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J. Biol. Chem., Vol. 279, Issue 33, 34794-34801, August 13, 2004

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

Mark Berryman, Jonathan Bruno¶, Jessica Price, and John C. Edwards¶

From the Department of Biomedical Sciences, Ohio University College of Osteopathic Medicine and the Molecular and Cellular Biology Program, Athens, Ohio 45701 and the ^¶St. Louis Veterans Affairs Medical Center and the Department of Medicine, St. Louis University, St. Louis, Missouri 63106

CLIC-5A is a member of the chloride intracellular channel 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.

Received for publication, March 12, 2004 , and in revised form, May 24, 2004.

^* This work was supported by funds from Ohio University (to M. B.), National Institutes of Health Grant RO1 DK060551, and a Department of Veterans Affairs Merit Award (to J. C. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biomedical Sciences, Ohio University College of Osteopathic Medicine, 237 Life Sciences Bldg., Athens, OH 45701. Tel.: 740-593-2364; Fax: 740-597-2778; E-mail: berryman{at}ohiou.edu.

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