Articles Effects of Deglycosylation of Sodium Channels on Their Structure and Function ² Nora B. Cronin, ‡ Andrias O’Reilly, ‡ Herve ´ Duclohier, §,| and B. A. Wallace* ,‡,⊥ Department of Crystallography, Birkbeck College, UniVersity of London, London WC1E 7HX, U.K., BBSRC Centre for Protein and Membrane Structure and Dynamics, CCLRC Daresbury Laboratory, Warrington WA4 4AD, U.K., and UMR 6026 CNRS-UniVersite ´ de Rennes I, Laboratoire des Interactions Cellulaires et Mole ´ culaires, Campus de Beaulieu, Ba ˆ timent 13, 35042 Rennes Cedex, France ReceiVed June 17, 2004; ReVised Manuscript ReceiVed October 15, 2004 ABSTRACT: Voltage-gated sodium channels are important membrane proteins underlying electrical signaling in the nervous and muscular systems. They undergo rapid conformational changes between closed resting, activated, and inactivated states. Approximately 30% of the mass of the sodium channel is carbohydrate, present as glycoconjugate chains, mostly composed of N-acetylhexosamines and sialic acid. In this study, the effects of removing the carbohydrate on the functional and structural properties of highly purified sodium channels from Electrophorus electricus were investigated. After enzymatic deglycosylation, channels were reconstituted into planar lipid bilayers. In the presence of batrachotoxin, substates became evident and the single-channel conductance of the deglycosylated channels was slightly reduced relative to that of native channels, consistent with electrostatic effects due to the reduction in negative charge at the extracellular vestibule of the channel. The previously reported state-dependent changes in the circular dichroism spectra that are associated with the binding of the anticonvulsant drug Lamotrigine and batrachotoxin are also seen in the modified channels. Synchrotron radiation circular dichroism (SRCD) spectroscopy on the type of sugars found in the sodium channel showed that unlike most carbohydrates, these sugars produce a significant dichroic signal in the far- ultraviolet region. This can account for all of the measured SRCD-detected spectral differences between the native and deglycosylated channels, thereby indicating that no net change in protein secondary structure results from the deglycosylation procedure. Furthermore, thermal denaturation studies detected no significant differences in stability between native and deglycosylated channels. In summary, while the sugars of the voltage-gated sodium channels from electroplax are not essential for functional or structural integrity, they do appear to have a modulating effect on the conductance properties of these channels. Voltage-gated sodium channels underlie action potential initiation and propagation in nerves and muscles and are also involved in various pathophysiologies and channelopathies due to inherited mutations (1). Their functions, whether in situ or after heterologous expression and reconstitution into ² This work was funded by Project Grant B15499 from the BBSRC (to B.A.W.), a seed grant from the CNRS and the Royal Society (to H.D. and B.A.W.), an international cooperation grant from the Wellcome Trust (to B.A.W. and H.D.), and an equipment grant from the CNRS (to H.D.). The circular dichroism instrumentation was supported, in part, by Grant B14225 from the BBSRC (to B.A.W.). The SRCD beam time was provided by a grant from the CCLRC (to B.A.W.). A.O’R. was supported by the H. W. Fletcher Studentship from the British Heart Foundation. * To whom correspondence should be addressed. Phone: +44-207- 631-6857. Fax: +44-207-631-6803. E-mail: ubcg25a@ mail.cryst.bbk.ac.uk. ‡ University of London. § UMR 6026 CNRS-Universite ´ de Rennes I. | Present address: Institut de Physiologie et de Biologie Cellulaires, UMR 6187 CNRS-Universite ´ de Poitiers, 86022 Poitiers Cedex, France. ⊥ CCLRC Daresbury Laboratory. © Copyright 2005 by the American Chemical Society Volume 44, Number 2 January 18, 2005 10.1021/bi048741q CCC: $30.25 © 2005 American Chemical Society Published on Web 12/16/2004