Ca^<2+> Channels at the Presynaptic Terminal; Distributions and Functions

1. cacophony（cac）缺失突变体胚胎中的突触传递研究表明，由cacophony（cac）基因编码的Ca^<2 +>内流通过果蝇N型Ca^<2+>通道触发快速突触传递。剩下的问题是：cac Ca^<2+>通道是快速突触传递的唯一Ca^<2+>通道吗？由于cac^<null>突变是致命的，这个问题只能通过检查cac^胚胎中的突触传递来回答<null>。在cac神经<null>肌肉接头（NMJ），神经刺激时未检测到快速同步突触传递，而观察到罕见的延迟量子突触事件。当野生型cac基因被引入cac^背景中时<null>，快速突触传递恢复到杂合对照胚胎（cac^<null>/+）的50%。因此，cac Ca^2+通道对于快速突触传递是必需的和充分的。出乎意料的是，即使是在CAC胚胎中<null>，神经刺激也会诱导延迟性流产。 关于我们 在HL 3溶液（1.5 mM Ca^2+）中少数细胞和在5 mM [Ca^2+]_e中检测的所有细胞中突触囊泡的e。这些延迟事件在刺激后约8 ms开始出现，频率迅速增加至约13 ms的峰值，并在约80 ms后逐渐降低至基线。（一种蜘蛛毒素类似物）表明电压门控Ca^&lt;2+&gt;通道，而不是cac Ca^&lt;2+&gt;通道，有助于延迟释放，但不受50 μM La^&lt;3+&gt;的影响，La ^&lt;3+&gt;优先阻断果蝇突触前末梢的一种类型的Ca^&lt;2+&gt;通道。综上所述，cac Ca^&lt;2+&gt;通道是快速突触传递的唯一Ca^&lt;2+&gt;通道，而另一种类型的Ca^&lt;2+&gt;通道，即非cac、PLTXII敏感的Ca^&lt;2+&gt;通道，有助于延迟Ca^^胚胎中的递质释放<null>。利用表达EGFP标记的cac Ca^2+通道的果蝇细胞，我们已经可视化了突触前末梢处的通道簇的分布。通过检测荧光强度的漂白过程来估计每个簇中Ca^&lt;2+&gt;的数量。在共聚焦激光显微镜的线扫描模式下，由于漂白，团簇的荧光强度下降。在这个过程中，我们发现强度下降的步骤，这表明每个EGFP分子的贡献。通过计算步骤的数量，我们应该能够估计每个簇中Ca^2+通道的数量。事实证明，这个数字相对较小，约为10，与以前使用完全不同的方法在其他系统中获得的结果一致。这些信息对于模拟每个释放位点的胞吐过程是必不可少的。少

1.Synaptic transmission in cacopholy(cac)-null mutant embryosIt has been demonstrated that Ca^<2+> influx through Drosophila N-type Ca^<2+> channels, encoded by the cacophony (cac) gene, triggers fast synaptic transmission. A remaining question is ; Is the cac Ca^<2+> channel the sole type of Ca^<2+> channel for fast synaptic transmission? Since the cac^<null> mutation is lethal, this question can only be answered by examining synaptic transmission in cac^<null> embryos. At the cac^<null> neuromuscular junction (NMJ), no fast synchronous synaptic transmission was detected upon nerve stimulation while infrequent delayed quantal synaptic events were observed. When the wild-type cac gene was introduced in the cac^<null> background, fast synaptic transmission recovered to 50% of heterozygous control embryos (cac^<null>/+). Thus, cac Ca^<2+> channels are necessary and sufficient for fast synaptic transmission. Unexpectedly, even in cac^<null> embryos nerve stimulation induced delayed releas … More e of synaptic vesicles in the minority of cells in HL3 solution (1.5 mM Ca^<2+>) and all cells examined in 5 mM [Ca^<2+>]_e. These delayed events started to appear at around 8 ms after stimulation, quickly increased in frequency to a peak at around 13 ms and gradually decreased to the baseline after 〜80 ms. The delayed events were abolished by 10 nM PLTXII (a spider toxin analog) suggesting that voltage-gated Ca^<2+> channels, other than cac Ca^<2+> channels, are contributing to the delayed release, but were not affected by 50 μM La^<3+> that preferentially blocks one type of Ca^<2+> channel in the Drosophila presynaptic terminal. Taken together, the cac Ca^<2+> channel is the sole Ca^<2+> channel for fast synaptic transmission and another type of Ca^<2+> channel, non-cac, PLTXII-sensitive Ca^<2+> channel, is contributing to delayed transmitter release in cac^<null> embryos.2.Distribution of cac Ca^<2+> channel clusters and the number of channels in each cluster at the presynaptic terminal.Using a Drosophila transformant that expresses EGFP-tagged cac Ca^<2+> channels, we have visualized the distribution of channel clusters at the presynaptic terminal. The number of Ca^<2+> in each cluster was estimated by examining the bleaching process of fluorescence intensity. In the line scan mode of confocal laser microscopy, the fluorescence intensity of a cluster declined due to bleaching. During this process, we found that the intensity declines in steps, which indicates a contribution of each EGFP molecule. By counting the number of steps we should be able to estimate the number of Ca^<2+> channels in each cluster. It turned out that the number is relatively small, around 10, in agreement with previous results in other systems obtained using completely different methods. This information is indispensable for simulation of the process of exocytosis at each release site. Less