The Neuronal Priming Complex

描述(申请人提供)：尽管我们在了解神经元中依赖钙离子的胞吐作用方面已经取得了很大进展，但对钙离子和胞吐作用之间的关系仍不是很清楚。对这一过程的准确认识是了解大脑功能的基础，也是未来神经疾病治疗的关键。N型钙离子通道与胞吐蛋白Synaxin和SNAP-25相互作用，但这些相互作用的结构基础尚不清楚。通过详细的一级和二级结构分析，我们发现N型Ca+2通道具有一个类似陷阱的基序，可能介导其与Synaxin和SNAP-25的相互作用。这个R21项目的目标1将检验这样的假设，即N型钙离子通道作为Q-SNARE，与Synaxin和SNAP-25的相互作用遵循一些在顺-SNARE中观察到的相同结构模式。这种Ca+2通道“陷阱”并不参与Synaxin-SNAP-25-synaptobrevin复合体的融合活性，但可能在胞吐之前将这些成分定位于Ca+2通道。利用重组SNARE蛋白的独特性质，我们希望证明N型Ca+2通道的Synprint结构域与Synaxin和SNAP-25以类似于其他SNARE蛋白的方式相互作用。目的2为进一步研究胞吐中钙离子感受器突触素1C2a-C2b的结构奠定基础。该蛋白还与钙离子通道、合成素和SNAP-25以多蛋白复合体相互作用。作为最终多蛋白分析的前奏，我们将使用各种创新的结晶学技术来优化突触素的结晶条件，这些技术旨在利用突触素独特的脂质缔合特性。一旦完成，这两个目标将为我们提供工具和理解，以进一步确定该蛋白在钙依赖的胞吐作用中的作用。在未来的R01资助下，我们将能够将这项工作扩展到对这一独特的SNARE复合体进行更广泛的生物物理和结晶学研究，并完成人类突触素1C2a-C2b的高分辨率晶体结构。

DESCRIPTION (provided by applicant): Although we have made great strides in understanding Ca+2-dependent exocytosis in the neuron, there is still not a clear understanding of the relationship between Ca+2 and exocytosis. An accurate view of this process is fundamental to understanding brain function, and crucial to the future treatment of neurological diseases. The N-type Ca+2 channel interacts with the exocytosis proteins syntaxin and SNAP-25; however, the structural basis of these interactions is still not clear. Through detailed primary and secondary structural analysis, we found that the N-type Ca+2 channel possesses a SNARE-like motif that may mediate its interactions with syntaxin and SNAP-25. Aim 1 of this R21 project will test the hypothesis that the N-type Ca+2 channel acts as a Q-SNARE, and that the interactions with syntaxin and SNAP-25 follow some of the same structural patterns observed in the cis-SNARE. This Ca+2 channel "SNARE" is not implicated in the fusogenic activities of the syntaxin-SNAP-25.synaptobrevin complex, but may serve to localize these components to the Ca+2 channel immediately before exocytosis. Taking advantage of the unique properties of the recombinant SNARE proteins, we expect to show that the synprint domain of the N-type Ca+2 channel interacts with syntaxin and SNAP-25 in a similar manner as do other SNARE proteins. Aim 2 will lay the foundation for further structural work on human synaptotagmin 1 C2A-C2B, the Ca+2 sensor in exocytosis. This protein also interacts with the Ca+2 channel, syntaxin and SNAP-25 in a multiprotein complex. As a prelude to eventual multiprotein analysis, we will refine the crystallization conditions of synaptotagmin using a variety of innovative crystallographic techniques designed to take advantage of synaptotagmin's unique lipid-associating properties. Once completed, these two aims will provide us with the tools and understanding to further define this protein's role in Ca+2-dependent exocytosis. Under future R01 funding we will be able to extend this work to more extensive biophysical and crystallographic studies of this unique SNARE complex, and complete the high-resolution crystal structure of human synaptotagmin 1 C2A-C2B.