Coevolution of tetrodotoxin and voltage-gated sodium channels

这项研究计划的长期目标是了解毒素及其目标的共同进化。我们用来理解毒素/靶标协同进化的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特异性的; 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被充分记录，并且尽管与TTX结合相关的NaV结构已被理解，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相互作用的性质只有有限的了解。我们发表的NSERC资助的研究表明，NaVs已经独立地进化出不同的结构机制来降低它们对TTX的敏感性，尽管这些适应对它们进化的生物体具有生理后果。这表明TTX/NaV相互作用的动态性质，可能反映在关系两侧的结构变化中。因此，我们研究计划的这一阶段的目的是关注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亚型中钠电流的能力，以及改变这些亚型中门控电流的能力。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。本发明的具体目的是：1）制备TTX变体以测试TTX中每个OH基团、胍链段和脲基团的作用：2）研究TTX变体在NaV 1.4、NaV 1.5和tsNaV 1.4中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偏移的浓度依赖性。我们计划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。

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.