Biophysics of Norepinephrine and Serotonin Transporters

描述（由申请人提供）：这个竞争性的更新集中在一个基本问题上：宏观输运现象和分子事件之间的关系。在过去的十年中，膜片钳、电流计和荧光显微镜等新技术提供了大量关于共转运体的信息。然而，这些新数据意味着什么，以及它们如何与现有的共转运体文献相结合，都存在不确定性。为了深入了解这些问题，我们将在宏观和微观层面比较转运蛋白作为载体和通道的双重功能。特异性Aim I结合了经典的摄取和结合技术与使用荧光底物的新数据。我们的目标是在亚细胞水平上实时解决结合和运输问题。特异性Aim II将全细胞和膜片钳电生理学相关联，并将这些数据与转运体的冷冻电镜图像进行比较。我们使用哺乳动物细胞来实现前两个目标，并利用诱导细胞系和功能获得和功能丧失突变体。特异性目的III使用整个细胞和切开结构的青蛙卵母细胞来测试前两个目的引起的共运输的机制预测。蛙卵母细胞适用于同时放射配体摄取和双微电极电压钳分析。在切开卵母细胞制备中，可以同时进行电压钳和内外溶液交换。因此，本建议强调新的实验方案与传统方法相结合，在分子水平上关注转运体生物物理学。

DESCRIPTION (provided by applicant): This competing renewal focuses on one fundamental question: the relationship between macroscopic transport phenomena and molecular events. During the past decade new techniques such as patch clamp, amperometry, and fluorescent microscopy have provided a wealth of information about co-transporters. Yet uncertainly exists about what these new data mean and how they come together with the existing literature on co-transporters. To get to the bottom of these issues we will compare transporters in their dual function as carriers and channels at both the macroscopic and microscopic level. Specific Aim I combines classical uptake and binding techniques with new data using fluorescent substrates. Our goal is to resolve binding and transport in real time at the sub-cellular level. Specific Aim II correlates whole-cell and patch-clamp electrophysiology and compares these data with cryo-EM images of transporters. We use mammalian cells for these first two aims, and take advantage of inducible cell lines and gain-of-function and loss-of-function mutants. Specific Aim III uses frog oocytes in the whole cell and cut-open configuration to test mechanistic predictions of co-transport arising from the first two aims. Frog oocytes are suitable for simultaneous radioligand-uptake and two-microelectrode voltage clamp analysis. In the cut-open oocyte preparation, simultaneous voltage clamp with internal and external solution exchange are possible. This proposal thus emphasizes new experimental protocols in combination with traditional approaches to focus on transporter biophysics at the molecular level.