神经肌接头上突触前乙酰胆碱自受体及其意义

在神经肌接头上存在着突触前ｎ型和ｍ型乙酰胆碱自受体，两者可能都既介导下在反馈也介导负反馈调节。ｎ型受体与书知的ｎ１和ｎ２受体不同，ｍ型受体与ｍ１型受体类似，突触前乙酰胆碱自受体介导的反馈调节如何影响递质释放尚待阐明。     

高血压与突触后肾上腺素受体

高血压发病过程中伴随着肾上腺素受体的变化,其中α２受体性质和功能的改变可能与高血压发病的遗传因素有关,而β受体功能下降与高血压的维持和发展有一定关系。     

突触前受体

外周和中枢的各类神经元几乎普遍存在着突触前受体,它受到生理学、药理学及临床医学工作者的广泛重视。本文就突触前受体的概念、确证和意义,以及已发现的突触前受体的概况作一简要概述。     

乙酰胆碱受体与自身免疫疾病

乙酰胆碱受体 (ＡｃｈＲ)是一个配体介导的离子通道蛋白。由 5个同源亚基组成 ,亚基在内质网组成一个环 ,其中 β亚基参与组装序列 ,而未组装的亚基易被降解。肌肉乙酰胆碱受体是神经传导的重要媒介 ,是神经突触处化学信号与电信号相互转换的关键物质。通常肌体内不产生乙酰胆碱受体的抗体 ,但重症肌无力患者由于自身免疫系统紊乱 ,该抗体与乙酰胆碱结合 ,削弱了骨骼肌间的神经传导 ,引起肌无力。因为肌无力症具有典型的自身免疫疾病特点 ,因而对它的研究与自身免疫疾病紧密相关。1 .肌肉乙酰胆碱受体与自体免疫应答反应早在 6 0年代 ,科学…     

AMPA受体的磷酸化与突触可塑性

突触可塑性在学习记忆中发挥了重要作用,AMPA(α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid,AMPA)受体功能和运输的调节是突触可塑性机制研究的重要环节。在突触可塑性发生过程中,激酶和磷酸酶能够调节AMPA受体C末端的磷酸化水平,进而影响AMPA受体运输。对于AMPA受体磷酸化的研究能够加深我们对突触可塑性机制的理解。     

乙酰胆碱对培养大鼠皮层神经元兴奋性及抑制性突触电流的相反作用

在分散培养的新生大鼠皮层神经元标本上,用全细胞电压箝技术分析了自发兴奋性及抑制性突触后电流的性质并观察了乙酰胆碱对它们的影响。     

烟碱型乙酰胆碱受体参与乙酰胆碱调控的气孔运动

动物细胞中 ,乙酰胆碱功能的发挥要求乙酰胆碱受体的参与 ,烟碱型受体的激活剂可以直接影响膜对离子的通透性 .在乙酰胆碱诱导的气孔开放过程中 ,可能同样涉及到烟碱型受体的作用 ,药理学的证据表明烟碱型乙酰胆碱受体参与乙酰胆碱调控的气孔运动 ,而且烟碱型乙酰胆碱受体介导的气孔开放与介质中的离子组成密切相关 ,只有在含K⁺的介质中烟碱才可以诱导气孔开放而在含Ca^{2 +}的介质中没有作用 ;同样 ,烟碱型乙酰胆碱受体的抑制剂只有在含K⁺的介质中才能抑制乙酰胆碱诱导的气孔开放 .进一步利用荧光定位技术证明烟碱型受体存在于蚕豆气孔保卫细胞中 ,而且主要分布在保卫细胞原生质体的表面 .免疫印迹实验初步证明在保卫细胞原生质体的微粒体中存在着能与动物烟碱型乙酰胆碱受体的α和β亚基发生免疫交叉反应的蛋白条带 .以上结果表明烟碱型乙酰胆碱受体存在于保卫细胞中 ,而且介导了乙酰胆碱诱导的气孔在含K+介质中的开放 .     

烟碱样乙酰胆碱受体的离子通道特性

烟碱样乙酰胆碱受体(AChR)是一种配基门控性离子通道,由5个亚单位(α_2βγα)构成。利用非洲蟾蜍卵母细胞的表达系统可以研究AChR的通道特性和各亚单位所起的作用。电鳗电器官AChR和小牛肌AChR之间门控特性的差别,主要是由δ亚单位决定的;而小牛肌成年型AChR与胚胎型AChR之间的差别,则由ε亚单位决定。     

The actin-driven movement and formation of acetylcholine receptor clusters

A new method was devised to visualize actin polymerization induced by postsynaptic differentiation signals in cultured muscle cells. This entails masking myofibrillar filamentous (F)-actin with jasplakinolide, a cell-permeant F-actin-binding toxin, before synaptogenic stimulation, and then probing new actin assembly with fluorescent phalloidin. With this procedure, actin polymerization associated with newly induced acetylcholine receptor (AChR) clustering by heparin-binding growth-associated molecule-coated beads and by agrin was observed. The beads induced local F-actin assembly that colocalized with AChR clusters at bead-muscle contacts, whereas both the actin cytoskeleton and AChR clusters induced by bath agrin application were diffuse. By expressing a green fluorescent protein-coupled version of cortactin, a protein that binds to active F-actin, the dynamic nature of the actin cytoskeleton associated with new AChR clusters was revealed. In fact, the motive force generated by actin polymerization propelled the entire bead-induced AChR cluster with its attached bead to move in the plane of the membrane. In addition, actin polymerization is also necessary for the formation of both bead and agrin-induced AChR clusters as well as phosphotyrosine accumulation, as shown by their blockage by latrunculin A, a toxin that sequesters globular (G)-actin and prevents F-actin assembly. These results show that actin polymerization induced by synaptogenic signals is necessary for the movement and formation of AChR clusters and implicate a role of F-actin as a postsynaptic scaffold for the assembly of structural and signaling molecules in neuromuscular junction formation.     

Ultrastructure of acetylcholine receptor clusters on cultured muscle fibers

The structure of regions with a high concentration of ACh receptors (clusters) on cultured skeletal muscle myotubes was examined by immunoperoxidase staining of bound alphaBT. The clusters did not appear to differ from the other regions except in their higher concentration of receptor.     

Actin at receptor-rich domains of isolated acetylcholine receptor clusters

Acetylcholine receptor (AChR) clusters of cultured rat myotubes, isolated by extraction with saponin (Bloch, R. J., 1984, J. Cell Biol. 99:984-993), contain a polypeptide that co-electrophoreses with purified muscle actins. A monoclonal antibody against actin reacts in immunoblots with this polypeptide and with purified actins. In indirect immunofluorescence, the antibody stains isolated AChR clusters only at AChR domains, strips of membrane within clusters that are rich in receptor. It also stains the postsynaptic region of the neuromuscular junction of adult rat skeletal muscle. Semiquantitative immunofluorescence analyses show that labeling by antiactin of isolated analyses show that labeling by antiactin of isolated AChR clusters is specific and saturable and that it varies linearly with the amount of AChR in the cluster. Filaments of purified gizzard myosin also bind preferentially at AChR-rich regions, and this binding is inhibited by MgATP. These experiments suggest that actin is associated with AChR-rich regions of receptor clusters. Depletion of actin by extraction of isolated clusters at low ionic strength selectively releases the actin-like polypeptide from the preparation. Simultaneously, AChRs redistribute within the plane of the membrane of the isolated clusters. Similarly, brief digestion with chymotrypsin reduces immunofluorescence staining and causes AChR redistribution. Treatments that deplete AChR from clusters in intact cells also reduce immunofluorescent staining for actin in isolated muscle membrane fragments. Upon reversal of these treatments, cluster reformation occurs in regions of the membrane that also stain for actin. I conclude that actin is associated with AChR domains and that changes in this association are accompanied by changes in the organization of isolated AChR clusters.     

Lipid domains of acetylcholine receptor clusters detected with saponin and filipin

The acetylcholine receptor (AChR) clusters of cultured rat myotubes contain two distinct, interdigitating, membrane domains, one enriched in AChR, the other poor in AChR but associated with sites of myotube- substrate contact (Bloch, R.J., and B. Geiger, 1980, Cell, 21:25-35). We have used two cholesterol-specific cytochemical probes, saponin and filipin, to investigate the lipid nature of these membrane domains. When studied with freeze-fracture electron microscopy or fluorescence microscopy, these reagents reacted moderately and preferentially with the AChR-rich domains of AChR clusters. Little or no reaction with the membrane in "contact" domains was seen. In contrast, membrane regions surrounding the AChR clusters reacted extensively with filipin. These results suggest that, in rat myotubes, the composition or the state of the lipids differs between the two membrane domains of the AChR clusters, and between clusters and surrounding membrane. In chick myotubes, AChR clusters do not appear to react with filipin or saponin, although surrounding membrane reacts extensively with these reagents.     

Participation of calcium and calmodulin in the formation of acetylcholine receptor clusters

We recently reported the detection of multiple classes of calmodulin-binding proteins in subcellular fractions of chicken embryo fibroblasts by using a gel binding procedure (Van Eldik, L.J., and W.H. Burgess, 1983, J. Biol. Chem., 258:4539-4547). In this report we identify many of these calmodulin-binding proteins and provide further evidence for the existence of multiple classes of calmodulin-binding proteins based on the interaction of these proteins with calmodulin and other calcium-modulated proteins. The fact that, in some cases, the same calmodulin-binding protein can bind troponin C and S100 alpha suggests that similar functional domains may be present in these distinct calcium-modulated proteins. We also have used protocols based on purification steps for calmodulin-binding proteins and calmodulin-regulated activities from other systems, in conjunction with enzymatic assays and various immunological methods, to identify many of the calmodulin-binding proteins in chicken embryo fibroblasts. The identities of these proteins suggest in vivo roles for calmodulin in the regulation of cell shape and motility, cyclic nucleotide metabolism, and possibly nucleic acid and protein turnover in fibroblasts.     

Formation of acetylcholine receptor clusters at neuromuscular junction in Xenopus cultures

The formation of acetylcholine receptor (AChR) clusters at the neuromuscular junction was investigated by observing the sequential changes in AChR cluster distribution on cultured Xenopus muscle cells. AChRs were labeled with tetramethylrhodamine-conjugated alpha-bungarotoxin (TMR-alpha BT). Before innervation AChRs were distributed over the entire surface of muscle cells with occasional spots of high density (hot spots). When the nerve contacted the muscle cell, the large existing hot spots disappeared and small AChR clusters (less than 1 micron in diameter) initially emerged from the background along the area of nerve contact. They grew in size, increased in number, and fused to form larger clusters over a period of 1 or 2 days. Receptor clusters did not migrate as a whole as observed during "cap" formation in B lymphocytes. The rate of recruitment of AChRs at the nerve-muscle junction varied from less than 50 binding sites to 1000 sites/hr for alpha BT. In this study the diffusion-trap mechanism was tested for the nerve-induced receptor accumulation. The diffusion coefficient of diffusely distributed AChRs was measured using the fluorescence photobleaching recovery method and found to be 2.45 X 10(-10) cm2/sec at 22 degrees C. There was no significant difference in these values among the muscle cells cultured without nerve, the non-nerve-contacted muscle cells in nerve-muscle cultures, and the nerve-contacted muscle cells. It was found that the diffusion of receptors in the membrane is not rate-limiting for AChR accumulation.     

Microtubules and the formation of acetylcholine receptor clusters in chick embryonic muscle cells

We have used the microtubule-stabilizing drug taxol to examine the relationship between microtubules and the appearance and cell surface distribution of acetylcholine receptors (AChRs) in primary cultures of chick embryonic muscle cells. Taxol at a 5-microM concentration induced the large scale polymerization of tubulin in muscle cells that was most obvious as intermittent bundles of microtubules along the myotube. Prominent bundles of microtubules were also clearly visible in the fibroblasts. This concentration of taxol had no significant effect on the incorporation rate, increased synthesis induced by brain extract or the total cell surface number of AChRs measured over a 24-h period. Thus, excess polymerization of microtubules does not affect the movement of receptors to the cell surface. However, when cell surface AChR distribution was examined using rhodamine-conjugated alpha-bungarotoxin, taxol treatment of myotubes was shown to induce the aggregation of receptors. If receptors were labeled before taxol addition, aggregation of these prelabeled receptors was also seen, a result indicating that taxol can induce the movement of receptors already in the membrane. We believe this evidence further implicates microtubules as being involved in the movement of these cell surface receptors in the plane of the myotube membrane.     

Microfilaments and actin-associated proteins at sites of membrane-substrate attachment within acetylcholine receptor clusters

Rat myotubes in tissue culture form broad areas of close contact with the substrate. These areas often display two distinct, interdigitating sets of membrane domains. One, the "contact domain", is close to the substrate; the other, termed the "AChR domain", is further from the substrate and is rich in acetylcholine receptors (AChR). We have used fluorescence techniques to study the organization of the cytoskeleton in these areas. Substrate-apposed membrane of the myotubes was exposed either by shearing or by permeabilizing the cells with a neutral detergent. Phalloidin derivatives and affinity-purified polyclonal or monoclonal antibodies specific for cytoskeletal proteins were then applied to the samples. Sheared samples were observed by epifluorescence microscopy; detergent-permeabilized samples were observed by total internal reflection fluorescence microscopy. We found that, like antivinculin, fluorescent phalloidin derivatives and antibodies to alpha-actinin, filamin, and talin preferentially labeled the contact domains. This suggests that bundles of microfilaments associate with the membrane at sites of myotube-substrate attachment. In contrast, a 43K protein, closely associated with AChR, was present only at AChR domains. A monoclonal antibody to actin labeled both AChR and contact domains, suggesting that actin is enriched over both regions. Our results suggest that, like the plasma membrane of AChR clusters, the underlying membrane skeleton is organized into at least two distinct domains.     

Reversible loss of acetylcholine receptor clusters at the developing rat neuromuscular junction

Exposure of sternomastoid muscles excised from 16-day embryonic rats to medium depleted of Ca²⁺ or containing high concentrations of KCl leads to extensive loss of aggregates of acetylcholine receptors newly formed at the motor end plate region. Upon restoration of Ca²⁺ or removal of excess KCl, receptor accumulations reappear in the central regions of about one-third of the muscle fibers. This susceptibility of junctional AChR aggregates lasts only a short while during development of the neuromuscular junction. By the time of birth, end plate receptor aggregates have become resistant to these treatments.