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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 111; no. 7; pp. 2758 - 2763
• Main Authors: Gajewiak, Joanna, Azam, Layla, Imperial, Julita, Walewska, Aleksandra, Green, Brad R., Bandyopadhyay, Pradip K., Raghuraman, Shrinivasan, Ueberheide, Beatrix, Bern, Marshall, Zhou, H. Mimi, Minassian, Natali A., Hagan, Rebecca H., Flinspach, Mack, Yi Liu, Bulaj, Grzegorz, Wickenden, Alan D., Olivera, Baldomero M., Yoshikami, Doju, Zhang, Min-Min
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 18.02.2014; National Acad Sciences
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Binding sites; Biological Sciences; Chromatography, High Pressure Liquid; Conotoxins - genetics; Conotoxins - metabolism; Conotoxins - toxicity; crosslinking; cysteine; Cysteine - metabolism; Disulfides; Disulfides - metabolism; DNA Primers - genetics; DNA, Complementary - genetics; Electric potential; Molecular Sequence Data; mutants; NAV1.2 Voltage-Gated Sodium Channel - metabolism; Neurons; Neurons - metabolism; Oocytes; Oocytes - metabolism; Oxidation; Patch-Clamp Techniques; peptides; Pharmacology; post-translational modification; Rats; RNA; Sequence Analysis, DNA; snails; sodium; Sodium channels; Tandem Mass Spectrometry; Thiols; Toxins; venoms; Voltage-Gated Sodium Channel Blockers - metabolism; Voltage-Gated Sodium Channel Blockers - toxicity; tethered toxin; NaVβ-subunit; S-cysteinylation; voltage-gated sodium channel; disulfide exchange
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1324189111

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.