Evolution of maurotoxin conformation and blocking efficacy towards Shaker B channels during the course of folding and oxidation in vitro.
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.
di Luccio
Eric
E
CNRS UMR 6560, Boulevard Pierre Dramard, 13916 Marseille Cedex 20, France.
Matavel
Alessandra
A
Opi
Sandrine
S
Regaya
Imed
I
Sandoz
Guillaume
G
M'barek
Sarrah
S
Carlier
Edmond
E
Estève
Eric
E
Carrega
Louis
L
Fajloun
Ziad
Z
Rochat
Hervé
H
Loret
Erwann
E
de Waard
Michel
M
Sabatier
Jean-Marc
JM
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
D004220:Disulfides; D015221:Potassium Channels; D012604:Scorpion Venoms; C104498:maurotoxin