神经递质释放过程中的可溶性NSF附着蛋白受体(SNARE)和相关蛋白

近年来,对突触小泡释放神经递质分子机制的研究迅速发展,发现了大量位于神经末梢的蛋白质.它们之间的相互作用与突触小泡释放神经递质相关,特别是位于突触小泡膜上的突触小泡蛋白/突触小泡相关膜蛋白(synaptobrevin/VAMP),位于突触前膜上的syntaxin和突触小体相关蛋白(synaptosome-associated protein of 25 ku),三者聚合形成的可溶性NSF附着蛋白受体(SNARE)核心复合体在突触小泡的胞裂外排、释放递质过程中有重要作用.而一些已知及未知的与SNARE蛋白有相互作用的蛋白质,可通过调节SNARE核心复合体的形成与解离来影响突触小泡的胞裂外排,从而可以调节突触信号传递的效率及强度,在突触可塑性的形成中起重要作用.     

突触小泡膜蛋白与神经递质释放的局部调节

突触小泡膜蛋白及其在神经递质释放过程中的作用已取得若干研究进展.突触素I、SY蛋白、SO蛋白、SB蛋白、SG蛋白等都是突触小泡膜的重要蛋白质,这些蛋白质在突触小泡的贴靠、膜融合及胞吐作用中起着局部自主性调节作用.     

胰岛素分泌及调节的分子机制

胰岛素是机体最重要的激素之一,它调节机体的血糖稳定、促进同化代谢、调节细胞的分裂分化和生长发育.胰岛β细胞的胰岛素分泌受到营养物质、神经递质和激素的精确调控.它们的作用部位可分为改变胞内第二信使物质水平的近端调节步骤（钙依赖性）,和直接作用于胞吐分子构件的末端调节步骤（钙非依赖性）.胰岛素的胞吐过程与神经递质的释放机制类似.葡萄糖等营养物质主要通过升高胞内的ATP/ADP比率,导致ATP敏感钾通道关闭、细胞膜去极化、钙内流这一途径增加胰岛素的分泌.神经递质和部分激素通过其G蛋白偶联受体-G蛋白系统的跨膜信号转换后,影响胞内IP₃、DAG、Ca²⁺等第二信使物质水平,主要通过PKA、PKC等蛋白激酶途径,调节胰岛素的分泌.胞内单体G蛋白参与了对囊泡运输和胞吐过程的调控,G蛋白也可能直接作用于胞吐过程,在分泌过程中发挥了重要的调节作用.     

神经递质释放机制的实验研究-即发态突触小泡假说

突触小泡（ＳｙｎａｐｔｉｃＶｅｓｉｃｌｅ）在神经递质释放过程中起关键性作用。采用膜片钳技术对突触前后细胞同步钳位研究了爪蟾胚胎神经元突触递质释放的过程,提出了如下小泡释放的假说：锚定在突触前膜的小泡中包含两类小泡：锚定态小泡和即发态小泡（即发态小泡定义为钙离子依赖性即时释放的小泡）。后者在动作电位到达时立即释放,而处于锚定态的小泡只有转换为钙离子依赖性释放的即发态时才能进入递质释放程序     

蛋白磷酸化去磷酸化与突触囊泡循环

神经递质合成酶、胞吐相关蛋白、神经递质受体,以及离子通道等蛋白的磷酸化和去磷酸化对神经系统的功能具有重要作用。神经递质的释放往往伴随众多蛋白的磷酸化或去磷酸化过程,包括突触蛋白磷酸化引起突触囊泡从细胞骨架上解离、突触囊泡通过复合体SNARE和Ca2 的介导与突触前膜发生锚靠、融合和神经递质释放,以及以网格蛋白依赖的形式实现突触囊泡从突触前膜上内陷、出芽和缢缩后,从膜上裂解到胞浆中重新形成突触囊泡。因此,蛋白磷酸化和去磷酸化对于神经系统完成神经信号传递具有重要的意义。     

Ca2+依赖和Ca2+不依赖的囊泡分泌研究进展

Ca2 是促发囊泡胞吐的关键调节因子.最近的研究表明,分泌囊泡和通道之间的空间距离调节囊泡分泌的过程和性质.Ca2 通道开口附近形成的Ca2 微区和Ca2 钠区和囊泡快速递质释放有非常紧密的联系.SNARE蛋白和钙离子传感器synaptotagmins等在触发分泌中起调控作用.同时另有一类不依赖于Ca2 的囊泡分泌存在.Latrotoxin和mastoparan等可以激活这一类不依赖于Ca2 的信号通路,从而触发囊泡释放.本文主要从ca2 对囊泡胞吐的调控作用着手,综述了Ca2 依赖和Ca2 不依赖的囊泡分泌过程和可能的调控机制.     

高锶离子亲和力传感蛋白介导CALYX突触囊泡异步和自发释放

突触囊泡在钙离子(Ca2+)触发下释放神经递质普遍存在着同步和异步两种形式.突触囊泡膜蛋白(synaptotagmin 2,Syt-2)已被证实是Calyx of Held突触囊泡同步释放的Ca2+传感蛋白,而相关的异步释放Ca2+传感蛋白还有待于探索.虽然锶离子(Sr2+)因其物理和化学性质都接近Ca2+,且能触发更多的囊泡异步释放成分而成为研究异步释放机制的常用工具,但有关Sr2+触发异步释放的机制存在着争议.本文在胞外以Sr2+替换Ca2+的条件下,通过对野生型(WT)和Syt-2敲除型(Z2B-/-)小鼠Calyx突触囊泡自发和诱发释放的电生理特性分析,发现Syt-2是介导Sr2+诱发的突触囊泡快速释放的传感蛋白,但不是介导Sr2+相关神经递质异步释放和自发释放的传感蛋白;而未知的触发囊泡异步释放的传感蛋白相比Syt-2对Sr2+具有更高的亲和力,同时也介导突触囊泡的自发释放.这一研究为探索并最终发现触发囊泡异步释放的未知传感蛋白提供了新的线索.     

质膜的损伤修复及相关模型

生物膜的磷脂双分子层将细胞与外界环境分开。大部分细胞会在机械损伤或化学应激下引发质膜损伤,如果不及时修复将会导致细胞死亡。胞外钙离子通过伤口进入细胞,作为损伤的最初信号,会诱发一系列的修复反应。随后,胞内细胞器也释放钙离子,并产生系列细胞行为来应对损伤,维护质膜的完整性。本文介绍了在损伤修复过程的胞吞作用、胞吐作用、胞外小泡脱落等细胞行为。综述了补丁模型、修复帽模型和大损伤修复的模型特点。补丁模型是最早的修复模型,提出后不断得到完善。细胞除了需要在损伤处聚集小泡、融合形成补丁外,还需通过胞吐、胞吞和出芽（小泡脱落）等方式参与伤口修复。本文简要介绍参与质膜修复的重要蛋白质如钙蛋白酶、dysferlin、MG53、膜联蛋白、突触结合蛋白(Syt-VⅡ)、ESCRTⅢ、酸性鞘磷脂酶、细胞骨架蛋白质等在修复过程中的作用。     

植物免疫应答过程中 Ca2 ＋及 CaM/CML 的功能

钙离子是一个多功能的第二信使,在植物响应各种生理刺激时,Ca2＋参与调节植物的多种生长发育和胁迫适应过程。在这些过程中,Ca2＋信号带有特异性标签,通过Ca2＋结合蛋白及其下游靶蛋白感知不同刺激并翻译成响应的细胞反应。钙调素（CaM）和钙调素类蛋白（CML）是Ca2＋主要感受器,通过调节不同靶蛋白的活性调控多种细胞功能。最近在植物对抗病原菌的防卫反应中有关Ca2＋／CaM信号转导系统的研究取得了一定进展。重点关注植物免疫应答过程中受CaM／CML调控的信号组分的研究,包括参与Ca2＋信号产生和Ca2＋依赖的表达基因组分调控。     

细胞胞吐的分子基础

近年来对神经末梢突触小泡膜蛋白的蛋白质化学,药理学研究及酵母细胞蛋白运输及分泌的遗传学的研究,发现了胞吐蛋白复合体的存在。ＳＮＡＲＥ假说的提出使得对神经递质释放,内外分泌细胞分泌机制的研究有了突飞猛进的进展。     

Trk is a calmodulin-binding protein: implications for receptor processing

The tyrosine kinase receptors for the neurotrophins (Trk) are a family of transmembrane receptors that regulate the differentiation and survival of different neuronal populations. Neurotrophin binding to Trk leads to the activation of several signalling pathways including a rapid, but moderate, increase in intracellular calcium levels. We have previously described the role of calcium and its sensor protein, calmodulin, in Trk-activated intracellular pathways. Here we demonstrate that calmodulin is able to precipitate TrkA from PC12 cell lysates. Using recombinant GST-fusion proteins containing the complete intracellular domain of TrkA, or fragments of this region, we show that calmodulin binds directly to the C-terminal domain of TrkA in a Ca2+-dependent manner. We have also co-immunoprecipitated endogenous Trk and calmodulin in primary cultures of cortical neurones. Moreover, we provide evidence that calmodulin is involved in the regulation of TrkA processing in PC12 cells. Calmodulin inhibition results in the generation of a TrkA-derived p41 fragment from the cytosolic portion of the protein. This fragment is autophosphorylated in tyrosines and can recruit PLCgamma and Shc adaptor proteins. These results suggest that calmodulin binding to Trk may be important for the regulation of Trk intracellular localization and cleavage.     

Neurotransmitters as ion shuttles in regulation of neural activity.

A molecular framework is described within which a single neural cell can modulate its excitability, or the quantity of transmitter released upon stimulation, in relationship to past stimulation. The key elements in this regulating system are complexation in the synaptic area by the transmitter molecule with extracellular ions, interaction of the complexed transmitter with the presynaptic receptor, possibly followed by reuptake of complexed transmitter.A number of mechanisms are suggested by which the transmitter/metal ion complex can regulate cellular function. Calculations are made to estimate the possible degree of change in the interior calcium concentration of a catecholaminergic cell by a calcium ion complex formed in the synapse. Experimental evidence is cited, which(a) documents the existence in the catecholaminergic cell of the necessary machinery for a calcium-ion-regulated cell-use registry device.(b) supports the hypothesis that catecholamines transport metal ions in neural systems, and(c) indicates that the ionic shuttle function of neurotransmitters plays a significant, but not exclusive, role in the transport of calcium. Calcium transported in this manner may be uniquely distinguishable from that derived from other sources of intracellular calcium in its temporal or spatial distribution. The existing evidence is discussed and rationalized with respect to the hypothesis that one of the chief presynaptic functions of many neurotransmitters is to feedback regulate cell function by performance as an ion shuttle.     

植物感受盐胁迫及相关钙信号的研究进展

盐胁迫对植物的生长和发育造成严重影响,其危害包括渗透胁迫、离子毒害等,严重损害了农业生产和粮食安全。在盐胁迫下,植物相关感受器接受刺激,使得Ca²⁺通过细胞膜以及细胞内钙库膜上打开的Ca²⁺通道进入细胞质基质,导致细胞质内Ca²⁺浓度升高,产生钙信号。钙离子作为重要的第二信使,在植物细胞内和细胞间传递信号,信号往下游传递,在不同生长和发育阶段引起植物一系列的生理响应来应对盐胁迫影响。钙信号主要通过钙调蛋白（CaM）、钙调素样蛋白（CML）、钙依赖性蛋白激酶（CDPK）、钙调磷酸酶B样蛋白（CBL）和CBL互作蛋白激酶（CIPK）感知并将特异的钙信号信息传递到下游;从而激活植物盐胁迫生理响应。本文主要综述植物如何感知盐胁迫刺激,以及钙信号产生与传导机制,并对该研究领域需解决的问题进行了展望。     

Control of erythrocyte shape by calmodulin

Erythrocytes are deformable cells whose shapes can be altered by treatments with a variety of drugs. The forms the erythrocyte may assume vary continuously from the spiny "echinocytes" or crenated cells at one extreme to highly folded and dented "cupped" cells at the other extreme. Examination of 39 compounds for cup-forming activity revealed a remarkable correlation between their ability to form cupped cells and their inhibitory activity against the calcium regulatory protein, calmodulin. Calmodulin is known to interact with several erythrocyte proteins including spectrin, spectrin kinase, and the Ca++ ATPase calcium pump of the membrane. These proteins regulate the form of the cytoskeleton as well as intracellular calcium and ATP levels. It is proposed that calmodulin is required to maintain normal erythrocyte morphology and that in the presence of calmodulin inhibitors, the cell assumes a cupped shape.     

Compartmentalization of the submembrane calcium activity during calcium influx and its significance in transmitter release.

Quantitative modeling indicates that, in presynaptic terminals, the intracellular calcium concentration profile during inward calcium current is characterized by discrete peaks of calcium immediately adjacent to the calcium channels. This restriction of intracellular calcium concentration suggests a remarkably well specified intracellular architecture such that calcium, as a second messenger, may regulate particular intracellular domains with a great degree of specificity.     

Role of the growth-associated protein B-50/GAP-43 in neuronal plasticity

The neuronal phosphoprotein B-50/GAP-43 has been implicated in neuritogenesis during developmental stages of the nervous system and in regenerative processes and neuronal plasticity in the adult. The protein appears to be a member of a family of acidic substrates of protein kinase C (PKC) that bind calmodulin at low calcium concentrations. Two of these substrates, B-50 and neurogranin, share the primary sequence coding for the phospho- and calmodulin-binding sites and might exert similar functions in axonal and dendritic processes, respectively. In the adult brain, B-50 is exclusively located at the presynaptic membrane. During neuritogenesis in cell culture, the protein is translocated to the growth cones, i.e., into the filopodia. In view of many positive correlations between B-50 expression and neurite outgrowth and the specific localization of B-50, a role in growth cone function has been proposed. Its phosphorylation state may regulate the local intracellular free calmodulin and calcium concentrations or vice versa. Both views link the B-50 protein to processes of signal transduction and transmitter release.     

Inhibition of mitosis in PtK2 cells by CAPP1-calmodulin

Various indirect evidence has indicated that calcium ions and the calcium-binding regulator protein, calmodulin, may regulate mitosis in higher eukaryotes. We have used the competitive antagonist, CAPP1-calmodulin, to antagonize intracellular calmodulin and test the hypothesis that calmodulin serves as a regulator of mitosis. We find that CAPP1-calmodulin inhibits the transit of cells through metaphase at estimated intracellular concentrations up to that of native calmodulin; beyond that level, the inhibition of mitosis vanishes. The membrane-permeant anticalmodulin agents, W7 and calmidazolium, also inhibit the progress of cells through metaphase. The similarity of the inhibitory curves for CAPP1-calmodulin, W7, and calmidazolium suggests that all these agents inhibit mitosis by antagonizing intracellular calmodulin. In order to test whether this inhibition of metaphase transit is due to an effect of the agents on intracellular free calcium, we used the calcium indicator Fura-2 to measure intracellular calcium levels after CAPP1-calmodulin injection or during calmidazolium treatment. We found that, while intracellular calcium levels are modestly elevated during calmidazolium treatment, they were unaffected by CAPP1-calmodulin, a result suggesting that mitosis inhibition was not due to an effect on intracellular free calcium. The reasons for the anomalous dose-response behavior of these drugs are not known; however, the behavior of cells at drug levels below the point of anomaly supports the hypothesis that calmodulin acts as a regulator of mitosis in these cells.     

A compact intermediate state of calmodulin in the process of target binding

Calmodulin undergoes characteristic conformational changes by binding Ca(2+), which allows it to bind to more than 300 target proteins and regulate numerous intracellular processes in all eukaryotic cells. We measured the conformational changes of calmodulin upon Ca(2+) and mastoparan binding using the time-resolved small-angle X-ray scattering technique combined with flash photolysis of caged calcium. This measurement system covers the time range of 0.5-180 ms. Within 10 ms of the stepwise increase in Ca(2+) concentration, we identified a distinct compact conformational state with a drastically different molecular dimension. This process is too fast to study with a conventional stopped-flow apparatus. The compact conformational state was also observed without mastoparan, indicating that the calmodulin forms a compact globular conformation by itself upon Ca(2+) binding. This new conformational state of calmodulin seems to regulate Ca(2+) binding and conformational changes in the N-terminal domain. On the basis of this finding, an allosteric mechanism, which may have implications in intracellular signal transduction, is proposed.     

Old proteins,developing roles: The regulation of calcium channels by synaptic proteins

Coupling of presynaptic voltage-gated calcium channels to synaptic release machinery is critical for neurotransmission. It was traditionally believed that anchoring calcium channels close to the calcium micro-domain dependent release machinery was the main reason for the physical interactions between channels and synaptic proteins, however in recent years, it is becoming clear that these proteins additionally regulate channel activity, and such processes as channel targeting and alternative splicing, to orchestrate a much broader regulatory role in controlling calcium channel function, calcium influx, and hence neurotransmission. Calcium signalling serves a multitude of cellular functions and therefore requires tight regulation. Specific, often calcium-dependent interactions between synaptic proteins and calcium channels appear to play a significant role in fine-tuning of the synaptic response over development. While it is clear that investigation of a few of the multitude of synaptic proteins will not provide a complete understanding of calcium channel regulation, consideration of the emerging mechanisms by which synaptic protein interactions might regulate calcium channel function is important in order to understand their possible contributions to synaptic transmission. Here, we review the current state of knowledge of the molecular mechanisms by which synaptic proteins regulate presynaptic calcium channel activity.     

Old proteins, developing roles: The regulation of calcium channels by synaptic proteins

Coupling of presynaptic voltage-gated calcium channels to the synaptic release machinery is critical for neurotransmission. It was traditionally believed that anchoring calcium channels close to the calcium microdomain dependent release machinery was the main reason for the physical interactions between channels and synaptic proteins, however in recent years, it is becoming clear that these proteins additionally regulate channel activity, and such processes as channel targeting and alternative splicing, to orchestrate a much broader regulatory role in controlling calcium channel function, calcium influx and hence neurotransmission. Calcium signalling serves a multitude of cellular functions and therefore requires tight regulation. Specific, often calcium-dependent interactions between synaptic proteins and calcium channels appear to play a significant role in fine-tuning of the synaptic response over development. While it is clear that investigation of a few of the multitude of synaptic proteins will not provide a complete understanding of calcium channel regulation, consideration of the emerging mechanisms by which synaptic protein interactions might regulate calcium channel function is important in order to understand their possible contributions to synaptic transmission. Here, we review the current state of knowledge of the molecular mechanisms by which synaptic proteins regulate presynaptic calcium channel activity.