COMPARISON OF VOLTAGE-ACTIVATED CL- CHANNELS IN RAT PAROTID ACINAR-CELLS WITH CLC-2 IN A MAMMALIAN EXPRESSION SYSTEM

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 ani on channel when expressed in Xenopus oocytes. In the present study, we found that cDNA derived from individual parotid acinar cells containe d sequence identical to that reported for ClC-2 in rat brain and heart . A polyclonal antibody generated against the N-terminal cytoplasmic d omain of ClC-2 recognized an approximately 100 kD protein on western b lots of both brain and parotid gland. ClC-2 expressed in oocytes has d ifferent kinetics from the currents found in parotid acinar cells. Sin ce the ClC-2 channel was cloned from and its transcripts are expressed in mammalian tissue, we compared the channel properties of acinar cel ls to a mammalian expression system. We expressed ClC-2 channels in hu man embryonic kidney cells, HEK 293, using recombinant ClC-2 DNA and C lC-2 DNA fused with DNA coding for jellyfish green fluorescent protein (GFP). Confocal microscopy revealed that the expressed ClC-2-GFP chim era 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 expres sed in HEK 293 cells were quite similar to those in acinar cells. Chan nels in parotid acinar and HEK 293 cells activated at more positive me mbrane potentials and with a faster time course than the channels expr essed in Xenopus oocytes. In summary, we found that ClC-2 message and protein are expressed in salivary cells and that the properties of vol tage-activated, inward rectifying Cl- channels in acinar cells are sim ilar to those generated by the ClC-2-GFP construct expressed in HEK 2. 93 cells. The properties of the ClC-2 anion channel seem to be depende nt on the type of cell background in which it is expressed.