The voltage-dependent Cl- channel ClC-5 and plasma membrane Cl- conductances of mouse renal collecting duct cells (mIMCD-3)

1. We have tested the hypothesis that the voltage-dependent Cl- channel, CI C-5 functions as a plasma membrane Cl- conductance in renal inner medullary collecting duct cells. 2. Full-length mouse kidney CIC-5 (mClC-5) was cloned and transiently expre ssed in CHO-KI cells. Fast whole-cell patch-clamp recordings confirmed that mClC-5 expression produces a voltage-dependent, strongly outwardly rectify ing Cl- conductance that was unaffected by external DIDS. 3 Slow whole-cell recordings, using nystatin-perforated patches from transf ected CHO-KI cells, also produced voltage-dependent Cl- currents consistent with CIC-5 expression. However, under this recording configuration an endo genous DIDS-sensitive Ca2+-activated Cl- conductance was also evident, whic h appeared to be activated by green fluorescent protein (GFP) transfection. 4. A mClC-5-GFP fusion protein was transiently expressed in CHO-KI cells; c onfocal laser scanning microscopy (CLSM) showed localization at the plasma membrane, consistent with patch-clamp experiments. 5. Endogenous expression of mClC-5 was demonstrated in mouse renal collecti ng duct cells (mIMCD-3) by RT-PCR and by immunocytochemistry. 6. Using slow whole-cell current recordings, mIMCD-3 cells displayed three biophysically distinct Cl--selective currents, which were all inhibited by DIDS. However, no cells exhibited whole-cell currents that had mClC-5 chara cteristics. 7. Transient transfection of mIMCD-3 cells with antisense mClC-5 had no eff ect on the endogenous Cl- conductances. Transient transfection with sense m ClC-5 failed to induce the Cl- conductance seen in CHO-K1 cells but stimula ted levels of the endogenous. Ca2+-activated Cl- conductance 24 h post-tran sfection. 8. Confocal laser scanning microscopy of mIMCD-3 cells transfected with mCI C-5-GFP showed that the protein was absent from the plasma membrane and was instead localized to acidic endosomal compartments. 9. These data discount a major role for ClC-5 as a cells but suggest a role in endosomal function.