title: Probing the Redox States of Sodium Channel Cysteines at the Binding Site of μO§-Conotoxin GVIIJ.
abstract: μO§-Conotoxin GVIIJ is a 35-amino acid peptide that readily blocks six of eight tested NaV1 subunit isoforms of voltage-gated sodium channels. μO§-GVIIJ is unusual in having an S-cysteinylated cysteine (at residue 24). A proposed reaction scheme involves the peptide-channel complex stabilized by a disulfide bond formed via thiol-disulfide exchange between Cys24 of the peptide and a Cys residue at neurotoxin receptor site 8 in the pore module of the channel (specifically, Cys910 of rat NaV1.2). To examine this model, we synthesized seven derivatives of μO§-GVIIJ in which Cys24 was disulfide-bonded to various thiols (or SR groups) and tested them on voltage-clamped Xenopus laevis oocytes expressing NaV1.2. In the proposed model, the SR moiety is a leaving group that is no longer present in the final peptide-channel complex; thus, the same koff value should be obtained regardless of the SR group. We observed that all seven derivatives, whose kon values varied over a 30-fold range, had the same koff value. Concordant results were observed with NaV1.6, for which the koff was 17-fold larger. Additionally, we tested two μO§-GVIIJ derivatives (where SR was glutathione or a free thiol) on two NaV1.2 Cys replacement mutants (NaV1.2[C912A] and NaV1.2[C918A]) without and with reduction of channel disulfides by dithiothreitol. The results indicate that Cys910 in wild-type NaV1.2 has a free thiol and conversely suggest that in NaV1.2[C912A] and NaV1.2[C918A], Cys910 is disulfide-bonded to Cys918 and Cys912, respectively. Redox states of extracellular cysteines of sodium channels have hitherto received scant attention, and further experiments with GVIIJ may help fill this void.
authors.affiliation: †Department of Biology, University of Utah, Salt Lake City, Utah 84112, United States.
authors.forename: Min-Min
authors.initials: MM
authors.lastname: Zhang
authors.affiliation: †Department of Biology, University of Utah, Salt Lake City, Utah 84112, United States.
authors.forename: Joanna
authors.initials: J
authors.lastname: Gajewiak
authors.affiliation: †Department of Biology, University of Utah, Salt Lake City, Utah 84112, United States.
authors.forename: Layla
authors.initials: L
authors.lastname: Azam
authors.affiliation: ‡Department of Medicinal Chemistry, College of Pharmacy, University of Utah, Salt Lake City, Utah 84108, United States.
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Bulaj
authors.affiliation: †Department of Biology, University of Utah, Salt Lake City, Utah 84112, United States.
authors.forename: Baldomero M
authors.initials: BM
authors.lastname: Olivera
authors.affiliation: †Department of Biology, University of Utah, Salt Lake City, Utah 84112, United States.
authors.forename: Doju
authors.initials: D
authors.lastname: Yoshikami
mesh_terms: D000818:Animals; D001665:Binding Sites; D020916:Conotoxins / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D062551:NAV1.2 Voltage-Gated Sodium Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009865:Oocytes; D010084:Oxidation-Reduction; D051381:Rats; D014982:Xenopus laevis
chemical_list: D020916:Conotoxins; D004220:Disulfides; D062551:NAV1.2 Voltage-Gated Sodium Channel; C568253:Scn2A protein, rat; D003545:Cysteine