Toggle Main Menu Toggle Search

Open Access padlockePrints

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

Lookup NU author(s): Professor John Sayer, Dr Gavin Stewart, Dr Stefan Boese, Dr Michael Gray, Professor Simon Pearce, Professor Tim Goodship, Professor Nicholas Simmons

Downloads

Full text for this publication is not currently held within this repository. Alternative links are provided below where available.


Abstract

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.


Publication metadata

Author(s): Sayer JA; Stewart GS; Gray MA; Goodship THJ; Pearce SHS; Boese SH; Simmons NL

Publication type: Article

Publication status: Published

Journal: Journal of Physiology

Year: 2001

Volume: 536

Issue: 3

Pages: 769-783

ISSN (print): 0022-3751

ISSN (electronic): 1469-7793

Publisher: Wiley-Blackwell Publishing Ltd.

URL: http://dx.doi.org/10.1111/j.1469-7793.2001.00769.x

DOI: 10.1111/j.1469-7793.2001.00769.x

PubMed id: 11691871


Altmetrics

Altmetrics provided by Altmetric


Actions

Find at Newcastle University icon    Link to this publication


Share