NMDA RECEPTOR--AGONIST AFFINITY, EFFICACY/TRANSDUCTION

该项目的长期目标是确定分子 NMDA受体激动剂亲和力和功效的基础，配体门控离子 通道，属于谷氨酸受体家族。NMDA的激活 受体需要结合两种共激动剂，甘氨酸和L-谷氨酸， NR 1和NR 2亚基中的受体结构域。由两种激动剂占据 引发了一系列分子事件，最终导致 相关离子通道。本建议的目的是确定具体的 激动剂与NMDA受体相互作用的分子决定因素。 最近发表的配体结合结构域的晶体结构， 相关谷氨酸受体（GluR 2）预测哪些氨基酸直接参与 与激动剂接触。我们实验室的初步数据表明 甘氨酸结合口袋中的转导元件和高度保守的 在M3跨膜区段中的区域。因此，以下具体目标 建议： (1)鉴定决定激动剂亲和力的氨基酸残基， 功效。 定点诱变已经鉴定了许多氨基酸残基， 突变引起激动剂剂量-反应曲线的偏移。但这种 激动剂敏感性的变化不能明确地解释。一个新 因此，将使用可以区分突变的方法， 影响激动剂亲和力或效力。通过使用半胱氨酸取代 诱变和巯基特异性修饰试剂，相同的群体 可以在修改之前和之后研究通道。全部和部分 激动剂将被用于明确解释疗效的改变 和亲和力。使用GluR 2受体的平行实验将用于 确认结构分配。 (2)为了检验M3节段是转导节段的假设 偶联配体结合至通道开口。 谷氨酸受体的M3跨膜段含有严格的 保守氨基酸序列。在该区域鉴定的半胱氨酸取代 巯基修饰导致组成型活性NMDA的残基 受体。由于这种修饰需要激动剂的存在，因此， 假设M3在受体活化后发生构象变化 巯基修饰将受体锁定在活性状态。这些 研究将导致详细的分子图像的动态变化， 结构伴随NMDA受体的活化。

The long term goal of this project is to define the molecular basis of agonist affinity and efficacy in the NMDA receptor, a ligand-gated ion channel that belongs to the glutamate receptor family. Activation of NMDA receptors requires binding of two co-agonists, glycine and L-glutamate, to receptor domains in the in the NR1 and NR2 subunits. Occupancy by both agonists initiates a series of molecular events that culminates in opening of the associated ion channel. The objective of this proposal is to identify specific molecular determinants of the interaction of agonists with the NMDA receptor. The recently published crystal structure of the ligand binding domains of a related glutamate receptor (GluR2) predicts which amino acids are in direct contact with the agonists. Preliminary data from our lab suggest the existence of transduction elements in the glycine binding pocket and a highly conserved region in the M3 transmembrane segment. Therefore, the following specific aims are proposed: (1) To identify amino acid residues that determine agonsist affinity and efficacy. Site-directed mutagenesis has identified many amino acid residues whose mutation caused shifts in the agonist dose-response curves. However, such shifts in agonist sensitivity cannot be interpreted unambiguously. A new approach will therefore be used which can distinguish between mutations that affect agonist affinity or efficacy. By using of cysteine-substitution mutagenesis and thiol-specific modifying reagents, the same population of channels can be studied before and after modification. Full and partial agonists will be employed to unequivocally interpret alterations in efficacy and affinity. Parallel experiments using the GluR2 receptor will be used to confirm the structural assignments. (2) To test the hypothesis that the M3 segment is a transduction segment coupling ligand binding to channel opening. The M3 transmembrane segment of glutamate receptors contains a strictly conserved amino acid sequence. Cysteine substitutions in this region identified a residue for which thiol modification results in constitutively active NMDA receptors. Since this modification requires the presence of agonists, it was hypothesized that M3 undergoes a conformational change upon receptor activation and that thiol modification locks the receptor in the active state. These studies will result in a detailed molecular picture of the dynamic change in structure that accompany activation of the NMDA receptor.