A Disulfide Tether Stabilizes The Block Of Sodium Channels By The Conotoxin Mu O-Gviij

A cone snail venom peptide, mu O -conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligand-binding site. mu O -conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJ(SSG)). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) Na(V)1.2. A mutant channel of rNa(V)1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10(3)-fold less sensitive to GVIIJ(SSG) than was wild-type rNa(V)1.2. In contrast, although rNa(V)1.5 was >10(4)-fold less sensitive to GVIIJ(SSG) than Na(V)1.2, an rNa(V)1.5 mutant with a cysteine in the homologous location, rNa(V)1.5[L869C], was >10(3)-fold more sensitive than wildtype rNa(V)1.5. The susceptibility of rNa(V)1.2 to GVIIJ(SSG) was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNa(V)beta 2 or rNa(V)beta 4, but not that of rNa(V)beta 1 or rNa(V)beta 3, protected rNa(V)1.1 to -1.7 (excluding Na(V)1.5) against block by GVIIJ(SSG). Thus, GVIIJ-related peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which Na-V beta- and Na-V alpha-subunits are present in native neurons.