Functional Cycle of a Mechanosensitive Channel

描述（由申请人提供）：小的机械敏感通道MscS，一种普遍存在的细菌β调节剂，是一种用于机械转导中初始事件的生物物理研究的先进模型系统。解决的晶体结构和真核同源物的存在使MscS特别有吸引力。我们的初步数据，实验和计算，奠定了基础，一个新的假设的门控机制的MSCS，我们现在提出的一系列构象状态和过渡来自晶体结构。尽管以前的概念，MscS是一个紧张和电压激活的通道，我们发现它的激活紧张，而电压无关。然而，去极化强烈促进失活过程。晶体结构的计算评估表明，孔是脱水的，其构象代表了非导电的，可能是失活状态。使用有针对性的能量最小化，我们设想了一个门控循环，其开始于桶的紧凑的静止构象，跨膜螺旋紧密地堆积在孔周围。通过与孔收缩的润湿和扩张相关的螺旋的协同向外运动来实现打开。当成孔的TM 3螺旋与面向脂质的TM 1和TM 2螺旋分离并塌缩成窄的（晶体样）构象时，发生失活。为了验证这一假设，我们将（1）进行操纵分子动力学模拟，并生成闭合和开放状态的精确模型;（2）通过二硫键交联验证预测的关键残基的邻近性，并在膜片钳实验中测试桥形成的功能后果;（3）使用半胱氨酸取代和MTS试剂测试孔和缝隙中残基的可接近性;（4）评估孔隙水化对浇口能量的贡献，验证先前提出的“气锁”机制。这项工作一旦完成，将使我们更接近于理解不断增长的感觉通道家族的机制。

DESCRIPTION (provided by applicant): The small mechanosensitive channel MscS, a ubiquitous bacterial osmoregulator, is an advanced model system for biophysical studies of the initial events in mechanotransduction. The solved crystal structure and the existence of eukaryotic homologs make MscS especially attractive. Our preliminary data, both experimental and computational, lay the foundation for a new hypothesis about the gating mechanism of MscS which we now present as a series of conformational states and transitions derived from the crystal structure. Despite previous notions that MscS is a tension and voltage-activated channel, we found its activation by tension rather voltage-independent. However, the process of inactivation was strongly promoted by depolarization. Computational assessment of the crystal structure suggested that the pore is dehydrated and its conformation represents a non-conducting, likely inactivated state. Using targeted energy minimizations we have envisioned a gating cycle which begins with a compact resting conformation of the barrel with transmembrane helices tightly packed around the pore. Opening is achieved through a concerted outward movement of helices associated with wetting and expansion of the pore constriction. Inactivation occurs when the pore-forming TM3 helices decouple from the lipid-facing TM1 and TM2 helices and collapse into a narrow (crystal-like) conformation. To test this hypothesis we will (1) perform steered molecular dynamics simulations and generate accurate models for the closed and open states; (2) verify the predicted proximities of critical residues by disulfide cross-linking and test the functional consequences of bridge formation in patch-clamp experiments; (3) test accessibilities of residues in the pore and crevices using cysteine substitutions and MTS reagents; (4) evaluate the contribution of the pore hydration to the gating energetics and validate the previously proposed Vapor lock' mechanism. The work, when accomplished, will move us closer toward understanding the mechanisms of the growing families of sensory channels.