title: Evolution of maurotoxin conformation and blocking efficacy towards Shaker B channels during the course of folding and oxidation in vitro.
abstract: Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K(+) channels, including the voltage-gated Shaker B subtype. In the present study, we have investigated over 80 h: (1) the time-course of folding of synthetic MTX (sMTX) by CD analysis; (2) the kinetics of disulphide bridge formation by MS; and (3) the potency of MTX in blocking Shaker B currents during the combined process of its in vitro folding and oxidation. From the CD data, we show that stable secondary structures of sMTX evolve sequentially over time, with the appearance of the alpha-helix within 5 h, followed by the formation of the beta-sheet within 22 h. Using MS analysis, the sMTX intermediates were also found to appear sequentially from the least (one-disulphide-bridged sMTX) to the most oxidized species (native-like, four-disulphide-bridged sMTX). The time course of formation of secondary structures coincides mainly with the occurrence of one-disulphide-bridged sMTX for the alpha-helix and two- or three-disulphide-bridged sMTX for the beta-sheet. On-line electrophysiological recordings, which measure sMTX blocking efficacy on K(+) currents during its folding and oxidation, were performed on Shaker B channels expressed in Xenopus oocytes. Unexpectedly, the results demonstrate that sMTX is highly potent at the initial stage of oxidation, whereas its blocking activity can be transiently and dramatically reduced at later stages during the course of folding/oxidation before it reaches full bioactivity. These data suggest that formation of disulphide bridges can both physically stabilize and alter the bioactive three-dimensional structure of sMTX.
authors.affiliation: CNRS UMR 6560, Boulevard Pierre Dramard, 13916 Marseille Cedex 20, France.
authors.forename: Eric
authors.initials: E
authors.lastname: di Luccio
authors.forename: Alessandra
authors.initials: A
authors.lastname: Matavel
authors.forename: Sandrine
authors.initials: S
authors.lastname: Opi
authors.forename: Imed
authors.initials: I
authors.lastname: Regaya
authors.forename: Guillaume
authors.initials: G
authors.lastname: Sandoz
authors.forename: Sarrah
authors.initials: S
authors.lastname: M'barek
authors.forename: Edmond
authors.initials: E
authors.lastname: Carlier
authors.forename: Eric
authors.initials: E
authors.lastname: Estève
authors.forename: Louis
authors.initials: L
authors.lastname: Carrega
authors.forename: Ziad
authors.initials: Z
authors.lastname: Fajloun
authors.forename: Hervé
authors.initials: H
authors.lastname: Rochat
authors.forename: Erwann
authors.initials: E
authors.lastname: Loret
authors.forename: Michel
authors.initials: M
authors.lastname: de Waard
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Sabatier
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D010084:Oxidation-Reduction; D015221:Potassium Channels / Q000187:drug effects*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012604:Scorpion Venoms / Q000737:chemistry* / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D014982:Xenopus laevis
chemical_list: D004220:Disulfides; D015221:Potassium Channels; D012604:Scorpion Venoms; C104498:maurotoxin