The long term goal of this proposal is to develop an understanding of the complex interactions between molecular and cellular events which contribute to measured drug efficacy, using the 5-HT1A receptor system initially s a model. This investigation is an integrated approach at different levels of biological organization: tissue, cellular and molecular. In vivo, the 5- HT1A receptor is negatively linked to adenylyl cyclase (AC) activity and mediates opening of K+ channels. Since cells are subject to simultaneous actions of neurotransmitters, hormones and modulators, activation of a receptor system rarely, if ever, occurs in isolation. Therefore, in Specific Aim 1, we propose to study alterations of drug efficacy at the 5- HT1A receptor in response to activation of second messengers known to be activated by phospholipase C (PLC)-linked pathways and the mechanisms by which such alterations may occur. Effects of increases in [Ca2+]i levels, protein kinase C activity or receptor-mediated activation of PLC on 5-HT1A responsiveness will be studied in two preparations; the rat hippocampal slice where the 5-HT1A receptor is expressed naturally and clonal cell lines where the 5-HT1A receptor has been transfected stably. The hippocampal slice preparation, which most closely approximates an in vivo environment, enables us to measure 5-HT1A receptor-mediated inhibition of AC as well as the opening of K+ channels. Studies in clonal cells will be done with several different cell types to control for (and to take advantage of) cell-type specific effects (due to differences in enzyme isoforms and G-protein complement). Such differences can offer insight into mechanisms involved in treatment-induced changes in drug efficacy. We will assess changes in 5-HT1A drug efficacy, produced through these PLC- linked cellular events using both receptor-binding and functional assays and mechanisms for these changes will be identified.
In Specific Aim 2. the contribution of structural components of the 5-HT1A receptor to drug efficacy will be assessed by site-directed mutagenesis. These studies will be guided by computational modeling of the 3-dimensional structure of the receptor and by the outcome of large scale molecular dynamics simulations of the mutants and their complexes with ligands. These computational studies will be complemented with receptor binding and functional studies in cell lines transfected with the cDNA of mutated receptors to identify determinants for drug recognition and for receptor activation. In summary, the multi-faceted approach proposed here will identify some of the key mechanisms underlying measured drug efficacy at the 5-HT1A receptor system. This information is crucial for the understanding of drug efficacy which is one of the fundamental principles in pharmacology. Knowledge of the molecular and cellular events which contribute to drug efficacy is required for rational design of selective drugs at an optimal efficacy level and for their rational therapeutic application.
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