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

J. A. Sayer, G. S. Stewart, S. H. Boese, M. A. Gray, S. H. S. Pearce *, T. H. J. Goodship † and N. L. Simmons

1. We have tested the hypothesis that the voltage-dependent Cl^- channel, ClC-5 functions as a plasma membrane Cl^- conductance in renal inner medullary collecting duct cells.
2. Full-length mouse kidney ClC-5 (mClC-5) was cloned and transiently expressed in CHO-K1 cells. Fast whole-cell patch-clamp recordings confirmed that mClC-5 expression produces a voltage-dependent, strongly outwardly rectifying Cl^- conductance that was unaffected by external DIDS.
3. Slow whole-cell recordings, using nystatin-perforated patches from transfected CHO-K1 cells, also produced voltage-dependent Cl^- currents consistent with ClC-5 expression. However, under this recording configuration an endogenous DIDS-sensitive Ca²⁺-activated Cl^- conductance was also evident, which appeared to be activated by green fluorescent protein (GFP) transfection.
4. A mClC-5-GFP fusion protein was transiently expressed in CHO-K1 cells; confocal 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 collecting 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 characteristics.
7. Transient transfection of mIMCD-3 cells with antisense mClC-5 had no effect on the endogenous Cl^- conductances. Transient transfection with sense mClC-5 failed to induce the Cl^- conductance seen in CHO-K1 cells but stimulated levels of the endogenous Ca²⁺-activated Cl^- conductance 24 h post-transfection.
8. Confocal laser scanning microscopy of mIMCD-3 cells transfected with mClC-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 plasma membrane Cl^- conductance in mIMCD-3 cells but suggest a role in endosomal function.

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