Biophysical Basis of Organization and Dynamics of Prestin Membrane Complexes

描述(申请人提供)：耳蜗外毛细胞通过由膜蛋白Prestin提供动力的主动机械反馈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Prestin与膜相互作用的机制，并对跨膜电位的变化做出反应。初步研究表明，prestin分子是自结合的，prestin-prestin相互作用的变化改变了prestin在膜中的功能和侧向组织。我们建议扩展这些研究，进一步阐明prestin的功能与膜中prestin复合体的形成之间的关系。第一个具体目标将是确定Prestin齐聚与功能之间的关系，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)、光漂白后荧光恢复(FRAP)和单分子成像。通过测量非线性电容来测试预充电的功能。将研究prestin的天然和突变形式，以描述介导prestin-prestin和prestin-膜相互作用的分子基序。突变研究的一个重点将是C-末端STAS结构域中预测参与蛋白质-蛋白质相互作用的区域。在第二个特定目标中，我们将确定prestin-prestin和prestin-膜的相互作用是否依赖于电压。这将通过在电压钳制单元中进行FRET、FRAP和FLIM实验来实现。这些目标的成功完成将代表着首次直接确定电压诱导的Prestin组织中的分子变化，并为Prestin的研究提供一个强大的新工具。我们还将研究干扰prestin功能和改变膜特性的药物对prestin-prestin和prestin-膜相互作用的影响，包括非类固醇抗炎药(NSAIDs)，这是已知的导致听力损失和耳鸣的药物。人类听力的敏感度取决于位于感觉外毛细胞中的一种名为prestin的运动蛋白的适当功能。耳毒性化合物可能通过改变prestin-prestin的相互作用而导致prestin的故障或缺失，从而导致高频听力损失。由于我们将研究的prestin分子的一部分是保守的，我们的研究对于理解其他疾病的分子基础也有相关性，如先天性腹泻和包括侏儒症在内的骨骼异常。

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.