Interaction of Anesthetics with Neuronal PDZ Domains

描述(申请人提供)：麻醉药通过改变突触传递来影响中枢神经系统，但其机制尚不清楚。PSD-95/SAP90是最近发现的通过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中的蛋白质家族之一。我们发现PSD-95/SAP90与脊髓中的NMDA受体和神经元一氧化氮合酶相互作用。抑制PSD-95/SAP90的表达减少了吸入麻醉所需的剂量。初步数据显示，临床相关浓度的麻醉药剂量依赖性地抑制PSD-95或PSD-93与NMDA受体或神经元型NOS之间由PDZ结构域介导的蛋白质相互作用。这些抑制作用是直接的、有效的、可逆的，并且发生在PSD-95的第二个PDZ结构域表面的疏水肽结合槽中。该建议的重点是了解吸入麻醉剂与神经元信号通路中的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。目前提案的目的将：1)确定吸入麻醉剂对神经元PDZ结构域介导的与NMDA受体的蛋白质-蛋白质相互作用的影响是否可以推广到其他神经元信号通路，使用酵母2-杂交、共IP、GST下拉和等离子共振方法。2)通过研究PSD-93/PSD-95的敲除、敲除和直接抑制对吸入麻醉药的MAC和对包括放置反射、抓握反射和翻正反射在内的运动活动的影响，明确吸入麻醉剂破坏含有PDZ结构域的蛋白相互作用在麻醉生物状态中的重要性。确定吸入麻醉剂对PSD-95或PSD-93基因敲除和未敲除PSD-95或PSD-93的小鼠脊髓和皮质NMDA介导的神经生理反应的影响。3)表征麻醉药与PDZ结构域的生物物理相互作用。为此，将使用等离子激元共振、亲和光标记法和量热法研究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结合的影响。

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.