Analysis of the protein nano-environment of voltage-activated N-type Ca2+ channels Cav2.2 in the brain

电压门控型N型钙通道(Cav2.2)在中枢神经系统中触发多种过程，包括突触的功能、形成和可塑性、活性依赖的基因表达以及电信号的兴奋性和传播的调节。利用对这些通道的分子纳米环境的全面和定量蛋白质组学，我们已经确定了它们在功能上显著多样性的框架：Cav2.2通道整合到扩展的蛋白质网络中，该网络可能由大约160个蛋白质池组装而成(具有明显的丰度和结合稳定性)，并反映了Cav2.2通道可以促进的细胞过程。下一个资助期旨在了解(I)Cav2.2通道网络的分子结构和组织，(Ii)它们的局部特异性和组装动力学(网络是否取决于突触/脑区域的类型、细胞边界，如神经元活动和/或“中枢蛋白”的存在？)，以及(Iii)对Cav2.2纳米环境的运行起决定性作用的蛋白质-蛋白质相互作用。为了实现这些目标，我们将采用多学科方法，包括交互应用一系列技术，包括高分辨率定量质谱学、生物化学、标准分子生物学、免疫细胞化学和免疫电子显微镜以及电生理学。

Voltage-gated N-type calcium channels (Cav2.2) trigger a variety of process in the CNS including function, formation and plasticity of synapses, activity-dependent gene expression and regulation of excitability and propagation of electrical signaling. Using comprehensive and quantitative proteomics on the molecular nano-environment of these channels we have identified the framework for their marked diversity in function: Cav2.2 channels are integrated into extended protein networks that may be assembled from a pool of ~160 proteins (at distinct abundance and stability of association) and reflect the cellular processes that can be fueled by Cav2.2 channels. The next funding period aims at the understanding (i) of the molecular structure and organization of the Cav2.2 channel networks, (ii) their local specificity and dynamics of assembly (do networks depend on the types of synapse/brain regions, on cellular boundaries such as neuronal activity and/or the presence of 'hub proteins'?), as well as on (iii) protein-protein interactions decisive for the operation of Cav2.2 nano-environments. For these goals we will apply a multidisciplinary approach comprising interactive application of an array of techniques including high-resolution quantitative mass spectrometry, biochemistry, standard molecular biology, immunocytochemistry and immunoelectron microscopy and electrophysiology.