Molekulare Identifizierung und funktionelle Charakterisierung der K+-Kanal-Ausstattung auditorischer Haarsinneszellen

Background: The precise processing of auditory stimuli is ensured by outer (OHC) and inner hair cells (IHC) in the organ of Corti. Since the physiological tasks (cochlear amplification and synaptic transmission) of these cells are driven by receptor potentials, the shape and the amplitude of these potential changes are essential for the function of the sensory cells. The characteristics of these receptor potentials depend on the activity of the apical mechano-electrical transduction channels and on basolateral (voltage-dependent) K+ conductances, which together define the biophysical membrane properties of the sensory cells. Due to their biophysical properties, the basolateral K+ currents were previously classified as IK,f, IK,n and IK,s. The underlying ion channels and their modulatory interaction partners are largely unknown. Aims of the thesis: The goals of this doctoral thesis were (I.) to investigate the role of a potential interaction partner (the enzyme β-secretase 1; BACE1) for K+ channel function in hair cells, and (II.) to unravel the channel subunits that make up the K+ channel repertoire in IHCs, as well as to elucidate the physiological relevance of these K+ channels by using in-silico simulations. Results on BACE1: I found that BACE1-/- mice exhibit significant hearing loss compared to wild-type mice, which is caused by disorganization of the synaptic connections between the IHC and the auditory nerve fibers and greatly reduced myelination of these nerve fibers. Noteworthy, BACE1 is important for the development of the cochlear nerve fibers, but not for the maintenance of their myelination in adulthood. I also identified Neuregulin-1 as the BACE1 substrate in the cochlea that is pivotal for normal organization and myelination of the cochlear nerve fibers. Contrary to our initial hypothesis, BACE1 thus is not an interaction partner of K+ channels in sensory hair cells. However, my results showed for the first time that BACE1 is indispensable for sensitive hearing. Because inhibiting BACE1 is considered a promising approach to treat Alzheimer's disease, it was important to determine whether such medication could affect hearing sensitivity. We did not detect any hearing impairment or abnormal changes in the cochlea of mice treated with the well-known BACE1 inhibitor NB-360. These results suggest that hearing loss may not be a side-effect of Alzheimer's disease therapy with BACE1 inhibitors. Results on the K+ channel repertoire of auditory hair cells: In a hypothesis-driven candidate approach, I succeeded in deciphering the K+ channel repertoire of IHCs. First, I extracted candidate channels from published single cell transcriptome analyses of IHCs. Then I confirmed expression of these channels in the cochlea using molecular biological and immunohistochemical methods. I employed electrophysiological techniques to elucidate whether these candidate channels are active in IHCs. To develop an approach towards identification of channel subtypes by their biophysical properties, I established protocols in heterologous expression system to characterize the candidate channels in detail. Thus, I identified unique properties and suitable measurement protocols for subsequent detection in native tissue. Surprisingly, these studies showed that despite close phylogenetic relationship and similarities in the overall architecture, the pharmacological properties of K+ channels can differ significantly. Based on the work in the expression system, I was able to show that Kv1.8, Kv11.1 and Kv12.1 channels are active in IHCs. Hence, for the first time I could assign a role in a sensory system to Kv11.1 channels, and my results constitute the first demonstration of relevance of Kv1.8 and Kv12.1. We then untangled the physiological importance of this extraordinary repertoire of five K+ channel subunits (Kv1.8, Kv7.4, Kv11.1, Kv12.1 and BKCa channels) in detail using in-silico simulations. These models showed that all channels together synergistically optimize the precision of sound processing in IHCs, while IHCs with fewer channel populations are limited in the processing of sound stimuli adequately. The channel repertoire thus represents an evolutionary adaptation, that enables the processing of complex sounds and sensitive hearing.