Identifying the mechanosensitive domains of the Piezo1 ion channel by application of localized force

 描述(申请人提供)：PIZO是一种机械门控离子通道家族，对各种压力敏感过程非常重要，如心血管发育和机械触摸和疼痛感觉。目前的战略试图解决导致疼痛和心血管疾病的各种原因，但往往伴随着脱靶效应和成瘾等风险。了解在可兴奋细胞中直接将物理力量转化为信号的感觉分子将有助于开发更准确的靶向治疗方法。因此，压电通道很有希望成为这类疗法的候选者。然而，必须阐明与通道机械敏感性有关的特定结构，才能推动药物开发的方向。该项目的长期目标是确定压电体中赋予机械敏感性的区域(S)，以便建立对压力感觉和伤害性感觉的分子理解。我假设，在Piezo内部，特定的区域对机械力是唯一敏感的，而相比之下，其他区域则不那么敏感。为了验证这一假设，我将在离子通道的单个区域施加局域力，并同时使用膜片钳电生理学测量通道活动的调制。为了做到这一点，我将把顺磁纳米棒与预测的Piezo细胞外环结合起来，并在强磁场的拉力存在的情况下测量压力诱导的通道响应。我的初步数据显示，通过使用这种方法，我可以检测到可以直接归因于离子通道特定区域的机械敏感性的变化。本项目将使用一种新的方法来探测和识别Piezo中的单个机械传感器结构域(S)，为Piezo活性的化学调控提供特定的分子靶点。

 DESCRIPTION (provided by applicant): Piezo is a family of mechanically gated ion channels important for a variety of pressure sensitive processes such as cardiovascular development and the sense of mechanical touch and pain. Current strategies attempting to address a diverse range of causes for pain and cardiovascular disorders are often accompanied with risks such as off-target effects and addiction. Understanding the sensory molecules that directly transduce physical force into signals in excitable cells will allow for the development of more accurately targeted therapies. Piezo channels are therefore promising candidates for such therapies. However, the specific structures involved in channel mechanosensitivity must be elucidated to drive the direction of drug development. The long-term goal of this project is to identify the domain(s) within Piezo that confer mechanosensitivity in order to establish a molecular understanding for pressure sensation and nociception. I hypothesize that within Piezo, specific domains are uniquely sensitive to mechanical force, whereas others are less sensitive in comparison. To test this hypothesis, I will apply a localized force on single domains of the ion channel and simultaneously measure modulations in channel activity with patch clamp electrophysiology. To accomplish this, I will conjugate paramagnetic nanobeads to predicted extracellular loops of Piezo and measure pressure-induced channel responses in the presence of a pulling force by a strong magnetic field. My preliminary data show that by using this approach I can detect shifts in mechanosensitivity that can be directly attributed to specific domains of the ion channel. This project will use a novel method to probe and identify the individual mechanosensor domain(s) in Piezo, providing specific molecular targets for chemical regulation of Piezo activity.