NMDA RECEPTOR--AGONIST AFFINITY, EFFICACY/TRANSDUCTION

NMDA 受体——激动剂亲和力、功效/转导

• 批准号: 6539099
• 负责人: ANTONIUS M VANDONGEN
• 金额: $ 15.4万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6539099
• 关键词: AMPA receptors; NMDA receptors; X ray crystallography; Xenopus oocyte; biological signal transduction; complementary DNA; cysteine; excitatory aminoacid; gene mutation; protein structure function; receptor binding; voltage /patch clamp

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.

该项目的长期目标是定义分子 NMDA 受体（一种配体门控离子）激动剂亲和力和功效的基础 属于谷氨酸受体家族的通道。 NMDA的激活 受体需要结合两种共激动剂，甘氨酸和L-谷氨酸， NR1 和 NR2 亚基中的受体结构域。两种激动剂均占据 引发一系列分子事件，最终导致 相关离子通道。该提案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域的晶体结构 相关谷氨酸受体（GluR2）预测哪些氨基酸是直接的 与激动剂接触。我们实验室的初步数据表明存在 甘氨酸结合口袋中的转导元件和高度保守的 M3 跨膜段中的区域。因此，具体目标如下 建议： (1) 鉴定决定激动剂亲和力的氨基酸残基和 功效。 定点诱变已鉴定出许多氨基酸残基，其 突变引起激动剂剂量反应曲线的变化。然而，这样的 激动剂敏感性的变化不能明确解释。一个新的 因此，将使用可以区分突变的方法 影响激动剂亲和力或功效。通过使用半胱氨酸取代 诱变和硫醇特异性修饰试剂，相同的群体 可以研究修改前后的通道。全部和部分 激动剂将用于明确解释功效的变化 和亲和力。使用 GluR2 受体的平行实验将用于 确认结构分配。 (2) 检验M3片段是转导片段的假设 耦合配体结合至通道开放。 谷氨酸受体的 M3 跨膜片段包含严格的 保守的氨基酸序列。确定该区域的半胱氨酸取代 硫醇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 在受体激活后发生构象变化 并且硫醇修饰将受体锁定在活性状态。这些 研究将得出动态变化的详细分子图像 伴随 NMDA 受体激活的结构。