Functional Cycle of a Mechanosensitive Channel

小机械敏感通道MscS，一种普遍存在的细菌细胞增殖调节剂，是一种先进的模型 机械传导初始事件的生物物理学研究系统。解析的晶体结构和 真核同源物的存在使得MscS特别有吸引力。我们的初步数据， 实验和计算，奠定了基础，一个新的假设有关的门控机制， 我们现在将其呈现为一系列来自晶体的构象状态和转变 结构尽管以前的观念认为MscS是一种张力和电压激活的通道，但我们发现它的 张力激活而非电压依赖性。然而，失活过程强烈地 由去极化促进。对晶体结构的计算评估表明， 脱水的，并且其构象代表非导电的、可能失活的状态。使用目标能量 最小化，我们设想了一个门控循环，其开始于一个紧凑的静息构象的 具有紧密包裹在孔周围的跨膜螺旋的桶。开放是通过协调一致的 与孔收缩的润湿和扩张相关的螺旋向外运动。灭活 当成孔的TM 3螺旋与面向脂质的TM 1和TM 2螺旋分离并塌陷时， 变成一个狭窄的（晶体状）构象。为了验证这一假设，我们将（1）进行转向分子 动力学模拟，并生成精确的模型，为关闭和打开状态;（2）验证预测 通过二硫键交联的关键残基的接近度，并测试桥的功能后果 膜片钳实验中的形成;（3）使用 半胱氨酸取代和MTS试剂;（4）评估孔水合对门控的贡献 并验证了先前提出的“气锁”机制。这项工作一旦完成， 使我们更接近于理解不断增长的感觉通道家族的机制。

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.