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Functional role of amino terminus in ClC‐3 chloride channel regulation by phosphorylation and cell volume
Author(s) -
Rossow C. F.,
Duan D.,
Hatton W. J.,
Britton F.,
Hume J. R.,
Horowitz B.
Publication year - 2006
Publication title -
acta physiologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.591
H-Index - 116
eISSN - 1748-1716
pISSN - 1748-1708
DOI - 10.1111/j.1748-1716.2006.01550.x
Subject(s) - chloride channel , amino acid , c terminus , patch clamp , n terminus , intracellular , hek 293 cells , peptide , phosphorylation , biology , biochemistry , chemistry , microbiology and biotechnology , peptide sequence , gene , receptor
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.