Biophysical Basis of Organization and Dynamics of Prestin Membrane Complexes

Prestin 膜复合物的组织和动力学的生物物理基础

• 批准号: 8317688
• 负责人: ROBERT M RAPHAEL
• 金额: $ 30.32万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8317688
• 关键词: Address; Amplifiers; Anti-Inflammatory Agents; Anti-inflammatory; Auditory; Binding; Biochemical; Biological Assay; C-terminal; Cells; Cochlea; Complex; Data; Dependence; Diarrhea; Diffusion; Dimerization; Disease; Dwarfism; Electric Capacitance; Event; Feedback; Fluorescence; Fluorescence Anisotropy; Fluorescence Recovery After Photobleaching; Fluorescence Resonance Energy Transfer; Frequencies; Goals; Hearing; High-Frequency Hearing Loss; Human; Image; Imaging Techniques; Lateral; Lead; Life; Measures; Mechanics; Mediating; Membrane; Membrane Microdomains; Membrane Potentials; Membrane Proteins; Microscopy; Molecular; Molecular Conformation; Motor; Mutate; Non-Steroidal Anti-Inflammatory Agents; Outcome; Outer Hair Cells; Pharmaceutical Preparations; Principal Investigator; Process; Property; Proteins; Regulation; Relative (related person); Research; Role; Sensory; Signal Transduction; Structure; Testing; Tinnitus; Transgenic Organisms; base; hearing impairment; intermolecular interaction; optical imaging; programs; protein protein interaction; rat Pres protein; research study; single molecule; skeletal abnormality; solute; tool; voltage; voltage clamp

DESCRIPTION (provided by applicant): Cochlear outer hair cells enhance the sensitivity of mammalian hearing through active mechanical feedback powered by the membrane protein prestin. The long-term goal of our research efforts is to understand the mechanism by which prestin interacts with the membrane and responds to changes in the transmembrane potential. Preliminary research has established that prestin molecules self-associate, and that changes in prestin-prestin interactions alter the function and lateral organization of prestin in the membrane. We propose to extend these studies and further elucidate the relationship between the function of prestin and the formation of prestin complexes in the membrane. The first specific aim will be to determine the relationship between prestin oligomerization and function and establish if a particular oligomeric state represents the functional unit of prestin. To accomplish this aim we will utilize advanced optical imaging techniques, including fluorescence resonance energy transfer (FRET), fluorescence lifetime imaging (FLIM), fluorescence recovery after photobleaching (FRAP) and single molecule imaging. The function of prestin will be assayed by measuring the nonlinear capacitance. Native and mutated forms of prestin will be studied in order to delineate the molecular motifs that mediate prestin-prestin and prestin-membrane interactions. One focus of the mutational studies will be on regions in the C-terminal STAS domain that are predicted to participate in protein-protein interactions. In a second specific aim, we will determine whether prestin-prestin and prestin-membrane interactions depend on voltage. This will be accomplished by performing FRET, FRAP and FLIM experiments in voltage-clamped cells. The successful completion of these aims will represent the first direct determination of voltage-induced molecular changes in prestin organization and contribute a powerful new tool to prestin research. We will also investigate the effect of agents that perturb prestin function and alter membrane properties on prestin-prestin and prestin-membrane interactions, including non-steroidal anti-inflammatory agents (NSAIDs), which are known to induce hearing loss and tinnitus. The sensitivity of human hearing depends on the proper function of a motor protein called prestin located in sensory outer hair cells. The malfunction or absence of prestin, as may occur through altered prestin-prestin interactions by ototoxic compounds, results in high-frequency hearing loss. Because of the conservation of the part of the prestin molecule we will study, our research also has relevance for understanding the molecular basis of other diseases such as congenital diarrhea and skeletal abnormalities including dwarfism.

描述（由申请人提供）：人工耳蜗外毛细胞通过由膜蛋白prestin提供动力的主动机械反馈来增强哺乳动物听力的敏感性。我们研究工作的长期目标是了解普雷斯汀与膜相互作用的机制，并响应跨膜电位的变化。初步研究已经确定，普雷斯汀分子是自我关联的，而普雷斯汀 - 普雷斯汀相互作用的变化改变了膜中普雷斯汀的功能和横向组织。我们建议扩展这些研究，并进一步阐明普雷斯汀的功能与膜中普雷斯汀复合物的形成之间的关系。第一个具体目的是确定Prestin寡聚和功能之间的关系，并确定特定的寡聚状态是否代表Prestin的功能单位。为了实现这一目标，我们将利用先进的光学成像技术，包括荧光共振能量转移（FRET），荧光寿命成像（FLIM），光漂白后的荧光回收（FRAP）和单分子成像。 Prestin的功能将通过测量非线性电容来测定。为了描绘介导prestin-prestin和prestin-mmbrane相互作用的分子基序，将研究天然和突变形式的prestin。突变研究的一个重点将放在预测参与蛋白质蛋白相互作用的C端Stas结构域中的区域。在第二个特定目标中，我们将确定Prestin-Prestin和Prestin-Mmbrane相互作用是否取决于电压。这将通过在电压夹细胞中进行FRET，FRAP和FLIM实验来实现。这些目标的成功完成将代表Prestin组织中电压诱导的分子变化的首次直接确定，并为Prestin研究贡献强大的新工具。我们还将研究扰动Prestin功能并改变膜特性对Prestin-Prestin和Prestin-Mmbrane相互作用的药物的作用，包括非甾体类抗炎药（NSAID），这些抗炎药（NSAID）已知会诱导听力损失和耳鸣。人类听力的敏感性取决于一种称为Prestin的运动蛋白的正确功能，称为Prestin，位于感觉外毛细胞中。由于耳毒性化合物通过改变Prestin-Prestin的相互作用而发生故障或不存在Prestin，导致高频听力损失。由于我们将研究Prestin分子的一部分，我们的研究也与理解其他疾病的分子基础有关，例如先天性腹泻和骨骼异常，包括矮人。