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

Christopher H. Thompson; Pedro R. Olivetti; Matthew D. Fuller; Cody S. Freeman; Denis McMaster; Robert J. French; Jan Pohl; Julia Kubanek; Nael A. McCarty

doi:10.1074/jbc.M109.031724

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

From the ^‡School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332,
the ^§Program in Molecular and Systems Pharmacology,
the **Microchemical and Proteomics Facility, and
^§§Children's Healthcare of Atlanta and the Department of Pediatrics, Emory University, Atlanta, Georgia 30322,
the ^¶Peptide Synthesis Core Facility and
the ^‖Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada, and
the ^‡‡School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332

↵⁴ To whom correspondence should be addressed: 2015 Uppergate Dr., Atlanta, GA 30322. Tel.: 404-727-3654; Fax: 404-712-0920; E-mail: namccar{at}emory.edu.

↵¹ Present address: Div. of Genetic Medicine, Vanderbilt University, Nashville, TN 37232.
↵² Present address: Dept. of Pharmacology, University of Washington, Seattle, WA 98195.
↵³ Present address: Biotechnology Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333.

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 K_D 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.

Footnotes

↵* This work was supported, in whole or in part, by National Institutes of Health Grant DK066409. 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.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3 and supplemental text.