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: 10796256
• 负责人: THOMAS WALZ
• 金额: $ 10.74万
• 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/10796256
• 关键词: Adopted; Affect; Antibiotics; Bacteria; Binding; Cancerous; Carbon; Complement; Cryoelectron Microscopy; Cyclic AMP; Diameter; Electrophysiology (science); Environment; Equilibrium; Family; Hearing; Human; Ions; Life; Lipids; Membrane; Molecular Conformation; Nanotechnology; Osmosis; Pathogenicity; Pathway interactions; Play; Prokaryotic Cells; Role; Sensory Process; Stress; Structure; Testing; Tissues; Touch sensation; Visualization; blood pressure regulation; cyclic-nucleotide gated ion channels; insight; mechanical force; member; molecular dynamics; nano; nanodisk; novel; paralogous gene; particle; patch clamp; pressure

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）通道有意义并通过打开离子传导来响应机械力 路径。 MS频道都在生活的所有王国中都发现，在人类中，在许多人中扮演着重要角色 感觉过程，包括听力，触摸感，血压平衡和调节。第一个 MS通道可能在早期原核生物中演变为免受低湿度应激的保护。因为细菌MS 通道在细菌中无处不在，但没有在人类中表达，并且因为它们的不受控制的开口具有 对细菌的删除且经常致命的作用，大概是由于重要代谢物，细菌的丧失 MS通道是开发新型抗生素的有趣靶标。细菌表达两种类型的MS通道， 大电导率（MSCL）和小电导的MS通道（MSC）。 MSCL的成员 家庭是高度保守的，MSCL已成为理解MS频道的范例，因为 它的简单性和对不同实验方法的合理性。 MSCS渠道更多的潜水员， 细菌通常表达多个旁系同源物。两种细菌MS通道都是根据“力from- 脂质的原理并响应周围膜的跨膜压力曲线。然而， 即使在不同功能状态下可用于MSCL和MSC的结构， 膜张力打开这些通道仍然神秘。我们最近确定了冷冻电子 MSC在不同膜环境中的显微镜（冷冻EM）结构，包括纳米盘（包括） 一个模仿张力下的膜。通过分子动力学（MD）完成的结构 模拟和电生理研究使我们能够在不同的功能状态下可视化通道和 推断出与MSC相关的脂质在机理中起着哪些作用。我们将继续使用 单粒子冷冻EM，斑块钳电生理学和MD模拟的组合研究结构 和细菌MS通道的门控。在AIM 1中，我们将继续探索脂质在MSC功能中的功能， 特别是它是否适应本机脂质环境中定义的开放构象，调节剂如何影响 MSC通过改变其脂质环境，以及16碳酰基链在MSC门控中是否起着特定的作用。 在AIM 2中，我们将将研究扩展到生物周期性核苷酸门控（BCNG）通道，以阐明如何阐明 MSC折叠的适应性使通道响应营地绑定而不是膜张力。目标3意志 专注于MSCL。我们将在本地脂质环境中确定MSCL的结构，以确认（或反驳） 有人认为在门控中起着至关重要的作用，存在脂质填充的纳米袋。最后，我们将确定 MSCL的结构由不同的效果打开，以在开放状态和 为了测试我们的假设，即不同的影响会导致与孔径不同的开放构象。 这些研究的结果不仅将为细菌MS通道的门控机制提供新的见解，即 但也有助于利用这些渠道进行生物医学应用。