氯离子通道杀虫剂靶标的研究进展

配体门控氯离子通道和电压门控氯离子通道都是杀虫剂的作用靶标,配体门控氯离子通道是抑制剂激发膜兴奋性的基础元件,γ-氨基丁酸（GABA）受体是一种氯离子载体复合物,是目前应用的杀虫剂的主要作用位点。林丹、硫丹、氟虫腈等杀虫剂能阻断弘氨基丁酸门控氯离子通道,引起神经抑制,导致中枢神经系统过度兴奋、惊厥,甚至造成机体死亡。电压门控氯离子通道是氯离子通道的家族又一个大的成员,依赖于电压的氯离子通道与维持电兴奋性、氯离子释放和吸收、内囊酸化、细胞内容物调节等生理活性相关,目前电压门控氯离子通道逐步成为杀虫剂的靶标成为研究热点。本文综述了杀虫剂靶标氯离子通道与杀虫剂作用的研究进展。     

阿维菌素类杀虫剂与配体门控离子通道相互作用研究进展

介绍了受体对药物的选择性结合与受体中存在的一些关键位点的氨基酸残基有很大关系。氨基酸残基在通道内排成一个装满水的口袋似的结构,这个口袋结构充当着药物与哺乳类GABAA受体和甘氨酸受体的结合位点。阿维菌素的主要作用靶标谷氨酸门控氯离子通道与这两类受体具有密切的关系,因此,可推测阿维菌素与受体的作用方式也有相类似的模式。这对创制新的具有安全、高活性、高选择性的杀虫剂有重要意义。     

γ-氨基丁酸门控氯离子通道受体及其靶标杀虫剂

作为半胱氨酸环配体门控离子通道超家族成员之一,γ-氨基丁酸门控氯离子通道(GABA-Cl)主要介导神经系统快速抑制性神经传递.由于其在昆虫和脊椎动物体内存在显著差异,GABA-Cl受体成为了现代杀虫剂研发的理想靶标.对GABA-Cl受体的结构和功能进行介绍,并对其靶标杀虫剂的研发背景与现状、化学合成、作用机制、生物活性...     

鲫鱼GABAARβ_3亚基生物信息学分析与同源建模

A型γ-氨基丁酸(aminobutyric acid,GABA)是一类配体门控离子通道型受体,亦为脊椎动物和非脊椎动物的中枢神经系统内最主要的抑制性神经递质GABA的受体,其离子通道上存在氟虫腈等杀虫剂的作用靶点,是医药和农药工作者研究的重点。通过RACE-PCR技术成功获取新基因鲫鱼GABAARβ_3的全长(GenBank登录号:KC964110)。序列分析显示该基因核苷酸序列共2 767 bp,含有1个由502个氨基酸残基组成的开放阅读框,编码蛋白分子量约为56 KD。报道了该基因的生物信息学分析和三维结构模拟及其与氟虫腈作用情况,BLAST结果表明,氨基酸序列与其他已知GABAARβ_3基因家族成员间序列相似性介于76%~89%之间,并与其他亚族蛋白基因存在很高的同源性。以线虫体内对阿维菌素敏感的谷氨酸门控氯离子通道受体蛋白的α亚基(PDB ID:3RHW)作为模板,用DS(Discovery Studio)模拟鲫鱼GABAAβ_3亚基的同源五聚体结构,优化结构模型,与农药配体分子做分子对接,计算作用模式和结合能力。研究鱼类GABAAR亚基与农药分子的作用。     

以昆虫瞬时受体电位(TRP)通道作为杀虫剂及驱避剂的靶标

<正>昆虫瞬时受体电位(TRP)通道是离子通道的一大类别,在几乎所有的感觉系统中发挥关键的信号传导作用。其他离子通道类别,特别是电压依赖性离子通道、配体门控离子通道和细胞内钙释放通道(鱼尼汀受体)长期以来被认为是最成功的杀虫剂靶标。但是最近才发现,TRP通道是探索已久的某些取食抑制剂类杀虫剂的靶标,使这类通道和感觉过程成为杀虫剂靶标的开发热点。最近还发现驱避剂香茅醛可激活TRPA1通道。由于感受和响应外部和     

VNX-000440系列均三嗪类杀虫活性物质的发现和活性

ω-hexatoxin-Hv1a是蜘蛛体内存在的多肽毒素,是昆虫电压门控离子通道拮抗剂。建立了此物质的3D药效团模型,用计算机从已知的商品化物质数据库中筛选出了与药效团模型相匹配的物质,并进一步用Accelrys软件进行筛选。对选出的物质进行活性测定,均三嗪类物质VNX-000440是得到的一个活性物质。经结构修饰得到了对埃及伊蚊和甜菜夜蛾活性高的物质VNX-000443。经对蜚蠊DUM神经元离子通道的影响测定试验,发现此类物质对昆虫电压门控离子通道有抑制作用。     

电压门控钠通道作为杀虫剂靶标

作为生物系统中兴奋性的主要结构,钠通道是源于动植物异源信息系统的一系列神经毒素的作用靶标,根据其生理活性和结合特点,至少已命名了9类不同的钠通道神经毒素（表1）。这些毒素可干扰电压门控钠通道的全部三种基本功能。     

作用于烟碱乙酰胆碱受体的新颖杀虫剂Flupyrimin

<正>烟碱乙酰胆碱受体(nAChR)是配体门控氯离子通道,快速地传递神经的兴奋性,是杀虫剂的一个重要作用靶标。烟碱型杀虫剂和相关相似配体能够与软体动物烟碱乙酰胆碱结合蛋白(AChBP)结合,此蛋白是烟碱乙酰胆碱受体胞外配体结合域的适合替代物,可用于用高分辨率的化学/结构生物学方法来弄明白药物结合域的识别特性。随后用与烟碱配体结合的位点的三维结构成功设计出具有独特药效团的新颖杀虫化合物。     

膜片钳技术在杀虫剂神经毒性研究中的应用

膜片钳技术是研究细胞膜离子通道的电生理学技术,也是从细胞水平探讨杀虫剂神经毒性作用机制的重要研究方法,在杀虫剂的研制、合理使用等方面具有很好的指导作用。综述了利用膜片钳技术研究多种膜蛋白杀虫剂靶标(电压门控钠、钾、钙通道,氯通道,烟碱型乙酰胆碱受体通道,P2X通道,TRP通道)在杀虫剂毒性机理分析中的应用,为开发高效、安全的杀虫剂提供参考。     

GABAA受体的结构

正Paul Miller和Radu Aricescu报告了人GABAA受体的第一个X-射线晶体结构,该受体是一个五聚配体门控的离子通道和脑中迅速抑制性突触传输的主要中介物(介导物)。整体结构与其他Cys-环受体的结构相似,但也有一个独特特征,其中包括存在一个延伸的聚糖鞘,后者将限制与其他突触蛋白的相互作用。作     

Ion Selectivity in the ENaC/DEG Family: A Systematic Review with Supporting Analysis

The Epithelial Sodium Channel/Degenerin (ENaC/DEG) family is a superfamily of sodium-selective channels that play diverse and important physiological roles in a wide variety of animal species. Despite their differences, they share a high homology in the pore region in which the ion discrimination takes place. Although ion selectivity has been studied for decades, the mechanisms underlying this selectivity for trimeric channels, and particularly for the ENaC/DEG family, are still poorly understood. This systematic review follows PRISMA guidelines and aims to determine the main components that govern ion selectivity in the ENaC/DEG family. In total, 27 papers from three online databases were included according to specific exclusion and inclusion criteria. It was found that the G/SxS selectivity filter (glycine/serine, non-conserved residue, serine) and other well conserved residues play a crucial role in ion selectivity. Depending on the ion type, residues with different properties are involved in ion permeability. For lithium against sodium, aromatic residues upstream of the selectivity filter seem to be important, whereas for sodium against potassium, negatively charged residues downstream of the selectivity filter seem to be important. This review provides new perspectives for further studies to unravel the mechanisms of ion selectivity.     

狗心脏中I_(to)相关钾离子通道表达分析

目的探讨钾离子通道Kv4·3和Kv1·4在正常与心衰的心肌组织中的表达差别,为进一步阐明心衰的发病机理奠定基础。方法建立狗的心衰模型,用Western blot方法对正常与心衰的狗心肌组织中Kv4·3、Kv1·4及辅助亚基Mink的表达进行分析。结果在心衰时,Kv4·3表达有明显下降;Kv1·4表达有所上升,在心肌组织的不同部位,即外层和内层Kv1·4的表达明显不同;同时Mink的表达在心肌外层也有明显下降。结论心衰时,心肌组织中形成Ito的钾离子通道Kv4·3和Kv1·4以及辅助亚基Mink的蛋白表达变化之间可能存在着一些相关性。     

Negative Influence by the Force: Mechanically Induced Hyperpolarization via K2P Background Potassium Channels

The two-pore domain K_2P subunits form background (leak) potassium channels, which are characterized by constitutive, although not necessarily constant activity, at all membrane potential values. Among the fifteen pore-forming K_2P subunits encoded by the KCNK genes, the three members of the TREK subfamily, TREK-1, TREK-2, and TRAAK are mechanosensitive ion channels. Mechanically induced opening of these channels generally results in outward K⁺ current under physiological conditions, with consequent hyperpolarization and inhibition of membrane potential-dependent cellular functions. In the past decade, great advances have been made in the investigation of the molecular determinants of mechanosensation, and members of the TREK subfamily have emerged among the best-understood examples of mammalian ion channels directly influenced by the tension of the phospholipid bilayer. In parallel, the crucial contribution of mechano-gated TREK channels to the regulation of membrane potential in several cell types has been reported. In this review, we summarize the general principles underlying the mechanical activation of K_2P channels, and focus on the physiological roles of mechanically induced hyperpolarization.     

Anti-Inflammatory Effect of Licochalcone A via Regulation of ORAI1 and K+ Channels in T-Lymphocytes

Calcium signaling plays a vital role in the regulation of various cellular processes, including activation, proliferation, and differentiation of T-lymphocytes, which is mediated by ORAI1 and potassium (K⁺) channels. These channels have also been identified as highly attractive therapeutic targets for immune-related diseases. Licochalcone A is a licorice-derived chalconoid known for its multifaceted beneficial effects in pharmacological treatments, including its anti-inflammatory, anti-asthmatic, antioxidant, antimicrobial, and antitumorigenic properties. However, its anti-inflammatory effects involving ion channels in lymphocytes remain unclear. Thus, the present study aimed to investigate whether licochalcone A inhibits ORAI1 and K⁺ channels in T-lymphocytes. Our results indicated that licochalcone A suppressed all three channels (ORAI1, Kv1.3, and KCa3.1) in a concentration-dependent matter, with IC₅₀ values of 2.97 ± 1.217 µM, 0.83 ± 1.222 µM, and 11.21 ± 1.07 µM, respectively. Of note, licochalcone A exerted its suppressive effects on the IL-2 secretion and proliferation in CD3 and CD28 antibody-induced T-cells. These results indicate that the use of licochalcone A may provide an effective treatment strategy for inflammation-related immune diseases.     

Trifunctional Saxitoxin Conjugates for Covalent Labeling of Voltage-Gated Sodium Channels**

Voltage-gated sodium ion channels (Na_Vs) are integral membrane protein complexes responsible for electrical signal conduction in excitable cells. Methods that enable selective labeling of Na_Vs hold potential value for understanding how channel regulation and post-translational modification are influenced during development and in response to diseases and disorders of the nervous system. We have developed chemical reagents patterned after (+)-saxitoxin (STX) – a potent and reversible inhibitor of multiple Na_V isoforms – and affixed with a reactive electrophile and either a biotin cofactor, fluorophore, or ‘click’ functional group for labeling wild-type channels. Our studies reveal enigmatic structural effects of the probes on the potency and efficiency of covalent protein modification. Among the compounds analyzed, a STX-maleimide-coumarin derivative is most effective at irreversibly blocking Na⁺ conductance when applied to recombinant Na_Vs and endogenous channels expressed in hippocampal neurons. Mechanistic analysis supports the conclusion that high-affinity toxin binding is a prerequisite for covalent protein modification. Results from these studies are guiding the development of next-generation tool compounds for selective modification of Na_Vs expressed in the plasma membranes of cells.     

Comparative Analysis of Single-Molecule Dynamics of TRPV1 and TRPV4 Channels in Living Cells

TRPV1 and TRPV4, members of the transient receptor potential vanilloid family, are multimodal ion channels activated by various stimuli, including temperature and chemicals. It has been demonstrated that TRPV channels function as tetramers; however, the dynamics of the diffusion, oligomerization, and endocytosis of these channels in living cells are unclear. Here we undertook single-molecule time-lapse imaging of TRPV1 and TRPV4 in HEK 293 cells. Differences were observed between TRPV1 and TRPV4 before and after agonist stimulation. In the resting state, TRPV4 was more likely to form higher-order oligomers within immobile membrane domains than TRPV1. TRPV1 became immobile after capsaicin stimulation, followed by its gradual endocytosis. In contrast, TRPV4 was rapidly internalized upon stimulation with GSK1016790A. The selective loss of immobile higher-order oligomers from the cell surface through endocytosis increased the proportion of the fast-diffusing state for both subtypes. With the increase in the fast state, the association rate constants of TRPV1 and TRPV4 increased, regenerating the higher-order oligomers. Our results provide a possible mechanism for the different rates of endocytosis of TRPV1 and TRPV4 based on the spatial organization of the higher-order structures of the two TRPV channels.     

Gentamicin Blocks the ACh-Induced BK Current in Guinea Pig Type II Vestibular Hair Cells by Competing with Ca2+ at the l-Type Calcium Channel

Type II vestibular hair cells (VHCs II) contain big-conductance Ca²⁺-dependent K⁺ channels (BK) and l-type calcium channels. Our previous studies in guinea pig VHCs II indicated that acetylcholine (ACh) evoked the BK current by triggering the influx of Ca²⁺ ions through l-type Ca²⁺ channels, which was mediated by M2 muscarinic ACh receptor (mAChRs). Aminoglycoside antibiotics, such as gentamicin (GM), are known to have vestibulotoxicity, including damaging effects on the efferent nerve endings on VHCs II. This study used the whole-cell patch clamp technique to determine whether GM affects the vestibular efferent system at postsynaptic M2-mAChRs or the membrane ion channels. We found that GM could block the ACh-induced BK current and that inhibition was reversible, voltage-independent, and dose-dependent with an IC₅₀ value of 36.3 ± 7.8 μM. Increasing the ACh concentration had little influence on GM blocking effect, but increasing the extracellular Ca²⁺ concentration ([Ca²⁺]_o) could antagonize it. Moreover, 50 μM GM potently blocked Ca²⁺ currents activated by (−)-Bay-K8644, but did not block BK currents induced by NS1619. These observations indicate that GM most likely blocks the M2 mAChR-mediated response by competing with Ca²⁺ at the l-type calcium channel. These results provide insights into the vestibulotoxicity of aminoglycoside antibiotics on mammalian VHCs II.     

Calmodulin Interactions with Voltage-Gated Sodium Channels

Calmodulin (CaM) is a small protein that acts as a ubiquitous signal transducer and regulates neuronal plasticity, muscle contraction, and immune response. It interacts with ion channels and plays regulatory roles in cellular electrophysiology. CaM modulates the voltage-gated sodium channel gating process, alters sodium current density, and regulates sodium channel protein trafficking and expression. Many mutations in the CaM-binding IQ domain give rise to diseases including epilepsy, autism, and arrhythmias by interfering with CaM interaction with the channel. In the present review, we discuss CaM interactions with the voltage-gated sodium channel and modulators involved in CaM regulation, as well as summarize CaM-binding IQ domain mutations associated with human diseases in the voltage-gated sodium channel family.     

Computational Analyses of the AtTPC1 (Arabidopsis Two-Pore Channel 1) Permeation Pathway

Two Pore Channels (TPCs) are cation-selective voltage- and ligand-gated ion channels in membranes of intracellular organelles of eukaryotic cells. In plants, the TPC1 subtype forms the slowly activating vacuolar (SV) channel, the most dominant ion channel in the vacuolar membrane. Controversial reports about the permeability properties of plant SV channels fueled speculations about the physiological roles of this channel type. TPC1 is thought to have high Ca²⁺ permeability, a conclusion derived from relative permeability analyses using the Goldman–Hodgkin–Katz (GHK) equation. Here, we investigated in computational analyses the properties of the permeation pathway of TPC1 from Arabidopsis thaliana. Using the crystal structure of AtTPC1, protein modeling, molecular dynamics (MD) simulations, and free energy calculations, we identified a free energy minimum for Ca²⁺, but not for K⁺, at the luminal side next to the selectivity filter. Residues D269 and E637 coordinate in particular Ca²⁺ as demonstrated in in silico mutagenesis experiments. Such a Ca²⁺-specific coordination site in the pore explains contradicting data for the relative Ca²⁺/K⁺ permeability and strongly suggests that the Ca²⁺ permeability of SV channels is largely overestimated from relative permeability analyses. This conclusion was further supported by in silico electrophysiological studies showing a remarkable permeation of K⁺ but not Ca²⁺ through the open channel.     

Structure and Function of Ion Channels Regulating Sperm Motility—An Overview

Sperm motility is linked to the activation of signaling pathways that trigger movement. These pathways are mainly dependent on Ca²⁺, which acts as a secondary messenger. The maintenance of adequate Ca²⁺ concentrations is possible thanks to proper concentrations of other ions, such as K⁺ and Na⁺, among others, that modulate plasma membrane potential and the intracellular pH. Like in every cell, ion homeostasis in spermatozoa is ensured by a vast spectrum of ion channels supported by the work of ion pumps and transporters. To achieve success in fertilization, sperm ion channels have to be sensitive to various external and internal factors. This sensitivity is provided by specific channel structures. In addition, novel sperm-specific channels or isoforms have been found with compositions that increase the chance of fertilization. Notably, the most significant sperm ion channel is the cation channel of sperm (CatSper), which is a sperm-specific Ca²⁺ channel required for the hyperactivation of sperm motility. The role of other ion channels in the spermatozoa, such as voltage-gated Ca²⁺ channels (VGCCs), Ca²⁺-activated Cl-channels (CaCCs), SLO K⁺ channels or voltage-gated H⁺ channels (VGHCs), is to ensure the activation and modulation of CatSper. As the activation of sperm motility differs among metazoa, different ion channels may participate; however, knowledge regarding these channels is still scarce. In the present review, the roles and structures of the most important known ion channels are described in regard to regulation of sperm motility in animals.