Structural and functional analysis of Apo-states of the model Cys-loop receptor GluCl from Caenorhabditis elegans

化学突触的神经传递通过神经递质对受体的作用以许多不同的方式进行调节。Cys-loop受体是一个五聚体配基门控离子通道大家族，介导快速的神经传递。尽管它们具有共同的体系结构，但根据它们的离子选择性，它们可以引起兴奋和抑制效应。由于这种广泛的功能，它们参与了各种神经系统疾病，并成为许多临床药物和药物滥用物质的靶标。尽管对这些受体的结构和功能的了解取得了很大进展，但它们的激活和门控机制的细节仍不清楚。最近发表的秀丽线虫阴离子选择性Cys-loop受体GluCl与变构激动剂伊维菌素结合的结构为该受体的机制提供了重要的初步见解。然而，为了完全理解，来自蛋白质不同构象状态的额外结构信息是必需的。所有当前的结构都显示了受体的配体结合构象。了解蛋白质的载脂蛋白状态将极大地提高对受体激活时结构变化的理解。因此，我想获得模型蛋白GluCl在不同构象状态下的高分辨率结构信息。初步结果表明，脂类可能在受体的激活中发挥重要作用。因此，该提案的一个重点将是研究脂类在受体激活过程中的功能和结构作用。由于部分变构激动剂伊维菌素结合在受体的跨膜区内，有关与脂类等自然调节剂相互作用的结构信息也可能有助于开发新的治疗方法。此外，我还想了解通道孔的门控是如何实现的。为此，通过定点突变产生的大量野生型和突变型蛋白将在昆虫细胞中异源表达，并通过亲和层析和尺寸排除层析纯化为均一。蛋白质的均一性和稳定性将通过荧光检测尺寸排除层析进行监测。该蛋白的功能将通过闪烁邻近分析中的放射性配基结合、非洲爪哇卵母细胞的双电极电压钳电生理和进入重组蛋白脂质体的离子通量来评估。一旦有足够数量的均质和活性蛋白质可用，将建立高脂条件下突变体的3D结晶试验，以通过X射线衍射来解决蛋白质结构。

Neurotransmission at chemical synapses is modulated in many different ways by the action of neurotransmitters on receptors. Cys-loop receptors are a large family of pentameric ligand-gated ion channels that mediate fast neurotransmission. Despite sharing a common architecture they can evoke both excitatory and inhibitory effects depending on their ion selectivity. Due to this wide-spread function they are involved in various neurological diseases and serve as targets for many clinical drugs and substances of drug abuse.Despite great advances on the understanding of the structure and function of these receptors, the details of their activation and gating mechanism are still poorly understood. A recently-published structure of the anion-selective Cys-loop receptor GluCl from Caenorhabditis elegans with the allosteric agonist ivermectin bound provided important initial insights into the mechanisms of the receptor. Yet, for a complete understanding, additional structural information from different conformational states of the protein is required. All current structures exhibit a ligand-bound conformation of the receptor. Knowledge especially about the apo state of the protein will greatly improve the understanding of the structural changes upon receptor activation. I therefore want to obtain high-resolution structural information of the model protein GluCl in different conformational states.Preliminary results indicate that lipids might play an important role in the activation of the receptor. One focus of this proposal will therefore be to investigate the functional and structural role of lipids in the context of receptor activation. Because the partial allosteric agonist ivermectin binds within the transmembrane region of the receptor, structural information about the interaction with natural modulators like lipids might also help to develop new therapeutics. Additionally I want to find out how gating of the channel pore is accomplished.For this purpose large amounts of wild-type as well as mutant protein made by site-directed mutagenesis will be expressed heterologously in insect cells and purified to homogeneity by affinity and size-exclusion chromatography. Homogeneity and stability of the protein will be monitored by fluorescence-detection size exclusion chromatography. Functionality of the protein will be assessed by radioligand binding in scintillation proximity assay, two-electrode voltage clamp electrophysiology on Xenopus laevis oocytes and ion flux into reconstituted proteoliposomes. Once sufficient amounts of homogeneous and active protein are available, 3D crystallization trials of the mutants in high-lipid conditions will be set up to solve the protein structure by X-ray diffraction.