5-HT RECEPTORS AND THEIR EFFECTORS

本提案的长期目标是了解 分子和细胞事件之间的复杂相互作用 为了测量药物疗效，使用5-HT 1A受体系统最初是一种 模型 这项调查是在不同层次上的综合办法， 生物组织：组织、细胞和分子。 在体内，5- HT 1A受体与腺苷酸环化酶（AC）活性呈负相关， 介导K+通道的开放。 由于细胞同时受到 神经递质、激素和调节剂的作用， 受体系统很少单独发生。 因此在 具体目标1，我们建议研究在5- HT 1A受体对第二信使激活的反应，已知是 磷脂酶C（PLC）连接的途径激活， 这种变化可能发生。 [Ca 2 +]i水平升高的影响， 蛋白激酶C活性或受体介导的PLC对5-HT 1A的激活 将在两种制剂中研究反应性;大鼠海马 5-HT 1A受体天然表达的切片和克隆细胞 5-HT 1A受体已稳定转染的细胞系。 的 海马切片制备，其最接近于体内 环境，使我们能够测量5-HT 1A受体介导的抑制， AC以及K+通道的开放。 克隆细胞的研究将是 用几种不同的细胞类型来控制（并采取 细胞类型特异性效应（由于酶的差异 同种型和G蛋白补体）。 这种差异可以提供洞察力 涉及治疗引起的药物疗效变化的机制。 我们 将评估5-HT 1A药物疗效的变化，通过这些PLC产生- 使用受体结合和功能测定的关联细胞事件 并将确定这些变化的机制。 具体目标2。 5-HT 1A受体结构组分对药物的作用 将通过定点诱变来评估效力。 这些研究将 通过三维结构的计算建模来指导 受体和大规模分子动力学模拟的结果 突变体及其与配体的复合物。 这些计算 研究将补充受体结合和功能研究 在用突变受体的cDNA转染的细胞系中， 药物识别和受体激活的决定因素。 总的来说， 这里提出的多方面的方法将确定一些关键的 在5-HT 1A受体系统测量药物功效的潜在机制。 这些信息对于了解药物疗效至关重要， 药理学的基本原理之一 知识 需要有助于药物功效的分子和细胞事件 用于在最佳功效水平下合理设计选择性药物， 合理的治疗应用。

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.