Elucidating the gating mechanisms of bacterial mechanosensitive channels

机械敏感(MS)通道通过打开离子导体来感知机械力并对其做出响应 路径。MS通道在所有生命王国中都能找到，而在人类中，在许多 感觉过程，包括听觉、触觉、血压的平衡和调节。第一 MS通道可能在早期原核生物中进化，以保护其免受低渗胁迫。因为细菌多发性硬化症 通道在细菌中普遍表达，但在人类中不表达，因为它们不受控制的打开 细菌对细菌的有害且通常是致命的影响，可能是由于重要的代谢物，细菌 MS通道是开发新型抗生素的耐人寻味的目标。细菌表达两种类型的MS通道， 大电导毫秒通道(MSCL)和小电导毫秒通道(MSCS)。MSCL成员 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。MSCS渠道更加多样化， 细菌常常表现出不止一个并列基因。这两个细菌的MS通道都是基于“来自- 脂质的原理和对周围膜的跨膜压力分布的反应。然而， 尽管MSCL和MSCs在不同的功能状态下有结构可用，但其机制 膜张力打开这些通道一直是个谜。我们最近测定了低温电子 不同膜环境中间充质干细胞的显微镜(冷冻-EM)结构，由纳米盘提供，包括 一种在张力下模仿薄膜的物体。由分子动力学(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和 推测与间充质干细胞相关的脂类在机械感觉中的作用。我们将继续使用 结合单粒子冷冻-EM、膜片钳电生理和MD模拟来研究结构 以及细菌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类环境中采用定义的开放构象，调节剂如何影响 通过改变MSCs的脂质环境，以及16碳酰链是否在MSCs门控中发挥特定的作用。 在目标2中，我们将把我们的研究扩展到细菌环核苷酸门控(BCNG)通道，以阐明 MSCs折叠被调整为使通道对cAMP结合而不是对膜张力做出反应。目标3将 关注MSCL。我们将在天然脂质环境中确定MSCL的结构，以证实(或反驳) 存在被认为在门控中起关键作用的充满脂质的纳米口袋。最后，我们将确定 MSCL的结构由不同的效应器打开，以可视化该通道在打开状态下的结构和 来验证我们的假设，即不同的效应器会导致具有不同孔径的开放构象。这个 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渠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.