Molecular Interactions of the Carboxy Terminus of Prestin

描述（由申请人提供）：五年前，蛋白质普雷斯廷（SLC 26 A5）被鉴定为驱动耳蜗外毛细胞中机电转导的运动蛋白。 随后的实验证实，普雷斯廷是外毛细胞（OHC）的电活动和正常的听觉功能所必需的。 这种独特的多位膜蛋白有助于检测跨膜电位变化的电压传感器和OHC电运动的马达机制。 普雷斯廷是阴离子转运蛋白SLC 26 A家族的成员，其在离子和流体稳态以及pH和细胞体积调节中起关键作用。 普雷斯廷运动功能的分子基础目前尚不清楚。这一建议的目的是推进我们对普雷斯廷功能的理解。 特别是，我们将测试的假设，即普雷斯廷分子自缔合形成功能复合物。 SLC 26家族成员的C-末端含有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SLC 26 A蛋白的STAS结构域中的突变导致多种人类疾病，包括Pendred综合征（SLC 26 A4）、先天性氯化物腹泻（SLC 26 A3）和畸形性发育不良（SLC 26 A2）。 来自较低门的STAS结构域已经证明与膜的相互作用。 基于这些和其他发现，我们假设普雷斯廷的STAS结构域介导了普雷斯汀-普雷斯廷相互作用以及普雷斯廷和细胞膜之间的相互作用。 重要的是，我们的初步数据表明，改变膜微环境改变普雷斯廷功能。在本研究中，我们将利用生物化学、细胞、生物物理和光学（FRET和FRAP）方法来研究普雷斯廷、普雷斯廷与细胞膜以及STAS结构域与细胞膜之间的分子相互作用。 此外，我们将确定SLC 26 A STAS域是否可以在功能上交换。 我们的研究结果将导致更深入的了解，不仅普雷斯廷功能和电运动的分子基础，但也将提供深入了解SLC 26 A家族的STAS结构域的功能。

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.