Redox regulation of CLIC1 by cysteine residues associated with the putative channel pore.
Chloride intracellular channels (CLICs) are putative pore-forming glutathione-S-transferase homologs that are thought to insert into cell membranes directly from the cytosol. We incorporated soluble, recombinant human CLIC1 into planar lipid bilayers to investigate the associated ion channels, and noted that channel assembly (unlike membrane insertion) required a specific lipid mixture. The channels formed by reduced CLIC1 were similar to those previously recorded from cells and "tip-dip" bilayers, and specific anti-CLIC1 antibodies inhibited them. However, the amplitudes of the filtered single-channel currents were strictly regulated by the redox potential on the "extracellular" (or "luminal") side of the membrane, with minimal currents under strongly oxidizing conditions. We carried out covalent functional modification and site-directed mutagenesis of this controversial ion channel to test the idea that cysteine 24 is a critical redox-sensitive residue located on the extracellular (or luminal) side of membrane CLIC1 subunits, in a cysteine-proline motif close to the putative channel pore. Our findings support a simple structural hypothesis to explain how CLIC1 oligomers form pores in membranes, and suggest that native channels may be regulated by a novel mechanism involving the formation and reduction of intersubunit disulphide bonds.
Singh
Harpreet
H
Biomedical Sciences, University of Edinburgh Medical School, Edinburgh, United Kingdom.
Ashley
Richard H
RH
D001665:Binding Sites; D003198:Computer Simulation; D003545:Cysteine / Q000737:chemistry*; D006801:Humans; D015640:Ion Channel Gating; D008051:Lipid Bilayers / Q000737:chemistry*; D008560:Membrane Fluidity; D008564:Membrane Potentials; D008954:Models, Biological; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D016062:Porosity; D011485:Protein Binding; D013329:Structure-Activity Relationship
D008051:Lipid Bilayers; D003545:Cysteine