Molecular Sites of Neurosteroid Binding

神经类固醇是神经元兴奋性和神经系统发育的重要调节剂 作为抗抑郁剂、麻醉剂和神经保护剂具有巨大的治疗潜力。校长 神经类固醇的分子靶点是GABAA受体。我们已经证明了有多个亚基- 神经类固醇在GABAA受体上的特定结合部位，每一个都参与了 神经类固醇对受体表达和功能的影响。该项目将使用光标记技术来定义 神经类固醇在最丰富形式的突触和突触外GABA上的精确结合部位 感受器。新型神经类固醇类似物光标记试剂将被开发并用于测定 每个亚基上的神经类固醇结合位点数，以识别结合部位并确定 神经类固醇在这些部位的定位。为了实现这些目标，我们将利用最先进的蛋白质 化学和表达技术结合尖端的质谱学(MS)方法， 包括中间向下的完整蛋白质和天然MS，然后我们将突变每一个已鉴定的氨基酸 结合位点，并使用功能读数来确定哪些结合位点介导了 神经类固醇对GABAA受体表达和功能的影响。该项目有两个具体目标： 在目标1中，将使用病毒表达系统(BacMam)来表达大量突触(α1β2γ2) 和突触外(α4β3δ)GABA受体和一套神经类固醇类似物光标记试剂将 其中光标记部分被放置在神经类固醇周围的不同位置 我的脊梁。表达的受体将与光标记试剂一起使用来确定 使用完整蛋白MS的每个亚基和使用五聚体受体的每个亚基上的标记位点数 天然MS通过光标记修饰的特定氨基酸将使用中下层MS进行鉴定。 然后，光标记数据将与分子建模和对接一起使用，以确定 神经类固醇在每个结合口袋中的优先取向，并识别可能 对于神经类固醇的结合或作用是必要的。在AIM 2受体中将表达这些关键的 残基被诱变。突变受体将用于功能分析(通道门控、运输 表面受体和直位配基结合的调节)以确定每个受体的贡献 神经类固醇作用的结合部位(S)。神经类固醇的标记和定位随后将在 突变的受体(如目标1)，以确定突变如何改变神经类固醇的结合和/或影响。这个 这些研究的数据将提供对神经类固醇潜在的特定分子相互作用的洞察 作用于其在GABAA受体上的每个结合部位。这些数据将为以下项目提供结构模板 亚单位和功能特异性神经类固醇类似物的发展以及对 神经类固醇生物学。

Neurosteroids are important modulators of neuronal excitability and nervous system development with enormous therapeutic potential as anti-depressants, anesthetics and neuro-protectants. The principal molecular target of neurosteroids is the GABAA receptor. We have shown that there are multiple, subunit- specific binding sites for neurosteroids on GABAA receptors, each of which contributes to the effects of neurosteroids on receptor expression and function. This project will use photolabeling techniques to define the precise sites of neurosteroid binding on the most abundant forms of synaptic and extrasynaptic GABAA receptors. Novel neurosteroid analogue photolabeling reagents will be developed and used to determine the number of neurosteroid binding sites on each subunit, to identify the binding sites and to determine the orientation of the neurosteroids in these sites. To achieve these goals we will utilize state-of-the-art protein chemistry and expression techniques in conjunction with cutting edge mass spectrometry (MS) methods, including middle-down, intact protein and native MS. We will then mutate amino acids in each of the identified binding sites and use functional readouts to determine which binding sites mediate the various effects of neurosteroids on GABAA receptor expression and function. The Project has two specific aims: In Aim 1, a viral expression system (BacMam) will be used to express large quantities of synaptic (α1β2γ2) and extrasynaptic (α4β3δ) GABAA receptors and a suite of neurosteroid analogue photolabeling reagents will be synthesized in which the photolabeling moieties are placed at various positions around the neurosteroid backbone. The expressed receptors will be used in conjunction with the photolabeling reagents to determine the number of labeling sites on each subunit using intact protein MS and on each pentameric receptor using native MS. The specific amino acids modified by photolabeling will be identified using middle-down MS. The photolabeling data will then be used in conjunction with molecular modeling and docking to determine the preferred orientation of neurosteroids in each of the binding pockets and to identify critical residues which may be necessary for neurosteroid binding or effect. In Aim 2 receptors will be expressed in which these critical residues are mutagenized. The mutant receptors will be used in functional assays (channel gating, trafficking of receptors to the surface and modulation of orthosteric ligand binding) to determine the contribution of each binding site to neurosteroid action(s). Neurosteroid labeling and orientation will be then be assessed in the mutated receptors (as in Aim 1) to determine how the mutations alter neurosteroid binding and/or effect. The data from these studies will provide insight into the specific molecular interactions underlying neurosteroid actions at each of its binding sites on GABAA receptors. These data will provide a structural template for development of subunit- and function-specific neurosteroid analogues as well as novel insights into neurosteroid biology.