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Thermal and chemical unfolding and refolding of a eukaryotic sodium channel

Charalambous, K., O'Reilly, A.O., Bullough, P.A. and Wallace, B.A. (2009) Thermal and chemical unfolding and refolding of a eukaryotic sodium channel. Biochimica et Biophysica Acta - Biomembranes, 1788 (6). pp. 1279-1286. ISSN 0005-2736


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Voltage-gated sodium channels are dynamic membrane proteins essential for signaling in nervous and muscular systems. They undergo substantial conformational changes associated with the closed, open and inactivated states. However, little information is available regarding their conformational stability. In this study circular dichroism spectroscopy was used to investigate the changes in secondary structure accompanying chemical and thermal denaturation of detergent-solubilised sodium channels isolated from Electrophorus electricus electroplax. The proteins appear to be remarkably resistant to either type of treatment, with "denatured" channels, retaining significant helical secondary structure even at 77 degrees C or in 10% SDS. Further retention of helical secondary structure at high temperature was observed in the presence of the channel-blocking tetrodotoxin. It was possible to refold the thermally-denatured (but not chemically-denatured) channels in vitro. The correctly refolded channels were capable of undergoing the toxin-induced conformational change indicative of ligand binding. In addition, flux measurements in liposomes showed that the thermally-denatured (but not chemically-denatured) proteins were able to re-adopt native, active conformations. These studies suggest that whilst sodium channels must be sufficiently flexible to undergo major conformational changes during their functional cycle, the proteins are highly resistant to unfolding, a feature that is important for maintaining structural integrity during dynamic processes. (c) 2009 Elsevier B.V. All rights reserved.

Item Type: Article
Copyright, Publisher and Additional Information: © 2009 Elsevier. This is an author produced version of a paper subsequently published in Biochimica et Biophysica Acta-Biomembranes. Uploaded in accordance with the publisher's self-archiving policy.
Keywords: Voltage-gated sodium channel; Protein folding; Membrane protein; Secondary structure; Circular dichroism spectroscopy; Toxin binding
Institution: The University of Sheffield
Academic Units: The University of Sheffield > University of Sheffield Research Centres and Institutes > The Krebs Institute for Biomolecular Research (Sheffield)
The University of Sheffield > Faculty of Science (Sheffield) > School of Biological Sciences (Sheffield) > Department of Molecular Biology and Biotechnology (Sheffield)
Depositing User: Miss Anthea Tucker
Date Deposited: 06 Jul 2009 16:10
Last Modified: 08 Feb 2013 16:58
Published Version: http://dx.doi.org/10.1016/j.bbamem.2009.02.005
Status: Published
Publisher: Elsevier
Refereed: Yes
Identification Number: 10.1016/j.bbamem.2009.02.005
URI: http://eprints.whiterose.ac.uk/id/eprint/8751

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