT-PCR indicates a low expression in tracheal epithelial cells. Heterologous expression of mCaCC in Xenopus oocytes elicits membrane currents that are anion-selective and inhibited by DIDS and by niflumic acid, a blocker of the endogenous chloride current in oocytes. The identification of genes belonging to the CaCC family will help to evaluate their role as ion channels or channel regulators and their actual contribution to epithelial chloride transport.

Introduction

Cl channels in epithelial cells are key elements in the process of transepithelial electrolyte and fluid absorption or secretion (Anderson et al., 1992). In the airways, Cl secretion occurring at the surface epithelium or in the submucosal gland lumen contributes to the hydration of the mucous layer which covers the epithelium (Boucher, 1994; Wu et al., 1998). The relevance of this function is evidenced by the fact that cystic fibrosis patients, who have a defective Cl transport in epithelial cells, display insippated mucous secretions and suffer from recurrent bacterial infections and chronic obstructive pulmonary disease (Boucher, 1994). Besides CFTR, which is the channel coded by the gene altered in CF patients, other Cl channels have been described in epithelial cells (Anderson et al., 1992). In particular, the increase of intracellular Ca2+, such as that caused by various agonists, is followed by the activation of Cl channels different from CFTR. The contribution of these channels to transepithelial ion transport and their relevance in CF, is not clear. However, recent studies, performed on transgenic mice, suggest that the activity of Ca2+-dependent Cl channels might reduce the severity of the CF phenotype (Rozmahel et al., 1996), and a modifier gene locus, which could contain a gene for a Cl channel, has been mapped to mouse chromosome 7 (Rozmahel et al., 1996).

The identification of new genes coding for Cl channels could therefore reveal new aspects of physiological and pathological processes involving ion transport in epithelial cells. Recently, a gene coding for a putative epithelial Cl channel has been purified and cloned with a biochemical approach from the bovine tracheal epithelium (Cunningham et al., 1995). This protein, termed CaCC, has novel structural features and seems to act as a Ca2+-regulated Cl channel. Since the bovine CaCC may represent the member of a new gene family, we aimed at identifying the corresponding murine and human homologs. The interest in CaCC is also increased by the finding that a highly identical bovine gene, Lu-ECAM-1, has the functional features of a cell adhesion molecule (Elble et al., 1997). In the present paper, we describe at the molecular and functional level a murine cDNA highly homologous to the bovine gene.

Section snippets

EST identification, sequencing and analysis

A homology search of the EST database was performed with the BLAST E-mail Server ([email protected]) using the bovine CaCC (Accession No. U36445) as the query sequence. The clone containing the EST AA245677, identified by this search, was obtained from the UK HGMP Resource Centre (Cambridge). The entire nucleotide sequence of the clone was determined using both ‘end sequences’ and ‘primer walking’ strategies. Cycle sequencing was carried out with the −21M13 and M13 Rev dye primers

Results

The availability of an increasing number of expressed sequence tags (EST) in sequence databases allows identification of new genes belonging to a given protein family. We performed a BLAST search using the bovine CaCC as the query sequence. The search showed several murine sequences, some with a high sequence identity (>80%). The EST AA245677 appeared to contain the full-length coding sequence. Determination of the entire sequence contained in this clone revealed an insert of 3474 bp with an

Discussion

Electrophysiological studies indicate that different Cl channels exist in epithelial cells. Some of these channels, such as CFTR and ClC2, have been cloned (Fong and Jentsch, 1995). Others are only known at the biophysical and pharmacological level. The bovine CaCC (Cunningham et al., 1995) represents an interesting candidate gene for Ca2+-dependent Cl channels in airway epithelial cells. Taking advantage of the EST database, we have identified a mouse cDNA with a strong homology to the

Acknowledgements

The financial support of Telethon-Italy (Grant no. E.593) and Minisan 98.512.01F is gratefully acknowledged. We also thank C.M. Fuller for providing us with the bovine CaCC clone, E. Gaggero for making the voltage-clamp amplifier, F. Caroli for DNA sequencing, and G. Borsani (Tigem) for helpful discussion and for providing us with some of the EST clones.

References (17)

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