Probing the Redox States of Sodium Channel Cysteines at the Binding Site of 渭O搂-Conotoxin GVIIJ

详细信息    查看全文

• 作者：Min-Min Zhang ; Joanna Gajewiak ; Layla Azam ; Grzegorz Bulaj ; Baldomero M. Olivera ; Doju Yoshikami
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：June 30, 2015
• 年：2015
• 卷：54
• 期：25
• 页码：3911-3920
• 全文大小：436K
• ISSN：1520-4995

文摘

渭O搂-Conotoxin GVIIJ is a 35-amino acid peptide that readily blocks six of eight tested Na_V1 subunit isoforms of voltage-gated sodium channels. 渭O搂-GVIIJ is unusual in having an S-cysteinylated cysteine (at residue 24). A proposed reaction scheme involves the peptide鈥揷hannel complex stabilized by a disulfide bond formed via thiol鈥揹isulfide 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 Na_V1.2). To examine this model, we synthesized seven derivatives of 渭O搂-GVIIJ in which Cys24 was disulfide-bonded to various thiols (or SR groups) and tested them on voltage-clamped Xenopus laevis oocytes expressing Na_V1.2. In the proposed model, the SR moiety is a leaving group that is no longer present in the final peptide鈥揷hannel complex; thus, the same k_off value should be obtained regardless of the SR group. We observed that all seven derivatives, whose k_on values varied over a 30-fold range, had the same k_off value. Concordant results were observed with Na_V1.6, for which the k_off was 17-fold larger. Additionally, we tested two 渭O搂-GVIIJ derivatives (where SR was glutathione or a free thiol) on two Na_V1.2 Cys replacement mutants (Na_V1.2[C912A] and Na_V1.2[C918A]) without and with reduction of channel disulfides by dithiothreitol. The results indicate that Cys910 in wild-type Na_V1.2 has a free thiol and conversely suggest that in Na_V1.2[C912A] and Na_V1.2[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.