Molecular Genetic Dissection of Synaptic Transmission

为了进行突触传递，含有神经递质的突触前末端的囊泡必须融合到膜上，并将内容释放到突触间隙。已知多种蛋白质参与了这种融合过程。一个假说，圈套假说，被提出来解释这些蛋白质的排列。我们利用缺乏关键分子的果蝇突变幼虫研究了这一过程的分子机制。神经元突触缩短蛋白(n-syb)是囊泡膜蛋白，也是破伤风毒素的靶标。在缺乏n-syb的突变幼虫中，没有观察到神经诱发的突触电流。然而，微小突触电流(MSC)仍保持在较低的频率。在高钾盐溶液中，味精的产生频率依赖于外界的钙离子浓度。此外，黑寡妇蜘蛛蛇毒提取物和钙离子载体A23187使MSC频率增加。这些结果表明，n-syb突变体中剩余的MSC是钙依赖的。在野生型…中腺苷环化酶激动剂Forsklin或cAMP的膜通透性类似物db-cAMP使更多的幼虫突触前神经末梢的cAMP升高，导致MSC的增加。然而，在n-syb突变体中，这两种药物都没有增加MSG的频率。因此，我们得出结论：囊泡融合的n-syb非依赖途径对cAMP不敏感(Yoshihara et at.，1999)。半胱氨酸串蛋白(CSP)也是囊泡蛋白，被认为与末端膜上的电压门控钙通道相互作用。在CSP的温度敏感突变体中，在不允许的温度(32ﾟC)下没有观察到神经诱发的突触电流。这可能是由于高温下电压门控钙通道的功能缺失所致。为了测试这种可能性，我们用一种对钙敏感的染料来观察末端的钙离子。在室温下，重复刺激神经可使细胞内钙离子浓度升高。然而，在不允许的温度下，钙通道信号显著减弱，提示钙通道功能失效。由于CSP位于突触小泡膜上，因此可能钙通道必须与突触小泡相联系才能正常工作。为了测试这一可能性，我们使用了另一个名为shibire的突变体，在该突变体中，内吞作用在不允许的温度下被阻止。我们还在Shibire中发现，在不允许的温度下，Ca^&lt；2+&gt；信号较低。总之，为了使突触前终末中的电压门控钙通道正常工作，小泡可能必须对接到释放部位(Umbach等人，1998)。较少

For synaptic transmission vesicles in the presynaptic terminal containing neurotransmitter have to fuse to the membrane and release the content to the synaptic cleft. Various proteins are known to be involved in this fusion process. A hypothesis, SNARE hypothesis, is proposed to account for the arrangement of those proteins. We have investigated the molecular mechanism of this process by using Drosophila mutant larvae which lack key molecules.Neuronal synaptobrevin (n-syb) is vesicle membrane protein and a target of tetanus toxin. In mutant larvae which lack n-syb no nerve-evoked synaptic currents were observed. However, miniature synaptic currents (mSC) remained in lower frequencies. The frequency of mSG's was dependent on external Ca^<2+> concentrations in high potassium saline. Furthermore, the mSG frequency increased with black widow spider venom extract and Ca^<2+> ionophore, A23187.These results indicate that the remaining mSC's in n-syb mutants are Ca^<2+> dependent. In wild typ … More e larvae an elevation of cAMP in the presynaptic nerve terminal either by an activator of adenylate cyclase, forskolin, or by a membrane permeable analog of cAMP, db-cAMP, resulted in an increase in msc's. However, in n-syb mutants neither of these drugs increased the mSG frequency. Thus we conclude that the n-syb independent pathway for vesicle fusion is insensitive to cAMP (Yoshihara et at., 1999).Cysteine-string protein (csp) is also vesicle protein and supposed to interact with voltage-gated Ca^<2+> channels in the terminal membrane. In a temperature sensitive mutant of csp no nerve-evoked synaptic currents were observed in non-permissive temperature (32 ﾟC). This could be due to lack of function of voltage-gated Ca^<2+> channels at high temperature. To test this possibility we visualized terminal Ca^<2+> by a Ca^<2+> sensitive dye. At room temperature the Ca^<2+> concentration increase upon repetitive stimulation of nerve. However, at non-permissive temperature the Ca^<2+> signal decreased significantly, thus suggesting a failure of Ca^<2+> channel function.Since csp is on the synaptic vesicle membrane it is possible that Ca^<2+> channels have to be associated with synaptic vesicles to operate properly. To test this.possibility we used another mutant call shibire in which endocytosis is blocked at non-permissive temperature. We also found in shibire that Ca^<2+> signals were lower in non-permissive temperature. In conclusion, for voltage-gated Ca^<2+> channels in the presynaptic terminal to work properly vesicles might have to be docked to release sites (Umbach et al., 1998). Less