Functional Cycle of a Mechanosensitive Channel

小型机械敏感通道MSCs是一种普遍存在的细菌渗透压调节因子，是一种先进的模型。 机械转导中初始事件的生物物理研究系统。解出的晶体结构和 真核细胞同源物的存在使骨髓间充质干细胞尤其具有吸引力。我们的初步数据，都是 实验和计算，为一种新的假说奠定了基础。 我们现在展示的MSCs是由晶体衍生的一系列构象状态和跃迁 结构。尽管之前认为间充质干细胞是一种张力和电压激活的通道，但我们发现其 通过张力而不是与电压无关的方式激活。然而，失活的过程是强烈的 由去极化推动。对晶体结构的计算评估表明，孔洞是 脱水的，它的构象代表非导电的，可能是失活的状态。使用目标能量 最小化我们已经设想了一个门控循环，它以紧凑的静止构象 毛孔周围紧密堆积着跨膜螺旋的圆桶。开放是通过协调一致的 螺旋向外运动，与毛孔收缩的湿润和扩张有关。失活 当造孔的TM3螺旋与面向脂质的TM1和TM2螺旋解偶联并坍塌时发生 形成一种狭窄的(晶体状)构象。为了验证这一假设，我们将(1)执行Steed分子 对闭合状态和开合状态进行动力学仿真，生成准确的模型；(2)验证预测 通过二硫化物交联法接近临界残基，并测试桥的功能后果 膜片钳实验中残留物的形成；(3)使用 半胱氨酸取代和MTS试剂；(4)评价气孔水化对浇注的贡献 能量学，并验证先前提出的汽相锁定机制。这项工作一旦完成，将 让我们更深入地了解感觉通道家族不断壮大的机制。

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.