Trapping the activation mechanism of AMPA-type glutamate receptors

在整个大脑中，支撑思想和行为的快速兴奋性突触传递由AMPA-离子型谷氨酸受体亚型(IGluRs)处理。AMPA受体具有快速的动力学，在数百微秒内激活和失活。谷氨酸与四个亚基中的每一个结合，驱动一个完整的跨膜离子通道的打开。为了绘制全长AMPA受体激活过程中的构象变化图，我们将使用一种最近开发的技术：化学修饰与快速溶液交换电生理学相结合。这种方法使我们能够在与天然突触相同的时间尺度上激活受体，跟踪它们的活动，同时化学地将它们捕捉到与它们在大脑中的信号作用相关的构象中。我们将从检查各个子单元内的运动开始。随后，我们将把我们的研究扩展到新发现的配体结合域之间的亚基间接触。由金属离子结合在引入的位置或通过二硫键形成的捕获“桥”将与一系列药理工具结合使用。因此，我们将在全长受体的背景下直接阐明神经递质结合部位的动力学。为了补充这些功能研究，我们将对选定的捕获突变体进行结构和生化研究，以研究它们的构象和多聚体排列。最后，我们将使用我们开发的陷阱桥作为工具来定义不同亚基在混合复合体中所占据的位置。这些研究有望为正常脑活动和疾病中AMPA通道的门控提供新的见解，并阐明药物调节这些通道的新机制。

Throughout the brain, the fast excitatory synaptic transmission that underpins thought and deed is handled by the AMPA-subtype of ionotropic glutamate receptors (iGluRs). AMPA-receptors have rapid kinetics, activating and deactivating within hundreds of microseconds. Glutamate binding to each of the four subunits drives the opening of an integral transmembrane ion channel. To map conformational changes during activation in full-length AMPA receptors, we will use a recently-developed technique: chemical modification coupled to rapid solution exchange electrophysiology. This method allows us to activate receptors on the same timescale as those at native synapses, tracking their activity whilst chemically trapping them in conformations relevant to their signaling role in the brain. We will begin by examining the motions within individual subunits. Subsequently, we will extend our studies to newly-discovered intersubunit contacts between the ligand binding domains. Trapping ‘bridges’ formed by metal ions bound at introduced sites, or by disulfide bonds, will be employed in combination with a battery of pharmacological tools. We will thus directly elucidate the dynamics of the neurotransmitter-binding site in the context of the full-length receptor. To complement these functional studies, we will perform structural and biochemical studies on selected trapped mutants to investigate their conformations and multimeric arrangement. Finally, we will use the trapping bridges we develop as tools to define the positions occupied by different subunits in mixed complexes. These studies are expected to provide novel insights into the gating of AMPA channels during normal brain activity and in disease, and elucidate new mechanisms for the modulation of these channels by drugs.