Voltage-gated CaV1.3 (L-type) Ca2+ channels mediate Ca2+ influx and exocytosis at the auditory inner hair cell (IHC) ribbon synapse and are required for hearing. These channels undergo a negative feedback regulation by incoming Ca2+ ions (Ca2+-dependent inactivation) that is mediated by calmodulin. The action of calmodulin is prevented by Ca2+ binding proteins (CaBPs) that, together with calmodulin, belong to the large family of EF-hand-motif-containing proteins. Previously, an expression of CaBP1, 2, 4, and 5 in mouse cochlear IHCs has been demonstrated. Whereas genetic deletion of CaBP4 causes only a minor alteration of IHC Ca2+ influx leaving hearing unperturbed, mutations in CaBP2 have recently been identified to underlie a nonsyndromic form of autosomal recessive deafness (DFNB93) in human patients. An ongoing study currently investigates the cellular disease mechanisms of this hearing disorder in a mutant mouse model. In the new project, we plan to build on our recent findings and perform further in-depth analysis of the role of CaBP2 in the regulation of mouse hair cell Ca2+ influx and exocytosis, and sound encoding in the cochlea. Using recently generated knock-out mouse models and virus-mediated gene transfer into the embryonic otocyst, we will study the regulation of CaV1.3 channels by wild-type and selected mutant CaBP2s, related to pathologic mutations found in humans. The hearing of knock-out mice expressing the CaBP2 constructs will be investigated in detail on the systems and the cellular level. We will perform IHC patch-clamp recordings of Ca2+ and Ba2+ currents to investigate current inactivation kinetics, measure changes in membrane capacitance to detect differences in IHC exocytosis, and perform Ca2+ imaging to examine presynaptic Ca2+ signals. We will examine sound encoding by single auditory nerve fiber recordings in “rescued” CaBP1/2 knock-outs expressing various CaBP2 constructs. Finally, in case of calmodulin, distinct roles of its N- and C-terminal lobes have been suggested; but data for CaBPs in this context are still lacking. The available CaBP2 mutant constructs will now allow us to assess the function of each of the two CaBP2 lobes in order to properly address this question.
A Priority Project of