Structure-function analysis of prestin, the cochlear amplifier protein

描述（由申请人提供）：我们的长期目标是了解prestin功能的分子基础，推动该领域更接近于设计某些类型听力损失的治疗方法。Prestin是耳蜗外毛细胞中的一种膜蛋白，参与了耳蜗放大导致频率敏感性的过程。几条线索的证据表明，prestin寡聚，但其功能意义尚不清楚。在这一建议中，我们的目的是剖析prestin单体和寡聚物种的功能作用。为此，我们将追求两个具体目标。在Aim 1中，我们将使用计算、诱变、生化和电生理技术来鉴定和表征全长prestin细胞质域中prestin-prestin相互作用残基。在目标2中，我们将对分离的prestin细胞质区域使用生化和生物物理技术，结合热力学分析构建一个描述各种单体和低聚prestin物种功能贡献的模型。由声波引起的机械刺激在耳蜗内被放大，从而产生对广泛频率范围的敏感性。这种放大至少部分是由耳蜗内一种叫做外毛细胞（ohc）的特殊细胞中的机械马达提供动力的。Prestin是ohc中的一种膜蛋白，于2000年被发现是这种马达的重要组成部分。缺乏prestin的小鼠表现出听力缺陷，某些类型的听力损失与prestin的突变有关。因此，了解prestin功能的基础对于理解和治疗某些类型的听力损失至关重要。在这个项目中，我们将使用计算分析来确定prestin中假定的功能残基，然后结合生物学、生物物理学和电生理学研究来确定和表征对功能至关重要的特定残基的作用。我们的研究是全面和系统的，并将产生关于prestin功能的重要信息，这将有助于设计针对某些类型听力损失的治疗方法。

DESCRIPTION (provided by applicant): Our long-term objectives are to understand the molecular basis of prestin function, to advance the field closer to designing therapeutics in certain types of hearing loss. Prestin, a membrane protein in outer hair cells in the cochlea, is involved in cochlear amplification leading to frequency sensitivity. Several lines of evidence indicate that prestin oligomerizes, but the functional significance of this is unclear. In this proposal, we aim to dissect the functional roles of prestin monomeric and oligomeric species. Towards this objective, we will pursue two Specific Aims. In Aim 1, we will use computational, mutagenesis, biochemical and electrophysiological techniques to identify and characterize the prestin-prestin interaction residues in the cytoplasmic domains of full-length prestin. In Aim 2, we will use biochemical and biophysical techniques on the isolated prestin cytoplasmic domains, combined with thermodynamic analysis to construct a model describing the functional contributions of various monomeric and oligomeric prestin species. Mechanical stimuli caused by sound waves are amplified within the cochlea, giving rise to sensitivity to a wide range of frequencies. This amplification is powered at least in part by a mechanical motor in specialized cells called outer hair cells (OHCs) in the cochlea. Prestin, a membrane protein in OHCs, was discovered in 2000 as an essential component of this motor. Mice that lack prestin show defects in hearing and certain types of hearing loss are associated with mutations in prestin. Knowledge of the basis of prestin function is therefore essential for understanding and treating certain types of hearing loss. In this project, we will use computational analyses to identify putative functional residues in prestin, and then use a combination of biological, biophysical and electrophysiological studies to identify and characterize the roles of specific residues that are essential for function. Our studies are comprehensive and systematic and will yield important information about prestin function that will aid in the design of therapeutics to certain types of hearing loss. PUBLIC HEALTH RELEVANCE: According to statistics from the NIDCD, approximately 15% (32.5 million) of Americans have a hearing impairment. Understanding the mechanistic basis of cochlear function is an important first step to treating the underlying causes of hearing loss. Our research proposal uses a combination of approaches to achieve this goal by investigating the molecular basis of function of prestin, an important cochlear component.