OPIOID RECEPTOR MECHANISMS

这项提议的总体目标是使用高分子技术 解决阿片受体机制的特征，因为它们发生在 原代培养完整神经细胞的膜环境。受体 受体-效应器偶联在整个细胞中的占有率和有效性 将与大鼠和猴子的离体膜中的细胞进行对比， 考虑膜结构不受扰动的影响，胞浆 因素，以及跨质膜的离子梯度。为了研究 激动剂和拮抗剂结合的动力学性质，纯化的GTP- 调控蛋白将通过膜融合的方式被整合，其 用百日咳毒素和碱处理调整浓度。 受体-效应器偶联也将在转移后进行评估 阿片受体从膜到原代去受体星形胶质细胞 文化。Mu受体、β受体和kappa受体的荧光标记 将进行和标记的受体用于评估侧支 膜迁移率是其碰撞偶联过程中的重要过程 效应物，G蛋白和腺苷环化酶。若要描述 配体-受体-效应器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 疏水性)，分离的脂转移蛋白将被 用来系统地改变神经元细胞膜的组成。 通过诱导受体和被掺入物之间的荧光能量转移 磷脂，具有重要功能的脂质边界的结构 将描述Mu受体、增量受体和kappa受体周围的层。 在神经细胞和神经元-神经胶质细胞共培养中，长期使用 特异性脂质掺入、受体占位及与G-受体偶联 将重点评估蛋白质/腺苷环化酶的作用 细胞耐受和依赖条件下的膜调节 到鸦片类药物。

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.