CLIC-5A Functions as a Chloride Channel in Vitro and Associates with the Cortical Actin Cytoskeleton in Vitro and in Vivo

• Published in: The Journal of biological chemistry Vol. 279; no. 33; pp. 34794 - 34801
• Main Authors: Berryman, Mark, Bruno, Jonathan, Price, Jessica, Edwards, John C
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 13-08-2004
• Subjects: Actins - metabolism; Anions; Biological Transport; Bridged Bicyclo Compounds, Heterocyclic - pharmacology; Cell Line, Tumor; Cell Membrane - metabolism; Chloride Channels - chemistry; Chloride Channels - metabolism; Chloride Channels - physiology; Chlorides - metabolism; Chlorine - metabolism; Cytoskeletal Proteins; Cytoskeleton - metabolism; Detergents - pharmacology; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Gene Deletion; Glutathione Transferase - metabolism; Humans; Immunoblotting; In Vitro Techniques; Iodides - chemistry; Ions; Liposomes - metabolism; Microfilament Proteins - metabolism; Microfilament Proteins - physiology; Microscopy, Fluorescence; Microvilli - metabolism; Models, Biological; Octoxynol - pharmacology; Phosphoproteins - metabolism; Placenta - metabolism; Precipitin Tests; Protein Binding; Recombinant Fusion Proteins - metabolism; Recombinant Proteins - metabolism; Subcellular Fractions; Thiazoles - pharmacology; Thiazolidines; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M402835200

CLIC-5A is a member of the c h l oride i ntracellular c hannel protein family, which is comprised of six related human genes encoding putative chloride channels. In this study, we
found that reconstitution of purified recombinant CLIC-5A into artificial liposomes resulted in a dose-dependent chloride
efflux that was sensitive to the chloride channel blocker IAA-94. CLIC-5A was originally isolated as a component of an ezrin-containing
cytoskeletal complex from human placental microvilli. Here we show that similar protein complexes can be isolated using either
immobilized CLIC-5A or the C-terminal F-actin-binding domain of ezrin and that actin polymerization is required for de novo assembly of these complexes. To investigate the behavior of CLIC-5A in vivo , JEG-3 placental choriocarcinoma cells were stably transfected with epitope-tagged CLIC-5A. In fixed cells, CLIC-5A displayed
a polarized distribution and colocalized with ezrin in apical microvilli. Microvillar localization of CLIC-5A was retained
after Triton X-100 extraction and was disrupted by treatment with latrunculin B. In transient transfections assays, we mapped
a region between residues 20 and 54 of CLIC-5A that is required for targeting of CLIC-5A to microvilli in JEG-3 cells. Interestingly,
expression of CLIC-5A in JEG-3 cells did not enhance the rate of iodide efflux in intact cells, suggesting that if CLIC-5A
is a chloride channel, its channel activity may be restricted to intracellular membrane compartments in these cells. Regardless
of its role in ion transport, CLIC-5A, like ezrin, may play an important role in the assembly or maintenance of F-actin-based
structures at the cell cortex.