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

Abstract

We previously reported the cloning of a calcium-activated chloride channel (CLCA) from rat brain (Biochem. Biophys. Res. Commun., 334, 569-576 (2005)), which we designated rbCLCA1. We further showed that rbCLCA1 is expressed in the central nervous system and peripheral organs, and may be functionally expressed in mammalian HEK293 cells. In the present study, we report the successful cloning of a second CLCA from rat cerebrum (designated rbCLCA2), using reverse transcription-PCR (RT-PCR) with primers specific for rbCLCA1. We have begun to clone this cDNA based on the rbCLCA1-like sequence. The full-length rbCLCA2 cDNA, obtained via 5' and 3' rapid amplification of cDNA ends (RACE), is 2900 bp long and encodes a putative polypeptide of 905 amino acids having at least two major transmembrane domains and showing 85.2% identity to rbCLCA1. RT-PCR analysis revealed that, similar to rbCLCA1, rbCLCA2 was predominantly expressed in the rat cerebrum, cerebellum, kidney, stomach, spinal cord, lung and small intestine, but not in the heart, large intestine, liver, orand spleen. Whole-cell patch clamp studies in HEK293 cells transiently co-transfected with expression vectors encoding rbCLCA2 and EGFP allowed us to identify the presence of niflumic acid (a CLCA channel blocker)-sensitive and voltage-dependent chloride currents in cells expressing rbCLCA2 but not EGFP alone. Treatment of these cells with ionomycin, a Ca2+ ionophore, significantly increased the novel currents in cells expressing rbCLCA2 and EGFP, but not those expressing EGFP alone, indicating that activation of the rbCLCA2 current is Ca2+-dependent. In sum, we herein report the cloning of a second member of the rbCLCA family from rat brain and its functional expression in vitro, thus adding to our knowledge of anion channels and facilitating future exploration of brain and other organ physiology.