Referenced in: none

Automatically associated channels: Nav1.2 , Nav1.6

Title: Probing the Redox States of Sodium Channel Cysteines at the Binding Site of μO§-Conotoxin GVIIJ.

Authors: Min-Min Zhang, Joanna Gajewiak, Layla Azam, Grzegorz Bulaj, Baldomero M Olivera, Doju Yoshikami

Journal, date & volume: Biochemistry, 2015 Jun 30 , 54, 3911-20

PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/26039939

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.