Permeation and Gating Mechanisms of Mechanosensitive PIEZO channels

项目摘要 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。 在此背景下对人类病理生理学的评估表明，这些疾病有着共同的根本原因： 异常的机械传导-细胞对物理压力和力量的反应过程。 机械敏感离子通道，细胞将外力转化为电的分子机器 因此，对于理解生物学过程和治疗， 发展 Piezo家族（Piezo 1和Piezo 2）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觉和疼痛（躯体感觉）、音量和听觉的关键传感器。 调节（触觉）、切应力（心血管紧张度）、压力感受、本体感受和呼吸 生理学，并可能有其他重要的功能尚未发现。在最后一个方面作出了巨大努力 在美国人口中识别压电相关疾病和事件的十年。到目前为止，Piezo 功能障碍与包括高血压、淋巴疾病和贫血在内的多种病理学有关， 躯体感觉和神经障碍、癌症和转移等。尽管他们的生物学和 尽管压电依赖性机械转导与医学相关，但压电依赖性机械转导背后的机制仍然难以捉摸。因此，我们认为， 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Piezo 1 在健康和疾病状态下发挥作用。 本研究提案重点关注Piezo 1通道的离子渗透和力依赖性门控机制， 在细胞中以及在重构的脂质双层系统中。我们将采用生物化学和生物物理技术 在努力了解脂质双层如何控制Piezo 1的门控和随后的跨膜离子传导中， 膜的此外，我们已经确定了强大的表达和蛋白质纯化方案，以检查 Piezo 1通道的功能。液滴脂质双层将被用来研究单通道电导和开放 纯化蛋白质在生物学相关脂质组合物中的概率。结构鉴定的孔结构域 Piezo 1将被用作了解压力敏感性和电压依赖性失活的模板- 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。 初步的数据是惊人的，并表明液滴双层方法与传统的细胞 膜片钳测定理想地适合于研究哺乳动物Piezo 1通道功能。我们相信， 全面了解压电的功能是对哺乳动物领域的及时贡献 机械传导我们独特的建议代表了单分子研究压电的应用。 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 靶向治疗神经性疼痛、脑缺血和神经胶质瘤等。

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.