Leipold Enrico, DeBie Herbert, Zorn Stefan, Borges Adolfo, Olivera Baldomero M, Terlau Heinrich, Heinemann Stefan H
Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University, Jena, Germany.
Channels (Austin). 2007 Jul-Aug;1(4):253-62. doi: 10.4161/chan.4847. Epub 2007 Aug 7.
The muO-conotoxins MrVIA and MrVIB are 31-residue peptides from Conus marmoreus, belonging to the O-superfamily of conotoxins with three disulfide bridges. They have attracted attention because they are inhibitors of tetrodotoxin-insensitive voltage-gated sodium channels (Na(V)1.8) and could therefore serve as lead structure for novel analgesics. The aim of this study was to elucidate the molecular mechanism by which muO-conotoxins affect Na(V) channels. Rat Na(V)1.4 channels and mutants thereof were expressed in mammalian cells and were assayed with the whole-cell patch-clamp method. Unlike for the M-superfamily mu-conotoxin GIIIA from Conus geographus, channel block by MrVIA was strongly diminished after activating the Na(V) channels by depolarizing voltage steps. Searching for the source of this voltage dependence, the gating charges in all four-voltage sensors were reduced by site-directed mutagenesis showing that alterations of the voltage sensor in domain-2 have the strongest impact on MrVIA action. These results, together with previous findings that the effect of MrVIA depends on the structure of the pore-loop in domain-3, suggest a functional similarity with scorpion beta-toxins. In fact, MrVIA functionally competed with the scorpion beta-toxin Ts1 from Tityus serrulatus, while it did not show competition with mu-GIIIA. Ts1 and mu-GIIIA did not compete either. Thus, similar to scorpion beta-toxins, muO-conotoxins are voltage-sensor toxins targeting receptor site-4 on Na(V) channels. They "block" Na(+) flow most likely by hindering the voltage sensor in domain-2 from activating and, hence, the channel from opening.
μO-芋螺毒素MrVIA和MrVIB是来自大理石芋螺的31个氨基酸残基的肽，属于具有三个二硫键的芋螺毒素O超家族。它们引起了人们的关注，因为它们是河豚毒素不敏感的电压门控钠通道（Na(V)1.8）的抑制剂，因此可以作为新型镇痛药的先导结构。本研究的目的是阐明μO-芋螺毒素影响Na(V)通道的分子机制。大鼠Na(V)1.4通道及其突变体在哺乳动物细胞中表达，并用全细胞膜片钳方法进行检测。与来自地纹芋螺的M超家族μ-芋螺毒素GIIIA不同，通过去极化电压阶跃激活Na(V)通道后，MrVIA对通道的阻断作用大大减弱。通过定点诱变降低所有四个电压传感器中的门控电荷，以寻找这种电压依赖性的来源，结果表明结构域2中电压传感器的改变对MrVIA的作用影响最大。这些结果，连同先前的发现，即MrVIA的作用取决于结构域3中孔环的结构，表明其与蝎β-毒素具有功能相似性。事实上，MrVIA在功能上与来自锯齿蝎的蝎β-毒素Ts1竞争，而它与μ-GIIIA没有竞争。Ts1和μ-GIIIA也不相互竞争。因此，与蝎β-毒素类似，μO-芋螺毒素是靶向Na(V)通道上受体位点4的电压传感器毒素。它们“阻断”Na(+)流动很可能是通过阻碍结构域2中的电压传感器激活，从而阻碍通道开放。
