A short CIC-2 mRNA transcript is produced by exon skipping

Abstract

CIC-2 is a voltage- and volume-regulated chloride channel expressed in many tissues. We have shown that CIC-2 in rat lung airways is significantly down-regulated after birth [Murray,C.B. et al. (1995) Am. J. Respir. Cell Mol. Biol., 12, 597-604]. During PCR amplification from rat lung cDNA, a second transcript was identified which is 60 bp shorter than the full length sequence. The peptide translated from this 60 bp sequence contains many positively charged amino acid residues. Rat genomic DNA sequencing showed that the 60 bp sequence is an intact exon. A 71% pyrimidine content and an AAG 3'-end splice site in the intron immediately upstream from the 60 bp sequence were identified which may account for the alternative splicing of the following exon. Human genomic sequence analyses demonstrated similar intron-exon arrangement. A high CT content and an AAG 3' acceptor site were conserved in the intron corresponding to the rat upstream intron. The presence of the full length short form transcript was confirmed in rat kidney by RT-PCR, and the ratio of the long and the short form transcripts varied significantly according to the tissues examined, with the lowest long/short form ratio found in the lung among the tissues studied. Our data demonstrated that the alternatively spliced short form (CIC-2S) is transcribed in many rat tissues, the ratio of the long/short form transcripts is lower in the lung compared with the brain, and the genomic organization in this area is conserved in rat and human.

Functional Characterization of Novel Alternatively Spliced ClC-2 ChlorideChannel Variants in theHeart

“…Transcript variants of the ClC-2 gene have been identified by a number of other investigators (4,5,7,10). Chu et al (4) reported the genomic structure of rat ClC-2 and identified that alternative mRNA splice variants of the rat ClC-2 gene are generated by exon skipping.…”

“…It is therefore not known whether the functional characteristics of heart ClC-2 are the same as those of brain ClC-2 in rat. In fact, short isoforms of ClC-2 mRNA transcript truncated at the C terminus due to alternative splicing were identified in multiple tissues in rat (4). Using RT-PCR, we cloned the putative full-length cDNA of ClC-2 from rat heart (rClC-2), which had 99.4% homology to the previously reported brain ClC-2 (3).…”

“…This deletion is not a complete "exon signature" alternative splicing event because it skipped only a portion of exon 15, which is 214 bp long. It may represent a new ClC-2 spliced isoform that has not been reported before in any other tissues of the rat (4,5,29). ClC-2 expression in rat heart was further analyzed by the use of real-time quantitative RT-PCR (Fig.…”

The Functional Role of Chloride Channels in Cardiac Pacemaker Activity

“…Later several alternatively spliced forms were cloned from several other tissues and species, including human (Furukawa et al, 1995;Malinowska et al, 1995;Cid et al, 1995;Chu et al, 1996;Chu & Zeitlin, 1997;Furukawa et al, 1998;Cid et al, 2000;Loewen et al, 2000;Furukawa et al, 2002). Expression of ClC-2 cDNA in Xenopus oocytes or mammalian cells resulted in hyperpolarization-activated inward-rectifying Cl -currents which are sensitive to changes in cell volume and extracellular pH (Jordt & Jentsch, 1997;Furukawa et al, 1998;Park et al, 1998;Stroffekova et al, 1998;Cid et al, 2000;Park et al, 2001;Furukawa et al, 2002).…”

Expression of novel isoforms of the CIC‐1 chloride channel in astrocytic glial cells in vitro

“…Although many chloride channels of the ClC family have been cloned and expressed functionally in heterologous systems, few studies have examined the regulation of their gene expression (Shimada et al, 2000). In the family of ClC channels, ClC-2, ClC-3 and ClC-6 have been reported to have multiple isoforms (Chu et al, 1996;Chu and Zeitlin, 1997;Eggermont et al, 1997;Shimada et al, 2000). The best characterized of these is ClC-2, for which tissue-specific exon skipping and alternative splicing have been shown to generate multiple isoforms of the channel (Chu et al, 1996;Chu and Zeitlin, 1997).…”