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: 10654863
• 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/10654863
• 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; Health; Hematological Disease; Homologous Gene; Human; Hypertension; Investigation; Ion Channel; Ions; Kinetics; Lateral; Link; Lipid Bilayers; Lipids; Liquid substance; Lymphatic Diseases; Malignant Neoplasms; 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; 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; Tissues; Touch sensation; Transmembrane Domain; United States; Vertebrates; 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.

项目概要 许多心血管和神经系统疾病以及肿瘤发生都是由细胞力学的变化引起的。 在这种情况下对人类病理生理学的评估表明，这些疾病有一个共同的根本原因： 异常机械转导——细胞对物理压力和力量做出反应的过程。 机械敏感离子通道，细胞将外力转化为电能的分子机器 因此，对于理解生物过程和治疗而言，它们是新兴的令人感兴趣的目标 发展。 Piezo 家族（Piezo1 和 Piezo2）于 2010 年被发现，是第一个兴奋性机械敏感离子通道 在脊椎动物中，压电通道现在被认为是触觉和疼痛（体感）、音量的关键传感器。 调节（渗透感觉）、剪切应力（心血管张力）、压力感受、本体感受和呼吸 生理学，并且可能还有其他重要功能尚待发现。 迄今为止，美国人口中识别压电相关疾病和事件的时间已长达十年。 功能障碍与多种病理有关，包括高血压、淋巴疾病和贫血， 体感和神经系统疾病、癌症和转移等，尽管它们具有生物学和功能性。 医学相关性，压电依赖性机械传导背后的机制仍然难以捉摸。 我们实验室的目标是了解压力和膜张力等物理力如何控制压电1 健康和患病状态下的功能。 该研究计划重点关注 Piezo1 通道的离子渗透和力依赖性门控机制， 在细胞以及重建的脂质双层系统中，我们将采用生物化学和生物物理技术。 了解脂质双层如何控制 Piezo1 的门控以及随后的离子传导 此外，我们还确定了强大的表达和蛋白质纯化方案来检查膜。 Piezo1 通道的功能将用于研究单通道电导和开放。 生物学相关脂质组合物中纯化蛋白质的概率。 Piezo1 将用作模板来了解压力敏感性和电压依赖性失活 - 压电通道的标志 - 通过构建各种缺失突变体 - 在 HEK 细胞中异源表达。 初步数据令人震惊，并表明液滴双层方法与传统细胞相结合 膜片钳检测非常适合研究哺乳动物 Piezo1 通道功能。 对压电功能的全面理解是对哺乳动物领域的及时贡献 我们独特的建议代表了压电单分子研究的应用。 该提案的完成将为剖析和启动有效疗法的开发提供一条途径 针对神经性疼痛、脑缺血和神经胶质瘤等。