Authors: Rossow, C. F.¹; Duan, D.²; Hatton, W. J.²; Britton, F.¹; Hume, J. R.²; Horowitz, B.

Source: Acta Physiologica, Volume 187, Numbers 1-2, May/June 2006 , pp. 5-19(15)

Publisher: Wiley-Blackwell

Abstract:

Aim: 

This study investigated the functional role of the ClC-3 amino-terminus in channel regulation in response to changes in cell volume. Methods: 

Wild-type sClC-3 tagged with a green fluorescence protein (GFP) at the C-terminus was used as a template to construct a number of deletion mutants which were functionally expressed in NIH-3T3 cells. Whole cell and single channel patch-clamp electrophysiology was used to determine the functional properties of heterologously expressed channels. Results: 

The first 100 amino acids of the ClC-3 N-terminus were removed and the truncated channel (sClC-3ΔNT) was functionally expressed. Immunocytochemistry confirmed membrane expression of both wtsClC-3 and sClC-3ΔNT channels in NIH/3T3 cells. sClC-3ΔNT yielded constitutively active functional channels, which showed no response to protein kinase C or changes in cell volume. Deletion of a cluster of negatively charged amino acids 16-21 (sClC-3Δ16-21) within the N-terminus also yielded a constitutively active open channel phenotype, indicating these amino acids are involved in the N-type regulation. Intracellular delivery of a thiol-phosphorylated peptide corresponding to N-terminal residues 12-61 (NT peptide) markedly inhibited sClC-3ΔNT whole-cell and single-channel currents, further confirming the essential role of the N-terminus in volume regulation of channel activity. Conclusions: 

These data strongly suggest the N-terminus of sClC-3 channels acts as a blocking particle inhibiting the flow of anions through the channel pore. This `N-type' regulation of sClC-3 channels may be an important transducing mechanism linking changes in cell volume and channel protein phosphorylation to channel gating.

Keywords: cardiac electrophysiology; cell volume regulation; VSOAC channel

Document Type: Research article

DOI: http://dx.doi.org/10.1111/j.1748-1716.2006.01550.x

Affiliations: 1: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, USA 2: Department of Pharmacology, Center of Biomedical Research Excellence, University of Nevada School of Medicine, Reno, NV, USA

Publication date: 2006-05-01

Related content

• In this: publication
• By this: publisher
• In this Subject: Anatomy & Physiology
• By this author: Rossow, C. F. ;  Duan, D. ;  Hatton, W. J. ;  Britton, F. ;  Hume, J. R. ;  Horowitz, B.

Website © Publishing Technology. Article copyright remains with the publisher, society or author(s) as specified within the article.

• Terms and conditions
• Privacy policy
• Information for advertisers