Single-Molecule Studies of Synaptic Vesicles

描述（由申请人提供）：获得关于囊泡递质摄取机制的工作的详细知识对于理解突触功能和可塑性是重要的，因为这种摄取过程直接控制释放到突触间隙中的递质的数量，从而控制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理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填充的囊泡的能量需求也是重要的。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囊泡实验的目标是破译的分子组织和机制，神经递质加载到突触囊泡。为此，我们的具体目标是：目标1：突触囊泡完整膜蛋白和囊泡内所含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：研究突触囊泡上递质摄取与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，并对神经系统疾病引起的功能障碍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。

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.