蛇三指环毒素家族的研究进展

蛇毒是其毒腺分泌的,主要由有生物活性的蛋白和多肽等混合物组成.这些蛋白和多肽大部分可归为两大类:酶类和非酶类.三指环毒素家族是其中最大的非酶类毒液蛋白类群,虽然该家族的蛋白质都是由β-折叠片组成的三指环结构,但是却呈现出了多样的功能活性.神经毒素是该家族中很重要的组分.三指环毒素家族中不同的功能群体结构的差异,导致了其生物功能的多样性以及每个功能类群的特殊性.蛇的三指环毒素家族很可能起源于无毒的三指环祖先蛋白.研究表明,正选择在该毒素家族的进化和多样性形成中起重要作用.     

蛇毒神经毒素和肌肉毒素研究进展

对蛇毒中神经毒素和肌肉毒素的分类、理化性质、结构及药理学活性进行了综述。当前研究表明,蛇毒神经毒素可分为3类:突触前神经毒素、突触后神经毒素和类神经毒素;前两者主要抑制运动终板处的神经肌肉传导,造成肌肉麻痹和呼吸衰竭;而后者可以阻断平滑肌收缩和离子通道,表现出类神经毒活性。蛇毒肌肉毒素主要分为3类:响尾蛇毒素、心脏毒素和肌肉毒PLA2;它们主要表现为破坏肌细胞膜,降解肌纤维,造成肌肉坏死。近年来,大量的蛇毒神经毒素和肌肉毒素的结构与功能得以确定,其在临床医学及研究领域中将有着重要的应用前景。随着在作用机制等方面研究的不断深入,蛇毒神经毒素和肌肉毒素的用途也将越来越广泛。     

蛇毒神经毒素和肌肉毒素研究进展

对蛇毒中神经毒素和肌肉毒素的分类、理化性质、结构及药理学活性进行了综述。当前研究表明，蛇毒神经毒素可分为3类：突触前神经毒素、突触后神经毒素和类神经毒素；前两者主要抑制运动终板处的神经肌肉传导，造成肌肉麻痹和呼吸衰竭；而后者可以阻断平滑肌收缩和离子通道，表现出类神经毒活性。蛇毒肌肉毒素主要分为3类：响尾蛇毒素、心脏毒素和肌肉毒 PLA2；它们主要表现为破坏肌细胞膜，降解肌纤维，造成肌肉坏死。近年来，大量的蛇毒神经毒素和肌肉毒素的结构与功能得以确定，其在临床医学及研究领域中将有着重要的应用前景。随着在作用机制等方面研究的不断深入，蛇毒蛇毒毒素和肌肉毒素的用途也将越来越广泛。
     

蝎毒素多肽与钠通道特异性结合位点研究进展

蝎毒素多肽是一种通道门控调节剂,早先研究发现其通过与离子通道的电压感受域结合影响通道的门控特性。新近研究表明,门控调解剂毒素与离子通道孔区也具有额外结合位点。因此,本文就蝎毒素多肽与钠通道特异性结合位点相关研究展开综述,以梳理门控调节剂毒素的结构与调节功能,为蝎毒素多肽与钠通道结合位点结构与功能的解析提供依据,也为靶向钠通道药物的设计与开发提供参考。     

蜘蛛多肽类毒素异源表达的研究进展

蜘蛛毒液是一个巨大的天然多肽和蛋白质分子组合库,具有多种生物学功能．但仅靠从天然的蜘蛛中提取出研究所需的蜘蛛多肽类毒素远不能满足对其的全面研究和开发利用．相比化学的固相合成法合成的低产量和较高成本,利用基因工程的方法异源表达蜘蛛毒素有较大的优势．综述了近年来利用大肠杆菌表达系统、酵母表达系统、昆虫表达系统和植物表达系统异源表达蜘蛛毒素的研究进展．     

虎纹捕鸟蛛神经毒素Huwentoxin-Ⅰ的二级结构研究

Huwentoxin-Ⅰ是一种小分子量的多肽类毒素,它能阻断脊椎动物的神经肌肉接头传递。该毒素分子内含33个氨基酸残基,有三对二硫键。本文报道用圆二色性光谱分析法对该毒素二级结构的测定和分析,并用Creenfield等介绍的方法计算其二级结构中各种成分的含量。结果表明,此多肽分子中,α螺旋约占22—28％,β折叠约占22—35％,而β转角和无规卷曲约占41—49％。实验还表明,此毒素在不同pH条件下的二级结构均比较稳定,将它加热到80℃保温20分钟后再测定其CD光谱,其二级结构成分变化不大。用各种不同的方法对其二级结构进行了预测,结果表明该分子一级结构序列N-末端有一小段β折叠,C-末端有一段α螺旋,分子中部的大部分残基为β转角和无规卷曲构象。     

从中国大陆产银环蛇毒纯化的第三个K－神经毒素Ⅱ．功能检测

从中国大陆产银环蛇毒纯化得到两个Ｋ－神经毒素：Ⅵ２６及Ⅷ１ｂ峰，其中Ⅵ２ｄ峰的部分氨基酸顺序与Ｋ－银环蛇毒素结构均一。Ⅵ２６及Ⅷ１ｂ峰在浓度分别为１５０ｎｍｏｌ／Ｌ及２００ｎｍｏｌ／Ｌ经３０分钟可完全阻断小鸡睫状神经节的烟碱传递，表明这两个Ｋ－神经毒素均为高效神经元烟碱乙酰胆碱受体拮抗剂。以Ａｆｆｉ－ｇｅｌ４０１亲和层析柱纯化的电鳐烟碱受体在严格的脂－蛋白比例下重构至人工微团上，以￣（８６）Ｒｂ￣＋内流至荷有烟碱乙酰胆碱受体的囊泡来衡量离子通道功能．测定了α－银环蛇毒素及Ｋ－神经毒素对受体离子内流的抑制作用．毒素（μｍｏｌ）与受体（μｍｏｌ）比值为０．５时，α－银环蛇毒素１００％阻断氨甲酰胆碱作用下的受体离子内流。１０倍于此浓度的Ｋ－神经毒素对离子通道活动无影响。     

蜈蚣粗毒中多肽毒素组分生理活性初步分析

几乎所有蜈蚣都是肉食动物,都依靠其毒液来捕杀和消化猎物,蜈蚣粗毒为混合物,其主要成分为蛋白质.本文以蜈蚣粗毒为原料,通过反相高效液相色谱进行分离纯化,将纯化产物进行MALDI-TOF-MS鉴定,发现蜈蚣粗毒中具有多肽组分.收集多肽组分在DRG细胞上进行全细胞膜片钳电生理实验研究,发现其中4个组分具有抑制电压门控钠通道电流活性、4个组分具有抑制电压门控钾通道电流活性.表明蜈蚣毒素具有很好的开发应用前景,为后续工作打下了良好的实验基础.     

抗昆虫蝎毒素多肽的重组表达研究

蝎毒素是蝎子自我防御和捕食的有力工具,属于活性短肽。抗昆虫蝎毒素多肽作用于昆虫细胞膜离子通道,调控通道动力学特性,是潜在的生物杀虫剂,具很高的研究价值。序列比对分析发现,抗昆虫蝎毒素多肽有高保守性,序列同源性达67%,其中有4个保守的半胱氨酸残基,这可能与其抗昆虫作用的结构有关。但天然蝎毒素蛋白获取较困难,致使抗昆虫毒素多肽的结构与功能研究受限,因此,利用基因重组技术获得高纯度高活性重组蝎毒素是有效途径之一。本文综述了抗昆虫蝎毒素多肽外源重组表达的研究现状,旨在更深入全面理解其结构及抗昆虫机理等,以助推后续相关研究。     

抗痛蝎毒素多肽的重组表达研究

蝎毒素多肽作用于众多细胞膜离子通道,调控通道动力学特性。显具抗痛活性的蝎毒素多肽呈现较高的学术研究价值和医药应用前景。序列比对分析发现,抗痛蝎毒素多肽具较高保守型,序列同源性达45%,均具8个半胱氨酸残基,且其位置相对保守,这可能与抗痛功能有关。天然蝎毒素蛋白的获得、结构与功能研究受限,而高纯度、高活性重组毒素多肽是一条有效解决途径。本文综述抗痛蝎毒素多肽的重组表达,藉此推进其结构与功能相关性的理解。     

Spider toxins activate the capsaicin receptor to produce inflammatory pain

Bites and stings from venomous creatures can produce pain and inflammation as part of their defensive strategy to ward off predators or competitors. Molecules accounting for lethal effects of venoms have been extensively characterized, but less is known about the mechanisms by which they produce pain. Venoms from spiders, snakes, cone snails or scorpions contain a pharmacopoeia of peptide toxins that block receptor or channel activation as a means of producing shock, paralysis or death. We examined whether these venoms also contain toxins that activate (rather than inhibit) excitatory channels on somatosensory neurons to produce a noxious sensation in mammals. Here we show that venom from a tarantula that is native to the West Indies contains three inhibitor cysteine knot (ICK) peptides that target the capsaicin receptor (TRPV1), an excitatory channel expressed by sensory neurons of the pain pathway. In contrast with the predominant role of ICK toxins as channel inhibitors, these previously unknown 'vanillotoxins' function as TRPV1 agonists, providing new tools for understanding mechanisms of TRP channel gating. Some vanillotoxins also inhibit voltage-gated potassium channels, supporting potential similarities between TRP and voltage-gated channel structures. TRP channels can now be included among the targets of peptide toxins, showing that animals, like plants (for example, chilli peppers), avert predators by activating TRP channels on sensory nerve fibres to elicit pain and inflammation.     

Functional analysis of an archaebacterial voltage-dependent K+ channel

All living organisms use ion channels to regulate the transport of ions across cellular membranes. Certain ion channels are classed as voltage-dependent because they have a voltage-sensing structure that induces their pores to open in response to changes in the cell membrane voltage. Until recently, the voltage-dependent K+, Ca2+ and Na+ channels were regarded as a unique development of eukaryotic cells, adapted to accomplish specialized electrical signalling, as exemplified in neurons. Here we present the functional characterization of a voltage-dependent K+ (K(V)) channel from a hyperthermophilic archaebacterium from an oceanic thermal vent. This channel possesses all the functional attributes of classical neuronal K(V) channels. The conservation of function reflects structural conservation in the voltage sensor as revealed by specific, high-affinity interactions with tarantula venom toxins, which evolved to inhibit eukaryotic K(V) channels.     

Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR

The active site of potassium (K+) channels catalyses the transport of K+ ions across the plasma membrane--similar to the catalytic function of the active site of an enzyme--and is inhibited by toxins from scorpion venom. On the basis of the conserved structures of K+ pore regions and scorpion toxins, detailed structures for the K+ channel-scorpion toxin binding interface have been proposed. In these models and in previous solution-state nuclear magnetic resonance (NMR) studies using detergent-solubilized membrane proteins, scorpion toxins were docked to the extracellular entrance of the K+ channel pore assuming rigid, preformed binding sites. Using high-resolution solid-state NMR spectroscopy, here we show that high-affinity binding of the scorpion toxin kaliotoxin to a chimaeric K+ channel (KcsA-Kv1.3) is associated with significant structural rearrangements in both molecules. Our approach involves a combined analysis of chemical shifts and proton-proton distances and demonstrates that solid-state NMR is a sensitive method for analysing the structure of a membrane protein-inhibitor complex. We propose that structural flexibility of the K+ channel and the toxin represents an important determinant for the high specificity of toxin-K+ channel interactions.     

P-type calcium channels blocked by the spider toxin omega-Aga-IVA.

Voltage-dependent calcium channels mediate calcium entry into neurons, which is crucial for many processes in the brain including synaptic transmission, dendritic spiking, gene expression and cell death. Many types of calcium channels exist in mammalian brains, but high-affinity blockers are available for only two types, L-type channels (targeted by nimodipine and other dihydropyridine channel blockers) and N-type channels (targeted by omega-conotoxin). In a search for new channel blockers, we have identified a peptide toxin from funnel web spider venom, omega-Aga-IVA, which is a potent inhibitor of both calcium entry into rat brain synaptosomes and of 'P-type' calcium channels in rat Purkinje neurons. omega-Aga-IVA will facilitate characterization of brain calcium channels resistant to existing channel blockers and may assist in the design of neuroprotective drugs.     

IS4 peptide forms ion channel in rat skeletal muscle cell membrane

A 22-mer peptide, identical to the primary sequence of domain I segment 4 (IS4) of rat brain sodium channel I, has been synthesized. IS4 peptide can incorporate into cultured rat skeletal myotube membranes and form ion channels. With patch clamp cell-attached technique single channel currents through IS4 channels can be recorded. The single channel conductances of IS4 channels are distributed heterogeneously. With different holding potentials, the mean open time, the mean closed time and the mean open probability are different respectively. IS4 channels are selective for Na⁺, Li⁺ and K⁺, but not for Cl⁻.     

Portability of paddle motif function and pharmacology in voltage sensors

Voltage-sensing domains enable membrane proteins to sense and react to changes in membrane voltage. Although identifiable S1-S4 voltage-sensing domains are found in an array of conventional ion channels and in other membrane proteins that lack pore domains, the extent to which their voltage-sensing mechanisms are conserved is unknown. Here we show that the voltage-sensor paddle, a motif composed of S3b and S4 helices, can drive channel opening with membrane depolarization when transplanted from an archaebacterial voltage-activated potassium channel (KvAP) or voltage-sensing domain proteins (Hv1 and Ci-VSP) into eukaryotic voltage-activated potassium channels. Tarantula toxins that partition into membranes can interact with these paddle motifs at the protein-lipid interface and similarly perturb voltage-sensor activation in both ion channels and proteins with a voltage-sensing domain. Our results show that paddle motifs are modular, that their functions are conserved in voltage sensors, and that they move in the relatively unconstrained environment of the lipid membrane. The widespread targeting of voltage-sensor paddles by toxins demonstrates that this modular structural motif is an important pharmacological target.     

Single apamin-blocked Ca-activated K+ channels of small conductance in cultured rat skeletal muscle

Action potentials in many excitable cells are followed by a prolonged afterhyperpolarization that modulates repetitive firing. Although it is established that the afterhyperpolarization is produced by Ca-activated K+ currents, the basis of these currents is not known. The large conductance (250 pS) Ca-activated K+ channel (BK channel) is not a major contributor to the afterhyperpolarization in non-innervated skeletal muscle and some nerve cells, because apamin, a neurotoxic component of bee venom, abolishes the afterhyperpolarization but does not block BK channels, and 5 mM extracellular tetraethylammonium ion (TEA) blocks BK channels but does not reduce the afterhyperpolarization. We now report single-channel currents from small conductance (10-14 pS) Ca-activated K+ channels (SK channels) with the necessary properties to account for the afterhyperpolarization. SK channels are blocked by apamin but not by 5 mM external TEA (TEAo). They are also highly Ca-sensitive at the negative membrane potentials associated with the afterhyperpolarization.     

Bilayer-dependent inhibition of mechanosensitive channels by neuroactive peptide enantiomers

The peptide GsMTx4, isolated from the venom of the tarantula Grammostola spatulata, is a selective inhibitor of stretch-activated cation channels (SACs). The mechanism of inhibition remains unknown; but both GsMTx4 and its enantiomer, enGsMTx4, modify the gating of SACs, thus violating a trademark of the traditional lock-and-key model of ligand-protein interactions. Suspecting a bilayer-dependent mechanism, we examined the effect of GsMTx4 and enGsMTx4 on gramicidin A (gA) channel gating. Both peptides are active, and the effect increases with the degree of hydrophobic mismatch between bilayer thickness and channel length, meaning that GsMTx4 decreases the energy required to deform the boundary lipids adjacent to the channel. GsMTx4 decreases inward SAC single-channel currents but has no effect on outward currents, suggesting it is located within a Debye length of the outer vestibule of the SACs, but significantly farther from the inner vestibule. Likewise, GsMTx4 decreases gA single-channel currents. Our results suggest that modulation of membrane proteins by amphipathic peptides--mechanopharmacology--involves not only the protein itself but also the surrounding lipids. The surprising efficacy of the d form of GsMTx4 peptide has important therapeutic implications, because d peptides are not hydrolysed by endogenous proteases and may be administered orally.     

Molecular characterization of a K+ channel blocker in the scorpionButhus martensii Karsch

K⁺ channel blockers of scorpion venoms are of important value in studying pharmacology and physiology of specific K⁺ channel of cells. Based on the amino acid sequences of BmP01 previously characterized as a small-conductance Ca²⁺-activated K⁺ channel blocker, two “back to back” degenarate primers have been designed and synthesized for inverse PCR strategy, its full-length cDNA has been cloned from the venom gland of the Chinese scorpionButhus martensii. The cDNA is composed of 3 parts: 5′ UTR, ORF and 3′ UTR. The flanking sequence of translation initiation codon ATG is AAAATGA, which is highly conserved in scorpion Na⁺ channel toxin and protozoan genes, suggesting that these genes may have followed a common mechanism for translation initiation. The 3′ UTR contains poly(A) signal AATAAA. The open reading frame encodes a precursor of 57 residues with a signal peptide of 28 residues and a mature peptide of 29 residues. The signal peptide is rich in hydrophobic amino acid residues and its length is significantly different from that of the determined scorpion Na⁺ channel toxin. The deduced amino acid sequence of mature peptide is completely consistent with BmP01 previously determined by primary structure analysis.     

Cell-free expression of functional Shaker potassium channels.

The functional activity of ion channels and other membrane proteins requires that the proteins be correctly assembled in a transmembrane configuration. Thus, the functional expression of ion channels, neurotransmitter receptors and complex membrane-limited signalling mechanisms from complementary DNA has required the injection of messenger RNA or transfection of DNA into Xenopus oocytes or other target cells that are capable of processing newly translated protein into the surface membrane. These approaches, combined with voltage-clamp analysis of ion channel currents, have been especially powerful in the identification of structure-function relationships in ion channels. But oocytes express endogenous ion channels, neurotransmitter receptors and receptor-channel subunits, complicating the interpretation of results in mRNA-injected eggs. Furthermore, it is difficult to control experimentally the membrane lipids and post-translational modifications that underlie the regulation and modulation of ion channels in intact cells. A cell-free system for ion channel expression is ideal for good experimental control of protein expression and modulatory processes. Here we combine cell-free protein translation, microsomal membrane processing of nascent channel proteins, and reconstitution of newly synthesized ion channels into planar lipid bilayers to synthesize, glycosylate, process into membranes, and record in vitro the activity of functional Shaker potassium channels.