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.
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