Fine-tuning the pentameric ligand-gated ion channel response via the lipid bilayer

我的研究项目主要是了解脂质影响膜蛋白功能的机制。具体来说，我感兴趣的是一个超家族的蛋白质称为五聚体配体门控离子通道（pLGIC）的脂质敏感性。这些离子通道通常存在于突触后膜中，在那里它们将化学信号（神经递质）转化为电反应（膜电位的变化）。pLGIC的一个迷人特征是通道门控发生在毫秒时间尺度上，即使门控中的微小变化也具有深远的生物学效应。此外，这些离子通道的活性对脂质高度敏感，表明脂质可能在pLGIC功能中发挥作用。 由于低温电子显微镜的最新进展，我的研究计划发生了重大转变，现在使我们能够解决嵌入脂质双层（纳米盘）中的膜蛋白的高分辨率结构。在我最近的休假期间，我开发了将功能性烟碱乙酰胆碱受体（nAChR）重建成脂质纳米盘的方法，并以足够高的分辨率解决了结构，以确定11个脂质结合位点。通过合作，我最近还解决了原核pLGIC，ELIC的相对高分辨率的X射线晶体结构，并确定了在ELIC功能中发挥作用的脂质结合位点。该结合位点的功能特征导致脂质结合如何影响ELIC脱敏的相对详细的模型。这项研究资助提案中描述的工作将建立在这些令人兴奋的开创性发现的基础上。本提案的目的是阐明和比较脂质改变三种pLGIC（nAChR、GLIC和ELIC）功能的机制。具体来说，我们将重点关注脂质结合所有三个pLGIC改变功能的机制。我们将1）定义存在于每个pLGIC周围的微环境中的内源性脂质，2）表征这些和其他脂质如何影响通道门控，3）使用分子动力学（MD）模拟探索这些和其他脂质与每个pLGIC的结合，4）求解不同膜中的pLGIC结构以探索脂质结合特异性和脂质结合对结构的影响，（5）采用诱变和电生理学相结合的方法来验证机理假说。 我们现在可以第一次解决脂质环境中膜蛋白的结构。我们可以可视化脂质结合位点，然后使用复杂的生物物理技术探索脂质结合的功能后果。我的实验室准备在我们对脂质-蛋白质相互作用的理解上取得重大突破。拟议的研究不仅将导致我们对脂质-pLGIC相互作用的理解取得重大突破，而且还将为研究生，研究生和本科学员的培训提供一个特殊的环境。

My research program is focused on understanding the mechanisms by which lipids influence membrane protein function. Specifically, I am interested in the lipid sensitivities of a super-family of proteins called pentameric ligand-gated ion channels (pLGICs). These ion channels are typically found in post-synaptic membranes, where they convert a chemical signal (the neurotransmitter) into an electrical response (change in membrane potential). One fascinating feature of pLGICs is that channel gating occurs on the millisecond time scale with even subtle changes in gating having profound biological effects. In addition, the activities of these ion channels are highly sensitive to lipids suggesting that lipids may play a role in pLGIC function. My research program has undergone a major shift due to recent advances in cryo-electron microscopy, which now allow us to solve high resolution structures of membrane proteins embedded in lipid bilayers (nanodiscs). During my recent sabbatical, I developed methods for reconstituting functional nicotinic acetylcholine receptors (nAChRs) into lipid nanodiscs and solved structures at high enough resolution to identify 11 lipid binding sites. Through collaborations, I also recently solved a relatively high-resolution x-ray crystal structure of the prokaryotic pLGIC, ELIC and identified a lipid binding site that plays a role in ELIC function. This binding site was functionally characterized leading to a relatively detailed model of how lipid binding influences ELIC desensitization. The work described in this research grant proposal will build on these exciting seminal findings. The goal of this proposal is to elucidate and compare the mechanisms by which lipids alter the function of three pLGICs, the nAChR, GLIC and ELIC. Specifically, we will we focus on the mechanisms by which lipid binding to all three pLGICs alters function. We will 1) define the endogenous lipids that exist in the microenvironments surrounding each pLGIC, 2) characterize how these and other lipids influence channel gating, 3) explore the binding of these and other lipids to each pLGIC using molecular dynamics (MD) simulations, 4) solve pLGIC structures in different membranes to explore both lipid binding specificity and the effects of lipid binding on structure, and 5) use a combination of mutagenesis and electrophysiology to validate mechanistic hypothesis. We can now for the first time solve structures of membrane proteins in lipid environments. We can visualize lipid binding sites and then explore the functional consequences of lipid binding using sophisticated biophysical techniques. My lab is poised to make major breakthroughs in our understanding of lipid-protein interactions. The proposed studies will not only lead to major breakthroughs in our understanding of lipid-pLGIC interactions but will also provide an exceptional environment for the training of post-graduate, graduate, and undergraduate trainees.