Single-Molecule Studies of Synaptic Vesicles

描述(申请人提供)：获得囊泡递质摄取机制的详细知识对于了解突触功能和可塑性是重要的，因为这个摄取过程直接控制释放到突触间隙的递质的数量，从而决定突触后受体的激活程度。确定囊泡递质摄取的机制和速率对于了解突触处囊泡再循环的动力学以及在突触前末端加载和维持充满的囊泡所需的能量也是重要的。由于突触传递依赖于囊泡内神经递质的摄取和储存，因此它们是许多药物的作用部位也就不足为奇了，因此了解这一重要分子机制的功能也将具有重要的药理学意义。我们提出的单分子和单小泡实验旨在破译神经递质被加载到突触小泡中的分子组织和机制。为此，我们的具体目标是：目标1：突触小泡完整膜蛋白和囊泡内小分子的旋转测量目标2：囊泡H+ATPase的单分子研究目标3：囊泡谷氨酸转运体的单分子研究目标4：研究递质摄取与突触小泡上的糖酵解酶的偶联以及摄取机制的空间组织。通过这些实验，我们将深入了解这种具有单分子分辨率的复杂分子机制的工作原理，并对神经疾病造成的故障或药物对这一机制的靶向破坏如何影响突触传递提供新的见解。项目简介：为了协调神经传递，超过1000种蛋白质存在于突触前终端，它们直接或间接与突触小泡相互作用，最终结果是神经递质被调节释放到突触间隙。突触前功能向突触小泡的高度汇聚导致了一种以突触小泡为中心的神经传递观点，认为突触小泡是突触功能的中心细胞器。这项建议描述了用单分子分辨率研究突触小泡的计划，因为辨别这个重要细胞器的详细工作方式对于实现对突触传递和神经疾病引起的功能障碍的下一个水平的定量理解至关重要。

DESCRIPTION (provided by applicant): Gaining detailed knowledge on the workings of the vesicular transmitter uptake machinery is important in understanding synaptic function and plasticity, because this uptake process governs directly the number of transmitters released into the synaptic cleft and thus the degree of activation of the postsynaptic receptors. Determining the mechanism and rates of vesicular transmitter uptake also is important towards understanding the kinetics of vesicle recycling at the synapse and the energy requirements for loading and maintaining the filled vesicle at the presynaptic terminal. Given synaptic transmission is dependent on the uptake and storage of neurotransmitters within vesicles, it is not surprising that they are sites of action for many drugs, and thus understanding the functioning of this important molecular machinery will also have important pharmacological implications. Our proposed single-molecule and single-vesicle experiments are targeted towards deciphering the molecular organization and mechanism by which neurotransmitters are loaded into the synaptic vesicle. Towards this end, our specific aims are: Aim 1: Rotational measurements of synaptic vesicle integral membrane proteins and of small molecules contained within the vesicle Aim 2: Single-molecule studies of the vesicular H+ATPase Aim 3: Single-molecule studies of the vesicular glutamate transporters Aim 4: Investigation of the coupling of transmitter uptake to the glycolytic enzymes on synaptic vesicles and the spatial organization of the uptake machinery. From these experiments, we will develop an in-depth understanding of the workings of this complex molecular machinery with single-molecule resolutions, and offer new insight into how malfunctioning caused by neurological diseases or targeted disruption of this machinery with drugs can affect synaptic transmission. Project Narrative: To orchestrate neurotransmission, more than one thousand proteins are present at the presynaptic terminal and which either directly or indirectly interact with the synaptic vesicle, with the end result being the regulated release of neurotransmitters into the synaptic cleft. This high degree of convergence of presynaptic functions onto the synaptic vesicle has led to a "vesicocentric" view of neurotransmission that focuses on the synaptic vesicle as the central organelle in synaptic function. This proposal describes plans to study the synaptic vesicle with single-molecule resolutions, because discerning the detailed workings of this important organelle is critical in achieving the next level of quantitative understanding of synaptic transmission and malfunctioning caused by neurological diseases.