中国科学院昆明动物研究所机构知识库

电压门控钠通道控制动作电位的起始和传导，是细胞兴奋性的重要分子基础。因此，它们与疼痛、癫痫、心率失常等临床疾病息息相关，也成为了许多生物毒素的直接作用靶点。对应于膜电位的变化，钠通道总是处于静息、激活、失活三种状态的循环中。快失活是钠通道从激活态到失活态的过程，其时程仅为几个毫秒，有多个结构元件参与其中，是一复杂的门控过程。由于时程极短，快失活发生的结构基础和变构过程尚不十分清楚。钠通道的快失活异常会导致一些严重的疾病如家族性周期瘫痪综合征（高血钾型）、先天性肌强直等。本论文第一章首先从组成形式、生理功能、工作机制、门控特性、三维结构等方面对钠通道近几十年的研究进展做了简要介绍。这些工作是我们认识钠通道并就感兴趣的问题展开思考的重要基础。其次，对东亚钳蝎及其毒素系列工作的总结，表明蝎毒是富含离子通道调控物质的优质资源库，其中一些活性成分已成为生物药物开发的先导分子。随着东亚钳蝎基因组测序工作的完成，更方便了毒液成分的研究及新药前体分子的筛选。因此我们采用东亚钳蝎作为研究材料，在分析其毒素分子多样性的同时寻找一个钠通道毒素探针，以研究钠通道的相关门控过程。在第二章中，通过对东亚钳蝎毒液的解析，我们发现了一个能高效调控钠通道快失活的α毒素BmK-M9。使用原核表达的方法解决了进行各类实验所需样品量的问题。两个简单的动物实验表明BmK-M9能影响两种蜚蠊和两种中华按蚊的正常运动，且对它们均具有致死活性，这佐证了电生理实验中BmK-M9可以抑制昆虫钠通道快失活的结果；同时提示了BmK-M9与经典杀虫剂拟除虫菊酯在钠通道上作用位点的不同及在生物农药应用方面的可能性。在第三章中，我们比较了BmK-M9对哺乳动物不同钠通道，钾通道及钙通道的选择性，发现在可供我们研究的受体中，它专一性作用于电压门控钠通道，且对NaV1.4效应最强，对NaV1.7最弱。这里引入了一个参数I5 ms/Ipeak，I5 ms代表给予测试电位5 ms后的电流，Ipeak代表该测试电位下的峰值电流。I5 ms/Ipeak的变化可以反映毒素对不同类型钠通道及不同浓度毒素对某一钠通道功能的差异，有助于更科学地判断毒素的药学活性。随后描述了BmK-M9与BgNaV1.1结合引起的通道动力学改变，其中一个重要变化是BgNaV1.1的电压敏感性增强，即开放所需的膜电位阈值向复极化方向移动。与已报道的其他α-蝎毒相比，BmK-M9与钠通道的亲和力很高，特别是和BgNaV1.1的结合几近是不可逆的。基于此，我们在判断NaV1.4上与结合相关的残基时，除观察毒素功能在各通道突变体上是否有显著性变化外，还比较了不同通道突变体上毒素的洗脱速率。一系列嵌合体和突变体功能和结合实验发现，NaV1.4 DIV胞外一个带电的Asp-Lys-Tyr链形成了BmK-M9的结合区域，同时决定了它的钠通道亚型选择性。另外，BmK-M9分子上维持生物活性的关键残基也是其抑制钠通道快失活的结构要素。因此找到毒素分子和通道分子上决定功能的关键残基并通过双突变循环实验确定一一对应关系有助于我们对这个高亲和力结合的理解及相应结合模型的建立。已有的电生理实验数据证明，DIV VSD在钠通道快失活过程中发挥关键作用。在第四章中，我们探索了BmK-M9操控钠通道DIV电压敏感元件的分子机制。通过在不同钳制电位下给药的方式，确定了BmK-M9更易结合静息态的钠通道。借助荧光电压钳技术，进一步证实BmK-M9在通道静息态时结合到DIV的voltage sensor而阻滞了其应对去极化膜电位的向胞外的门控运动，最终导致了孔区的持续性开放及延长的Na+流。但当DIV的voltage sensor处于运动过程中或已经被激活时，BmK-M9的结合力变弱。有趣的是，膜电位复极化时这个结合力又会逐渐增强，而且还能加速DIV voltage sensor向静息态的运动。最后，本文对目前已有的研究结果进行了总结和讨论。BmK-M9的极高亲和力使我们有机会将钠通道稳定在开放态并获取毒素通道复合物，进而通过冷冻电镜展示功能钠通道某种状态的结构。特别是随着一些真核钠通道结构的解析，更为我们进行相关实验，如提高通道在细胞系上的表达水平等，提供了有益的借鉴。以上工作不仅有助于钠通道工作原理和结构模型的确定，还可以指导以钠通道为靶点的相关药物的设计开发。 

Voltage-gated sodium channels dominate the initiation and propagation of action potentials and are the molecular basis of cellular excitability. Therefore, they are closely related to clinical diseases such as pain, epilepsy, arrhythmia, and are direct targets for many biotoxins. A sodium channel is always in a cycle of resting state, activated state and inactivated state in correspondence to changes in membrane potential. Fast inactivation is the course from the activated state to the inactivated state with a duration of only few milliseconds. It is a complicated gating process in which multiple structural elements are involved. Due to the extremely short time span, structural elements and conformation changes in fast inactivation are still not fully understood. Abnormal fast inactivation could lead to serious diseases such as the familial periodic paralysis syndromes hyperkalemic periodic paralysis and paramyotonia congenita.The first chapter briefly introduces the research progress of sodium channels from aspects of composition, physiological functions, working mechanism, gating characteristics, and three-dimensional structures in recent decades. These efforts are essential to explain sodium channels and may be utilized in researching on some new issues of interest. Next, the summary of a series of work on Mesobuthus martensii and its toxins shows that scorpion venom is a high-quality resource library rich in ion channel regulators, and some active components have become leading molecules in the development of pharmaceuticals. Along with completion of the genome work, it is much more convenient to conduct a research on the venom components and screen some new drug precursors. Consequently, we hope to search a probe to focus on the gating process of sodium channels from Mesobuthus martensii, while analyzing the molecular diversity of its venom.In the second chapter, an α-toxin called BmK-M9, which can effectively regulate fast inactivation was identified through purifying the venom gradually. Prokaryotic expression was utilized to obtain adequate sample quantities required for various experiments. Two simple animal assays showed that BmK-M9 could affect the normal movement of two kinds of cockroaches and two types of Anopheles sinensis and had lethal activity against all of them, which were also confirmed by the result that BmK-M9 could inhibit the inactivation of insect sodium channels. The analyses indicate not just different binding sites of BmK-M9 and pyrethroids on sodium channels but the possibility of BmK-M9 in biological pesticides application.In the third chapter, we compared the selectivity of BmK-M9 on different mammalian sodium, potassium, and calcium channels. It was found that BmK-M9 specifically acts on voltage-gated sodium channels among the receptors available for us. A parameter named I5 ms/Ipeak was introduced, whereby Ipeak represented the pore peak current at a test potential, and I5 ms meant the current in the 5th millisecond after exertion of the same test potential. The changes in I5 ms/Ipeak could be used to quantify the distinct effects of toxins on different sodium channels and different concentrations on a certain channel, thereby evaluating the activity of BmK-M9 more scientifically. Next, we described the sodium channel kinetics changes caused by the interaction between BmK-M9 and BgNaV1.1 in detail. One of the most important distinctions is a decrease in the voltage threshold required for channel opening, which means it shifts toward repolarization direction. Compared with other reported alpha-toxins, the affinity of BmK-M9 to sodium channels is much more potent. Particularly to BgNaV1.1, it is almost irreversible. Based on the high affinity, we could screen for the binding sites on NaV1.4 in two ways. Besides identifying a channel mutant possesses significant weaker binding affinity to toxins, we monitored the wash out rates of BmK-M9 on each mutant. A series of function and binding experiments reveal that a charged Asp-Lys-Tyr chain in the extracellular linker on DIV forms the binding region of BmK-M9 and determines its channel subtype selectivity at the same time. Certainly, the key residues that maintain biological activity on BmK-M9 molecule are also the structural basis for the inhibition of sodium channel fast inactivation. Overall, key residues determined the function on both toxin and channel molecule and their corresponding information derived from mutation cycle analysis promote our comprehension about this high affinity binding and establishment of relevant binding model. Published electrophysiological data proves that DIV VSD plays a critical role in fast inactivation. In the fourth chapter, we explored the molecular mechanism underlies the control of DIV voltage sensor by BmK-M9. When we applied BmK-M9 at different holding potentials, the binding affinity seemed to be modified and BmK-M9 preferred binding to resting sodium channels. With the help of VCF, it was further confirmed that BmK-M9 inhibits DIV voltage sensor outward movement in response to the depolarizing membrane potentials, resulting in continuous opening of the pore domain and persistent Na+ flux. But if DIV voltage sensor is in motion or has been activated, the binding affinity will become weaker. Interestingly BmK-M9 can seem to accelerate inward movement of DIV voltage sensor to resting state by virtue of the increasing affinity during the repolarization of membrane potentials.Finally, we concluded and discussed the findings in the context of the already existing research. The extremely high affinity of BmK-M9 gives us an opportunity to stabilize sodium channels in open state and acquire the complex, which in turn shows the structure of a functional sodium channel in a specific state through cryo-electron microscopy. Especially with published eukaryotic structures of NaVPaS and EeNaV1.4, more helpful references have been provided for us to perform comparative experiments, such as improving the expression level of channels in cell lines. The above work not only contributes to the existing sodium channel functional principles and structural model, but also guides the design and development of drugs targeting the sodium channels.