中国科学院昆明动物研究所机构知识库

蜈蚣是现存最古老的陆生节肢动物之一，在数亿年的进化过程中进化出了毒液系统用来防御和捕食。蜈蚣毒液成分极其复杂，其中一些多肽毒素能够与离子通道互作进而影响神经系统。由于这些多肽毒素与离子通道的结合具有高效性和特异性，因而可以将其作为研究离子通道结构与功能的重要工具分子。在本研究中，我们通过葡聚糖凝胶层析、反相高效液相色谱以及膜片钳等技术从少棘蜈蚣毒液中分离纯化到一条具有电压门控钾通道Kv1.1抑制功能的多肽SsTx，SsTx由53个氨基酸残基组成，氨基酸序为EVIKKDTPYKKRKFPYKSECLKACATSFTGGDESRIQEGKPGFFKCTCYFTTG，其中Cys20-Cys46、Cys24-Cys48配对形成两对二硫键，MALDI-TOF鉴定其分子量为6018 Da，核磁共振（NMR）实验结果表明SsTx 是一个结构紧凑的极性蛋白质。通过膜片钳技术的进一步研究我们发现该多肽分子对Kv1.1的抑制作用具有浓度依赖性，其半效抑制浓度IC50为1.25 ± 0.12 μM。为了研究SsTx与Kv1.1相互作用的分子机制，我们进行了Kv1.1及SsTx的突变扫描和双突变循环实验，实验结果表明SsTx通过K13位点与Kv1.1的E353位点相互作用，从而将SsTx锚定在Kv1.1的外孔区，阻止钾离子的通过。 另外，在突变体扫描实验中，我们还发现D6, E33, E38这三个氨基酸残基突变成A后能提高SsTx与Kv1.1的亲和力。相较于SsTx的半效抑制浓度（1.25 ± 0.12 μM），SsTx-D6A, SsTx-E33A和SsTx-E38A的IC50分别为0.82 ± 0.10 μM, 0.7 ± 0.08 μM, 0.44 ± 0.05 μM。从SsTx的溶液结构上看，这3个残基分别位于三维结构的β折叠、β转角、β折叠，因此丙氨酸突变可能对SsTx的化学微环境产生影响，进而改变了SsTx对Kv1.1的亲和力。 由于蜈蚣毒液是一个pH约为6.2的酸性混合物，我们比较了不同pH条件下SsTx 对Kv1.1的抑制活性，以观察蜈蚣毒液的酸性环境是否对SsTx的亲和力有所贡献。实验结果表明SsTx在pH 6.2的酸性条件下对电压门控钾通道的半效抑制浓度IC50为0.527 ± 0.03 μM，较pH 7.4条件下其Kv1.1抑制活性有所增强，从目前已有的SsTx-Kv1.1相互作用分子机制上看，这一现象可能是由于在电压门控钾通道Kv1.1的孔区附近存在氢离子结合位点，在酸性环境条件下，氢离子与Kv1.1孔区结合，导致孔区化学微环境向更加有利于结合SsTx的构象变化，最终表现为SsTx对Kv1.1的抑制活性增强。因此，蜈蚣毒液的酸性环境对蜈蚣的捕食防御有着重要的生物学意义，具体表现为：氢离子本身就能抑制电压门控钾通道电流；氢离子的存在能够增加多肽毒素与离子通道的亲和力。本文的研究取得了3个方面的进展：1）我们发现了一个能够抑制电压门控钾通道Kv1.1的新型多肽毒素SsTx，该分子与所有已知的钾通道抑制剂没有序列同源性。2）SsTx通过K13位点与Kv1.1的E353位点相互作用实现其孔区阻断的分子功能。3）SsTx与Kv1.1的相互作用具有高度的pH依赖性，这种依赖性系毒素-离子通道相互作用研究中的首例报道。基于这些研究进展，本论文的结论及意义具体体现为3方面：1）对SsTx结构和功能的探究拓展了我们对产毒动物毒素分子多态性的认识，为研究Kv通道的门控特性提供了一个结构简单、功能高效的分子工具。2）SsTx-Kv1.1相互作用的研究为我们认识Kv孔区阻断剂的工作机制提供了一例详实的证据，为未来基于Kv孔区的药物设计增加了一个新的结合热点E353。 3）酸性环境增强SsTx的亲和力说明了产毒动物不仅仅利用毒素的结构特征来优化其捕食防御过程，其毒液的化学环境甚至一些我们目前尚未考虑到的因素也可能是产毒动物在进化中利用到的重要策略，这些策略与毒素-受体的分子相互作用策略具有同样重要的地位。 综上所述，本文以SsTx-Kv1.1相互作用的分子机制研究为基础，阐明了SsTx通过K13与Kv1.1的E353位点相互作用实现其抑制Kv1.1钾电流的分子机制，提示了有毒动物毒液酸性环境能有效的提高毒素与受体的亲和力，对今后深入研究Kv通道结构与功能以及靶向Kv通道的药物研发都具有重要的意义。

Centipede is one of the oldest terrestrial arthropods, which has evolved venom system used for defense and prey during billions of years. The compounds of centipede venoms are extremely complex, including some ion channel-interacting peptide toxins which can impact function of ion channels and cecentral nervous system. Due to binding to ion channels efficiently and specifically, peptide toxins become important tools for the study of the function and structure of ion channels.In this study, we separated and purified a peptide toxin SsTx from Scolopendra subspinipes mutilans by gel filtration and reverse phase high performance liquid chromatography. SsTx is composed of 53 amino acid residues (EVIKKDTPYKRKFPYKSECLKACATSFTGGDESRIQEGKPGFFKCTCYFTTG). Four cysteine residues from SsTx form two disulfide bonds, Cys20-Cys46 and Cys24- Cys48. The molecular weight of SsTs is 6018 Da. The results of NMR indicate that the structure of SsTx is compact and polar. Patch clamp experiment indicates SsTx is an inhibitor of voltage-gated potassium channel.The inhibition of SsTx is concentration-dependent with an IC50 of 1.25 ± μM. To study molecular basis of SsTx interacting with Kv1.1, we performed mutant scaning and thermodynamic double mutant cycle on Kv1.1 and SsTx. Our study found that K13 of SsTx binded to E353 on the out pore region of Kv1.1 and anchored SsTx on Kv1.1, thus blocking potassium ion influx in the pore.Besides, during SsTx mutant scaning, we also found that the affinity of SsTx-Kv1.1 was enhanced after replacement of three amino acid residues (D6, E33 and E38) by Ala. Comparing with IC50 of SsTx (1.25 ± 0.12 μM), IC50 of SsTx_D6A, SsTx_E33A and SsTx_E38A are 0.82 ± 0.10 μM, 0.7 ± 0.08 μM and 0.44 ± 0.05 μM, respectively. These three negative amino acid residues (D6, E33 and E38) respectively locate at the beta sheet, beta turn and beta sheet of solution structure of SsTx. Alanine mutations may affect SsTx chemical microenviroment, and then change the SsTx-Kv1.1 affinity. Because the centipede venom is a mixture of around pH 6.2, we compared inhibitory activity of SsTx at different pH to indicate the contribution of acid environment of centipede venom to SsTx-Kv1.1 affinity. Results showed that the IC50 of SsTx was enhanced to 0.527 ± 0.03 μM at pH6.2 environment. From molecular mechanisms of interaction between SsTx and Kv1.1, we speculate there is a proton binding site near pore region of Kv1.1. In the acidic environment, proton binds to pore region of Kv1.1. Therefore chemical microenvironment is changed and inhibitory activity of SsTx is enchanced. Thus the acidic environment of centipede venom plays an important role in predation and defense for centipede, For example, proton can inhibit voltage-gated potassium channel current by itself; Proton enhances SsTx-Kv1.1 affinity.This study includes three progresses: 1) We find a new peptide toxin which can inhibit voltage-gated potassium channel Kv1.1 and has no sequence homology with known Kv1.1 inhibitors. 2) SsTx interacts with E353 of Kv1.1 by K13 and blocks pore region of Kv1.1. 3) The interaction between SsTx with Kv1.1 is highly pH- dependent and this pH- dependence is first reported.Based on those research progresses, this paper reflects three main conclusions and significances: 1) the structure-function of SsTx expands our knowledge of peptide toxins. SsTx provide a simple and efficient molecular tool for the study of Kv1.1 gating properties. 2) The research of interaction between SsTx and Kv1.1 provides a detailed evidence for us to understand the molecular mechanism of Kv inhibitor and a new binding site for drugs targeting Kv pore region. 3) Acidic environment enhances SsTx affinity, indicating that venomous animals not only optimize defense by toxins’ structures, but also by evolving chemical environment of the venoms. They may be equally significant.In summary, based on molecular mechanism of interaction between SsTx and Kv1.1, this study demonstrates that SsTx inhibits Kv1.1 by K13 interacting with E353 of Kv1.1 and the acidic environment of venom enhances toxin-receptor affinity. This study is great significance for future research of the structure-function of Kv channels and development of Kv inhibitors.