The Role of Individual Disulfide Bonds of μ-Conotoxin GIIIA in the Inhibition of NaV1.4.

Penggang Han,Chongxu Fan,Shangyi Liu,Jisheng Chen,Hui Jiang,Xiandong Dai,Kang Wang,Ying Cao,Wenjian Wu

doi:10.3390/md14110213

Abstract

μ-Conotoxin GIIIA, a peptide toxin isolated from Conus geographus, preferentially blocks the skeletal muscle sodium channel NaV1.4. GIIIA folds compactly to a pyramidal structure stabilized by three disulfide bonds. To assess the contributions of individual disulfide bonds of GIIIA to the blockade of NaV1.4, seven disulfide-deficient analogues were prepared and characterized, each with one, two, or three pairs of disulfide-bonded Cys residues replaced with Ala. The inhibitory potency of the analogues against NaV1.4 was assayed by whole cell patch-clamp on rNaV1.4, heterologously expressed in HEK293 cells. The corresponding IC50 values were 0.069 ± 0.005 μM for GIIIA, 2.1 ± 0.3 μM for GIIIA-1, 3.3 ± 0.2 μM for GIIIA-2, and 15.8 ± 0.8 μM for GIIIA-3 (-1, -2 and -3 represent the removal of disulfide bridges Cys3–Cys15, Cys4–Cys20 and Cys10–Cys21, respectively). Other analogues were not active enough for IC50 measurement. Our results indicate that all three disulfide bonds of GIIIA are required to produce effective inhibition of NaV1.4, and the removal of any one significantly lowers its sodium channel binding affinity. Cys10–Cys21 is the most important for the NaV1.4 potency.

Highlights

Conotoxins are small, cysteine-rich bioactive peptides derived from the venom of tropical marine cone snails, genus Conus
GIIIA preferentially blocks skeletal muscle sodium channel subtype NaV 1.4 by a rather complex toxin–pore interaction, with several residues in the toxin contributing to the high-affinity binding [8]
This research investigated the role of individual disulfide bonds of GIIIA in the inhibition of

Summary

Introduction

Conotoxins are small, cysteine-rich bioactive peptides derived from the venom of tropical marine cone snails, genus Conus. Conotoxins have a wide range of pharmacological targets, including different isoforms of ion channels and membrane receptors [1]. Μ-Conotoxins belong to the M superfamily of conopeptides and comprise 16 to 26 residues with six. Μ-Conotoxins are the only venom peptides that selectively inhibit voltage-gated sodium channels by sterically and electrostatically blocking the ion flux via binding to the outer vestibule of the channel [6]. The μ-conotoxin GIIIA isolated from Conus geographus was among the first to be characterized. GIIIA preferentially blocks skeletal muscle sodium channel subtype NaV 1.4 by a rather complex toxin–pore interaction, with several residues in the toxin contributing to the high-affinity binding [8]

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Conclusion