Dynamics of ligand gated ion channels

项目摘要/摘要 谷氨酸受体介导哺乳动物中枢神经系统的兴奋性反应，并最终 控制运动和认知功能。谷氨酸受体可分为三个亚家族 几种合成和天然激动剂：N-甲基-D-天冬氨酸、-氨基-5-甲基-3-天冬氨酸 羟基-4-异恶唑丙酸酯(AMPA)和海人藻酸受体。所有三个子类型都大致相似，都具有 具有相似拓扑的二聚体体系结构的二聚体。然而，它们的选通特性和机制是 独特的结果导致在突触传递中扮演独特的角色。我实验室的工作重点是了解 这些子类型的微调背后的机制。AMPA受体介导快速突触 传输和它们的选通特性也由辅助亚单位(Tarp)的存在来调制 通过翻译后的修改。我的实验室的工作提供了对这种机制的洞察 结合生物物理和生物化学研究AMPA受体的激活和脱敏 方法：研究方法。在这里，我们建议研究如何将这些机制转化为Tarp和TARP的调制 翻译后修饰，如磷酸化。为此，我们将使用smFRET和 LRET以确定动力学和构象变化，并验证这些结构-动态变化 通过对类十字受体的生化操作引起的变化进行功能表征 链接和突变。另一方面，NMDA受体介导了钙离子可渗透的长去极化 信号并通过小分子调节剂、磷酸化和相互作用的伙伴如 细胞内羧基末端的钙调蛋白和α-肌动蛋白。SMFRET和LRET调查来自 我的实验室以及X射线和EM结构提供了对构象的重要洞察 有序的胞外结构域内的变化。然而，跨域的通信和角色 无序的C-末端结构域在很大程度上还没有被探索。在这里，我们建议研究相互作用的作用 跨域控制受体的动态和构象变化以及调节剂的作用 以及C-末端结构域在这些相互作用和构象动力学上的变化。这些研究将 然后与功能后果相关联，从而提供了对结构-动力学途径的洞察 这些受体的激活、脱敏和调节。

PROJECT SUMMARY/ABSTRACT Glutamate receptors mediate excitatory responses in the mammalian central nervous system, and ultimately control motor and cognitive functions. Glutamate receptors are classified into three subfamilies based on affinity profiles for several synthetic and natural agonists: N-methyl-D-aspartate (NMDA), -amino-5-methyl-3- hydroxy-4-isoxazole propionate (AMPA) and kainate receptors. All three subtypes are broadly similar having a dimer of dimer architecture with similar topologies. However, their gating characteristics and mechanisms are unique resulting in unique roles in synaptic transmission. The work in my laboratory focuses on understanding the mechanisms underlying the fine-tuning of each of these subtypes. AMPA receptors mediate fast synaptic transmission and their gating properties are modulated by the presence of auxiliary subunits (TARPs) as well as through post-translational modifications. Work from my laboratory has provided insight into the mechanism of activation and desensitization in AMPA receptors using a combination of biophysical and biochemical methods. Here, we propose to study how these mechanisms can be translated to modulation by TARPs and post-translational modifications such as phosphorylation. For this we will use a combination of smFRET and LRET to determine the dynamics and conformational changes, and validate these structure-dynamic changes through functional characterization of changes elicited by biochemical manipulations of the receptor like cross linking and mutations. The NMDA receptors, on the other hand, mediate Ca2+ permeable long depolarizing signals and are modulated through small molecule modulators, phosphorylation and interacting partners such as calmodulin and alpha-actinin at the intracellular carboxy terminus. smFRET and LRET investigations from my laboratory as well as the X-ray and EM structures have provided significant insight into conformational changes within the ordered extracellular domains. However, the communication across domains and the role of the disordered C-terminal domain are largely unexplored. Here we propose to study the role of interactions across domains in controlling dynamics and conformational changes in the receptor, and effects of modulators and changes at the C-terminal domain on these interactions and conformational dynamics. These studies will then be correlated to functional consequences thus providing insight into the structure-dynamic pathway of activation, desensitization, and modulation in these receptors.