海南捕鸟蛛毒素-Ⅵ，一种新型的抑制昆虫电压门控钠通道失活的狼蛛神经毒素

通过阳离子交换和反相HPLC柱层析从海南捕鸟蛛（Ornithoconus hainana）粗毒中分离到一种新型的神经毒素,海南捕鸟蛛毒素-Ⅵ(HNTX-Ⅵ), 由34个氨基酸残基组成,含有6个保守的半胱氨酸残基. 运用全细胞膜片钳技术,研究了HNTX-Ⅵ对电压门控钠通道的影响.先前从海南捕鸟蛛粗毒中分离到的几种毒素,具有抑制哺乳动物钠通道激活的特性.本文研究结果表明,HNTX-Ⅵ能以类似于δ-atractoxins作用方式延缓蜚蠊背侧不成对中间(dorsal unpaired median,DUM）神经细胞的钠通道的失活,且导致钠通道稳态失活变得不完全,在预钳制电压大于-55 mV时形成不完全失活结构. HNTX-Ⅵ的这种新的功能不仅为探索钠通道的门控机制提供了有用的工具,也为开发新的安全的杀虫剂提供理论基础.     

海南捕鸟蛛毒素-IV对大鼠背根神经节细胞钠通道的抑制影响(英文)

海南捕鸟蛛毒素 IV(HNTX IV)是从中国捕鸟蛛Seleconosmiahainana粗毒中分离得到的一种肽类神经毒素 ,在成年大鼠背根神经节 (DRG)细胞上观察了该毒素对电压门控钠通道的影响。在全细胞膜片钳条件下 ,HNTX IV能明显抑制哺乳动物神经性河豚毒敏感型 (TTX S)钠电流 ,但不影响河豚毒不敏感型 (TTX R)钠电流。HNTX IV对DRG细胞TTX S钠电流的抑制作用具有浓度依从性 ,其有效半抑制浓度 (IC50 )为 44 .6nmol/L。该毒素不影响DRG钠电流的激活与失活时间特征 ,但能导致钠通道的半数稳态失活电压向超极化方向漂移约 10 .1mV。结果表明HNTX IV是一种新型的蜘蛛毒素 ,其影响电压门控钠通道的机制可能有别于那些结合于通道位点 3来延缓钠电流失活时间特征的蜘蛛毒素如δ 澳洲漏斗网蛛毒素、μ 美洲漏斗网蛛毒素I VI等。     

海南捕鸟蛛毒素—Ⅳ对大鼠背根神经节细胞钠通道的抑制影响

海南捕鸟蛛毒素-Ⅳ（HNTX-Ⅳ）是从中国捕鸟蛛Seleconosmia hainana粗毒中分离得到的一种肽类神经毒素,在成年大鼠背根神经节（DRG）细胞上观察了该毒素对电压门控钠通道的影响。在全细胞膜片钳条件下,HNTX-Ⅳ能明显抑制哺乳动物神经性河豚毒敏感型（TTX-S）钠电流,但不影响河豚毒不敏感型（TTX-R）钠电流,HNTX-Ⅳ对DRG细胞TTX-S钠电流的抑制作用具有浓度依从性。其有效半抑制浓度（IC50）为44.6nmol/L。该毒素不影响DRG钠电流的激活与失活时间特征,但能导致钠通道的半数稳态失活电压向超极化方向漂移约10.1mV。结果表明HNTX-Ⅳ是一种新型的蜘蛛毒素,其影响电压门控钠通道的机制可能有别于那些结合于通道位点3来延缓钠电流失活时间特征的蜘蛛毒素如δ-澳洲漏斗网蛛毒素,μ-美洲漏斗网蛛毒素I-Ⅵ等。     

Thr28突变对虎纹捕鸟蛛毒素-Ⅳ钠通道活性的影响

虎纹捕鸟蛛毒素-Ⅳ(HWTX-Ⅳ)是从虎纹捕鸟蛛粗毒中分离纯化到的一种新型多肽类神经毒素,能明显抑制表达于大鼠背根神经节细胞的河豚毒素敏感型(TTX-S)钠通道.为了更好地研究该毒素的结构与功能之间的关系,采用芴甲氧羰基(Fmoc)固相多肽化学合成法合成了用谷氨酸(Glu)替代HWTX-Ⅳ第28位苏氨酸残基的突变体T28D-HWTX-Ⅳ,线性多肽合成产物经反相高效液相色谱(HPLC)分离纯化后进行谷胱甘肽氧化复性.复性产物采用基质辅助激光解析飞行时间质谱(MALDI-TOF/TOF MS)技术鉴定分子质量,通过全细胞膜片钳电生理技术测定其电压门控钠通道药理学活性.当第28位Thr残基被Glu取代后,突变体T28D-HWTX-Ⅳ对表达于大鼠DRG细胞膜上的TTX-S钠通道的IC50值约为362 nmol/L,对TTX-S钠通道的抑制活性比天然HWTX-Ⅳ(IC50值=30 nmol/L)下降了约12倍,显示第28位的Thr残基是HWTX-Ⅳ与TTX-S型钠通道相互作用的关键活性残基.目前的研究为进一步探索HWTX-Ⅳ的结构与功能关系及新型镇痛药物的研发奠定了基础.     

虎纹捕鸟蛛神经细胞急性分离培养及其电压门控通道膜片钳

探索了虎纹捕鸟蛛(Ornithoctonus huwena)食道下神经细胞急性分离培养条件,并利用全细胞膜片钳技术对虎纹捕鸟蛛食道下神经细胞电压门控性钠、钾和钙通道的基本电生理学特性进行了研究.适合虎纹捕鸟蛛神经细胞离体培养的培养基为(g/L):葡萄糖0.7,果糖0.4,琥珀酸0.06,咪唑0.06,L-1513.7,Hepes 2.38,酵母粉2.8,乳白蛋白2.5,青霉素200 IU/ml,链霉素200 mg/ml,小牛血清15%;pH 6.8.该培养基非常适合虎纹捕鸟蛛神经节神经细胞离体培养,细胞在温度(27±2)℃的培养箱中培养2～4h,培养的细胞数目多、结构完整、贴壁效果好,细胞近似汤勺形,有一个长的单极突起,大部分细胞在10～30μm之间.全细胞模式下可以记录到钠、钾和钙三种电压门控离子通道电流.钙电流为高电压激活电流,该电流能够被NiCl2完全抑制;钾电流为瞬时钾电流和延迟整流钾电流,这两类钾电流分别被细胞外液中的4-氨基吡啶和氯化四乙胺所阻断;钠电流为TTX敏感型电流.     

苦皮藤素Ⅳ和Ⅴ对棉铃虫幼虫神经细胞钠通道的影响

电压门控钠通道是神经细胞兴奋传导的基础,也是杀虫剂最主要的作用靶标。具有二氢沉香呋喃多元酯骨架的苦皮藤素Ⅳ和Ⅴ是卫矛科植物苦皮藤的主要杀虫活性成分,苦皮藤素Ⅳ和Ⅴ处理后昆虫的中毒症状分别表现为麻醉和兴奋。本实验应用全细胞膜片钳技术就苦皮藤素Ⅳ和Ⅴ对棉铃虫Helicoverpa armigera幼虫离体培养神经细胞钠离子通道的影响进行了比较。结果表明：苦皮藤素Ⅳ对TTX-敏感钠通道电流的抑制明显具有浓度和时间依赖性,高浓度（10 μmol/L和1 μmol/L）条件下,峰值电流迅速减小而被抑制,在较中间浓度（0.1 μmol/L）时缓慢降低,而在低浓度（0.01 μmol/L）下,峰值电流先增加然后再缓慢降低;苦皮藤素Ⅳ对激活电压无明显影响,但使峰值电压向正电位方向移动,在高浓度移动迅速,低浓度移动缓慢。苦皮藤素Ⅴ对TTX-敏感钠通道电流峰值有明显的增大作用,也有一定的浓度依赖性;对激活电压无明显影响,峰值电压在高浓度下变化不明显,在较低浓度(0.1 μmol/L和 0.01 μmol/L)下向正电位方向移动明显。结果说明,苦皮藤素Ⅳ和Ⅴ可能在钠通道上有一个相同的靶标位点,但由于它们化学结构上的差异,可能对钠通道动力学的修饰 不同,导致不同的生理效应,昆虫表现出不同的神经中毒症状。     

敬钊毒素-Ⅲ对Nav1.8通道的抑制作用

敬钊毒素-Ⅲ(JingzhaotoxinⅢ,JZTX-Ⅲ)是从敬钊缨毛蛛毒液中分离到的一种门控调节型毒素,能选择性抑制钠通道亚型Nav1.5激活,但对其他6种钠通道亚型(Nav1.1 Nav1.4 Nav1.6和Nav1.7)无抑制作用。为了更好地研究钠通道结构与功能之间的关系,采用全细胞膜片钳技术检测了JZTX-Ⅲ对表达在ND7123细胞上的Nav1.8画道的影响。结果显示,JZTX-Ⅲ抑制Nav1.8电流,并且这种抑制作用具有时间和浓度依赖性,抑制时间常数和IC_(50)值分别为41.15±0.6 s和1.4±0.23μmol/L;1μmol/JZTX-Ⅲ使Nav1.8画道的电流-电压关系曲线和激活曲线分别向去极化方向漂移10 mV和9mV,使Nav.1.8通道的稳态失活曲线向超极化方向漂移16 mV,明显改变Nav1.8通道的激活和稳态失活动力学。此外,钠通道序列比对结果提示JZTX-Ⅲ可能通过结合Nav1.8通道DIIS3~S4连接环上的Lys(K)残基抑制Nav1.8通道。以上研究结果为进一步探索钠通道结构与功能之间的关系奠定了基础。     

乌拉坦对大鼠海马CA1神经元电压门控钠通道和动作电位的作用

乌拉坦对兴奋性和抑制性配体门控通道具有广泛的可检测的作用.作者运用全细胞膜片钳技术研究乌拉坦对wistar大鼠海马CA1神经元电压门控钠通道和动作电位的作用.结果发现乌拉坦可逆并剂量依赖性地抑制钠电流和动作电位,其中,在10mmol/L浓度时可减小钠电流强度达38%,使激活曲线向去极化方向移动,并延长钠通道失活后的恢复时间,降低动作电位的幅值.这些结果表明乌拉坦对电压门控钠通道的抑制作用可能是乌拉坦全身麻醉作用的机制之一.     

美洲大蠊中枢DUM神经元的分离和电压门控Na+电流的记录

【目的】建立美洲大蠊Periplaneta americana中枢神经系统背侧不成对中间神经元（dorsal unpaired median neurons, DUM neurons）的分离方法和DUM神经元电生理实验模型。【方法】IA型胶原酶法消化美洲大蠊末端腹神经节, 机械吹打得到DUM神经元细胞, 运用膜片钳技术记录DUM神经元细胞电压门控Na+电流。【结果】分离得到的DUM神经元细胞状态良好, 具有DUN神经元典型的梨状形态和表面特征。以膜片钳全细胞方式记录到的Na+电流符合钠通道电流特征。【结论】IA型胶原酶消化得到美洲大蠊DUM神经元细胞的方法可靠, 能稳定地记录到Na+电流。本文描述的方法为昆虫神经细胞的电生理机制研究提供一个可用的实验模型。     

棉铃虫幼虫神经细胞的急性分离培养及其电压门控通道的膜片钳研究

探索了棉铃虫Helicoverpa armigera幼虫神经细胞的急性分离与体外培养的条件,并利用全细胞膜片钳技术首次对棉铃虫幼虫急性分离神经细胞的电压门控性钠、钾和钙通道的基本电生理学特性进行了研究。结果表明,棉铃虫幼虫中枢神经细胞在TC-100、L-15和Grace培养基中均可贴壁生长,在DMEM培养基中基本不能存活。在TC-100培养基分别与其它三种培养基按一定比例混合形成的培养液中,TC-100与L-15等量混合培养液更适合于神经细胞的生长。全细胞电压钳条件下,可分别记录到电压门控性钠、钾和钙通道电流。钙电流特征为高电压激活、缓慢失活;钠电流对河豚毒素敏感;钾电流可被细胞外液中的氯化四乙胺和4-氨基吡啶抑制。     

Jingzhaotoxin-I, a novel spider neurotoxin preferentially inhibiting cardiac sodium channel inactivation

Jingzhaotoxin-I (JZTX-I), a 33-residue polypeptide, is derived from the Chinese tarantula Chilobrachys jing-zhao venom based on its ability to evidently increase the strength and the rate of vertebrate heartbeats. The toxin has three disulfide bonds with the linkage of I-IV, II-V, and III-VI that is a typical pattern found in inhibitor cystine knot molecules. Its cDNA determined by rapid amplification of 3'- and 5'-cDNA ends encoded a 62-residue precursor with a small proregion of eight residues. Whole-cell configuration indicated that JZTX-I was a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3. Although JZTX-I also exhibits the interaction with channel isoforms expressing in mammalian and insect sensory neurons, its affinity for tetrodotoxin-resistant subtype in mammalian cardiac myocytes (IC50 = 31.6 nm) is approximately 30-fold higher than that for tetrodotoxin-sensitive subtypes in latter tissues. Not affecting outward delay-rectified potassium channels expressed in Xenopus laevis oocytes and tetrodotoxin-resistant sodium channels in mammal sensory neurons, JZTX-I hopefully represents a potent ligand to discriminate cardiac sodium channels from neuronal tetrodotoxin-resistant isoforms. Furthermore, different from any reported spider toxins, the toxin neither modifies the current-voltage relationships nor shifts the steady-state inactivation of sodium channels. Therefore, JZTX-I defines a new subclass of spider sodium channel toxins. JZTX-I is an alpha-like toxin first reported from spider venoms. The result provides an important witness for a convergent functional evolution between spider and other animal venoms.     

The effects of huwentoxin-I on the voltage-gated sodium channels of rat hippocampal and cockroach dorsal unpaired median neurons

Huwentoxin-I (HWTX-I) is a 33-residue peptide isolated from the venom of Ornithoctonus huwena and could inhibit TTX-sensitive voltage-gated sodium channels and N-type calcium channels in mammalian dorsal root ganglion (DRG) neurons. However, the effects of HWTX-I on mammalian central neuronal and insect sodium channel subtypes remain unknown. In this study, we found that HWTX-I potently inhibited sodium channels in rat hippocampal and cockroach dorsal unpaired median (DUM) neurons with the IC₅₀ values of 66.1 ± 5.2 and 4.80 ± 0.58 nM, respectively. Taken together with our previous work on DRG neurons (IC₅₀ ≈ 55 nM), the order of sodium channel sensitivity to HWTX-I inhibition was insect central DUM ? mammalian peripheral > mammalian central neurons. HWTX-I exhibited no effect on the steady-state activation and inactivation of sodium channels in rat hippocampal and cockroach DUM neurons.     

Solution structure of two insect-specific spider toxins and their pharmacological interaction with the insect voltage-gated Na+ channel

Delta-paluIT1 and delta-paluIT2 are toxins purified from the venom of the spider Paracoelotes luctuosus. Similar in sequence to mu-agatoxins from Agelenopsis aperta, their pharmacological target is the voltage-gated insect sodium channel, of which they alter the inactivation properties in a way similar to alpha-scorpion toxins, but they bind on site 4 in a way similar to beta-scorpion toxins. We determined the solution structure of the two toxins by use of two-dimensional nuclear magnetic resonance (NMR) techniques followed by distance geometry and molecular dynamics. The structures of delta-paluIT1 and delta-paluIT2 belong to the inhibitory cystine knot structural family, i.e. a compact disulfide-bonded core from which four loops emerge. Delta-paluIT1 and delta-paluIT2 contain respectively two- and three-stranded anti-parallel beta-sheets as unique secondary structure. We compare the structure and the electrostatic anisotropy of those peptides to other sodium and calcium channel toxins, analyze the topological juxtaposition of key functional residues, and conclude that the recognition of insect voltage-gated sodium channels by these toxins involves the beta-sheet, in addition to loops I and IV. Besides the position of culprit residues on the molecular surface, difference in dipolar moment orientation is another determinant of receptor binding and biological activity differences. We also demonstrate by electrophysiological experiments on the cloned insect voltage-gated sodium channel, para, heterologuously co-expressed with the tipE subunit in Xenopus laevis oocytes, that delta-paluIT1 and delta-paluIT2 procure an increase of Na+ current. delta-PaluIT1-OH seems to have less effect when the same concentrations are used.     

昆虫钠离子通道的研究进展

昆虫只有一个或两个电压门控钠离子通道α亚基基因,但两种转录后修饰(选择性剪切和RNA编辑)实现了昆虫钠离子通道的功能多样性。昆虫β辅助亚基TipE和TEH1-4在钠离子通道表达和调控中也起着重要作用。电压门控钠离子通道在动作电位的产生和传递中至关重要,是多种天然和人工合成神经毒素及杀虫剂的作用靶标,包括广泛使用的拟除虫菊酯类、茚虫威和氰氟虫腙等杀虫剂。其中,拟除虫菊酯类杀虫剂通过调控昆虫钠离子通道的失活和去激活,延长跨膜钠离子流的时间,引起神经兴奋性传导障碍;茚虫威和氰氟虫腙阻断昆虫中枢和外周神经系统神经元的动作电位传导,这些神经毒剂都能干扰昆虫钠离子通道的正常功能。昆虫钠离子通道一般存在两个拟除虫菊酯类杀虫剂结合位点,但不同物种钠离子通道与拟除虫菊酯的结合位点存在一定差异。据此,本文就昆虫钠离子通道及其与杀虫剂的相互作用加以综述,有望推动昆虫神经受体研究,且对鉴定昆虫抗药性相关突变位点和研发高效的杀虫剂均具有重要参考价值。     

BjalphaIT: a novel scorpion alpha-toxin selective for insects--unique pharmacological tool

Long-chain neurotoxins derived from the venom of the Buthidae scorpions, which affect voltage-gated sodium channels (VGSCs) can be subdivided according to their toxicity to insects into insect-selective excitatory and depressant toxins (beta-toxins) and the alpha-like toxins which affect both mammals and insects. In the present study by the aid of reverse-phase HPLC column chromatography, RT-PCR, cloning and various toxicity assays, a new insect selective toxin designated as BjalphaIT was isolated from the venom of the Judean Black Scorpion (Buthotus judaicus), and its full primary sequence was determined: MNYLVVICFALLLMTVVESGRDAYIADNLNCAYTCGSNSYCNTECTKNGAVSGYCQWLGKYGNACWCINLPDKVPIRIPGACR (leader sequence is underlined). Despite its lack of toxicity to mammals and potent toxicity to insects, BjalphaIT reveals an amino acid sequence and an inferred spatial arrangement that is characteristic of the well-known scorpion alpha-toxins highly toxic to mammals. BjalphaITs sharp distinction between insects and mammals was also revealed by its effect on sodium conductance of two cloned neuronal VGSCs heterloguously expressed in Xenopus laevis oocytes and assayed with the two-electrode voltage-clamp technique. BjalphaIT completely inhibits the inactivation process of the insect para/tipE VGSC at a concentration of 100 nM, in contrast to the rat brain Na(v)1.2/beta1 which is resistant to the toxin. The above categorical distinction between mammal and insect VGSCs exhibited by BjalphaIT enables its employment in the clarification of the molecular basis of the animal group specificity of scorpion venom derived neurotoxic polypeptides and voltage-gated sodium channels.     

Native Pyroglutamation of Huwentoxin-IV: A Post-Translational Modification that Increases the Trapping Ability to the Sodium Channel

Huwentoxin-IV (HWTX-IV), a tetrodotoxin-sensitive (TTX-s) sodium channel antagonist, is found in the venom of the Chinese spider Ornithoctonus huwena. A naturally modified HWTX-IV (mHWTX-IV), having a molecular mass 18 Da lower than HWTX-IV, has also been isolated from the venom of the same spider. By a combination of enzymatic fragmentation and MS/MS de novo sequencing, mHWTX-IV has been shown to have the same amino acid sequence as that of HWTX-IV, except that the N-terminal glutamic acid replaced by pyroglutamic acid. mHWTX-IV inhibited tetrodotoxin-sensitive voltage-gated sodium channels of dorsal root ganglion neurons with an IC₅₀ nearly equal to native HWTX-IV. mHWTX-IV showed the same activation and inactivation kinetics seen for native HWTX-IV. In contrast with HWTX-IV, which dissociates at moderate voltage depolarization voltages (+50 mV, 180000 ms), mHWTX-IV inhibition of TTX-sensitive sodium channels is not reversed by strong depolarization voltages (+200 mV, 500 ms). Recovery of Nav1.7current was voltage-dependent and was induced by extreme depolarization in the presence of HWTX-IV, but no obvious current was elicited after application of mHWTX-IV. Our data indicate that the N-terminal modification of HWTX-IV gives the peptide toxin a greater ability to trap the voltage sensor in the sodium channel. Loss of a negative charge, caused by cyclization at the N-terminus, is a possible reason why the modified toxin binds much stronger. To our knowledge, this is the first report of a pyroglutamic acid residue in a spider toxin; this modification seems to increase the trapping ability of the voltage sensor in the sodium channel.     

A spider toxin that induces a typical effect of scorpion alpha-toxins but competes with beta-toxins on binding to insect sodium channels

Delta-palutoxins from the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) are 36-37 residue long peptides that show preference for insect sodium channels (NaChs) and modulate their function. Although they slow NaCh inactivation in a fashion similar to that of receptor site 3 modifiers, such as scorpion alpha-toxins, they actually bind with high affinity to the topologically distinct receptor site 4 of scorpion beta-toxins. To resolve this riddle, we scanned by Ala mutagenesis the surface of delta-PaluIT2, a delta-palutoxin variant with the highest affinity for insect NaChs, and compared it to the bioactive surface of a scorpion beta-toxin. We found three regions on the surface of delta-PaluIT2 important for activity: the first consists of Tyr-22 and Tyr-30 (aromatic), Ser-24 and Met-28 (polar), and Arg-8, Arg-26, Arg-32, and Arg-34 (basic) residues; the second is made of Trp-12; and the third is made of Asp-19, whose substitution by Ala uncoupled the binding from toxicity to lepidopteran larvae. Although spider delta-palutoxins and scorpion beta-toxins have developed from different ancestors, they show some commonality in their bioactive surfaces, which may explain their ability to compete for an identical receptor (site 4) on voltage-gated NaChs. Yet, their different mode of channel modulation provides a novel perspective about the structural relatedness of receptor sites 3 and 4, which until now have been considered topologically distinct.     

Expression of the voltage-gated sodium channel NaV1.5 in the macrophage late endosome regulates endosomal acidification

Voltage-gated sodium channels expressed on the plasma membrane activate rapidly in response to changes in membrane potential in cells with excitable membranes such as muscle and neurons. Macrophages also require rapid signaling mechanisms as the first line of defense against invasion by microorganisms. In this study, our goal was to examine the role of intracellular voltage-gated sodium channels in macrophage function. We demonstrate that the cardiac voltage-gated sodium channel, NaV1.5, is expressed on the late endosome, but not the plasma membrane, in a human monocytic cell line, THP-1, and primary human monocyte-derived macrophages. Although the neuronal channel, NaV1.6, is also expressed intracellularly, it has a distinct subcellular localization. In primed cells, NaV1.5 regulates phagocytosis and endosomal pH during LPS-mediated endosomal acidification. Activation of the endosomal channel causes sodium efflux and decreased intraendosomal pH. These results demonstrate a functionally relevant intracellular voltage-gated sodium channel and reveal a novel mechanism to regulate macrophage endosomal acidification.