Interaction of Anesthetics with Neuronal PDZ Domains

麻醉药与神经元 PDZ 结构域的相互作用

基本信息

批准号：
7850412
负责人：
Roger A Johns
金额：
$ 11.33万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-17 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7850412
关键词：
AMPA Receptors Address Affect Affinity Affinity Labels Alveolar Anesthesia procedures Anesthetics Anterior Area Attenuated Behavioral Binding Binding Sites Biological Brain Breathing C-terminal Calorimetry Chimeric Proteins Co-Immunoprecipitations Complex Data Detergents Dose Event Excitatory Postsynaptic Potentials Figs - dietary Fingers Funding GluR2 subunit AMPA receptor Halothane Hybrids In Vitro Ions Isoflurane Kinetics Knock-out Knockout Mice Label Learning Ligands Link Mediating Memory Methods Molecular Molecular Target Motor Activity Mus Mutate Mutation N-Methyl-D-Aspartate Receptors N-Methylaspartate Neuraxis Neurons Nitric Oxide Synthase Type I Peptides Persistent pain Play Potassium Channel Process Prosencephalon Protein Family Proteins Publishing Reflex action Relative (related person)Research Research Personnel Resistance Role Scaffolding Protein Signal Pathway Signal Transduction Signaling Protein Slice Spinal Cord Spinal cord posterior horn Stroke Structure Subcellular structure Surface Surface Plasmon Resonance Synapses Synaptic Transmission Titrations Work Yeasts affinity labeling central sensitization chronic pain clinically relevant dimer discs, large (Drosophila) homolog 2 protein, rat grasp in vivo neurotoxicity neurotransmission novel patch clamp postsynaptic programs protein protein interaction receptor research study response sevoflurane yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Anesthetics affect the central nervous system by altering synaptic transmission, but the mechanisms are poorly understood. PSD-95/SAP90 is one of a family of proteins recently shown to physically link synaptic signaling proteins into macromolecular signal transduction structures via PDZ domain interactions. We discovered PSD-95/SAP90 interacts with NMDA receptors and neuronal NOS in the spinal cord. Suppression of PSD-95/SAP90 expression reduced the dose required for inhalational anesthesia. Preliminary data shows clinically relevant concentrations of anesthetics dose-dependently inhibit the PDZ domain-mediated protein interaction between PSD-95 or PSD-93 and the NMDA receptor or neuronal NOS. These inhibitory effects are immediate, potent, reversible, and occur at a hydrophobic peptide-binding groove on the surface of the second PDZ domain of PSD-95. The focus of this proposal is to understand the mechanism by which inhaled anesthetics interact with PDZ domains in neuronal signaling pathways and the biological consequences of these interactions. The aims of the current proposal will: 1) Determine if the effect of inhalational anesthetics on neuronal PDZ domain-mediated protein-protein interactions observed with the NMDA receptor can be generalized to other neuronal signaling pathways using yeast 2-hybrid, co- IP, GST pulldown and plasmon resonance approaches. 2) Define the importance of inhalational anesthetic disruption of PDZ domain-containing protein interactions in the biologic state of anesthesia by investigating the effect of knockout, knockdown and direct inhibition of PSD-93/PSD-95 on MAC of inhalational anesthetics and on locomotor activity, including placing reflex, grasping reflex and righting reflex. Determine the effect of inhalational anesthetic on NMDA mediated neurophysiologic responses in the spinal cord and cortex of mice with and without knockout of PSD-95 or PSD-93. 3) Characterize the biophysical interaction of anesthetics with the PDZ domains. To do so, the effect of mutation of PDZ2 inhalational anesthetic-binding sites and other similar domains on inhalational anesthetic binding to the domains will be evaluated using plasmon resonance, affinity photolabeling, and calorimetry studies.

描述（由申请人提供）：麻醉剂通过改变突触传递来影响中枢神经系统，但对其机制知之甚少。 PSD-95/SAP90 是最近显示可通过 PDZ 结构域相互作用将突触信号蛋白物理连接到大分子信号转导结构的蛋白质家族之一。我们发现 PSD-95/SAP90 与脊髓中的 NMDA 受体和神经元 NOS 相互作用。抑制 PSD-95/SAP90 表达可减少吸入麻醉所需的剂量。初步数据显示临床相关浓度的麻醉剂剂量依赖性地抑制 PSD-95 或 PSD-93 与 NMDA 受体或神经元 NOS 之间 PDZ 结构域介导的蛋白质相互作用。这些抑制作用是立即、有效、可逆的，并且发生在 PSD-95 第二个 PDZ 结构域表面的疏水性肽结合沟处。该提案的重点是了解吸入麻醉药与神经元信号通路中 PDZ 结构域相互作用的机制以及这些相互作用的生物学后果。当前提案的目标是：1) 确定吸入麻醉剂对 NMDA 受体观察到的神经元 PDZ 结构域介导的蛋白质-蛋白质相互作用的影响是否可以推广到使用酵母 2-hybrid、co-IP、 GST 下拉和等离子共振方法。 2) 通过研究 PSD-93/PSD-95 的敲除、敲低和直接抑制对吸入麻醉药 MAC 和运动活性的影响，定义吸入麻醉药破坏麻醉生物状态下含 PDZ 结构域的蛋白质相互作用的重要性，包括放置反射、抓握反射和翻正反射。确定吸入麻醉剂对敲除 PSD-95 或 PSD-93 的小鼠脊髓和皮质中 NMDA 介导的神经生理反应的影响。 3) 表征麻醉剂与 PDZ 结构域的生物物理相互作用。为此，将使用等离子体共振、亲和光标记和量热研究来评估 PDZ2 吸入麻醉剂结合位点和其他类似结构域的突变对吸入麻醉剂与这些结构域的结合的影响。