NMDA RECEPTOR--AGONIST AFFINITY, EFFICACY/TRANSDUCTION

这个项目的长期目标是定义分子 配体门控离子NMDA受体激动剂亲和力和有效性的基础 属于谷氨酸受体家族的通道。激活NMDA 受体需要两种共同激动剂甘氨酸和L-谷氨酸结合才能 NR1和NR2亚基中的受体结构域。两种激动剂的占有率 引发了一系列分子事件，最终导致了 缔合离子通道。这项提案目标是确定具体的 激动剂与NMDA受体相互作用的分子决定因素。 最近发表的一种配体结合域的晶体结构 相关谷氨酸受体(GluR2)预测哪些氨基酸是直接 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 M3跨膜区。因此，以下是具体目标 现建议： (1)确定决定激动剂亲和力的氨基酸残基和 功效。 定点突变已鉴定出许多氨基酸残基，其 突变会导致激动剂的剂量-反应曲线发生变化。然而，这样的 激动剂敏感性的变化不能被明确地解释。一种新的 因此，将使用一种方法来区分 影响激动剂的亲和力或效力。通过使用半胱氨酸取代 诱变和硫醇特异性修饰试剂，相同的种群 可以对修改前后的通道进行研究。全部和部分 将使用激动剂来明确地解释疗效的变化。 和亲和力。使用GluR2受体的平行实验将用于 确认结构指定。 (2)检验M3片段为转导片段的假设 偶联配体结合到通道开口。 谷氨酸受体的M3跨膜片段含有严格的 保守的氨基酸序列。确定该区域的半胱氨酸替代 一种残基，其硫醇修饰可产生具有结构活性的NMDA 感受器。由于这种改造需要激动剂的存在，所以它是 假设M3在受体激活时经历构象变化 而硫醇修饰将受体锁定在激活状态。这些 研究将导致详细的分子图像的动态变化 伴随着NMDA受体激活的结构。

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.