STRUCTURAL DETERMINANTS OF GABA RECEPTOR FUNCTION

分子克隆研究揭示了许多哺乳动物 神经元GABA-A受体亚单位，并帮助解释了多样性， 在体外发现的受体。 某些亚基组合或突变导致 在不同的GABA亲和力、功效或药理学中产生受体; 然而，人们对这些现象背后的微观机制知之甚少， 观察这些受体在突触样条件下如何发挥作用 短暂的GABA暴露 假设是：受体组成和 特定的分子决定因素改变了参与的事件或过程， GABA结合、通道门控和受体的微观动力学 失活和变构调节与突触有关。 为了研究亚基组成和分子水平的作用， GABA受体功能的决定因素，提出了三个具体目标： #1）确定亚基组成对亲和力的功能后果， 表达受体的门控和药理学调节;#2）评估 参与表达的结合、门控和调节的特定氨基酸 #3）将GABA-A受体亚单位mRNA与 单个神经元中受体的电生理特性。 超快配体交换（100 SU）和电生理技术将 用于研究表达的定义亚基组合的切除斑块 HEK 293细胞 调查人员将直接解决流程 参与受体激活和门控，并研究受体功能 和药理学，以更好地概括激动剂活性的突触。 为了进一步将基因表达信息与受体 功能，单细胞RT-PCR技术将定性确定mRNA 存在于活的神经元和动力学指纹将获得与 通过<1ms GABA应用诱发的神经元受体系统响应。 这 该项目将提高我们对GABA分子相关性的理解 受体的结构和功能，可以导致更好的治疗 癫痫、焦虑和麻醉的干预。

DESCRIPTION: Molecular cloning studies have revealed numerous mammalian neuronal GABA-A receptor subunits and have helped explain the diversity of receptors found in vitro. Certain subunit combinations or mutations result in different GABA affinity, efficacy or pharmacology of resultant receptors; however little is known about what microscopic mechanisms underlie these observations or how these receptors function under synaptic-like conditions of transient GABA exposure. The hypothesis is: Receptor composition and specific molecular determinants alter events or processes involved in microscopic kinetics of GABA binding, channel gating and receptor deactivation and allosteric modulation that are relevant to the synapse. In an effort to study the role of subunit composition and molecular determinants for GABA receptor function, three specific aims are proposed: #1) Determine functional consequences of subunit composition on affinity, gating and pharmacological modulation of expressed receptors; #2) Evaluate specific amino acids involved in binding, gating and modulation of expressed GABA receptors; and #3) Correlate GABA-A receptor subunit mRNA and with electrophysiological properties of receptors in individual neurons. Ultrafast ligand exchange (100 su) and electrophysiological techniques will be used to study excised patches of defined subunit combinations expressed in HEK 293 cells. The investigators will directly resolve processes involved in receptor activation and gating and also study receptor function and pharmacology that better recapitulates agonist activity in the synapse. To further correlate information on genetic expression with receptor function, single cell RT-PCR techniques will qualitatively determine mRNA present in living neurons and kinetic fingerprints will be obtained with neuronal receptor system responses evoked by <1 ms GABA applications. This project will improve our understanding of the molecular correlates of GABA receptor structure and function which can result in better therapeutic intervention in epilepsy, anxiety and anesthesia.