Modes and Regulation of Endocytic Membrane Retrieval in Mouse Auditory Hair Cells

Synaptic vesicle recycling sustains high rates of neurotransmission at the ribbon-type active zones (AZs) of mouse auditory inner hair cells (IHCs), but its modes and molecular regulation are poorly understood. Electron microscopy indicated the presence of clathrin-mediated endocytosis (CME) and bulk endocytosis. The endocytic proteins dynamin, clathrin, and amphiphysin are expressed and broadly distributed in IHCs. We used confocal vglut1-pHluorin imaging and membrane capacitance (C-m) measurements to study the spatial organization and dynamics of IHC exocytosis and endocytosis. Viral gene transfer expressed vglut1-pHluorin in IHCs and targeted it to synaptic vesicles. The intravesicular pH was similar to 6.5, supporting only a modest increase of vglut1-pHluorin fluorescence during exocytosis and pH neutralization. Ca2+ influx triggered an exocytic increase of vglut1-pHluorin fluorescence at the AZs, around which it remained for several seconds. The endocytic C-m decline proceeded with constant rate (linear component) after exocytosis of the readily releasable pool (RRP). When exocytosis exceeded three to four RRP equivalents, IHCs additionally recruited a faster C-m decline (exponential component) that increased with the amount of preceding exocytosis and likely reflects bulk endocytosis. The dynamin inhibitor Dyngo-4a and the clathrin blocker pitstop 2 selectively impaired the linear component of endocytic C-m decline. A missense mutation of dynamin 1 (fitful) inhibited endocytosis to a similar extent as Dyngo-4a. We propose that IHCs use dynamin-dependent endocytosis via CME to support vesicle cycling during mild stimulation but recruit bulk endocytosis to balance massive exocytosis.