SUBUNIT ASSEMBLY DOMAINS--TARGETS TO LIMIT NEUROTOXICITY

与许多配体门控离子通道一样，谷氨酸受体（GluRs）是由多个跨膜亚基组成的寡聚糖蛋白复合物。GluRs的功能特性在很大程度上取决于组成受体的亚基。为了形成具有适当功能属性的glur，必须存在调节亚基组装的机制。因此，GluR亚基可能包含高度特异性的相互作用和识别域，这些域的功能是调节亚基组装，并可能调节受体的亚基化学计量。在本应用中提出的实验旨在确定GluR亚基的NMDA和非NMDA类型的关联和组装域。烟碱乙酰胆碱受体、抑制性甘氨酸和γ -氨基丁酸受体的亚基含有决定亚基化学计量的结构域，这是受体组装所必需的。对于构成这些通道的亚基，“关联域”位于亚基多肽的胞外氨基端。本申请中提出的研究的总体目标是确定NMDA和非NMDA GluR亚基的结构域，这些结构域相互作用，并且是准确组装功能性同质和异质受体所必需的。一旦确定了NMDA和AMPA受体亚基的组装域，组装域肽将在培养的小脑颗粒细胞中测试其在EAA激活的神经元死亡期间的神经保护能力，这是研究EAA诱导的神经毒性的一个成熟的模型系统。细胞外和细胞内传递GluR关联结构域肽的神经保护特性将被确定。通过将相关结构域肽连接到天线基同源结构域第三螺旋的16个氨基酸肽，从而促进了肽的细胞内递送，该结构域可迅速被神经元内化。抑制兴奋性毒性的神经保护结合结构域肽的鉴定，可能是开发针对GluRs个体亚型的新型临床重要神经保护剂的第一步。

Like many ligand gated ion channels, glutamate receptors (GluRs) are oligomeric glycoprotein complexes composed of multiple membrane spanning subunits. The functional properties of GluRs are largely determined by the subunits that compose the receptor. For GluRs with the appropriate functional properties to form, there must exist mechanisms to regulate subunit assembly. It seems likely therefore, that GluR subunits contain highly specific interaction and recognition domains that function to regulate subunit assembly and possibly subunit stoichiometry of the receptor. The experiments proposed with in this application are aimed at identifying the association and assembly domains of both the NMDA and non-NMDA types of GluR subunits. Subunits of the nicotinic acetylcholine receptor, the inhibitory glycine and gamma-aminobutyric acid receptors contain structural domains that determine subunit stoichiometry and that are necessary for assembly of the receptor. For the subunits composing those channels, the "association domains" are located within the extracellular amino-terminus of the subunit polypeptides. The overall goal of the research proposed in this application is to identify structural domains of NMDA and non-NMDA GluR subunits that interact and that are required for the accurate assembly of functional homomeric and heteromeric receptors. Once assembly domains for the NMDA and AMPA receptor subunits are defined, assembly domain peptides will be tested for their ability to be neuroprotective during periods of EAA activated neuronal death in cultured cerebellar granule cells, a well established model system to study EAA induced neurotoxicity. The neuroprotective properties of both extracellular and intracellularly delivered GluR association domain peptides will be determined. The intracellular delivery of peptides will be facilitated by linking the association domain peptides to a 16 amino acid peptide from the third helix of the antennapedia homeodomain that is rapidly internalized by neurons. The identification of neuroprotective association domain peptides that inhibit excitotoxicity, may be the first steps toward the development of a new class of clinically important neuroprotective agents that target individual subtypes of GluRs.