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首页> 外文学位 >Molecular interactions between sodium channels and a novel neurotoxin, ProTx-II: Implications for the structure and function of voltage-gated sodium channels.
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Molecular interactions between sodium channels and a novel neurotoxin, ProTx-II: Implications for the structure and function of voltage-gated sodium channels.

机译：钠通道与新型神经毒素ProTx-II之间的分子相互作用：对电压门控钠通道的结构和功能的影响。

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摘要

Spider venoms are a rich source of highly specific peptide ligands that bind to voltage-gated calcium, potassium and sodium channels. These toxins have been used to help elucidate the structure and physiological roles of these channels and can potentially be useful models for drug design. ProTx-II, a peptide toxin originally purified from the venom of the tarantula Thrixopelma pruriens, reversibly inhibits Nav channel isoforms (Middleton et al., Biochemistry, 2002). Unlike other toxins that inhibit Nav channel function by occluding the pore, ProTx-II is thought to interact with the voltage sensor region of the channel to modify activation gating. Upon toxin binding, the voltage-dependence of activation is shifted toward more depolarized potentials and current is reduced. ProTx-II is also unique in that it demonstrates promiscuity across voltage-gated ion channels by inhibiting T-type Cav channels, suggesting a conserved structural motif among Nav and Cav channels. ProTx-II conforms to the inhibitory cystine knot (ICK) motif described previously for some toxins interacting with voltage-gated ion channels. This structural motif consists of a ring formed by two disulfide bonds that is threaded by a third disulfide bond. We have cloned a synthetic ProTx-II gene and developed a recombinant expression system that allows us to produce properly folded peptide toxin. Characterization by MALDI-TOF and circular dichroism indicates that the toxin has the correct disulfide pairings. We have confirmed the activity of recombinant ProTx-II by testing its function on HEK cells stably transfected with Na v1.5 channels via a voltage-clamp assay.; Based upon studies using gating modifier toxins and artificial phospholipid membranes, it has been proposed that toxins exhibiting high affinity inhibition for voltage-gated ion channels bind their receptor sites by partitioning into the lipid membrane (Lee, SY & MacKinnon, R, Nature, 2004; Wang et al., JGP, 2004). This theory has important implications for the structure of the voltage sensors and the mechanism of channel activation and while it has been proposed for Kv channels and their ligands, the model should be consistent throughout the superfamily of ion channels. Therefore, to test this hypothesis for a Nav channel ligand, I have analyzed the phospholipid binding capabilities of ProTx-II and compared these results to the phospholipid binding properties of other functionally distinct gating modifier toxins. In addition, I have analyzed the interaction between Nav1.5 and single alanine mutants created at every non-cysteine position of ProTx-II to identify residues important in binding. Furthermore, in order to determine the channel binding site I created 72 Nav1.5 mutants and analyzed the interactions between channel mutants and wild-type ProTx-II.; Toxin-lipid interaction studies identified ProTx-II as a gating modifier toxin with an affinity for phospholipids and the alanine scan of ProTx-II identified several hydrophobic and cationic residues important in modification of Nav1.5. These findings are consistent with a membrane-access mechanism of inhibition but also implicate anionic channel residues in formation of the toxin-channel complex. Our channel mutagenesis established ProTx-II as a novel neurotoxin acting at an unknown receptor site but additional studies are necessary in order to fully define this site.
机译：蜘蛛毒液是与电压门控的钙，钾和钠通道结合的高度特异性肽配体的丰富来源。这些毒素已用于帮助阐明这些通道的结构和生理作用，并且可能是药物设计的有用模型。 ProTx-II是一种最初从狼蛛Thrixopelma pruriens毒液中纯化的肽毒素，可逆地抑制Nav通道亚型（Middleton等人，Biochemistry，2002）。与其他毒素通过闭塞孔抑制Nav通道功能的毒素不同，ProTx-II被认为与通道的电压传感器区域相互作用以修饰激活门控。毒素结合后，激活的电压依赖性将移向更多的去极化电势，电流减小。 ProTx-II的独特之处还在于，它通过抑制T型Cav通道展示了跨电压门控离子通道的混杂，暗示了Nav和Cav通道之间的结构保守。 ProTx-II符合先前针对某些与电压门控离子通道相互作用的毒素所描述的抑制性胱氨酸结（ICK）主题。该结构基序由由两个二硫键形成的环组成，该环被第三个二硫键穿过。我们已经克隆了一个合成的ProTx-II基因，并开发了一个重组表达系统，该系统可使我们产生适当折叠的肽毒素。通过MALDI-TOF和圆二色性进行表征表明该毒素具有正确的二硫键配对。我们已经通过电压钳测定法测试了其在稳定转染Na v1.5通道的HEK细胞上的功能，从而证实了重组ProTx-II的活性。基于使用门控修饰剂毒素和人工磷脂膜的研究，已提出对电压门控离子通道表现出高亲和力抑制作用的毒素通过分配进入脂质膜结合其受体位点（Lee，SY＆MacKinnon，R，Nature，2004）。 ； Wang等人，JGP，2004）。该理论对电压传感器的结构和通道激活机制具有重要意义，尽管已针对Kv通道及其配体提出了该理论，但该模型在整个离子通道超家族中应保持一致。因此，为了测试Nav通道配体的这一假设，我分析了ProTx-II的磷脂结合能力，并将这些结果与其他功能独特的门控修饰剂毒素的磷脂结合特性进行了比较。此外，我分析了Nav1.5与ProTx-II的每个非半胱氨酸位置处产生的单个丙氨酸突变体之间的相互作用，以鉴定对结合重要的残基。此外，为了确定通道结合位点，我创建了72个Nav1.5突变体，并分析了通道突变体与野生型ProTx-II之间的相互作用。毒素-脂质相互作用研究确定ProTx-II为对磷脂具有亲和力的门控修饰剂毒素，ProTx-II的丙氨酸扫描确定了对Nav1.5修饰很重要的几个疏水和阳离子残基。这些发现与抑制作用的膜进入机制一致，但也暗示了毒素通道复合物形成过程中的阴离子通道残基。我们的通道诱变将ProTx-II建立为一种作用于未知受体位点的新型神经毒素，但为了完全定义该位点，还需要进行其他研究。

著录项

  • 作者

    Smith, Jaime J.;

  • 作者单位

    State University of New York at Buffalo.$bBiochemistry.;

  • 授予单位 State University of New York at Buffalo.$bBiochemistry.;
  • 学科 Chemistry Biochemistry.; Biophysics General.
  • 学位 Ph.D.
  • 年度 2007
  • 页码 210 p.
  • 总页数 210
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;生物物理学;
  • 关键词

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