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。初步研究已经证实，prestin分子可以自我结合，prestin-prestin相互作用的改变改变了prestin在膜中的功能和横向组织。我们建议进一步扩展这些研究，进一步阐明prestin的功能与膜内prestin复合物形成之间的关系。第一个具体目标将是确定前蛋白寡聚化和功能之间的关系，并确定一个特定的寡聚状态是否代表前蛋白的功能单位。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分析prestin的作用。将研究原生和突变形式的prestin，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端STAS结构域的区域，该区域被预测参与蛋白质-蛋白质相互作用。在第二个具体目标中，我们将确定prestin-prestin和prestin-membrane相互作用是否依赖于电压。这将通过在电压箝位的细胞中进行FRET， FRAP和FLIM实验来完成。这些目标的成功完成将首次直接确定电压诱导prestin组织的分子变化，并为prestin研究提供有力的新工具。我们还将研究扰乱普司汀功能和改变膜特性的药物对普司汀-普司汀和普司汀-膜相互作用的影响，包括非甾体抗炎药（NSAIDs），已知会导致听力损失和耳鸣。人类听觉的敏感性取决于位于感觉外毛细胞中的一种叫做prestin的运动蛋白的正常功能。由于耳毒性化合物改变了普司汀与普司汀之间的相互作用，普司汀的功能障碍或缺失可能导致高频听力损失。由于我们将研究的部分prestin分子的保守性，我们的研究也与理解其他疾病的分子基础相关，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.