AMPAR Function in Synaptic and Extrasynaptic Membranes

AMPA受体(AMPAR)介导大部分兴奋性谷氨酸能突触传递 中枢神经系统。大多数AMPAR一旦与谷氨酸结合，就允许Na+和K+流过细胞 膜，导致神经元去极化。然而，缺乏GluR2亚单位的AMPAR也是可渗透的 到钙离子。这些钙通透性(CP)AMPAR在发育过程中高度表达，当它们 对于依赖活动的可塑性是必不可少的，这一功能在成年期的一些突触中持续存在。一个 生物物理特性称为整流，通常用于区分CP-AMPAR和不透钙(CI)AMPAR。虽然CP-AMPAR表现出很强的内向整流，但CI-AMPA受体 显示线性电流-电压关系。胞内CP-AMPAR的内向整流 作为开放通道阻滞剂的多胺，以防止外向电流通量。因此，向内整顿和 结合在多胺位点的拮抗剂的敏感性提供了AMPAR亚单位的生物物理特征 组成，因此钙离子渗透性，这些特征已被广泛用于建立规则 AMPAR亚基的可塑性。小脑分子层中间神经元提供了一个公认的模型 用于理解AMPAR定位和运输的系统，因为重复突触刺激或单个 恐惧的经历触发了一种称为亚单位转换的可塑性，其中突触上的CP-AMPAR是 替换为来自非突触AMPAR池的CI-AMPAR。虽然整流指数和对 多胺位点毒素被广泛用于区分含GluR2和缺乏AMPAR 是否有许多文献中的例子表明这些生物物理性质并不完全反映亚单位 组成。一篇单独的文献集中在AMPAR的门控模型上，该模型包括 电导状态，但其功能暗示尚不清楚。现在，我们的初步数据表明，CP-AMPAR整流和药理学对调节AMPAR电导状态的因素很敏感， 使用这些生物物理特性作为结果的唯一替代可能会使结果的解释复杂化 亚基组成。我们建议理解AMPAR的多个亚导状态是如何 有助于标志性生物物理特性CP-AMPAR。我们将使用高分辨率的钙离子成像， 异源表达系统和基因操作研究CP-AMPAR的调控 生物物理特性，并利用这一理解来批判性地评估CP-AMPAR的定位和可塑性 小脑分子层中间神经元。 Ampar亚基组成具有重要的功能后果， 从调节突触后细胞的能力到精确跟踪高频突触活动和 调节钙离子内流，从而触发可塑性或病理。成功完成拟议的研究将 揭示AMPAR的新特性，这些特性对于理解它们在突触和 正常和患病大脑中完整的回路。

AMPA receptors (AMPARs) mediate the majority of excitatory glutamatergic synaptic transmission in the central nervous system. Most AMPARs, once bound to glutamate, allow Na+ and K+ flux across the cell membrane, causing neurons to depolarize. However, AMPARs that lack the GluR2 subunit are also permeable to Ca2+. These Ca2-permeable (CP) AMPARs are highly expressed during development when they are essential for activity-dependent plasticity, and this function persists at some synapses throughout adulthood. A biophysical characteristic known as rectification is commonly used to differentiate CP-AMPARs from Ca2+-impermeable (CI) AMPARs. Whereas CP-AMPARs exhibit strong inward rectification, CI-AMPA receptors display linear current-voltage relationships. Inward rectification of CP-AMPARs results from intracellular polyamines that act as open channel blockers to prevent outward current flux. Thus, inward rectification and sensitivity to antagonists that bind at the polyamine site provide biophysical signatures of AMPAR subunit composition and hence Ca2+ permeability, and these characteristics have been widely used to establish rules of AMPAR subunit plasticity. Molecular layer interneurons of the cerebellum provide a well-established model system for understanding AMPAR localization and trafficking because repetitive synaptic stimulation or a single experience of fear triggers a form of plasticity called subunit-switching wherein CP-AMPARs at synapses are replaced by CI-AMPARs from a pool of extrasynaptic AMPARs. Although rectification index and sensitivity to polyamine site toxins are widely used to distinguish between GluR2-containing and -lacking AMPARs, there are many examples from the literature that show these biophysical properties do not exclusively reflect subunit composition. A separate literature has converged on gating models of AMPARs that include multiple conductance states, but the functional implications are unclear. Now, our preliminary data show that CP-AMPAR rectification and pharmacology are sensitive to factors that regulate AMPAR conductance states, potentially complicating the interpretation of results using these biophysical properties as sole proxies of subunit composition. We propose to understand how the multiple sub-conductance states of AMPARs contribute to the hallmark biophysical properties CP-AMPARs. We will use high resolution Ca2+ imaging, heterologous expression systems and genetic manipulation to understand regulation of CP-AMPAR biophysical properties and use that understanding to critically evaluate CP-AMPAR localization and plasticity in cerebellar molecular layer interneurons. AMPAR subunit composition has important functional consequences, ranging from regulating the ability of postsynaptic cells to precisely follow high-frequency synaptic activity and mediating Ca2+ influx that can trigger plasticity or pathology. Successful completion of the proposed studies will reveal novel properties of AMPARs that are essential for understanding their function within synapses and intact circuits in the normal and diseased brain.