Elucidating the gating mechanisms of bacterial mechanosensitive channels

阐明细菌机械敏感通道的门控机制

• 批准号: 10583324
• 负责人: THOMAS WALZ
• 金额: $ 49.01万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10583324
• 关键词: Adopted; Affect; Anacystisnidulans; Antibiotics; Bacteria; Binding; Biological Process; Cancerous; Carbon; Cells; Chimeric Proteins; Complement; Cryoelectron Microscopy; Cyclic AMP; Cyclic Nucleotides; Cytolysis; Diameter; Drug Delivery Systems; Electrophysiology (science); Environment; Equilibrium; Escherichia coli; Eukaryota; Exposure to; Family; Gadolinium; Hearing; Human; Incubated; Ions; Length; Life; Ligand Binding; Lipids; Lysophospholipids; Maps; Membrane; Molecular; Molecular Conformation; Nanotechnology; Osmosis; Pathogenicity; Pathway interactions; Pharmaceutical Preparations; Play; Probability; Prokaryotic Cells; Proteins; Reaction; Resolution; Role; Sensory Process; Stress; Structure; Styrenes; Synechocystis; Testing; Tissues; Touch sensation; Visualization; Work; beta-Cyclodextrins; blood pressure regulation; copolymer; cyclic-nucleotide gated ion channels; desensitization; falls; follow-up; imaging agent; insight; maleic acid; mechanical force; mechanical stimulus; member; molecular dynamics; mutant; nano; nanodisk; novel; novel antibiotic class; paralogous gene; particle; patch clamp; pressure; reconstitution; response

Mechanosensitive (MS) channels sense and respond to mechanical forces by opening an ion-conducting pathway. MS channels are found in all kingdoms of life, and in humans play essential roles in a number of sensory processes, including hearing, the sense of touch, balance and regulation of blood pressure. The first MS channels likely evolved in early prokaryotes as protection from hypoosmotic stress. Because bacterial MS channels are ubiquitously expressed in bacteria, but not in humans, and because their uncontrolled opening has a deleterious and often lethal effect on the bacteria, presumably due to the loss of important metabolites, bacterial MS channels are intriguing targets for developing novel antibiotics. Bacteria express two types of MS channels, MS channels of large conductance (MscL) and MS channels of small conductance (MscS). Members of the MscL family are highly conserved and MscL has become a paradigm for the understanding of MS channels because of its simplicity and amenability to different experimental approaches. MscS channels are more diverse, and bacteria often express more than one paralog. Both bacterial MS channels are gated based on the ‘force-from- lipids’ principle and respond to the transmembrane pressure profile of the surrounding membrane. However, even though structures are available for MscL and MscS in different functional states, the mechanism by which membrane tension opens these channels has remained enigmatic. We have recently determined cryo-electron microscopy (cryo-EM) structures of MscS in different membrane environments, provided by nanodiscs, including one mimicking a membrane under tension. The structures, complemented by molecular dynamics (MD) simulations and electrophysiological studies, allowed us to visualize the channel in different functional states and to deduce what roles lipids associated with MscS play in mechanosensation. We will continue to use a combination of single-particle cryo-EM, patch-clamp electrophysiology and MD simulations to study the structure and gating of bacterial MS channels. In Aim 1, we will continue to explore the function of lipids in MscS function, in particular whether it adopts a defined open conformation in a native lipid environment, how modulators affect MscS by changing its lipid environment, and whether 16-carbon acyl chains play a specific role in MscS gating. In Aim 2, we will expand our studies to bacterial cyclic nucleotide-gated (bCNG) channels to elucidate how the MscS fold was adapted to make the channel respond to cAMP binding rather than membrane tension. Aim 3 will focus on MscL. We will determine the structure of MscL in a native lipid environment to confirm (or disprove) the existence of lipid-filled nano-pockets that were suggested to play a critical role in gating. Finally, we will determine the structure of MscL opened by different effectors to visualize the structure of this channel in the open state and to test our hypothesis that different effectors result in open conformations with different pore diameters. The results of these studies will not only provide new insights into the gating mechanism of bacterial MS channels, but also help in exploiting these channels for biomedical applications.

机械敏感 (MS) 通道通过打开离子传导通道来感知机械力并做出响应 MS 通道存在于所有生命领域，并且在人类的许多方面发挥着重要作用。 感觉过程，包括听觉、触觉、平衡和血压调节。 MS 通道可能是在早期原核生物中进化出来的，因为细菌 MS 可以保护免受低渗应激。 通道在细菌中普遍表达，但在人类中却不然，因为它们不受控制的开放 对细菌产生有害且通常致命的影响，可能是由于重要代谢物、细菌的损失 MS 通道是开发新型抗生素的有趣目标细菌表达两种类型的 MS 通道， 大电导 MS 通道 (MscL) 和小电导 MS 通道 (MscS)。 家族高度保守，MscL 已成为理解 MS 通道的范例，因为 它的简单性和对不同实验方法的适应性更加多样化，并且 细菌通常表达多个旁系同源物，这两种细菌 MS 通道均基于“力”进行门控。 脂质的原理并对周围膜的跨膜压力曲线做出反应。 尽管结构可用于处于不同功能状态的 MscL 和 MscS，但其机制 膜张力打开这些通道仍然是个谜。我们最近确定了低温电子。 由纳米圆盘提供的不同膜环境中 MscS 的显微镜（冷冻电镜）结构，包括 一种模拟张力下膜的结构，并辅以分子动力学 (MD)。 模拟和电生理学研究使我们能够可视化不同功能状态下的通道 为了推断与 MscS 相关的脂质在机械感觉中的作用，我们将继续使用 结合单颗粒冷冻电镜、膜片钳电生理学和 MD 模拟来研究结构 和细菌 MS 通道的门控 在目标 1 中，我们将继续探索脂质在 MScS 功能中的作用， 特别是它是否在天然环境脂质中采用明确的开放构象，调节剂如何影响 MScS通过改变其脂质环境，以及16碳酰基链是否在MscS门控中发挥特定作用。 在目标 2 中，我们将研究扩展到细菌环核苷酸门控 (bCNG) 通道，以阐明细菌如何 MscS 折叠经过调整，使通道对 cAMP 结合而不是 Aim 3 的膜张力做出反应。 我们将重点关注 MscL 在天然脂质环境中的结构，以证实（或反驳）这一点。 脂质填充纳米袋的存在被认为在门控中发挥着关键作用。最后，我们将确定。 由不同效应器打开的 MscL 结构，以可视化该通道在打开状态下的结构 检验我们的假设，即不同的效应器会导致具有不同孔径的开放构象。 这些研究结果不仅将为细菌MS通道的门控机制提供新的见解， 而且还有助于利用这些渠道进行生物医学应用。