Development of endocytosis in cochlear inner hair cells

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Mammalian inner hair cells (IHC) indefatigably transduce mechanical sound stimuli into graded receptor potentials and graded transmitter release with excellent temporal precision over a wide dynamic range. Before the onset of hearing, IHCs undergo a developmental program during which they generate Ca2+-driven action potentials. Transmitter release differs in many regards between pre-hearing and mature IHCs, such as i) the kind of trigger (Ca2+ action potentials vs. graded receptor potentials), ii) Ca2+ efficiency, iii) crucial molecules and iv) morphology of the synapse. Whereas function and maturation of vesicle release is well studied in IHCs, this is not the case for endocytosis of previously inserted vesicle membrane from the plasma membrane. Preliminary data strongly suggest that endocytosis undergoes a process of maturation that parallels the developmental modifications of exocytosis. In this project I aim to characterise endocytosis in pre-mature compared to mature IHCs. The specific aims are: 1) to characterise the kinetics and Ca2+ dependence of endocytosis; 2) to identify the molecular components of the endocytic machinery; 3) to analyse the role of endocytic proteins. Endocytosis will be measured as membrane capacitance changes using the patch clamp technique. We will identify components of the endocytic machinery on mRNA level by transcript analysis and on protein level by immunolabelling using light microscopy and transmission electron microscopy. Finally, we will elucidate the role of endocytic proteins in IHCs by blocking their function and studying the resulting effect on endocytosis via membrane capacitance recordings and imaging of membrane uptake using a fixable membrane dye. The knowledge gained here is essential to understand final maturation of the IHC and how IHCs indefatigably transfer sound information to the auditory pathway. This study will further serve as a basis for future projects involving mutant mouse models of endocytic proteins for understanding human deafness.