Biophysical Basis of Organization and Dynamics of Prestin Membrane Complexes

描述（由申请人提供）：耳蜗外毛细胞通过由膜蛋白普雷斯廷提供动力的主动机械反馈增强哺乳动物听觉的灵敏度。我们研究工作的长期目标是了解普雷斯廷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，普雷斯廷分子自缔合，并且普雷斯蛋白-普雷斯廷相互作用的变化改变了普雷斯廷在膜中的功能和侧向组织。我们建议扩展这些研究，并进一步阐明普雷斯廷的功能和膜中普雷斯廷复合物的形成之间的关系。第一个具体目标是确定普雷斯廷寡聚化和功能之间的关系，并确定特定的寡聚状态是否代表普雷斯廷的功能单元。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），荧光恢复后光漂白（FRAP）和单分子成像。普雷斯廷的功能将通过测量非线性电容来分析。天然和突变形式的普雷斯廷将进行研究，以描绘介导的分子基序，普雷斯丁-普雷斯廷和普雷斯丁-膜相互作用。突变研究的一个重点将是预测参与蛋白质-蛋白质相互作用的C-末端STAS结构域中的区域。在第二个具体的目标，我们将确定是否prestin-prestin和prestin-membrane相互作用依赖于电压。这将通过在电压钳位细胞中进行FRET、FRAP和FLIM实验来实现。这些目标的成功完成将代表电压诱导的普雷斯廷组织中分子变化的首次直接测定，并为普雷斯廷的研究贡献一个强有力的新工具。我们还将研究干扰普雷斯廷功能和改变膜特性的药物对普雷斯汀-普雷斯廷和普雷斯汀-膜相互作用的影响，包括已知可诱导听力损失和耳鸣的非甾体抗炎药（NSAID）。人类听觉的灵敏度取决于位于感觉外毛细胞中的一种名为普雷斯廷的运动蛋白的正常功能。普雷斯廷的功能障碍或缺乏，如可能通过耳毒性化合物改变的普雷斯汀-普雷斯廷相互作用而发生的，导致高频听力损失。由于我们将要研究的部分普雷斯廷分子的保守性，我们的研究也与理解其他疾病的分子基础有关，如先天性腹泻和骨骼异常，包括侏儒症。

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.