The Effects Of Disulfide Bonds On The Structure And Toxicity Of The Voltage-gated Sodium Channel Inhibitor Conotoxin μ-GⅢA

Voltage-gated sodium channels(VGSCs) are multi-molecular transmembrane protein complexes expressed in excitable cells such as nerve and muscle. VGSCs play an essential role in the initiation and propagation of action potentials in these cells. They are composed of an α-subunit that is associated with one or more regulatory β-subunits(β1-β4). Nine different mammalian α-subunit isoforms(Nav1.1-Nav1.9) have been characterized and each of them has distinct tissue distribution and biophysical properties. Conotoxins isolated from the venom of predatory marine cone snails have a diverse range of pharmacological targets, including membrane ion channels like VGSCs. As small-size, disul?de-rich, high bioactivity and rapid effect, conotoxins are important in the research of VGSCs.This research chooses μ-GⅢA for the study. μ-GⅢA isolated from the vendom of C. Geographus was the first μ-conotoxin that have been characterised. GⅢA consists of 22-residue peptide amides with three hydroxyproline residues and three disulfide bonds(Cys3-Cys15,Cys4-Cys20,Cys10-Cys21). It has been established that GIIIA has strong selectivity for the muscle subtype Nav1.4 by interacting with the site1 and blocking the pore. The three disulfide bonds maybe important in the peptide structure and toxicity of GⅢA. The main purpose of the research is to evaluate the three disulfide bonds in the peptide structure and toxicity. Natural GⅢA and seven derivative peptides of it which contain two, one or none disulfide bond(GⅢA-1,GⅢA-2,GⅢA-3,GⅢA-1,2,GⅢA-2,3,GⅢA-1,2,3)were synthesised by solid phase peptide synthesis(SPPS) and disulfide bond oxidations. Then the high structure of these peptides were scaned by circular dichroism(CD) to analyze the effect of disulfide bonds on the peptide structure. And acute toxicity experiments of mice were made respectively to assess their toxicity. The results show that all the three disulfide bonds are important in the toxicity of GⅢA, the absense of any one will cause large decrease in toxicity. Relatively, the importance of three disulfide bonds can be ranked as: Cys10-Cys21> Cys4-Cys20> Cys3-Cys15. The research provide the basis to develop new VGSC-targeted peptides with simplier structure and higher bioactivity by structure modification.