5-HT RECEPTORS AND THEIR EFFECTORS

这项建议的长期目标是加深对 分子和细胞事件之间的复杂相互作用 为了衡量药物疗效，最初使用5-HT1a受体系统的S a 模特。这项调查是在不同层面上的综合方法 生物组织：组织、细胞和分子。在体内，5- HT1a受体与腺酰环化酶(AC)活性负相关，并且 调节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，激活 受体系统很少单独出现，如果有的话。因此，在 具体目标1，我们建议研究药物疗效的变化在5- HT1a受体对已知的第二信使激活的反应 磷脂酶C(PLC)激活途径及其作用机制 这样的变化可能会发生。[Ca~(2+)]i升高的影响， 蛋白激酶C活性或受体介导的PLC在5-HT1A上的激活 反应性将在两种准备中进行研究；大鼠海马区 5-HT1a受体自然表达的切片和克隆细胞 5-HT1a受体已稳定转染的细胞系。这个 海马片的制备，它最接近于体内的 环境，使我们能够测量5-HT1A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 使用几种不同的单元格类型进行控制(和获取 优势)细胞类型特异性效应(由于酶的差异 异构体和G蛋白补体)。这样的差异可以提供洞察力 研究治疗引起的药效变化的机制。我们 将评估通过这些PLC产生的5-HT1A药物疗效的变化- 使用受体结合和功能分析的关联细胞事件 并将确定这些变化的机制。在具体目标上2. 5-HT1a受体的结构成分对药物的贡献 疗效将通过定点突变进行评估。这些研究将 以三维结构的计算模型为指导 受体和大尺度分子动力学模拟的结果 突变体及其与配体的络合物。这些计算量 研究将与受体结合和功能研究相补充。 在转染突变受体基因的细胞系中鉴定 药物识别和受体激活的决定因素。总而言之， 这里提出的多方面方法将确定一些关键 在5-HT1a受体系统中测量药物疗效的潜在机制。 这些信息对于理解药物的有效性是至关重要的 药理学的基本原理之一。了解以下内容 需要有助于药物疗效的分子和细胞事件 合理设计具有最佳疗效水平的选择性药物 他们合理的治疗应用。

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.