Functional Expression of an Arachnid Sodium Channel Reveals Residues Responsible for Tetrodotoxin Resistance in Invertebrate Sodium Channels
Functional Expression of an Arachnid Sodium Channel Reveals Residues Responsible for Tetrodotoxin Resistance in Invertebrate Sodium Channels
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DOI：
10.1074/jbc.m109.045690
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发表时间：
2009-12-04
影响因子：
4.8
通讯作者：
Dong, Ke
中科院分区：
文献类型：
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作者：
Du, Yuzhe;Nomura, Yoshiko;Dong, Ke
河豚毒素 (TTX) 是电压门控钠通道的有效阻断剂，但并非所有钠通道对 TTX 的抑制作用都同样敏感。哺乳动物钠通道 TTX 敏感性差异的分子基础已基本阐明。相比之下，我们对无脊椎动物钠通道对 TTX 敏感性的了解仍然很少，部分原因是这些通道的功能表达的成功有限。在这项研究中，我们报告了非洲爪蟾卵母细胞中第一个非昆虫、无脊椎动物电压门控钠通道的功能特征，该钠通道来自瓦螨（瓦螨，一种蜜蜂的体外寄生虫）。该蜘蛛钠通道快速激活和失活，在 -18 mV 时半最大激活，在 -29 mV 时半最大快速失活。有趣的是，这种蜘蛛通道表现出令人惊讶的 TTX 抗性。 TTX 以 1 mu M 的 IC50 阻断该通道。随后的定点诱变揭示了分别位于结构域 III 和 IV 的孔形成区域中的两个残基 Thr-1674 和 Ser-1967，这两个残基是观察到的对 TTX 抑制的抗性的原因。此外，序列比较和额外的氨基酸取代表明，这两个位置的序列多态性可能是调节不同无脊椎动物钠通道 TTX 敏感性的广泛机制。
Tetrodotoxin (TTX) is a potent blocker of voltage-gated sodium channels, but not all sodium channels are equally sensitive to inhibition by TTX. The molecular basis of differential TTX sensitivity of mammalian sodium channels has been largely elucidated. In contrast, our knowledge about the sensitivity of invertebrate sodium channels to TTX remains poor, in part because of limited success in functional expression of these channels. In this study, we report the functional characterization in Xenopus oocytes of the first non-insect, invertebrate voltage-gated sodium channel from the varroa mite (Varroa destructor), an ecto-parasite of the honeybee. This arachnid sodium channel activates and inactivates rapidly with half-maximal activation at -18 mV and half-maximal fast inactivation at -29 mV. Interestingly, this arachnid channel showed surprising TTX resistance. TTX blocked this channel with an IC50 of 1 mu M. Subsequent site-directed mutagenesis revealed two residues, Thr-1674 and Ser-1967, in the pore-forming region of domains III and IV, respectively, which were responsible for the observed resistance to inhibition by TTX. Furthermore, sequence comparison and additional amino acid substitutions suggested that sequence polymorphisms at these two positions could be a widespread mechanism for modulating TTX sensitivity of sodium channels in diverse invertebrates.