蛇毒蛋白Mambalgin与酸敏感离子通道ASIC复合物的结构解析及相互作用机制研究

Acid-sensing ion channels (ASICs) are voltage-independent, proton-activated receptors that belong to the epithelial sodium channel/degenerin family of ion channels and expressed throughout vertebrate central and peripheral nervous systems. These ubiquitous eukaryotic ion channels have essential roles in biological activities as diverse as sodium homeostasis, taste and pain. Polypeptide toxins have played a central part in understanding physiological and physiopathological functions of ion channels. In the field of pain, they led to important advances in basic research and even to clinical applications. Recently, a new class of three-finger peptides from the black mamba venom was found to be able to abolish pain. These peptides, which are called mambalgins, specifically inhibit a set of ASICs expressed either in central or peripheral neurons to produce potent analgesic effects in vivo in mice. Their analgesic effects can be as strong as morphine but are resistant to naloxone and do not involve opioid receptors. Mambalgins have no apparent toxicity and seem to produce fewer unwanted side effects than morphine, illustrating the potential therapeutic value of these ASIC-inhibitory peptides. However, the structural and chemical principles underlying the binding and the inhibitory mechanism of these peptides on ASICs are still not known, although it has been recently suggested that mambalgin-2 may bind in close proximity of the acidic pocket of the ASIC1a channel. Our overall goal of this project is to uncover the molecular mechanism of channel inhibition by the mambalgins pain-relieving peptides by means of solving the complex structure of chicken ASIC1a–Mambalgin-1.These data will help to understand inhibition by mambalgins and provide clues for the development of new optimized blockers of ASIC channels. Based on the complex structure, a series of chemical modifications will be adjusted on Mambalgins to strengthen the analgesic effect and prolong the serum stability.

ASIC（Acid-Sensing Ion Channel）是高等生物神经系统中一类重要的离子通道蛋白，能够感知胞外H+浓度变化并作出响应，在痛觉的产生和维持过程中起着关键作用。蛇毒蛋白Mambalgin是一类新发现的ASIC抑制物，具有很高的亲和力和特异性，生理上展现出有效镇痛效果，具有药物开发的潜力。目前，mambalgin毒素特异性识别并结合ASIC的结构基础并不清楚，毒素抑制ASIC通道活性的作用机制也有待深入研究。针对这些问题，本项目将采用化学合成手段获取具有天然活性的mambalgin蛋白，解析ASIC-mambalgin复合物高分辨率的三维结构，捕获ASIC活性调控中的构象变化并进行相关功能的深入研究，在阐明多肽类毒素调控离子通道活性的分子机制上有重要的科学意义；也将基于复合物结构进行毒素衍生分子的设计与改造，为镇痛药物的开发提供有价值的线索。

高等生物内许多生理过程与pH密切相关。酸感应离子通道（ASICs）神经元上的pH传感器，广泛存在于中枢神经系统和外周神经系统中。ASIC是一类非电压依赖性的阳离子通道，与学习，恐惧行为，缺血性中风后的神经退行性变和疼痛感知等生理病理过程密切相关。因此，ASIC已经成为精神疾病，神经退行性疾病和疼痛治疗中的新的潜在药物靶标。前人的研究已经报道了处于不同状态的鸡源ASIC1（cASIC1）的结构，包括非活性状态，低pH脱敏状态和静息状态的cASIC1结构，与PcTx1结合的脱敏状态，以及与MitTx结合的开放状态的结构。但是，人源ASIC（hASIC1a）的结构及其通过毒素的调节机制仍然未知。Mambalgin1是从黑曼巴蛇毒中分离出来的一种毒素。它是一种有效，快速和可逆的针对ASIC1a或ASIC1b通道抑制剂。动物实验表明Mambalgin1具有类似于吗啡的镇痛作用，但其镇痛效应不涉及阿片类药物受体，因此与传统阿片类药物相关的不良副作用较少，表明其具有很高的治疗价值。.本项目的研究中，我们运用冷冻电镜技术解析了cASIC1-Mambalgin1复合物， hASIC1a, hASIC1a-Mambalgin1复合物的，分辨率分别为5.6 Å，3.6 Å，3.9 Å。结合膜片钳及点突变功能分析，主要有以下发现：（1）首次报道了人源酸敏感离子通道的结构。（2）详细揭示了Mambalgin1与ASIC1离子通道之间相互作用的结构机制。（3）提出了一种新的Mambalgin1抑制hASIC1a的构像锁定机制，而不是先前提出的变构机制。发现Mambalgin1倾向于结合并锁定静息状态下的hASIC1a通道。Mambalgin1通过阻止质子诱导的从静息状态到活性和/或脱敏状态的转变来抑制hASIC1a通道活性。（4）发现了Mambalgin1对hASIC1a的抑制作用强于cASIC1, ASIC1通道胞外结构域某些残基之间的相互作导致了Mambalgin1对cASIC1和hASIC1a抑制效果的差异。上述发现不仅在概念上具有新颖性，对于理解蛇毒素多肽Mambalgin1抑制ASIC的机制具有重要意义，而且对物种特

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