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

通过施加局部力来识别 Piezo1 离子通道的机械敏感域

• 批准号: 8977179
• 负责人: Jason Wu
• 金额: $ 4.31万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8977179
• 关键词: Address; Adult; Affect; Afferent Neurons; Analgesics; Blood Vessels; Bradykinin; Cardiovascular Diseases; Cardiovascular system; Cells; Charge; Chemicals; Cyclic AMP; Data; Development; Disease; Drug Design; Drug Targeting; Electrophysiology (science); Esthesia; Exhibits; Family; Gated Ion Channel; Goals; Hyperalgesia; Individual; Inflammation; Inflammation Mediators; Intervention; Ion Channel; Ion Channel Protein; Mammals; Measures; Mechanics; Mediating; Membrane; Methods; Molecular; Molecular Target; Mus; Nociception; Organ; Outcome; Pain; Pain Disorder; Physiology; Population; Process; Protein Family; Proteins; Regulation; Research Support; Risk; Role; Sensory; Signal Transduction; Stimulus; Structure; Testing; Touch sensation; Translating; Transmembrane Domain; Vertebrates; Work; Zebrafish; addiction; allodynia; base; design; detector; drug development; effective therapy; extracellular; magnetic beads; magnetic field; mechanical allodynia; medical complication; novel; painful neuropathy; patch clamp; pressure; protein complex; public health relevance; receptor; response; sensor; small molecule; targeted treatment; voltage

 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.

 描述（由申请人提供）：Piezo是一种机械门控离子通道家族，对于各种压力敏感过程（如心血管发育以及机械触觉和疼痛感）非常重要。目前试图解决疼痛和心血管疾病的各种原因的策略通常伴随着脱靶效应和成瘾等风险。了解直接将物理力转化为可兴奋细胞信号的感觉分子将有助于开发更准确的靶向治疗。因此，压电通道是用于这种疗法的有希望的候选者。然而，参与通道机械敏感性的特定结构必须阐明，以推动药物开发的方向。该项目的长期目标是确定Piezo内赋予机械敏感性的结构域，以建立对压力感觉和伤害感受的分子理解。我假设，在压电，特定的域是唯一敏感的机械力，而其他人是比较不敏感。为了验证这一假设，我将在离子通道的单域上施加局部力，同时用膜片钳电生理学测量通道活性的调制。为了实现这一点，我将顺磁性纳米珠结合到预测的压电细胞外环，并在强磁场的拉力存在下测量压力诱导的通道响应。我的初步数据表明，通过使用这种方法，我可以检测到机械敏感性的变化，可以直接归因于离子通道的特定域。该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，为Piezo活性的化学调节提供特定的分子靶标。