CLIC4 (p64H1) and its putative transmembrane domain form poorly selective, redox-regulated ion channels.
Despite being synthesized in the cytosol without a leader sequence, the soluble 253-residue mammalian protein CLIC4 (Chloride Intracellular Channel 4, or p64H1), a structural homologue of Omega-type glutathione-S-transferase, autoinserts into membranes to form an integral membrane protein with ion channel activity. A predicted transmembrane domain (TMD) near the N-terminus of CLIC4 could mediate membrane insertion, and contribute to oligomeric pores, with minimal reorganization of the soluble protein structure. We tested this idea by reconstituting recombinant CLIC4 in planar bilayers containing phosphatidyethanolamine, phosphatidylserine and cholesterol, recording ion channels with a maximum conductance of approximately 15 pS in KCl under both oxidizing and reducing conditions. The channels discriminated poorly between anions and cations, incompatible with the current "CLIC" nomenclature, and their conductance was modified by the trans (external or luminal) redox potential, as previously observed for CLIC1. We then reconstituted a truncated version of the protein, limited to the first 61 residues containing the predicted TMD. This included a single trans cysteine residue in the putative pore-forming subunits, at the external entrance to the pore. The truncated protein formed non-selective channels with a reduced conductance, but they retained their trans-redox sensitivity, and could still be blocked or inactivated by trans (not cis) thiol-reative dithiobisnitrobenzoic acid. We suggest that oligomers containing the putative TMD are essential components of the CLIC4 pore. However, the pore is inherently non-selective, and any ionic selectivity in CLIC4 (and other membrane CLICs) may be attributable to other regions of the protein, including the channel vestibules.
Singh
Harpreet
H
Biomedical Sciences, College of Medicine, University of Edinburgh, Edinburgh, UK.
Ashley
Richard H
RH
D000595:Amino Acid Sequence; D000818:Animals; D002021:Buffers; D018118:Chloride Channels / Q000737:chemistry* / Q000378:metabolism*; D004553:Electric Conductivity; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000378:metabolism*; D008051:Lipid Bilayers; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D051381:Rats
D002021:Buffers; D018118:Chloride Channels; C467578:Clic4 protein, rat; D007473:Ion Channels; D008051:Lipid Bilayers; D006861:Hydrogen Peroxide; D005978:Glutathione