Cloning and expression of Ca2+-activated chloride channel from rat brain

To clone the gene product responsible for the calcium-activated chloride channel (CLCA) in rat brain cerebrum, we performed a reverse transcription-PCR (RT-PCR) with gene-specific primers of a rat EST clone. We successfully cloned a rat brain CLCA (rbCLCA). The full-length cDNA is 2895 bp long and codes for a 902 amino acid protein. The clone consists of four transmembrane domains and shows a 79.1% of significant homology with previously reported mouse smooth muscle chloride channel sequence. We also performed RT-PCR using single neuron and glia, and various tissues to determine the tissue expression of rbCLCA. We found that rbCLCA was expressed in both neuron and glia. In peripheral organs, rbCLCA showed the predominant expressions in cerebrum, cerebellum, kidney, small intestine, and stomach but not in heart, large intestine, liver, lung, and spleen. Whole-cell patch clamp studies in HEK293 cells transfected with the clone identified a niflumic acid (a CLCA channel blocker)-sensitive and voltage-dependent chloride current but we could not observe this chloride current in mock-transfected cells. The identification of genes belonging to the CLCA family from rat brain and its functional expression will help to evaluate its physiological role in brain as anion channel.