Isolation and Characterization of a High Affinity Peptide Inhibitor of ClC-2 Chloride Channels

• Type of Material: Article
• Authors: Christopher H. Thompson, Georgia Institute of Technology; Pedro R. Olivetti, Georgia Institute of Technology; Matthew D. Fuller, Emory University; Cody S. Freeman, Georgia Institute of Technology; Denis McMaster, University of Calgary; Robert J. French, University of Calgary; Jan Pohl, Emory University; Julia Kubanek, Georgia Institute of Technology; Nael McCarty, Emory University
• Language: English
• Date: 2009-09-18
• Publisher: American Society for Biochemistry and Molecular Biology
• Publication Version: Final Published Version
• Copyright Statement: © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.
• Final Published Version (URL): http://www.jbc.org/content/284/38/26051
• Title of Journal or Parent Work: Journal of Biological Chemistry
• ISSN: 0021-9258
• Volume: 284
• Issue: 38
• Start Page: 26051
• End Page: 26062
• Grant/Funding Information: This work was also supported by the National Institutes of Health National Center for Research Resources, the Canadian Institutes of Health Research, the Cystic Fibrosis Foundation, and Children's Healthcare of Atlanta, Inc.; This work was supported, in whole or in part, by National Institutes of Health Grant DK066409.
• Research Categories: Chemistry, Biochemistry

Abstract

The ClC protein family includes voltage-gated chloride channels and chloride/proton exchangers. In eukaryotes, ClC proteins regulate membrane potential of excitable cells, contribute to epithelial transport, and aid in lysosomal acidification. Although structure/function studies of ClC proteins have been aided greatly by the available crystal structures of a bacterial ClC chloride/proton exchanger, the availability of useful pharmacological tools, such as peptide toxin inhibitors, has lagged far behind that of their cation channel counterparts. Here we report the isolation, from Leiurus quinquestriatus hebraeus venom, of a peptide toxin inhibitor of the ClC-2 chloride channel. This toxin, GaTx2, inhibits ClC-2 channels with a voltage-dependent apparent KD of ∼20 pm, making it the highest affinity inhibitor of any chloride channel. GaTx2 slows ClC-2 activation by increasing the latency to first opening by nearly 8-fold but is unable to inhibit open channels, suggesting that this toxin inhibits channel activation gating. Finally, GaTx2 specifically inhibits ClC-2 channels, showing no inhibitory effect on a battery of other major classes of chloride channels and voltage-gated potassium channels. GaTx2 is the first peptide toxin inhibitor of any ClC protein. The high affinity and specificity displayed by this toxin will make it a very powerful pharmacological tool to probe ClC-2 structure/function.