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的巨大治疗潜力。校长 神经类固醇的分子靶点是GABAA受体。我们已经证明有多个亚单位- GABAA受体上神经类固醇的特异性结合位点，每个位点都有助于 神经类固醇对受体表达和功能的影响。本项目将使用光标记技术来定义 神经类固醇结合在突触和突触外GABAA最丰富形式上的精确位点 受体。新型神经甾体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的数量，以鉴定结合位点并确定 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质谱（MS）方法， 包括中间向下，完整的蛋白质和天然MS。然后，我们将在每一个确定的突变氨基酸。 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）GABAA受体和一套神经类固醇类似物光标记试剂将 其中光标记部分位于神经甾体周围的不同位置 骨干表达的受体将与光标记试剂结合使用，以确定 使用完整蛋白质MS在每个亚基上的标记位点的数目和使用蛋白质MS在每个五聚体受体上的标记位点的数目。 通过光标记修饰的特定氨基酸将使用中-下MS来鉴定。 然后将光标记数据与分子建模和对接结合使用，以确定 神经甾体在每个结合口袋中的优选取向，并鉴定关键残基， 是神经类固醇结合或作用所必需的。在目标2受体将表达，其中这些关键的 残留物被诱变。突变体受体将用于功能测定（通道门控、运输 受体与表面的结合和正构配体结合的调节）以确定每个受体的贡献。 神经类固醇作用的结合位点。然后将评估神经类固醇标记和方向， 突变的受体（如在目的1中）以确定突变如何改变神经类固醇结合和/或作用。的 这些研究的数据将为深入了解神经甾体的特定分子相互作用提供依据 作用于GABAA受体上的每个结合位点。这些数据将提供一个结构模板， 亚基和功能特异性神经甾体类似物的开发以及对以下方面的新见解 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.