Single-Molecule Studies of Synaptic Vesicles

描述（由申请人提供）：获得泡状递质摄取机制的详细工作知识对于理解突触功能和可塑性非常重要，因为这一摄取过程直接控制释放到突触间隙的递质数量，从而控制突触后受体的激活程度。确定囊泡传递素摄取的机制和速率对于理解突触中囊泡循环的动力学以及在突触前末端装载和维持充满的囊泡所需的能量也很重要。鉴于突触传递依赖于神经递质在囊泡内的摄取和储存，因此它们是许多药物的作用位点并不奇怪，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囊泡实验旨在破译神经递质被装载到突触囊泡中的分子组织和机制。为此，我们的具体目标是：目标1：突触囊泡整体膜蛋白和囊泡内小分子的旋转测量；目标2：囊泡H+ atp酶的单分子研究；目标3：囊泡谷氨酸转运蛋白的单分子研究；目标4：突触囊泡上的糖酵解酶与递质摄取耦合的研究以及摄取机制的空间组织。从这些实验中，我们将以单分子分辨率深入了解这种复杂分子机制的工作原理，并为神经系统疾病引起的故障或药物对这种机制的靶向破坏如何影响突触传递提供新的见解。项目描述：为了协调神经传递，超过一千种蛋白质存在于突触前末端，它们直接或间接地与突触囊泡相互作用，最终结果是神经递质被调节释放到突触间隙中。突触前功能在突触囊泡上的高度趋同导致了神经传递的“囊泡中心”观点，即突触囊泡是突触功能的中枢细胞器。该提案描述了用单分子分辨率研究突触囊泡的计划，因为辨别这种重要细胞器的详细工作对于实现对神经系统疾病引起的突触传递和功能障碍的定量理解的下一个水平至关重要。

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.