异聚AMPA型受体中亚基组成和装配的研究

AMPA receptors (AMPARs) mediate fast excitatory neurotransmission in central nervous system and play critical roles in inducing and maintaining LTP and LTD. The subunit composition and tetrameric assembly of AMPARs determine the dynamics of fast glutamatergic signaling. Previous reports suggested the amino-terminal domains (ATDs) and ligand binding domains (LBDs) were essential for forming this transmembrane receptor/channel by assembling subunits as a dimer of dimers, but the detailed mechanism is still unclear. Our preliminary data indicated that the ATDs regulated tetrameric assembly of GluA1/2 AMPARs and were highly correlated with the stoichiometry of subunits. Thus, we hypothesize that there would be certain stoichiometry of tetrameric assembly of AMPARs, which is guided by certain element in the ATDs. To delineate the stoichiometry and assembly of heteromeric AMPARs architecture, we will conduct a series of disulfide-bond cross-linking experiments and patch-clamp related electrophysiological analyses. More importantly, we will also verify the key element guiding the assembly process by swapping critical domains of the ATDs. Our study will shed light on exploring AMPARs related neurological disease nosogenesis, as well as provide a possible new insight into the development of novel therapeutic strategies.

AMPA型谷氨酸受体(AMPAR)介导中枢神经系统快速兴奋性突触传递，在长时程增强和长时程抑制的诱发和维持中起重要作用。研究证实AMPAR主要以异源四聚体复合物形式存在，其亚基组成决定其突触靶向和通道特性。氨基末端结构域（ATD）和配体结合域（LBD）通过介导亚基二聚化后二聚化装配成有功能的四聚体跨膜受体通道，但具体机制尚不清楚。本课题前期研究表明ATD参与调节装配GluA1/2异源四聚体，并与其中亚基的排列定位高度相关。因此，我们提出：异源四聚体AMPAR中各亚基的组成配比和空间排列定位具有特定规律，ATD上存在特定元件调节装配过程。在这个假说驱动下，我们拟从蛋白结构水平和电生理功能方面对异源四聚体形成规则和其通道特性进行评估，阐明AMPAR的亚基组成比例、空间排列定位和装配机制。本课题的完成将为研究AMPAR有关神经类疾病的致病机理和相关药物设计提供实验依据。

AMPA型谷氨酸受体（AMPAR）在大脑中主要以异源四聚体形式存在并介导快速兴奋性突触传递。最近对GluA2同源四聚体晶体结构的解析证明了AMPAR由两对构象上有差异的亚单位以“二聚体的二聚体”形式组成。然而，异源聚合AMPAR的结构目前还不清楚。为了阐明异源聚合AMAPR的构建形式，本课题根据GluA2结构，对全长GluA1/GluA2、GluA2/GluA3和GluA2/GluA4进行了半胱氨酸突变体交联实验。我们发现氨基末端结构域决定了第一级异源二聚体的形成。当二聚体进一步装配成四聚体，GluA1和GluA2亚单位会有各自的优先位置，形成1-2-1-2的空间结构；而GluA2/GluA3和GluA2/GluA4受体则分别为3-2-3-2和4-2-4-2结构。通过交换重要的序列，我们惊喜地发现空间装配形式由可切割的信号肽控制。将原有信号肽换成无关的GluK2，我们明确了GluA1信号肽在亚单位空间优先性中起到至关重要的作用。因此，本课题阐明了大脑中重要形式谷氨酸受体的空间结构，同时揭示了信号肽的全新作用。