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J Neurosci. 2014 Jan 15;34(3):705-16. doi: 10.1523/JNEUROSCI.3313-13.2014.

Modes and regulation of endocytic membrane retrieval in mouse auditory hair cells.

Author information

1
InnerEarLab, Department of Otolaryngology, Molecular Architecture of Synapses Group, InnerEarLab, Department of Otolaryngology, and Department of Neurology, University Medical Center Göttingen, D-37099 Göttingen, Germany, Collaborative Research Center 889, International Max Planck Research Schools, Neuroscience, Göttingen Graduate School for Neuroscience, Biophysics, and Molecular Biosciences, Research Center for Nanoscale Microscopy and Molecular Physiology of the Brain, and Bernstein Focus for Neurotechnology, University of Göttingen, D-37073 Göttingen, Germany, and The Jackson Laboratory, Bar Harbor, Maine 04609.

Abstract

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 (Cm) 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 ∼6.5, supporting only a modest increase of vglut1-pHluorin fluorescence during exocytosis and pH neutralization. Ca(2+) influx triggered an exocytic increase of vglut1-pHluorin fluorescence at the AZs, around which it remained for several seconds. The endocytic Cm 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 Cm 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 Cm 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.

KEYWORDS:

clathrin; dynamin; endocytosis; hearing; inner hair cell; pHluorin

PMID:
24431429
PMCID:
PMC3891952
DOI:
10.1523/JNEUROSCI.3313-13.2014
[Indexed for MEDLINE]
Free PMC Article

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