电压依赖性钙通道 Cav3.1 的 cDNA 在爪蟾卵母细胞中的表达

Cav3.1是一种电压依赖性钙离子通道,cDNA全长7 625 bp.非洲爪蟾卵母细胞有表达外源基因的功能,被用于离子通道异源表达的研究中.pGEM-HE是卵母细胞的高效表达载体,但由于pGEM-HE多克隆位点处的酶切点较少,外源片段常不能直接插入.为了将Cav3.1的cDNA表达于卵母细胞中,首先对pGEM-HE的多克隆位点和β-Globin 3'端下游的切点进行了改造,引入需要的酶切点,删除了妨碍重组和mRNA转录的位点.并将Cav3.1的cDNA克隆到改造后的pGEM-HE载体中.将重组的质粒在爪蟾卵母细胞中表达,记录到了Cav3.1的通道电流,为以后的工作提供了基础.     

基于USB的卵母细胞电压钳同步数据采集和分析软件设计

介绍了基于USB接口的卵母细胞电压钳系统的数据采集分析系统,应用Delphi语言开发了系统的人机操作实验界面,探讨了电压钳的USB同步传输、数据采集和和显示的实现方法.在细胞模型测试中,电压钳放大器的跨膜电流分辨达到了0.01nA,噪声测试低于10pA.在非洲爪蟾卵母细胞表达Kv4.2钾通道实验中,记录跨膜电流波形清晰,数据分析可靠.与国外同类仪器相比:该系统操作简单,价格低,数据采集和分析方便,适用于卵母细胞表达实验.     

大黄素对辣椒素诱导的TRPV1通道电流的抑制作用

本研究通过显微注射技术将TRPV1 cRNA(1ng/nL)注射至非洲爪蟾卵母细胞(20nL/个)体内,放置于G-ORi培养液中,并在(19±1)℃的恒温培养箱内表达.同时利用灌流技术将不同浓度梯度的大黄素、辣椒素按照设计顺序依次灌流进入流动腔,同时控制流速保证非洲爪蟾卵母细胞完全浸没于流动腔内.使用双电极电压钳技术记录0.1,1.0,10.0,50.0μmol/L浓度梯度的大黄素对500nmol/L浓度的辣椒素激活TRPV1电流的影响.得到大黄素作用辣椒素激活TRPV1通道的抑制作用表现出浓度依耐性和电压非依赖性,IC50=38μmol/L,Hill系数n=0.5.结果表明了大黄素对TRPV1位点的相互作用是负协同的,在天然药物里面是一类比较弱的拮抗剂,且大黄素在ORi溶液中开始析出沉淀的浓度在50~60μmol/L之间.我们首次发现了大黄素能够抑制TRPV1通道电流,这可能为开发新的镇痛药物提供理论基础.     

H+对非洲爪蟾卵母细胞ATP-激活电流的调制作用

研究H^＋对非洲爪蟾卵母细胞内源性ATP受体的调制作用。采用双极电压钳技术记录卵母细胞膜ATP受体介导的电流。实验检测的细胞全部对ATP敏感，应用pH为7．4的ATP（10^-3mol／L）后引起一特征性电流，呈浓度依赖性。改用pH为6．5，6．0，5．5的ATP（10^-3mol／L）后，电流幅值明显减弱。结果表明，pH值降低，可抑制ATP诱导的卵母细胞膜电流。     

乙酰胆碱对非洲爪蟾卵母细胞ATP-激活电流的调制作用

研究乙酰胆碱对爪蟾卵母细胞上ATP激活电流的影响及其机理.用双电极电压钳技术记录爪蟾卵母细胞的细胞外液加入乙酰胆碱对ATP激活电流（IATP）的影响.结果显示,乙酰胆碱对大多数细胞IATP具有增强作用,而对少数细胞IATP具有抑制作用,且均呈现浓度依赖性;乙酰胆碱对IATP的调制作用是通过毒蕈碱样乙酰胆碱受体（M受体）激活实现的.     

模式生物——爪蟾在生物医学上的应用

随着生物医学的发展，模式生物——爪蟾研究系统在生物医学研究领域的应用越来越广泛.与其他模式生物相比，模式生物——爪蟾具有许多不可比拟的优点，适合建立多种人类疾病模型进行疾病发病机制和治疗等相关研究；还可以结合生物信息学分析进行高通量药物筛选及药物开发等研究；另外在发育生物学和基因功能研究方面也发挥重要作用.因此，爪蟾已经成为生物医学研究中的一种重要的模式生物，在医学基础及临床研究和药物筛选等研究领域中占有非常重要的地位.文中综述了爪蟾作为模式生物在生物医学研究中发挥的作用.     

非洲爪蟾卵母细胞内的螺旋钙波和靶波

该文应用定态线性化的Atri模型方程，描述非洲爪蟾(Xenopus Laevis)卵母细胞内钙波的生成和演化。在忽略细胞边界影响的近似下，得到了爪蟾卵母细胞内螺旋钙波和靶波的解析解。对于远离细胞中心的渐近情形，对应的螺旋波为Archimede型。由此得到的波速、波长和周期均与实验和数值模拟结果吻合。并指出IP3R失活常数必须大于2．43s，才能产生胞内周期钙波。     

运用爪蟾变态实验检测镉的甲状腺激素干扰效应

将NF51阶段非洲爪蟾(Xenopus laevis)蝌蚪暴露于Cd2+浓度为0.01,0.1和1mg.L-1的溶液中21d,观测Cd2+对爪蟾的生长、变态和甲状腺组织结构的影响.1mg.L-1Cd2+暴露组爪蟾蝌蚪与对照组相比,7d后,平均体重减少了0.39倍,体长和后肢长分别减少了0.17倍和0.27倍,发育阶段稍有延缓;21d后,1mg.L-1Cd2+暴露组爪蟾蝌蚪后肢长减小了0.11倍,延缓了1个发育阶段.其余各浓度组在7d和21d暴露后,体重、体长、后肢长和发育阶段与对照组无显著差异.21d后,Cd2+各浓度组甲状腺组织结构出现滤泡数目减少、胶质减少甚至空泡化等现象,1mg.L-1Cd2+暴露组甲状腺滤泡之间还出现溶通现象.结果表明CdCl2具有甲状腺激素干扰效应,并可能是由于该化合物直接作用于甲状腺组织引起.由此可见,爪蟾变态实验可以有效检测重金属盐的甲状腺激素干扰效应.     

爪蟾胚胎发生中角蛋白的表达

本文应用二株抗角蛋白单克隆抗体(MAb D10为抗爪蟾角蛋白,MAb AF5为抗人角蛋白)对爪蟾卵母细胞、受精卵、不同发育期胚胎作免疫转移印迹观察,研究其角蛋白多肽亚单位的组成。发现不同发育期胚胎角蛋白表达有变化。同时应用免疫组织化学染色技术研究角蛋白在胚胎中的分布。发现角蛋白分布于表皮、脊索、嗅板、垂体原基、粘腺、咽上皮、神经管内表面等部位。     

杭州西湖龙泓涧和北里湖水体及底泥毒性的生物学评价

以青海弧菌(Vibrio Qinghaiensis)、秀丽隐杆线虫(Caenorhabditis elegans)和热带爪蟾(Xenopus tropicalis)胚胎为研究对象,检测分析了龙泓涧和北里湖的水体及底泥的可能毒性.结果发现,绝大部分水样和底泥提取液对3种实验对象均未产生明显毒性效应.部分水体和底泥反而呈现出一定的正效应,表现为增强青海弧菌的发光强度,促进热带爪蟾胚胎发育.极个别水样对青海弧菌和秀丽线虫显示毒性,引起爪蟾胚胎轻微致畸效应,可能与污染物排放等偶然因素有关.结果表明,龙泓涧和北里湖的水体及底泥呈现较低的生态风险,西湖水体具有相对高的生物安全性.     

Rapid gating and anion permeability of an intracellular aquaporin

Aquaporin (AQP) water-channel proteins are freely permeated by water but not by ions or charged solutes. Although mammalian aquaporins were believed to be located in plasma membranes, rat AQP6 is restricted to intracellular vesicles in renal epithelia. Here we show that AQP6 is functionally distinct from other known aquaporins. When expressed in Xenopus laevis oocytes, AQP6 exhibits low basal water permeability; however, when treated with the known water channel inhibitor, Hg2+, the water permeability of AQP6 oocytes rapidly rises up to tenfold and is accompanied by ion conductance. AQP6 colocalizes with H+-ATPase in intracellular vesicles of acid-secreting alpha-intercalated cells in renal collecting duct. At pH less than 5.5, anion conductance is rapidly and reversibly activated in AQP6 oocytes. Site-directed mutation of lysine to glutamate at position 72 in the cytoplasmic mouth of the pore changes the cation/anion selectivity, but leaves low pH activation intact. Our results demonstrate unusual biophysical properties of an aquaporin, and indicate that anion-channel function may now be explored in a protein with known structure.     

Heteromultimeric channels formed by rat brain potassium-channel proteins

An important step towards understanding the molecular basis of the functional diversity of voltage-gated K+ channels in the mammalian brain has been the discovery of a family of genes encoding rat brain K+ channel-forming (RCK) proteins. All species of these RCK proteins form homomultimeric voltage-gated K+ channels with distinct functional characteristics in Xenopus laevis oocytes following injection of the respective cRNAs. RCK-specific mRNAs are coexpressed in several regions of the brain, suggesting that RCK proteins also assemble into heteromultimeric K+ channels. In addition expression experiments with fractionated poly(A)+ mRNA have suggested that heteromultimeric K+ channels may occur in mammalian brain. We report here that heteromultimeric K+ channels composed of two different RCK proteins (RCK1 and RCK4) assemble after cotransfection of HeLa cells with the corresponding cDNAs and after coinjection of the corresponding cRNAs into Xenopus oocytes. The heteromultimeric RCK1, 4 channel mediates a transient potassium outward current, similar to the RCK4 channel but inactivates more slowly, has a larger conductance and is more sensitive to block by dendrotoxin and tetraethylammonium chloride.     

Cloning by functional expression of a member of the glutamate receptor family

We have isolated a complementary DNA clone by screening a rat brain cDNA library for expression of kainate-gated ion channels in Xenopus oocytes. The cDNA encodes a single protein of relative molecular mass (Mr) 99,800 which on expression in oocytes forms a functional ion channel possessing the electrophysiological and pharmacological properties of the kainate subtype of the glutamate receptor family in the mammalian central nervous system.     

Messenger RNA from human brain induces drug- and voltage-operated channels in Xenopus oocytes

Sodium channels and receptors to serotonin and kainate were 'transplanted' from human brain into frog oocytes, by isolating messenger RNA from a fetal brain, and injecting it into Xenopus laevis oocytes. The mRNA was translated by the oocyte and induced the appearance of functional receptors and channels in its membrane. This approach renders drug- and voltage-operated channels of the human brain more amenable to detailed study.     

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.     

Expression of functional potassium channels from Shaker cDNA in Xenopus oocytes

The Shaker gene of Drosophila melanogaster encodes a potassium-selective ion channel, the 'A' channel, or one of its subunits. A single Shaker messenger RNA species suffices to direct the synthesis of functional A channels in Xenopus oocytes. Physiological characteristics of the A currents induced by two different mRNA species are compared.     

Functional characterization of a heteromeric NMDA receptor channel expressed from cloned cDNAs.

The glutamate receptor (GluR) channel plays a key part in brain function. Among GluR channel subtypes, the NMDA (N-methyl-D-aspartate) receptor channel which is highly permeable to Ca2+ is essential for the synaptic plasticity underlying memory, learning and development. Furthermore, abnormal activation of the NMDA receptor channel may trigger the neuronal cell death observed in various brain disorders. A complementary DNA encoding a subunit of the rodent NMDA receptor channel (NMDAR1 or zeta 1) has been cloned and its functional properties investigated. Here we report the identification and primary structure of a novel mouse NMDA receptor channel subunit, designated as epsilon 1, after cloning and sequencing the cDNA. The epsilon 1 subunit shows 11-18% amino-acid sequence identity with rodent GluR channel subunits that have been characterized so far and has structural features common to neurotransmitter-gated ion channels. Expression from cloned cDNAs of the epsilon 1 subunit together with the zeta 1 subunit in Xenopus oocytes yields functional GluR channels with high activity and characteristics of the NMDA receptor channel. Furthermore, the heteromeric NMDA receptor channel can be activated by glycine alone.     

New mammalian chloride channel identified by expression cloning.

Ion channels selectively permeable to chloride ions regulate cell functions as diverse as excitability and control of cell volume. Using expression cloning techniques, a complementary DNA from an epithelial cell line has been isolated, sequenced and its putative structure examined by site-directed mutagenesis. This cDNA, encoding a 235-amino-acid protein, gave rise to a chloride-selective outward current when expressed in Xenopus oocytes. The expressed, outwardly rectifying chloride current was calcium-insensitive and was blocked by nucleotides applied to the cell surface. Mutation of a putative nucleotide-binding site resulted in loss of nucleotide block but incurred dependence on extracellular calcium concentration. The unusual sequence of this putative channel protein suggests a new class of ion channels not related to other previously cloned chloride channels.     

Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor.

A variety of ligand-gated ion channels undergo a fast activation process after the rapid application of agonist and also a slower transition towards desensitized or inactivated closed channel states when exposure to agonist is prolonged. Desensitization involves at least two distinct closed states in the acetylcholine receptor, each with an affinity for agonists higher than those of the resting or active conformations. Here we investigate how structural elements could be involved in the desensitization of the acetylcholine-gated ion channel from the chick brain alpha-bungarotoxin sensitive homo-oligomeric alpha 7 receptor, using site-directed mutagenesis and expression in Xenopus oocytes. Mutations of the highly conserved leucine 247 residue from the uncharged MII segment of alpha 7 suppress inhibition by the open-channel blocker QX-222, indicating that this residue, like others from MII, faces the lumen of the channel. But, unexpectedly, the same mutations decrease the rate of desensitization of the response, increase the apparent affinity for acetylcholine and abolish current rectification. Moreover, unlike wild-type alpha 7, which has channels with a single conductance level, the leucine-to-threonine mutant has an additional conducting state active at low acetylcholine concentrations. It is possible that mutation of Leu 247 renders conductive one of the high-affinity desensitized states of the receptor.     

Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes

A complementary DNA encoding a voltage-gated chloride channel from Torpedo marmorata electric organ was cloned by expressing hybrid-depleted messenger RNA in Xenopus oocytes. The predicted protein has a sequence of 805 amino acids containing several putative membrane-spanning domains. Expression of the protein in Xenopus oocytes shows that it is sufficient for channel function.