Expression and canalicular localization of two isoforms of the ClC-3 chloride channel from rat hepatocytes

Abstract

The molecular identities of functional chloride channels in hepatocytes are largely unknown. We examined the ClC-3 chloride channel in rat hepatocytes and found that mRNA for two different isoforms is present. A short form is identical to the previously reported sequence for rat ClC-3, and a long form contains a 176-bp insertion immediately upstream of the translation initiation site. This predicts a 58-amino acid NH(2) terminal insertion. Both long and short form mRNA was expressed in diverse tissues of the rat. Transient transfection of the long form in CHO-K1 cells resulted in currents with an I(-) > B(-) > Cl(-) selectivity sequence, outward rectification, and inactivation at positive voltages. Short form currents had identical ionic selectivity but displayed a more extreme outward rectification and showed no voltage-dependent inactivation. Immunofluorescence and immunoblots localized native ClC-3 preferentially but not exclusively to the canalicular membrane. We have therefore identified a new isoform of rat ClC-3 and shown that expression of both isoforms produces functional channels. In hepatocytes, ClC-3 is located in association with the canalicular membrane.

AP-3-dependent Mechanisms Control the Targeting of a Chloride Channel (ClC-3) in Neuronal and Non-neuronal Cells

“…Although ClC-3 is a ubiquitously expressed protein (17,35), endogenous levels in fibroblasts were below the immunofluorescence detection level. To overcome this limitation, we transfected Flag-and GFP-tagged ClC-3 constructs into immortalized mocha fibroblasts rescued or not with the AP-3 delta subunit.…”

Biophysical Properties of ClC-3 Differentiate It from Swelling-activated Chloride Channels in Chinese Hamster Ovary-K1 Cells

“…This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. short form of rat ClC-3 with a FLAG epitope that was inserted at the 3Ј end of the open reading frame, was prepared by reverse transcriptasepolymerase chain reaction, ligated into pcDNA3.1 ϩ (Invitrogen), and sequenced using eight sequencing primers by the sequencing facility at the University of Texas Medical Branch as described previously (14). An additional expression vector consisting of ClC-3 without the FLAG epitope was prepared by introducing a stop codon before the FLAG epitope using the QuickChange TM site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol and confirmed by sequencing.…”

“…Patch Clamp-Whole cell patch clamp was performed by the methods previously described in detail (14,16). The bath solution contained (in mM) 114 NaCl, 5.4 CsCl, 1 MgSO 4 , 1.5 CaCl 2 , 10 HEPES, 10 glucose, pH 7.4, adjusted with NaOH.…”

“…The short form has 99.9% identity to the guinea pig ClC-3. Immunofluorescence and immunoblotting demonstrated that native ClC-3 in hepatocytes was localized both intracellularly and in association with the canalicular membrane (14). In the present study, we expressed a FLAG-tagged ClC-3 construct in Chinese hamster ovary (CHO) 1 -K1 cells, examined its activation by cell swelling, and compared its properties to those of endogenous swelling-activated chloride currents.…”

“…We recently cloned two isoforms of the ClC-3 channel from rat hepatocytes (14). The long form has the identical amino acid sequence as the mouse ClC-3 and has 98% identity to the human ClC-3.…”