The Role of Calcium Binding Proteins in Synaptic Sound Encoding

钙结合蛋白（Ca2+ binding proteins，CaBPs）在神经系统钙离子介导的信号转导中起重要作用。本课题旨在研究CaBPs对毛细胞内Ca2+内流和胞吐的调节作用。我们以前的工作证明了CaBPs 1，2，4和5蛋白在小鼠耳蜗内毛细胞（IHC）的表达和一个微小的改变后，基因消融CaBPs 4的IHC Ca 2+内流。最近，人类CaBP基因之一（CaBPx）的突变被鉴定为导致听力障碍。本课题旨在研究CaBPx在耳蜗声音编码中的作用，并探讨CaBPx相关性听力障碍的发病机制。我们将使用CD光谱研究纯化的野生型和突变体CaBPx的二级结构，并通过等温滴定量热法和荧光研究分析其Ca 2+结合特性。然后，我们将在HEK 293细胞中研究野生型和突变型CaBPx对CaV1.3通道的调节。对于听觉系统中CaBPx的功能分析，我们正在产生具有CaBPx基因（KO）的全局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探测潜在的形态学变化。我们将在财团内合作，在CaBPx突变体中记录单个听觉神经纤维。使用病毒介导的基因转移到胚胎耳囊中，我们将研究突变体CaBPx在CaBPx KO小鼠的IHC中的功能。我们将在毛细胞突触分析、病毒生产和病毒转导方面为该联盟提供专业知识。

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.