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1 Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago-7; and 2 Centro de Estudios Científicos, Valdivia, Chile
We identified two ClC-2 clones in a guinea pig
intestinal epithelial cDNA library, one of which carries a 30-bp
deletion in the NH2 terminus. PCR using primers
encompassing the deletion gave two products that furthermore were
amplified with specific primers confirming their authenticity. The
corresponding genomic DNA sequence gave a structure of three exons and
two introns. An internal donor site occurring within one of the exons
accounts for the deletion, consistent with alternative splicing.
Expression of the variants gpClC-2 and gpClC-2
77-86 in HEK-293
cells generated inwardly rectifying chloride currents with similar
activation characteristics. Deactivation, however, occurred with faster
kinetics in gpClC-277-86. Site-directed mutagenesis suggests
that a protein kinase C-mediated phosphorylation consensus site lost in
gpClC-277-86 is not responsible for the observed change. The
deletion-carrying variant is found in most tissues examined, and it
appears more abundant in proximal colon, kidney, and testis. The
presence of a splice variant of ClC-2 modified in its
NH2-terminal domain could have functional consequences in
tissues where their relative expression levels are different.
chloride secretion; alternative splicing; guinea pig; channel deactivation; intestinal epithelium
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