Investigating the role of lipid membrane in the cochlear hair cell mechanotransduction

研究脂质膜在耳蜗毛细胞机械转导中的作用

• 批准号: 10652895
• 负责人: Shefin Sam George
• 金额: $ 19.5万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10652895
• 关键词: Affect; Aging; Apical; Auditory; Auditory system; BODIPY; Biochemical; Biophysics; Body Temperature; Buffers; Calcium; Calibration; Cell physiology; Cells; Cholesterol; Cochlea; DNA Sequence Alteration; Data; Development; Disease; Electrophysiology (science); Environment; Extracellular Protein; Failure; Genetic Diseases; Goals; Hair; Hair Cells; Hearing; Human Genetics; Image; Individual; Ion Channel; Ions; Lead; Learning; Link; Lipid Bilayers; Lipids; Measures; Mechanics; Mediating; Medical; Membrane; Membrane Lipids; Microscopy; Molecular; Monitor; Noise; Organ of Corti; Persons; Phosphatidylinositol 4,5-Diphosphate; Phospholipids; Photobleaching; Play; Positioning Attribute; Presbycusis; Probability; Process; Property; Proteins; Recovery; Research Personnel; Resolution; Rest; Role; Sensory Hair; Signal Transduction; Site; Stereocilium; Stretching; Surface; System; Techniques; Technology; Testing; Time; Translating; Translations; Viscosity; career; deafness; experimental study; extracellular; fluorescence lifetime imaging; hearing impairment; hearing loss treatment; improved; insight; mechanotransduction; neuronal cell body; new technology; novel; response; sensor; sound; spatiotemporal; targeted treatment; technology/technique; time use; two-photon; voltage

Project Summary/Abstract The mechano-electrical transduction (MET) process allows the transduction of mechanical information from sound into electrical signals, and it is a fundamental step in cochlear system function. Failures in this process lead to hearing loss and deafness. Understanding the basic properties of MET will lead to a better understanding of deafness, leading to targeted treatments and therapies. MET takes place at the level of the hair bundle and is mediated by tip links, extracellular proteins connecting shorter stereocilia to adjacent taller stereocilia. Deflections of the hair bundle towards the tallest stereocilia row increase tip-link tension and open MET channels that reside at the top of the shorter stereocilia. Although there is a large body of work regarding lipid membrane modulation of mechanosensitive ion channels, there is a limited but growing body of data on lipid modulation of cochlear hair cell MET. The lipid environment can affect channels indirectly through changes in membrane mechanics, or directly through individual lipid/protein interactions. PIP2, an endogenous phospholipid, modulates MET channel properties, potentially through a direct interaction or indirectly by altering membrane mechanics. A stretch activated channel modifier, GsMTx4 reduces the resting open probability (Po) of MET channel while also blocking the increase in Po induced by lowering external calcium or depolarizing the hair cell, suggesting the lipid membrane may be involved in modulating MET channel Po. The effect of voltage and calcium could be mediated through changes in lipid packing due to multivalent ions interacting between adjacent lipids. Our recent direct assessment of membrane diffusivity of individual stereocilium at a time using two-photon Fluorescent Recovery after Photobleaching (FRAP) demonstrated that stereocilia membrane is sensitive to calcium and voltage but not the soma, and MET channel Po co-varies with membrane diffusivity, supporting the hypothesis that the MET channel can be modulated by membrane mechanics. However, due to spatial and temporal limitations of FRAP, we were unable to monitor stereocilia membrane locally and dynamically. To further test this hypothesis and overcome current technological limitations, I will combine electrophysiology with live-cell fluorescence lifetime imaging (FLIM) of a novel viscosity sensor to examine the membrane viscosity with improved spatio-temporal resolution for the first time in mammalian cochlea. I will assess local and temporal changes in the stereociliary membrane viscosity with voltage, calcium, and membrane components like cholesterol and PIP2 and correlate these effects to changes in MET channel Po. These studies will enhance our basic understanding of the importance of lipid membrane in hair cell mechanotransduction. Understanding the crucial components in the mechanical underpinnings of the stereocilia are both biophysically and biologically relevant. The development and use of these new technologies will greatly advance my career as an independent investigator and likely have broader applications in the auditory field and beyond.

项目总结/摘要 机械-电转换（MET）过程允许机械信息的转换 从声音到电信号，这是耳蜗系统功能的基本步骤。失败在此 导致听力损失和耳聋。了解MET的基本属性将有助于更好地 了解耳聋，从而有针对性的治疗和疗法。MET在国家一级进行， 毛束和介导的顶端链接，细胞外蛋白连接较短的静纤毛，以相邻的较高 静纤毛发束朝向最高静纤毛行的偏转增加了尖端连接张力和张开 位于较短静纤毛顶部的MET通道。虽然有大量的工作是关于 脂质膜调节机械敏感离子通道，有一个有限的，但越来越多的数据， 耳蜗毛细胞MET的脂质调节。 脂质环境可以通过膜力学的变化间接影响通道， 通过单个脂质/蛋白质相互作用。内源性磷脂PIP 2调节MET通道 通过直接相互作用或通过改变膜力学间接地改变膜的性质。拉伸 激活的通道修饰剂GsMTx 4降低MET通道的静息开放概率（Po），同时也阻断 通过降低外部钙或去极化毛细胞诱导的Po增加，表明脂质 膜可能参与调节MET通道Po。电压和钙离子可介导这种效应 通过由于多价离子在相邻脂质之间相互作用而引起的脂质堆积的变化。我们最近的直接 用双光子荧光恢复法评价单个静纤毛的膜扩散率 光漂白后（FRAP）表明，静纤毛膜对钙和电压敏感， 而不是索马，MET通道Po与膜扩散率共变，支持MET通道Po与膜扩散率共变的假设。 通道可以通过膜力学来调节。然而，由于FRAP的空间和时间限制， 我们不能局部和动态地监测静纤毛膜。 为了进一步验证这一假设并克服当前的技术限制，我将联合收割机 电生理学与活细胞荧光寿命成像（FLIM）的一种新的粘度传感器，以检查 膜粘度与改善的时空分辨率第一次在哺乳动物耳蜗。我会 用电压、钙和膜来评估静纤毛膜粘度的局部和时间变化 这些影响与MET通道Po的变化相关。这些研究 将加强我们对毛细胞机械转导中脂膜重要性的基本认识。 了解静纤毛的机械基础的关键组成部分， 和生物学相关。这些新技术的开发和使用将大大推进我的事业 作为一名独立研究者，可能在听觉领域及其他领域有更广泛的应用。