OPIOID RECEPTOR MECHANISMS

这项建议的总体目标是使用高分子技术， 分辨率，以表征阿片受体机制，因为它们发生在 原代培养中完整神经细胞的膜环境。 受体 整个细胞中受体-效应物偶联的占用率和功效 将与来自大鼠和猴的分离膜中的那些进行对比， 考虑到未扰动的膜结构的影响，胞质 因子和跨质膜的离子梯度。 研究 激动剂和拮抗剂结合的动力学特性，纯化的GTP- 调节蛋白将通过膜融合并入，并且其 用百日咳毒素和碱处理调节浓度。 受体-效应物偶联也将在以下转移后进行评估： 阿片受体从膜到受体缺乏的星形胶质细胞 文化 μ、δ和κ受体的荧光标记 将进行和标记的受体用于评估横向 膜流动性作为其碰撞耦合的基本过程， 效应物G蛋白和腺苷酸环化酶。 来描述 配体-受体-效应物相互作用对化学物质的依赖性 组成和/或物理化学性质（流动性和 由于膜的疏水性，分离的脂质转移蛋白将被 用于系统地改变神经细胞膜的组成。 通过诱导受体和掺入的受体之间的荧光能量转移， 磷脂，功能上重要的脂质边界的结构 将表征μ-、δ-和κ-受体周围的层。 在神经元细胞和神经元-神经胶质细胞共培养物中， 特异性脂质掺入、受体占有率和与G- 蛋白质/腺苷酸环化酶的作用将集中评估 细胞耐受性和依赖性条件下的膜调节 鸦片。

The overall goal of this proposal is to use techniques of high molecular resolution to characterize opioid receptor mechanisms as they occur in the membrane environment of intact neural cells in primary culture. Receptor occupancy and the efficacy of receptor-effector coupling in whole cells will be contrasted to those in isolated membranes from rat and monkey, considering the influence of unperturbed membrane structure, cytosolic factors, and ionic gradients across the plasma membranes. To study the kinetic properties of agonist and antagonist binding, purified GTP- regulatory protein will be incorporated by membrane fusion, and its concentration adjusted with pertussis toxin and alkaline treatment. Receptor-effector coupling will also be assessed following the transfer of opioid receptors from membranes to receptor-devoid astrocytes in primary culture. Fluorescent labeling of the mu-, delta-, and kappa-receptors will be carried out and the tagged receptors used to assess lateral membrane mobility as an essential process in their collision-coupling to the effectors, G-protein and adenylate cyclase. To describe the dependence of ligand-receptor-effector interactions on the chemical composition and/or physicochemical properties (fluidity and hydrophobicity) of the membrane, isolated lipid transfer proteins will be used to systematically alter the composition of neuronal cell membranes. By inducing fluorescent energy transfer between receptor and incorporated phospholipid, the structure of the functionally significant lipid boundary layer around the mu-, delta-, and kappa-receptor will be characterized. In neuronal cells and in neuron-glia co-cultures, chronically modified by specific lipid incorporation, receptor occupancy and coupling to G- protein/adenylate cyclase will be assessed focusing on the role of membrane modulation under conditions of cellular tolerance and dependence to opiates.