Functional Cycle of a Mechanosensitive Channel

机械敏感通道的功能循环

• 批准号: 7105290
• 负责人: SERGEI I SUKHAREV
• 金额: $ 28.22万
• 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/7105290
• 关键词: bacterial proteins; bioenergetics; chemical kinetics; chemical stability; conformation; cysteine; disulfide bond; hydropathy; lipid bilayer membrane; mechanoreceptors; membrane channels; model design /development; molecular dynamics; protein structure; protein structure function; site directed mutagenesis; voltage /patch clamp; water channel; bacterial proteins; bioenergetics; chemical kinetics; chemical stability; conformation; cysteine; disulfide bond; hydropathy; lipid bilayer membrane; mechanoreceptors; membrane channels; model design /development; molecular dynamics; protein structure; protein structure function; site directed mutagenesis; voltage /patch clamp; water channel

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（由申请人提供）：小型机械敏感通道MSC，一种无处不在的细菌渗透量调节剂，是一种用于机械转移初始事件的生物物理研究的高级模型系统。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，无论是实验性的还是计算的，都为MSC的门控机制奠定了基础，我们现在以一系列构象状态和源自晶体结构得出的过渡呈现。尽管以前认为MSC是张力和电压激活的通道，但我们发现其通过张力而不是电压独立的激活。然而，通过去极化强烈促进了失活的过程。晶体结构的计算评估表明，孔脱水，其构象代表了一种无导体，可能失活的状态。使用靶向能量最小化，我们设想了一个门控循环，该循环始于枪管对枪管的紧凑静止构象，其跨膜螺旋在孔周围紧紧地堆积。通过一致的向外运动与润湿和扩展孔隙收缩相关的螺旋向外运动。当孔形成的TM3螺旋从面向脂质的TM1和TM2螺旋中解散并塌陷成狭窄的（晶体样）构象时，发生灭活。为了检验该假设，我们将（1）执行转导的分子动力学模拟，并为封闭状态和开放状态生成准确的模型； （2）通过二硫键交联验证临界残基的预测邻近性，并测试斑块钳实验中桥梁形成的功能后果； （3）使用半胱氨酸取代和MTS试剂测试孔和缝隙中残基的可访问性； （4）评估孔隙水合对门控能源的贡献，并验证先前提出的蒸气锁机构。完成工作后，将使我们更加靠近理解不断增长的感官渠道家庭的机制。