disulfide tether stabilizes the block of sodium channels by the conotoxin μO§-GVIIJ

A cone snail venom peptide, μO§-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. μO§-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 V1.2. A mutant channel of rNa V1.2 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 V1.2. In contrast, although rNa V1.5 was >10 ⁴-fold less sensitive to GVIIJ SSG than Na V1.2, an rNa V1.5 mutant with a cysteine in the homologous location, rNa V1.5[L869C], was >10 ³-fold more sensitive than wild-type rNa V1.5. The susceptibility of rNa V1.2 to GVIIJ SSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNa Vβ2 or rNa Vβ4, but not that of rNa Vβ1 or rNa Vβ3, protected rNa V1.1 to -1.7 (excluding Na V1.5) 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 Vβ- and Na Vα-subunits are present in native neurons.