Elucidating the gating mechanisms of bacterial mechanosensitive channels

机械敏感（MS）通道通过打开离子传导通道来感测和响应机械力。 通路MS通道存在于所有生命王国中，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。第一 MS通道可能在早期原核生物中进化，作为对低渗胁迫的保护。因为细菌性MS 通道在细菌中普遍表达，但在人类中却没有，因为它们不受控制的开放， 对细菌的一种有害的，通常是致命的作用，可能是由于重要的代谢产物的损失，细菌 MS通道是开发新型抗生素的有趣靶点。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）。MscL成员 家族是高度保守的，MscL已经成为理解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。MSCS渠道更加多样化， 细菌通常表达一种以上的蛋白质。两种细菌MS通道都是基于“来自- 脂质的原理并响应于周围膜的跨膜压力分布。然而，在这方面， 尽管MscL和MscS在不同的功能状态下有不同的结构， 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 显微镜（cryo-EM）结构的MscS在不同的膜环境，提供了纳米盘，包括 一个是模仿张力下的膜。分子动力学（MD）补充的结构 模拟和电生理研究，使我们能够可视化通道在不同的功能状态， 以推断与MscS相关的脂质在机械感觉中起什么作用。我们将继续使用 结合单粒子冷冻EM，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脂质环境中采用确定的开放构象，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，我们将把我们的研究扩展到细菌的环核苷酸门控（bCNG）通道，以阐明如何在细菌的细胞中表达。 MscS折叠被调整以使通道响应cAMP结合而不是膜张力。目标3将 专注于MSCL。我们将在天然脂质环境中确定MscL的结构，以证实（或反驳） 存在脂质填充的纳米袋，其被认为在门控中起关键作用。最后，我们将确定 由不同效应物打开的MscL的结构，以可视化该通道在打开状态下的结构， 以检验我们的假设，即不同的效应物导致具有不同孔径的开放构象。的 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， 而且还有助于将这些通道用于生物医学应用。

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.