Molecular Interactions of the Carboxy Terminus of Prestin

描述（由申请人提供）：五年前，蛋白prestin （SLC26A5）被确定为驱动耳蜗外毛细胞机电转导的马达蛋白。随后的实验证实，prestin对外毛细胞（OHC）的电运动性和正常听觉功能都是必不可少的。这种独特的多聚膜蛋白有助于检测跨膜电位变化的电压传感器和OHC电运动性的马达机制。Prestin是SLC26A阴离子转运蛋白家族的一员，在离子和流体稳态以及pH和细胞体积调节中起关键作用。预防运动功能的分子基础目前尚不清楚。这一建议的目的是促进我们对prestin功能的理解。特别是，我们将测试的假设，prestin分子自结合形成功能复合物。SLC26家族成员的c端包含一个在原核生物和真核生物中高度保守的结构域，称为STAS结构域。几种SLC26A蛋白的STAS结构域突变可导致多种人类疾病，包括Pendred综合征（SLC26A4）、先天性氯化物腹泻（SLC26A3）和异型发育不良（SLC26A2）。来自较低门的STAS结构域已证明与膜相互作用。基于这些和其他发现，我们假设prestin的STAS结构域介导prestin-prestin相互作用以及prestin与膜之间的相互作用。重要的是，我们的初步数据表明，膜微环境的改变改变了prestin的功能。在本研究中，我们将使用生化、细胞、生物物理和光学（FRET和FRAP）方法来探索prestin、prestin与膜、STAS结构域与膜之间的分子相互作用。此外，我们将确定SLC26A STAS域是否可以功能交换。我们的研究结果不仅将使我们对prestin的功能和电动力的分子基础有更深入的了解，而且还将为SLC26A家族STAS结构域的功能提供见解。

DESCRIPTION (provided by applicant): Five years ago, the protein prestin (SLC26A5) was identified as the motor protein that drives electromechanical transduction in cochlear outer hair cells. Subsequent experiments have confirmed prestin is essential for both outer hair cell (OHC) electromotility and normal auditory function. This unique polytopic membrane protein contributes to the voltage sensor that detects changes in the transmembrane potential and to the motor mechanism of OHC electromotility. Prestin is a member of the SLC26A family of anion transporters which play critical roles in ion and fluid homeostasis as well as pH and cell volume regulation. The molecular basis of prestin motor function is presently unknown. The objective of this proposal is to advance our understanding of prestin function. In particular, we will test the hypothesis that prestin molecules self-associate to form functional complexes. The C-terminus of SLC26 family members contains a domain that is highly conserved throughout prokaryotes and eukaryotes, referred to as the STAS domain. Mutations in the STAS domain of several SLC26A proteins are responsible for a variety of human diseases including Pendred syndrome (SLC26A4), congenital chloride diarrhea (SLC26A3), and diastrophic dysplasia (SLC26A2). STAS domains from lower phyla have demonstrated interactions with the membrane. Based on these and other findings, we hypothesize that the STAS domain of prestin mediates prestin-prestin interactions and interactions between prestin and the membrane. Importantly, our preliminary data demonstrates that alterations in the membrane microenvironment alter prestin function. In this proposal, we will use biochemical, cellular, biophysical and optical (FRET and FRAP) approaches to probe the molecular interactions between prestin, prestin and the membrane, and the STAS domain and the membrane. Additionally, we will determine if SLC26A STAS domains can be functionally exchanged. The results from our studies will lead to a deeper understanding, not only of prestin function and the molecular basis of electromotility, but will also provide insights into the function of STAS domains from the SLC26A family.