TOPOLOGY & STOICHIOMETRY OF GLUTAMATE RECEPTOR SUBUNITS

谷氨酸受体在正常的大脑功能中起着重要作用。 它们与长期增强和长期抑郁有关， 思维过程是记忆和学习的基础。谷氨酸受体 也牵涉到许多病理生理状况 影响中枢神经系统功能，如癫痫，一些 神经退行性疾病，以及缺血和缺氧期间神经细胞的死亡 低血糖症。因此，尽管它们在突触功能中很重要， 人们对它们的整体结构知之甚少。从长远来看 拟议研究的目标是确定 谷氨酸受体亚单位序列暴露于 突触膜的胞外或胞浆表面 (拓扑学)，并确定每种亚基的数目 聚集形成重组谷氨酸受体(化学计量)。这个 谷氨酸受体亚基的拓扑结构将首先确定 在亚基内的不同位置引入报告表位， 然后在非洲爪哇卵母细胞中表达这些工程亚基，并 最终确定细胞内或细胞外的位置 表位，相对于卵母细胞的表膜，由 报告单抗的结合。确定拓扑 这些受体亚基中的一个应该开始揭示可能 与谷氨酸能细胞内结构成分相互作用 突触，类似于其他离子通道的突触，如 肌型乙酰胆碱受体的43K蛋白和吉普林 甘氨酸受体。可能有贡献的细胞外域 对已建立的内源性神经递质的结合部位，如 作为甘氨酸和L-谷氨酸等潜在的神经递质或 多胺和花生四烯酸等调节剂也可能是 已确认身份。一种重组NMDA受体的亚基化学计量 将会被确定。标记了表位的NMDA受体亚单位将 代谢性地标记[35S]蛋氨酸并表达为 亚基CRNAs中化学计量比不变的异构体 卵母细胞。亚基化学计量比将通过确定 分离的完全组装受体的放射性标记亚基比率 来自卵母细胞表面膜。亚基化学计量学的阐明 是确定NMDA完整结构的第一步 受体，以及单个亚单位结构域的贡献 形成配基结合部位和离子通道孔。 这些研究确定的胞外和胞内结构域 然后可以通过定点突变来更好地理解 他们的职能角色。通过仔细比较它们的特性 已知亚单位化学计量学的重组NMDA受体 来自表达相同组合的神经元的天然NMDA受体 亚基的化学计量学之间可能存在关联 某些重组受体与特定的天然亚型受体 感受器。

Glutamate receptors play an important role in formal brain function. They are implicated in long term potentiation and long term depression, processes thought to underlie memory and learning. Glutamate receptors have also been implicated in many pathophysiological conditions affecting central nervous system function such as epilepsy, some neurodegenerative diseases, and neuronal cell death during ischemia and hypoglycemia. Thus, despite their importance in synaptic function, very little is known about their overall structure. The long term objectives of the proposed study are to determine which parts of the sequence of glutamate receptor subunits are exposed to the extracellular or cytoplasmic surface of the synaptic membrane (topology) and to determine the number of each kind of subunit which assembles to form recombinant glutamate receptors (stoichiometry). The topology of glutamate receptor subunits will be determined by first introducing reporter epitopes at various locations within a subunit, then expressing these engineered subunits in Xenopus oocytes, and finally determining the intracellular or extracellular location of the epitopes, with respect to the surface membrane of oocytes, by the binding of reporter monoclonal antibodies. Determining the topology of these receptor subunits should begin to reveal domains that might interact with intracellular structural components of glutaminergic synapses, analogous to those found for other ion channels, such as the 43K protein for the muscle-type acetylcholine receptor, and gephyrin for the glycine receptor. Extracellular domains that might contribute to the binding sites of established endogenous neurotransmitters such as glycine and L-glutamate and other potential neurotransmitters or modulators such as polyamines and arachidonic acid also might be identified. The subunit stoichiometry of a recombinant NMDA receptor will be determined. NMDA receptor subunits tagged with epitopes will be metabolically labeled with [35S]methionine and expressed as a heteromer with an invariant stoichiometry from subunit cRNAs in oocytes. The subunit stoichiometry will be deduced by determining the ratio of radiolabeled subunits of fully assembled receptors isolated from oocyte surface membranes. Elucidating the subunit stoichiometry is a first step towards determining the complete structure of NMDA receptors, as well as the contributions of individual subunit domains to the formation of ligand binding sites and the ion channel pore. Extracellular and intracellular domains identified by these studies then can be targeted by site-directed mutagenesis to better understand their functional roles. By carefully comparing the properties of recombinant NMDA receptors of known subunit stoichiometries, with those of native NMDA receptors from neurons expressing the same combinations of subunits, it might be possible to correlate the stoichiometries of some recombinant receptors to those of specific subtypes of native receptors.