Permeation and Gating Mechanisms of Mechanosensitive PIEZO channels

机械敏感压电通道的渗透和门控机制

• 批准号: 10364203
• 负责人: RUHMA SYEDA
• 金额: $ 41万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10364203
• 关键词: Amino Acids; Anemia; Biochemical; Biological; Biological Assay; Biological Process; Biophysics; Brain Diseases; Brain Ischemia; Cardiovascular Diseases; Cardiovascular system; Cells; Chemicals; Complex; Coupled; Cryoelectron Microscopy; Data; Degenerative polyarthritis; Development; Disease; Drug Design; Elements; Environment; Evolution; Exhibits; Exposure to; Family; Functional disorder; Future; Glioma; Goals; Head; Health; Hematological Disease; Human; Hypertension; Investigation; Ion Channel; Ions; Kinetics; Lateral; Link; Lipid Bilayers; Lipids; Liquid substance; Lymphatic Diseases; Mechanics; Medical; Membrane; Membrane Proteins; Merkel Cells; Molecular; Molecular Biology; Molecular Machines; Neoplasm Metastasis; Neurons; Pain; Pathology; Physiological; Physiology; Piezo 1 ion channel; Piezo 2 ion channel; Piezo ion channels; Plant Roots; Play; Point Mutation; Population; Probability; Process; Property; Proprioception; Protein Region; Proteins; Protocols documentation; Publishing; Pulse Pressure; Regulation; Reporting; Research Proposals; Resolution; Respiratory physiology; Role; Sensory; Series; Somatosensory Disorders; Spinal Ganglia; Stimulus; Stress; Stretching; Structure; System; Tertiary Protein Structure; Testing; Time; Tissues; Touch sensation; Transmembrane Domain; United States; Vertebrates; base; biophysical techniques; direct application; disease-causing mutation; extracellular; insight; interest; mechanical properties; mechanotransduction; membrane assembly; monomer; mutant; nervous system disorder; painful neuropathy; patch clamp; pressure; protein purification; protein reconstitution; reconstitution; response; sensor; sensory system; shear stress; single molecule; structural biology; therapeutic development; therapeutic target; therapeutically effective; tumorigenesis; voltage

Project Summary Many cardiovascular and neurological disorders, and oncogenesis result from changes in cell mechanics. Assessment of human pathophysiology in this context reveals that these diseases share a common root cause: abnormal mechanotransduction – the process by which cells respond to physical stress and forces. Mechanosensitive ion channels, the molecular machines by which cells convert external forces into electrical response, are therefore emerging targets of interest, for understanding biological processes and for therapeutic development. Piezo family (Piezo1 and Piezo2) was discovered in 2010 as the first excitatory mechanosensitive ion channels in vertebrates. Piezo channels are now known to be critical sensors of touch and pain (somatosensation), volume regulation (osmosensation), shear stress (cardiovascular tone), baroreception, proprioception and respiratory physiology, and may have other important functions yet to be discovered. Substantial efforts are made in the last decade to identify Piezo related diseases and incidents within the United State population. So far, Piezo dysfunction is linked to diverse pathologies including hypertension, lymphatic disease and anemias, somatosensory and neurological disorders, cancer and metastasis, amongst others. Despite their biological and medical relevance, the mechanism behind Piezo-dependent mechanotransduction remains elusive. Therefore, our lab’s goal is to understand how physical forces such as pressure and membrane tension control Piezo1 function in health and diseased state. This research proposal focuses on ion permeation and force-dependent gating mechanisms of Piezo1 channels, in cells, as well as in reconstituted lipid bilayer systems. We will employ biochemical and biophysical techniques in efforts to understand how lipid bilayer control the gating of Piezo1 and subsequent ion conduction across the membrane. Moreover, we have identified robust expression and protein purification protocols to examine the function of Piezo1 channels. Droplet lipid bilayers will be used to study the single channel conductance and open probability of the purified protein in biologically relevant lipid compositions. Structurally identified pore domain of Piezo1 will be used as a template to understand the pressure sensitivity and voltage-dependent inactivation - hallmark of Piezo channels - by constructing various deletion mutants- heterologous expression in HEK cells. The preliminary data is striking, and shows that the droplet bilayer approach coupled with traditional cellular patch clamp assays are ideally suited to study mammalian Piezo1 channel function. We are convinced that a comprehensive understanding of Piezo’s function is a timely contribution to the field of mammalian mechanotransduction. Our unique proposal represents the application of single molecule investigation of Piezos. Completion of this proposal will provide a path to dissect and kick-start the development of effective therapeutics targeted towards neuropathic pain, brain ischemia and gliomas, amongst others.

项目摘要 许多心血管和神经系统疾病，以及细胞力学变化引起的肿瘤发生。 在这种情况下对人类病理生理学的评估表明，这些疾病具有共同的根本原因： 异常机械转导 - 细胞对身体应激和力反应的过程。 机械敏感的离子通道，即细胞将外力转化为电的分子机器 因此，反应是感兴趣的新兴靶标，用于理解生物学过程和治疗 发展。 在2010年发现了压电家族（压电1和压电2）作为第一个兴奋机械敏感的离子通道 在脊椎动物中。现在已知压电通道是触摸和疼痛的关键传感器（体积），体积 调节（渗透压），剪切应力（心血管张力），压力感受，本体感受和呼吸 生理学，并且可能具有其他重要功能有待发现。最后做出了巨大的努力 确定美国人口中与压电相关的疾病和事件的十年。到目前为止，压电 功能障碍与潜水病变有关，包括高血压，淋巴病和贫血， 体感和神经系统疾病，癌症和转移等。尽管它们的生物学和 医学相关性，压电依赖性机制背后的机制仍然难以捉摸。所以， 我们实验室的目标是了解压力和膜张力控制压电等物理力量如何 在健康和患病状态中起作用。 该研究建议着重于压电通道的离子渗透和力依赖力的门控机制， 在细胞以及重建的脂质双层系统中。我们将采用生化和生物物理技术 为了了解脂质双层如何控制压电1的门控和随后的离子传导 膜。此外，我们已经确定了鲁棒的表达和蛋白质纯化方案，以检查 压电1通道的功能。液滴脂质双层将用于研究单通道电导和打开 在生物学相关的脂质组成中纯化蛋白的概率。结构上鉴定出的孔结构域 压电1将用作模板，以了解压力敏感性和电压依赖性失活 - 压电通道的标志 - 通过在HEK细胞中构建各种缺失突变体 - 异源表达。 初步数据令人震惊，并表明液滴双层接近传统的蜂窝 斑块夹测定非常适合研究哺乳动物压电通道功能。我们坚信 对Piezo功能的全面了解是对哺乳动物领域的及时贡献 机械转导。我们的独特建议代表了压电的单分子研究的应用。 该提案的完成将为剖析和启动有效治疗的发展提供一条途径 针对神经性疼痛，脑缺血和神经胶质瘤等。