Comparison of voltage-activated Cl- channels in rat parotid acinar cells with ClC-2 in a mammalian expression system
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abstract
Rat parotid acinar cells express Cl- currents that are activated in a time-dependent manner by hyperpolarized potentials. ClC-2, a member of the ClC gene family, codes for a voltage-gated, inward rectifying anion channel when expressed in Xenopus oocytes. In the present study, we found that cDNA derived from individual parotid acinar cells contained sequence identical to that reported for ClC-2 in rat brain and heart. A polyclonal antibody generated against the N-terminal cytoplasmic domain of ClC-2 recognized an approximately 100 kD protein on western blots of both brain and parotid gland. ClC-2 expressed in oocytes has different kinetics from the currents found in parotid acinar cells. Since the ClC-2 channel was cloned from and its transcripts are expressed in mammalian tissue, we compared the channel properties of acinar cells to a mammalian expression system. We expressed ClC-2 channels in human embryonic kidney cells, HEK 293, using recombinant ClC-2 DNA and ClC-2 DNA fused with DNA coding for jellyfish green fluorescent protein (GFP). Confocal microscopy revealed that the expressed ClC-2-GFP chimera protein localized to the plasma membrane. Whole cell Cl- currents from HEK 293 cells expressing ClC-2-GFP were similar, if not identical, to the Cl- currents recorded from cells transfected with ClC-2 cDNA (no GFP). The voltage-dependence and kinetics of ClC-2 channels expressed in HEK 293 cells were quite similar to those in acinar cells. Channels in parotid acinar and HEK 293 cells activated at more positive membrane potentials and with a faster time course than the channels expressed in Xenopus oocytes. In summary, we found that ClC-2 message and protein are expressed in salivary cells and that the properties of voltage-activated, inward rectifying Cl- channels in acinar cells are similar to those generated by the ClC-2-GFP construct expressed in HEK 293 cells. The properties of the ClC-2 anion channel seem to be dependent on the type of cell background in which it is expressed.
