The Role of Calcium Binding Proteins in Synaptic Sound Encoding

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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.