Functional role of amino terminus in ClC-3 chloride channel regulation by phosphorylation and cell volume.
OBJECTIVE
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-3DeltaNT) was functionally expressed. Immunocytochemistry confirmed membrane expression of both wtsClC-3 and sClC-3DeltaNT channels in NIH/3T3 cells. sClC-3DeltaNT 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-3Delta16-21) 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-3DeltaNT 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.
Rossow
C F
CF
Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.
Duan
D
D
Hatton
W J
WJ
Britton
F
F
Hume
J R
JR
Horowitz
B
B
D016475:3T3 Cells; D000818:Animals; D048429:Cell Size; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D003062:Codon; D018274:Electroporation; D005347:Fibroblasts / Q000378:metabolism*; D017353:Gene Deletion; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000235:genetics; D006168:Guinea Pigs; D007150:Immunohistochemistry / Q000379:methods; D015640:Ion Channel Gating; D007473:Ion Channels / Q000378:metabolism; D051379:Mice; D009994:Osmolar Concentration; D018408:Patch-Clamp Techniques; D010766:Phosphorylation
D018118:Chloride Channels; C115376:ClC-3 channel; D003062:Codon; D007473:Ion Channels; D049452:Green Fluorescent Proteins