Functional Cycle of a Mechanosensitive Channel

机械敏感通道的功能周期

• 批准号: 7370990
• 负责人: SERGEI I SUKHAREV
• 金额: $ 27.4万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7370990
• 关键词: Accounting; Affect; Bacteria; Biological Assay; Biological Models; Biophysics; Caliber; Cell membrane; Charge; Cholinergic Receptors; Constriction procedure; Cysteine; Data; Data Analyses; Disulfides; Engineering; Equilibrium; Eukaryota; Eukaryotic Cell; Event; Family; Foundations; Helix (Snails); Homologous Gene; Hydration status; Kinetics; Life; Link; Lipid Bilayers; Lipids; Location; Measurement; Mechanics; Membrane; Modeling; Modification; Molecular; Molecular Conformation; Movement; Mutagenesis; Mutation; Play; Process; Prokaryotic Cells; Property; Proteins; Reagent; Regulation; Research; Rest; Role; Sensory; Series; Shapes; Simulate; Solutions; Staging; Stretching; Structure; Study models; System; Testing; Water; Work; aqueous; base; crosslink; desire; ear helix; gain of function; interest; models and simulation; molecular dynamics; mutant; novel; patch clamp; periplasm; pressure; research study; simulation; vapor; voltage

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.

小型机械敏感通道MSC是一种无处不在的细菌渗透量，是一种高级模型 机械转移中初始事件的生物物理研究系统。解决的晶体结构和 真核同源物的存在使MSC特别有吸引力。我们的初步数据 实验和计算，为关于门控机制的新假设奠定了基础 我们现在以一系列构象状态和从晶体衍生的过渡呈现的MSC 结构。尽管以前认为MSC是张力和电压激活的通道，但我们发现了它的 张力激活而不是电压无关。但是，失活的过程很强 通过去极化促进。晶体结构的计算评估表明孔是 脱水及其构象代表了一种无导体，可能被灭活的状态。使用目标能量 最小化，我们设想了一个门控循环，该循环始于紧凑的休息构象 枪管带有跨膜螺旋螺旋的孔紧紧地围在孔周围。开放是通过一致的 与润湿和扩展孔收缩相关的螺旋向外运动。失活 当孔形成的TM3螺旋从面向脂质的TM1和TM2螺旋分离并塌陷时，就会发生 进入狭窄的（晶体状）构象。为了检验该假设，我们将（1）执行转导的分子 动力学模拟并为封闭状态生成准确的模型； （2）验证预测 通过二硫键交联和测试桥梁的功能后果来接近临界残基 斑块钳实验中的形成； （3）使用孔和缝隙中残留物的测试可访问性 半胱氨酸取代和MTS试剂； （4）评估孔隙水合对门控的贡献 能量和验证先前提出的蒸气锁的机制。工作后， 使我们更加靠近了解不断增长的感觉渠道家族的机制。