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.

项目概要/摘要 机电转换 (MET) 过程可实现机械信息的转换 从声音到电信号，这是耳蜗系统功能的基本步骤。这方面的失败 过程导致听力损失和耳聋。了解 MET 的基本特性将有助于更好地 对耳聋的了解，导致有针对性的治疗和治疗。 MET 发生在 毛束，由尖端链接介导，细胞外蛋白质将较短的静纤毛连接到相邻较高的静纤毛 静纤毛。发束向最高的静纤毛行的偏转增加了尖端连接的张力并打开 MET 通道位于较短静纤毛的顶部。尽管有大量的工作涉及 机械敏感离子通道的脂膜调节，有关的数据有限但不断增长 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，或直接影响通道 通过个体脂质/蛋白质相互作用。 PIP2 是一种内源性磷脂，可调节 MET 通道 特性，可能通过直接相互作用或通过改变膜力学间接实现。伸展 激活通道修饰剂，GsMTx4 降低了 MET 通道的静息开放概率 (Po)，同时还阻塞 降低外部钙或去极化毛细胞引起的 Po 增加，表明脂质 膜可能参与调节 MET 通道 Po。电压和钙的影响可以被调节 由于相邻脂质之间的多价离子相互作用而导致脂质堆积发生变化。我们最近直接 使用双光子荧光恢复一次评估单个静纤毛的膜扩散率 光漂白 (FRAP) 证明静纤毛膜对钙和电压敏感，但 不是体细胞，并且 MET 通道 Po 与膜扩散率共同变化，支持 MET 的假设 通道可以通过膜力学进行调节。然而，由于FRAP的空间和时间限制， 我们无法局部动态监测静纤毛膜。 为了进一步检验这个假设并克服当前的技术限制，我将结合 利用新型粘度传感器的活细胞荧光寿命成像 (FLIM) 进行电生理学检查 首次在哺乳动物耳蜗中提高了时空分辨率的膜粘度。我会 评估静纤毛膜粘度随电压、钙和膜的局部和时间变化 胆固醇和 PIP2 等成分，并将这些影响与 MET 通道 Po 的变化相关联。这些研究 将增强我们对脂质膜在毛细胞机械转导中重要性的基本了解。 从生物物理角度理解静纤毛机械基础的关键组成部分 且具有生物学相关性。这些新技术的开发和使用将极大地促进我的职业生涯 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。