Analysis of Ca2+ -Triggered Neurotransmitter Release

描述（由申请人提供）：高效和快速的突触转导机制是脑回路活动的相干振荡和同步所必需的。突触前在< 1ms动作电位内转导成Ca2+触发的含神经递质囊泡的同步融合。两个蛋白家族，络合蛋白和突触tagmins，是将通用的SNARE蛋白膜融合装置调整为高速传感器的关键角色。已知这两种蛋白质都与SNARE复合物相互作用。本研究的目的是了解这些蛋白质如何完成快速融合，并确定哪些相互作用与它们的功能相关。我们假设络合蛋白和Synaptotagmin 1通过降低融合反应的能量屏障起作用，但它们通过不同的分子机制实现这一作用。在与SNARE复合体结合后，络合蛋白似乎引起了丰富复合体的稳定，或者作为一个适配器使Synaptotagmin 1与SNARE复合体结合。Synaptotagmin 1可能通过Ca2+依赖其C2A和C2B结构域与质膜的结合而触发快速的神经递质释放，这种膜穿透通过破坏灌注膜复合物的稳定来决定融合速率。为了解释Complexin和Synaptotagmin的功能，我们提出了一种结合电生理、结构和生化实验的综合方法。首先，我们将分析Complexin 1/2/Synaptotagmin 1敲除小鼠神经元的突触特性。然后，我们将探索使用拯救方法推测参与同步释放的蛋白质结构域的结构-功能关系。最后，我们将探索这两种蛋白质是独立作用还是相互作用，以及它们是否顺序起作用。更好地了解同步释放的机制可能有助于找到在大脑回路中表现出紊乱振荡和同步模式的疾病的原因和治疗方法，如注意缺陷综合征、自闭症、亨廷顿氏病或精神分裂症。

DESCRIPTION (provided by applicant): Efficient and rapid transduction mechanisms at synapses are required for coherent oscillations and synchronization of activity of brain circuits. The presynapse transduces within <1 ms action potentials into a Ca2+-triggered synchronous fusion of neurotransmitter containing vesicles. Two protein families, the Complexins and Synaptotagmins, are key players in tuning the generic SNARE protein-based membrane fusion apparatus into a high speed transducer. Both proteins are known to interact with the SNARE complex. The goal of this study is to understand how these proteins accomplish fast fusion, and to determine which interactions are relevant for their function. We hypothesize that Complexin and Synaptotagmin 1 act by reducing the energy barrier of the fusion reaction, but they accomplish this using different molecular mechanisms. Following binding to the SNARE complex, Complexins seem to cause stabilization of a profusion complex, or alternatively, serve as an adaptor to enable Synaptotagmin 1 binding to the SNARE complex. Synaptotagmin 1 may trigger fast neurotransmitter release by Ca2+-dependent binding of its C2A and C2B domains to the plasma membrane, and this membrane penetration determines fusion rates by destabilizing the profusion membrane complex. To decipher the function of Complexin and Synaptotagmin, we propose an integrated approach using electrophysiological, structural, and biochemical experiments. First, we will analyze synaptic properties in neurons derived from Complexin 1/2/Synaptotagmin 1 knockout mice. We will then explore the structure-function relationship of protein domains that are putatively involved in synchronous release using a rescue approach. Finally, we will probe whether both proteins act independently or in conjunction with each other, and whether they act sequentially. A better understanding of the mechanism of synchronous release may help to find the cause and treatment of diseases that show disturbed oscillations and synchronization patterns in brain circuits, like Attention Deficit Syndrome, Autism, Huntingdon's Disease or Schizophrenia.