Permeation and Gating Mechanisms of Mechanosensitive PIEZO channels

项目摘要 许多心血管和神经疾病，以及肿瘤的发生都是细胞力学改变的结果。 在这方面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--细胞对物理应力和力做出反应的过程。 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，反应是理解生物过程和治疗的新的关注目标 发展。 Piezo家族(Piezo1和Piezo2)是2010年发现的第一个兴奋性机械敏感离子通道 在脊椎动物中。现在已知，压电通道是触摸和疼痛(体感)、音量的关键传感器 调节(渗透感觉)、切应力(心血管张力)、压力感受、本体感觉和呼吸 生理学，可能还有其他重要的功能尚未发现。在过去的几年里，我们做出了很大的努力 十年，以确定美国人口中与Piezo有关的疾病和事件。到目前为止，Piezo 功能障碍与多种病理有关，包括高血压、淋巴疾病和贫血， 躯体感觉和神经障碍、癌症和转移等。尽管它们的生物性和 医学意义上，依赖于压电的机械转导背后的机制仍然难以捉摸。因此， 我们实验室的目标是了解压力和薄膜张力等物理力是如何控制Piezo1的 在健康和疾病状态下的功能。 本研究建议重点研究Piezo1通道的离子渗透和与力相关的门控机制， 在细胞中，以及在重组的脂质双层系统中。我们将使用生化和生物物理技术 为了了解脂质双层如何控制Piezo1的门控和随后的离子传导 薄膜。此外，我们已经确定了稳健的表达和蛋白质纯化方案来检查 Piezo1通道的功能。液滴脂双层膜将用于研究单通道的电导和开放 纯化蛋白在生物相关的脂类成分中的概率。经结构鉴定的孔结构域 PIEZO1将被用作了解压力敏感性和电压相关失活的模板- Piezo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的细胞 膜片钳技术是研究哺乳动物Piezo1通道功能的理想方法。我们确信，一个 全面了解Piezo的功能是对哺乳动物领域的及时贡献 机械转导。我们独特的方案代表了Piezos单分子研究的应用。 这项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 针对神经病理性疼痛、脑缺血和胶质瘤等。

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.