The Role of Calcium Binding Proteins in Synaptic Sound Encoding

钙结合蛋白在突触声音编码中的作用

• 批准号: 218185549
• 负责人: Professor Dr. Tobias Moser
• 金额: --
• 依托单位: Institut für Auditorische Neurowissenschaften
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/218185549?language=en
• 关键词: Role; Calcium; Binding; Proteins; Synaptic

Ca2+ binding proteins (CaBPs) play an important role in the Ca2+ mediated signaling in the nervous system. The goal of this project is to study the function of CaBPs in regulating hair cell Ca2+ influx and exocytosis. Our previous work demonstrated the expression of CaBPs 1, 2, 4 and 5 protein in cochlear inner hair cells (IHC) of the mouse and a minor alteration of IHC Ca2+ influx upon genetic ablation of CaBP4. Recently, mutations in one of the human CaBP genes (CaBPx) were identified to cause hearing impairment. In this project, we plan to investigate the role of CaBPx in sound encoding in the cochlea and to dissect the disease mechanism of the CaBPxrelated hearing disorder. We will study the secondary structure of purified wild-type and mutant CaBPx using CD spectroscopy and analyze their Ca2+ binding properties by isothermal titration calorimetry and fluorimetric studies. We will then study the regulation of CaV1.3 channels by wildtype and mutant CaBPx in HEK 293 cells. For functional analysis of CaBPx in the auditory system we are generating mice with global and hair cell specific inactivation of the CaBPx gene (KO). We will study hearing of these mice from the systems level to the cellular level (IHC patch-clamp measurements of Ca2+ and Ba2+ currents and exocytic capacitance changes, Ca2+ imaging). We will probe for potential morphological changes using light and electron microscopy. We will collaborate within the consortium for single auditory nerve fiber recordings in CaBPx mutants. Using virus-mediated gene transfer into the embryonic otocyst we will study the function of mutant CaBPx in IHCs of CaBPx KO mice. We will contribute expertise on analysis of hair cell synapses, virus production and viral transduction to the consortium.

Ca2+结合蛋白（CaBPs）在神经系统中Ca2+介导的信号传导中起重要作用。本项目的目的是研究CaBPs在调节毛细胞Ca2+内流和胞吐中的功能。我们之前的工作证明了cabp1、2、4和5蛋白在小鼠耳蜗内毛细胞（IHC）中的表达，以及CaBP4基因消融后IHC Ca2+内流的轻微改变。最近，一种人类CaBP基因（CaBPx）的突变被发现会导致听力障碍。在本项目中，我们计划研究CaBPx在耳蜗声音编码中的作用，并剖析CaBPx相关听力障碍的发病机制。我们将利用CD光谱研究纯化的野生型和突变型CaBPx的二级结构，并通过等温滴定量热法和荧光学研究分析它们的Ca2+结合特性。然后，我们将研究野生型和突变型CaBPx在HEK 293细胞中对CaV1.3通道的调节。为了分析CaBPx在听觉系统中的功能，我们制造了CaBPx基因（KO）全局失活和毛细胞特异性失活的小鼠。我们将从系统水平到细胞水平研究这些小鼠的听力（IHC膜片钳测量Ca2+和Ba2+电流和胞外电容变化，Ca2+成像）。我们将使用光学和电子显微镜来探测潜在的形态学变化。我们将在CaBPx突变体的单个听觉神经纤维记录的联盟中合作。利用病毒介导的基因转移到胚胎耳囊肿中，我们将研究CaBPx突变体在CaBPx KO小鼠ihc中的功能。我们将为联盟提供毛细胞突触分析、病毒产生和病毒转导方面的专业知识。