Coevolution of tetrodotoxin and voltage-gated sodium channels

河豚毒素和电压门控钠通道的共同进化

• 批准号: 342155-2012
• 负责人: Ruben, Peter
• 金额: $ 2.48万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=629591
• 关键词: Coevolution; tetrodotoxin; voltage; gated; sodium

The long-term goal of this research program is to understand the co-evolution of toxins and their targets. The model we use to understand the basis of toxin/target co-evolution is the interaction between tetrodotoxin (TTX) and the voltage-gated sodium channel (NaV). The interaction between these two entities is highly specific; NaV is the only known substrate for TTX. Although the physiological and biophysical consequences of TTX binding to NaVs are well documented, and although the NaV structures associated with TTX binding are understood, technical limitations have restricted the ability to manipulate the structure of TTX. We therefore have only a limited knowledge about the nature of the TTX/NaV interaction from the toxin perspective. Our published, NSERC-funded research shows that NaVs have independently evolved different structural mechanisms to reduce their sensitivity to TTX, although these adaptations have physiological consequences to the organisms in which they have evolved. This suggests a dynamic nature to the TTX/NaV interaction that may be reflected in structural changes on both sides of the relationship. Therefore, the aim of this phase of our research program is to focus on the structure of TTX. We will produce structural variants of TTX and test these variants for their ability to block the sodium current in different NaV isoforms, and to alter the gating current in these isoforms. To measure ion and gating currents, we will express sodium channels in Xenopus oocytes and record currents using the cut-open oocyte technique. The specific aims of the present proposal are: 1) produce TTX variants to test the role of each OH group, the guanidine segment, and urea groups in TTX; 2) study the concentration-dependence of ion current block of TTX variants in NaV1.4, NaV1.5, and tsNaV1.4; 3) study the concentration-dependence of shifts in the voltage-dependence of gating currents produced by the TTX variants in NaV1.4, NaV1.5, and tsNaV1.4. The significance of this phase of our program is that it will reveal the structural requirements of TTX for its effects on NaV and improve our understanding of how the structure of TTX may have co-evolved with sodium channels.

该研究计划的长期目标是了解毒素及其靶标的共同进化。我们用来理解毒素/靶标协同进化基础的模型是河豚毒素 (TTX) 和电压门控钠通道 (NaV) 之间的相互作用。这两个实体之间的相互作用是高度具体的； NaV 是 TTX 唯一已知的底物。尽管 TTX 与 NaV 结合的生理和生物物理后果已得到充分记录，并且尽管与 TTX 结合相关的 NaV 结构已被了解，但技术限制限制了操纵 TTX 结构的能力。因此，从毒素的角度来看，我们对 TTX/NaV 相互作用的性质了解有限。我们发表的、由 NSERC 资助的研究表明，NaV 独立进化了不同的结构机制，以降低它们对 TTX 的敏感性，尽管这些适应性对其进化的生物体产生生理影响。这表明 TTX/NaV 相互作用具有动态性质，这可能反映在关系双方的结构变化中。因此，我们这一阶段的研究目标是关注TTX的结构。我们将生产 TTX 的结构变体，并测试这些变体阻断不同 NaV 异构体中钠电流的能力，以及改变这些异构体中门控电流的能力。为了测量离子和门控电流，我们将在非洲爪蟾卵母细胞中表达钠通道，并使用切开卵母细胞技术记录电流。本提案的具体目标是：1）产生TTX变体以测试TTX中每个OH基团、胍片段和脲基团的作用； 2) 研究 NaV1.4、NaV1.5 和 tsNaV1.4 中 TTX 变体的离子电流阻断的浓度依赖性； 3) 研究 NaV1.4、NaV1.5 和 tsNaV1.4 中 TTX 变体产生的门控电流电压依赖性变化的浓度依赖性。我们项目这一阶段的意义在于，它将揭示 TTX 对 NaV 影响的结构要求，并提高我们对 TTX 结构如何与钠通道共同进化的理解。