Functional Cycle of a Mechanosensitive Channel

描述（由申请人提供）：小机械敏感通道MscS是一种普遍存在的细菌渗透调节剂，是一种先进的模型系统，用于机械转导初始事件的生物物理研究。已解决的晶体结构和真核同源物的存在使间充质干细胞特别有吸引力。我们的初步数据，包括实验和计算，为MscS的门控机制的新假设奠定了基础，我们现在将其作为晶体结构衍生的一系列构象状态和转变。尽管之前的观点认为间充质干细胞是一种张力和电压激活的通道，但我们发现它的张力激活与电压无关。而去极化则强烈地促进了失活过程。晶体结构的计算评估表明，孔隙是脱水的，其构象代表了一种非导电的，可能是失活的状态。利用目标能量最小化，我们设想了一个门控循环，它开始于一个紧凑的桶状静止构象，跨膜螺旋紧密地包裹在孔周围。开放是通过与润湿和孔收缩扩张相关的螺旋向外协调运动实现的。当形成孔隙的TM3螺旋与面向脂质的TM1和TM2螺旋分离并坍塌成狭窄的（晶体状）构象时，就会发生失活。为了验证这一假设，我们将(1)进行定向分子动力学模拟，并生成封闭和开放状态的精确模型；(2)通过二硫交联验证临界残留物的预测接近度，并在膜片钳实验中测试桥梁形成的功能后果；(3)利用半胱氨酸取代和MTS试剂测试孔隙和缝隙中残留物的可及性；(4)评价孔隙水化对门控能量的贡献，验证先前提出的汽锁机制。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.