Structural Requirements for Benzodiazepine Modulation of GABA-A Receptors

描述（由申请人提供）：γ -氨基丁酸A型受体（gabar）是配体门控离子通道，介导中枢神经系统中大多数快速抑制性神经传递。gabar介导的神经抑制的改变与几种疾病有关，包括癫痫、精神分裂症、焦虑和抑郁。一些临床上重要的药物包括苯二氮卓类药物（BZDs）、巴比妥类药物、麻醉剂和乙醇通过调节gaba介导的GABAR电流来发挥其治疗作用。然而，这些调节剂改变gaba受体激活的确切机制仍然存在争议。具体来说，BZD正调制和负调制的结构因素尚不清楚。本提案的主要目的是确定具有不同功能（正、负和零调制器）和结构特性（经典和非经典）的bzd如何调制GABAR。我将使用几种实验方法，包括位点定向诱变、双电极电压钳、半胱氨酸可及性、二硫捕获和放射性配体结合，来验证受体中的特定区域，即gamma2亚基的环路F，有助于BZD的有效性，并且对于BZD结合与gaba介导的电流调制的耦合是重要的。进一步的研究使用由栓系串联亚基、膜片夹紧和半胱氨酸可及性组成的受体，将深入了解bzd将其信息传递到GABA结合位点的机制。苯二氮卓类药物是最常用的处方类药物之一，用作抗焦虑药、抗惊厥药、助眠药、肌肉松弛剂和抗精神病药。本研究提出的研究将增强我们对苯二氮卓类药物选择性亲和力和疗效的结构因素的理解，并将有助于合理设计新的、更有效的药物，用于治疗从焦虑到癫痫等疾病。

DESCRIPTION (provided by applicant): Gamma-aminobutyric acid type A receptors (GABARs) are ligand-gated ion channels that mediate the majority of rapid inhibitory neurotransmission in the central nervous system. Alterations in GABAR-mediated neural inhibition have been linked to several disorders including epilepsy, schizophrenia, anxiety, and depression. Several clinically important drugs including benzodiazepines (BZDs), barbiturates, anethestics, and ethanol exert their therapeutic actions by modulating the GABA-mediated current of the GABAR. However, the exact mechanism by which these modulators alter GABA-activation of the receptor is still debated. Specifically, the structural elements underlying BZD positive and negative modulation remain unknown. The main objective of this proposal is to determine how BZDs with different functional (positive, negative, and zero modulators) and structural properties (classical and non-classical) modulate the GABAR. I will use several experimental approaches including site-directed mutagenesis, two-electrode voltage clamp, cysteine accessibility, disulfide trapping, and radioligand binding to test the hypothesis that a specific region in the receptor, Loop F of the gamma2 subunit, contributes to BZD efficacy and is important for coupling BZD binding to modulation of GABA-mediated current. Additional studies using receptors composed of tethered tandem subunits, patch clamping, and cysteine accessibility will provide insight into the mechanism by which BZDs relay their information to the GABA binding sites. Benzodiazepines are one of the most commonly prescribed classes of drugs and are used as anxiolytics, anticonvulsants, sleep aids, muscle relaxants, and antipsychotics. Research proposed in this study will enhance our understanding of the structural elements underlying the selective affinity and efficacy of benzodiazepines, and will be instrumental in the rational design of new, more effective drugs for the treatment of disorders ranging from anxiety to epilepsy.