Functional role of amino terminus in ClC-3 chloride channel regulation by phosphorylation and cell volume
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.
Document Type: Research Article
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: May 1, 2006