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Inhibition of ClC-2 Chloride Channels by a Peptide Component or Components of Scorpion Venom

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Abstract

ClC chloride channels play essential roles in membrane excitability and maintenance of osmotic balance. Despite the recent crystallization of two bacterial ClC-like proteins, the gating mechanism for these channels remains unclear. In this study we tested scorpion venom for the presence of novel peptide inhibitors of ClC channels, which might be useful tools for dissecting the mechanisms underlying ClC channel gating. Recently, it has been shown that a peptide component of venom from the scorpion L. quinquestriatus hebraeus inhibits the CFTR chloride channel from the intracellular side. Using two-electrode voltage clamp we studied the effect of scorpion venom on ClC-0, -1, and -2, and found both dose- and voltage-dependent inhibition only of ClC-2. Comparison of voltage-dependence of inhibition by venom to that of known pore blockers revealed opposite voltage dependencies, suggesting different mechanisms of inhibition. Kinetic data show that venom induced slower activation kinetics compared to pre-venom records, suggesting that the active component(s) of venom may function as a gating modifier at ClC-2. Trypsinization abolished the inhibitory activity of venom, suggesting that the component(s) of scorpion venom that inhibits ClC-2 is a peptide.

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Abbreviations

9-AC:

anthracene-9-carboxylic acid

CF:

cystic fibrosis

CFTR:

cystic fibrosis transmembrane conductance regulator;

CITx:

chlorotoxin

CPB:

2-(p-chlorophenoxy) butyric acid

CPP:

2-(p- chlorophenoxy) proprionic acid

DIDS:

4′4′-Diisothiocyano-2,2′-Stilbene disulfonate

DMSO:

dimethyl sulfoxide;

DNDS:

4,4′-Nitrostilbene- 2,2′-disulfonate

DPC:

diphenylamine 2-carboxylic Acid

NPPB:

5-nitro-2-(3-phenylpropylamino) benzoic acid

EGTA:

ethylene glycol-bis(β-aminoethyl ether)- N,N,N′,N′-tetraacetic acid;

VSTx:

voltage sensing toxin.

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Acknowledgements

We thank Kate Hubbard for contributing to initial experiments. We thank Dr. Steve Harvey and Dr. Robert Lee for critically reading an early version of the manuscript. This work was supported by the National Institute of Health (NIH DK 56481 and DK 066409). During the performance of this work, N.A.M. was an Established Investigator of the American Heart Association.

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Authors and Affiliations

  1. School of Biology, Georgia Institute of Technology, 310 Ferst Drive, Atlanta, Georgia, 30332-0230, USA

    C.H. Thompson, D.M. Fields, P.R. Olivetti, M.D. Fuller, Z.R. Zhang, J. Kubanek & N.A. McCarty

  2. Program in Molecular and Systems Pharmacology, Emory University, 5001 Rollins Research Center, Atlanta, Georgia, 30322-3090, USA

    M.D. Fuller

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  1. C.H. Thompson
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  2. D.M. Fields
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  3. P.R. Olivetti
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Thompson, C., Fields, D., Olivetti, P. et al. Inhibition of ClC-2 Chloride Channels by a Peptide Component or Components of Scorpion Venom. J Membrane Biol 208, 65–76 (2005). https://doi.org/10.1007/s00232-005-0818-8

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  • DOI: https://doi.org/10.1007/s00232-005-0818-8

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