中国科学院昆明动物研究所机构知识库

动物生存策略是复杂而精密的分子系统，目前对这一策略的认识大多停留在行为学、生理学、解剖学等阶段。而我们认为，探索动物生存策略的分子基础，是人类得以认识自然和改造自然的核心问题。离子通道是重要的膜蛋白受体，其基本功能包括调控动作电位和电信号，感知环境温度，调节体温，触发肌肉收缩、腺体分泌和神经突触传递等一系列生理反应，而离子通道活动的微小改变会直接带来机体的生理功能变化，因而与动物生存策略密切相关。本文提供了两项以离子通道功能为基础的动物生存策略研究进展。其一，离子通道TRPV1的结构功能进化是哺乳动物对环境温度适应的分子基础；其二，离子通道KCNQ是产毒动物生存策略中的重要干预靶点。动物对环境温度的适应策略：TRPV1通道是重要的伤害性感受器，多种物理和化学刺激能激活TRPV1从而引发疼痛。因此，它是感知外界刺激的重要元件。特别的是，TRPV1在高于42 °C的环境中被激活，是经典的热敏感通道。近期的研究进一步证实，TRPV1是哺乳动物感知伤害性热刺激的重要元件。TRPV1的门控机制，尤其是温度调控，一直是领域内的热点问题，但TRPV1热脱敏的生物学意义及其门控机制尚未得到揭示。在本论文中，我们首先提出了TRPV1热失活是哺乳动物温度适应分子基础的假说，并通过对一个种属特异性TRPV1分子的深入研究，阐明了热失活的分子机制，初步建立了TRPV1热失活与哺乳动物温度适应的关系。产毒动物的捕食策略：蜈蚣利用毒液进行高效的捕食和防御，而毒素的生物合成会大量消耗时间和生物体能量。因此，毒液系统在进化过程中不断优化，使其更有效地调控具有重要生理功能的受体，达到靶向多种生理系统的目的。本实验室的前期工作对蜈蚣叮咬导致急性疼痛的分子机制进行了详尽的描述，但尚未揭示其导致严重临床症状的分子基础（例如心肌缺血、心衰、呼吸抑制等）。本论文将详尽描述KCNQ通道在蜈蚣高效捕食和引发严重临床症状方面的核心作用，并提供靶向性的治疗策略。本论文主要分为三个章节进行阐述：第一章为本论文研究的基础背景，包括对现阶段TRPV1的研究进展的概述，以及简述已经探明的蜈蚣生存策略的分子基础。第二章中，我们结合遗传学分析和详尽的功能研究探索了TRPV1通道热失活的机制。通过遗传学分析，我们发现：（1）在进化过程中鸭嘴兽TRPV1通道 (pTRPV1) 基因受到强烈的正选择；（2）pTRPV1对已知的所有激活方式有正常的响应，例如辣椒素、温度、质子、Mg2+等二价阳离子、2APB和动物多肽毒素；（3）pTRPV1的热激活阈值显著低于其他哺乳动物TRPV1通道 (例如小鼠、人、骆驼等)，pTRPV1的热激活阈值仅为34 °C，低于其他哺乳动物的39 °C阈值；（4）pTRPV1不发生其他TRPV1具有的热失活现象；（5）TRPV1的N-和C-端的突变会影响TRPV1通道的热失活；（6）将mTRPV1的N-和C-端同时替换给pTRPV1使得构建的嵌合体pV1_mNC获得了热失活的性质；（7）C-末端单体可以竞争性结合TRPV1的N-末端并完全抑制TRPV1的热失活；（8）TRPV1 的N-和C-末端存在直接相互作用，并随着温度的升高亲和力越强；（9）TRPV1的 N-端和C-端在通道热失活过程中不断靠近，并特异性偶联于通道外孔区的构象变化而发生孔区关闭。该工作已完成的内容提示：1) TRPV1的N-和C-两端是通道热失活的“敏感元件”，它们之间直接相互作用是通道发生热失活的“分子开关”； 2) pTRPV1在温度门控中展示的特性可能是鸭嘴兽适应环境温度的分子基础。目前，我们正在利用pTRPV1基因敲入小鼠探索pTRPV1分子水平的热失活缺失与鸭嘴兽高温不耐受行为学的相互关系。第三章中，我们提供了蜈蚣围绕离子通道KCNQ建立捕食策略和引发严重临床症状的分子证据，并以此为依据，提出了靶向性临床干预手段。我们首先通过对蜈蚣毒液成分的活性追踪，找到了蜈蚣迅速征服巨大猎物的的分子机制。我们发现：（1）蜈蚣可以迅速制服体重大于自身15倍的小型哺乳动物；（2）通过对毒液活性成分追踪和分离纯化我们鉴定出一个多肽毒素SsTx，其溶液结构表面SsTx 结构紧凑，呈现明显的极性；（3）SsTx选择性的作用于KCNQ通道家族，对KCNQ4的IC50为2.5 μM；（4）SsTx与KCNQ的相互作用对孔区离子流敏感，提示SsTx可能作为孔区阻断剂发挥功能；（5）对KCNQ4胞外区域和SsTx带电氨基酸进行丙氨酸扫描，结果发现通道上的D266和D288，毒素上的K4、K10、K11、R12、K13和K45的突变影响了毒素对通道的结合，双突变循环和分子docking共同证实了D266-K13和D288-R12的直接相互作用形成盐键并保证SsTx-KCNQ复合物的构象稳定；（6）蜈蚣粗毒和SsTx均能引起大鼠胸主动脉环的收缩，将SsTx从粗毒中去除后收缩反应显著降低，说明SsTx是蜈蚣毒液中引起血管平滑肌收缩的关键因素；（7）小鼠或猕猴注射粗毒和SsTx能引起血压的急剧升高，同时出现心肌缺血症状；（8）SsTx可以引起大鼠的支气管平滑肌收缩，继而导致呼吸频率下降，呼吸幅度增加；（9）小鼠海马区注射SsTx导致癫痫和乙酰胆碱分泌；（10）SsTx还可以导致心肌细胞的动作电位时程延长；（11）将SsTx注射到小鼠皮下会引起皮肤组织坏死和炎症反应；（12）KCNQ激动剂RTG可以在组织水平和动物水平上有效干预粗毒和SsTx诱导的生理效应。本研究的意义在于：1)发现了蜈蚣毒液中的关键毒素分子SsTx；2)阐明了KCNQ通道家族是蜈蚣捕食和诱导严重临床症状的高效结合靶点；3)为蜈蚣叮咬导致的严重临床症状干预提供了候选药物RTG；4)揭示了离子通道是产毒动物生存策略中的重要结合靶点。本文通过对TRPV1和KCNQ的研究，反映了离子通道在动物生存策略中的重要功能：其一，离子通道的结构功能进化是动物环境适应的分子基础；其二，离子通道是产毒动物生存策略中的重要结合靶点。本论文分别以适应和捕食作为切入点，提供了离子通道结构、进化、门控、干预等方面的新信息，揭示了离子通道与动物生存策略的精密内在联系。

Animal survival strategy is a complex and precise molecular system, and the understanding of this strategy mostly stays in behavioral, physiological, anatomical and related levels. Based on the differences in animal's living habits and the study of the functional targets that affect animal's living habits, we can better understand animal's living strategies. Ion channel is an important molecular receptor, its function includes; regulating action potential and electrical signal, sensing environmental temperature, regulating body temperature, triggering a series of physiological reactions such as muscle contraction, gland secretion and neural synaptic transmission. An abnormal ion channel will seriously affect the body's environmental adaptation and normal physiological function, and consequently affect the survival of animals. Therefore, ion channels are closely related to animal survival strategies as the basis for maintaining the normal operation of the body. This paper provides two advances in animal survival strategies related to ion channels. First, the evolution of the structure and function of ion channels is the molecular basis for animal environmental adaptation; Second, ion channel is an important intervention target in the survival strategy of toxigenic animals. Animal environmental adaptation strategy: TRPV1 pathway is an important nociceptor. Physical and chemical stimuli activate TRPV1, causing pain, and therefore TRPV1 is an important element in the perception of external stimuli. TRPV1 is activated in an environment higher than 42 °C and is a classical heat sensitive channel. Recent studies have shown that TRPV1 is an important element in the mammalian perception of nociceptive thermal stimulation. The gating mechanism of TRPV1, especially temperature regulation, has been a hot issue in the field. In this thesis, we first put forward the hypothesis that thermal inactivation of TRPV1 is the molecular basis of animal temperature adaptation, and through the in-depth study of a species-specific TRPV1 molecule, clarify the molecular mechanism of thermal inactivation, and preliminarily establish the relationship between thermal inactivation of TRPV1 and animal temperature adaptation. Toxigenic animal survival strategy: centipede use venom for efficient predation and defense, and venom production will consume a lot of time and energy at the same time. Therefore, the venom system is constantly optimized in the process of evolution, so that it can better regulate and control the receptors with important physiological functions, so as to achieve the purpose of targeting multiple physiological systems. The molecular mechanism of acute pain caused by centipede bite has been described in detail, but the molecular basis for severe clinical symptoms, such as myocardial ischemia and heart failure, has not been revealed. In this paper, the key role of KCNQ channels in centipede predation and severe clinical symptoms will be described in detail, and targeted therapeutic strategies will be provided. This paper is mainly divided into three chapters: The first chapter is the basic background of this thesis, including the summary of the research progress of TRPV1 and the molecular basis of the known centipede survival strategy. In chapter 2, we explored the mechanism of TRPV1 pathway thermal desensitization by combining genome-wide positive selection analysis and patch clamp technique. Through target screening and genome-wide analysis, we found that during the evolution of platypus TRPV1 channel (pTRPV1) gene was positively selected. Then we synthesized the channel gene, and the function test showed that pTRPV1 could be activated by heat and reacted normally to capsaicin, and then reacted normally to the stimulation of proton, Mg2+, 2 APB and polypeptide toxin. The thermal activation threshold of pTRPV1 was only 34 °C compared with that of mouse TRPV1 channel (mTRPV1), and the thermal desensitization of pTRPV1 could not be induced by long-term thermal stimulation. Through truncated volume and point mutation experiments, we found that the change of NC end of mTRPV1 will affect the thermal desensitization of the channel. The new chimera pV1_mNC constructed by replacing the NC end of mTRPV1 with pTRPV1 respectively gives desensitization to the new chimera. The C-terminal and the WT plasmid of mTRPV1 were expressed together. It was found that the thermal desensitization of mTRPV1 was inhibited to a great extent. The addition of 1 mM C-terminal polypeptide to the internal electrode solution could completely inhibit the thermal desensitization, which indicated that the addition of mTRPV1 C-terminal fragment inhibited the thermal desensitization. Surface plasmon resonance (SPR) showed that the affinity of NC end of mTRPV1 was stronger with the increase in temperature, that is, there is a direct interaction between N and C terminal. The fluorescence resonance energy transfer showed that the N and C terminal of mTRPV1 were close to each other during desensitization. In the process of desensitization, it was found that the channel pore area was coupled with N and C terminal interaction. The significance of this work is as follows: 1) the first thermoinsensitive TRPV1 channel was discovered and cloned for the first time; 2) it is proved that NC is a channel desensitization ‘desensitization device’ by experimental evidence; 3) it is proved for the first time that the NC terminal of mTRPV1 are close to each other and combined with each other in desensitization process; 4) the structural and functional evolution of mTRPV1 is the molecular basis for animals’ adaptation to the environment. In chapter 3, we find the molecular mechanism of centipede rapidly conquering giant prey by tracking its venom activity and screening its ion channel activity. First, we found that centipede can quickly subdue the weight of more than 10 times its prey, through tracking and purification of venom active ingredients we identified a polypeptide toxin SsTx. The analysis of its nuclear magnetic resonance structure shows that SsTx has a compact structure and obvious polarity. Screening of channel activity showed that it selectively acted on KCNQ channel family, and the IC50 of KCNQ4 was 2.5 μΜ. The inhibition rate of SsTx on KCNQ channel decreased with the increase of voltage and intracellular potassium concentration, which suggested that SsTx might function as a pore blocking agent. Alanine scanning of the KCNQ4 extracellular region and SsTx charged amino acids showed that D266 and D288 of KCNQ4, and the K4, K10, K11, R12, K13 and K45 mutation of SsTx affected the binding of the toxin to the channel. The direct interaction between D266 and K13 was confirmed by double mutation cycle analysis. The structure simulation shows that D288 may interact with R12, which was verified by experiments. Both crude centipede venom and SsTx could induce contraction of thoracic aorta rings in rats, and the contraction response was significantly reduced after SsTx was removed from crude centipede venom, which indicated that SsTx was the key factor causing contraction of vascular smooth muscle in centipede venom. After injection of crude toxin and SsTx into the tail vein of mice, systolic blood pressure and diastolic blood pressure were increased. SsTx intravenous injection into rhesus monkeys can cause hypertension and myocardial ischemia in rhesus monkeys. SsTx could also constrict the bronchial smooth muscle of rats, and tail vein injection could decrease the respiratory rate and increase the respiratory amplitude of rats. The injection of SsTx into the hippocampus of mice caused rapid epilepsy and acetylcholine secretion. SsTx can also prolong the action potential duration of cardiomyocytes. Injection of SsTx subcutaneously into mice can cause skin necrosis and inflammatory response. These results are highly consistent with the clinical symptoms of centipede bites. The significance of this study is as follows: 1) the toxin SsTx which specifically acts on KCNQ family was found; 2) KCNQ channel family is the target of a series of physiological responses caused by centipede bite. 3) the candidate drug retigabine is provided for the intervention of centipede bite. Through the study of TRPV1 and KCNQ channels, this paper reflects the important function of ion channels in animal survival strategy. First, the evolution of the structure and function of ion channels is the molecular basis of animal environmental adaptation; Second, ion channel is an important intervention target in the survival strategy of toxigenic animals. In this thesis, adaptation and predation are taken as the breakthrough points to provide new information on ion channel structure, evolution, gating, intervention and other aspects, revealing the precise internal relationship between ion channel and animal survival strategy.