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A disulfide tether stabilizes the block of sodium channels by the conotoxin muO[section sign]-GVIIJ.

Gajewiak J., Azam L., Imperial J., Walewska A., Green B.R., Bandyopadhyay P.K., Raghuraman S., Ueberheide B., Bern M., Zhou H.M., Minassian N.A., Hagan R.H., Flinspach M., Liu Y., Bulaj G., Wickenden A.D., Olivera B.M., Yoshikami D., Zhang M.M.

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 GVIIJSSG). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) NaV1.2. A mutant channel of rNaV1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10(3)-fold less sensitive to GVIIJSSG than was wild-type rNaV1.2. In contrast, although rNaV1.5 was >10(4)-fold less sensitive to GVIIJSSG than NaV1.2, an rNaV1.5 mutant with a cysteine in the homologous location, rNaV1.5[L869C], was >10(3)-fold more sensitive than wild-type rNaV1.5. The susceptibility of rNaV1.2 to GVIIJSSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNaVβ2 or rNaVβ4, but not that of rNaVβ1 or rNaVβ3, protected rNaV1.1 to -1.7 (excluding NaV1.5) against block by GVIIJSSG. Thus, GVIIJ-related peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which NaVβ- and NaVα-subunits are present in native neurons.

Proc. Natl. Acad. Sci. U.S.A. 111:2758-2763(2014) [PubMed] [Europe PMC]