Functional Cycle of a Mechanosensitive Channel

机械敏感通道的功能周期

• 批准号: 7186658
• 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/7186658
• 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

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.

描述(申请人提供)：小的机械敏感通道MSCs，一种普遍存在的细菌渗透压调节器，是用于机械转导初始事件的生物物理研究的高级模型系统。已解决的晶体结构和真核同源物的存在使MSCs特别吸引人。我们的初步数据，包括实验和计算，为关于MSCs门控机制的新假说奠定了基础，我们现在将其描述为一系列源于晶体结构的构象状态和跃迁。尽管以前的观点认为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相当不依赖于电压。然而，去极化强烈地促进了这一失活过程。对晶体结构的计算评估表明，该孔是脱水的，它的构象代表了一种不导电的、可能是失活的状态。使用有针对性的能量最小化，我们设想了一个门控周期，从紧凑的桶休息构象开始，跨膜螺旋紧密地堆积在孔周围。开孔是通过与毛孔收缩的润湿和扩张相关的螺旋向外协调运动来实现的。当形成孔的TM3螺旋与面向脂质的TM1和TM2螺旋解偶联并坍塌成狭窄的(晶体状)构象时，就会失活。为了验证这一假说，我们将(1)执行操控分子动力学模拟，并为闭合和开放状态生成准确的模型；(2)通过二硫键交联验证关键残基的预测接近，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达性；(4)评估孔水化对门控能量学的贡献，并验证先前提出的Vapor lock机制。这项工作一旦完成，将使我们更接近于了解不断增长的感觉通道家族的机制。