Harmonin is a scaffolding protein required for normal mechanosensory function in hair cells. Here, we describe a novel presynaptic association of harmonin and Cav1.3 Ca2+ channels at the mouse inner hair cell synapse, which limits channel availability through a ubiquitin-dependent pathway.

The prevailing model of neurotransmitter release stipulates that Ca 2+ influx triggers the rapid fusion of vesicles that are docked at presynaptic active zones. Under this model, slower tonic release is supported by vesicles clustered nearby that have to translocate to the release sites before fusion. We have examined this hypothesis at the afferent synapse of saccular hair cells of the leopard frog, Rana pipiens. Detailed morphological measurements at this ribbon synapse show that on average 32 vesicles are docked at each active zone. We show that at this 'graded' synapse, depolarization produces an exocytotic 'burst' that is largely complete within 20 ms after fusion of 280 vesicles per active zone, almost an order of magnitude more than expected. Recovery from paired pulse depression occurs with a time constant of 29 ms, indicating that replenishment of this fast-fusing pool of vesicles is also fast. Our results suggest that non-docked vesicles are capable of fast fusion and that these vesicles constitute the vast majority of the fast-fusing pool. The view that the population of fast-fusing presynaptic vesicles is limited to docked vesicles therefore requires re-evaluation. We propose that compound fusion, i.e. the fusion of vesicles with each other before and/or after they fuse with the membrane can explain multivesicular release at this synapse.

Key points• Ca v 1.3 Ca 2+ channels mediate sound transmission by triggering presynaptic exocytosis of glutamate from cochlear inner hair cells (IHCs).• Harmonin is a PDZ-domain-containing protein in IHCs that is altered in Usher syndrome, a form of deaf-blindness in humans.• We show that harmonin enhances Ca v 1.3 voltage-dependent facilitation (VDF) in transfected HEK293T cells in a manner that depends on the identity of the auxiliary Ca 2+ channel β subunit.• Ca v 1.3 VDF is impaired, and synchronous exocytosis and the Ca 2+ efficiency of exocytosis are reduced, in IHCs from deaf-circler mice expressing a mutant form of harmonin (dfcr) that cannot interact with Ca v 1.3.• We conclude that harmonin regulates presynaptic function in mouse IHCs, which adds to our understanding of the factors that may influence hearing impairment in Usher syndrome.Abstract Ca v 1.3 channels mediate Ca 2+ influx that triggers exocytosis of glutamate at cochlear inner hair cell (IHC) synapses. Harmonin is a PDZ-domain-containing protein that interacts with the C-terminus of the Ca v 1.3 α 1 subunit (α 1 1.3) and controls cell surface Ca v 1.3 levels by promoting ubiquitin-dependent proteosomal degradation. However, PDZ-domain-containing proteins have diverse functions and regulate other Ca v 1.3 properties, which could collectively influence presynaptic transmitter release. Here, we report that harmonin binding to the α 1 1.3 distal C-terminus (dCT) enhances voltage-dependent facilitation (VDF) of Ca v 1.3 currents both in transfected HEK293T cells and in mouse inner hair cells. In HEK293T cells, this effect of harmonin was greater for Ca v 1.3 channels containing the auxiliary Ca v β 1 than with the β 2 auxiliary subunit. Ca v 1.3 channels lacking the α 1 1.3 dCT were insensitive to harmonin modulation. Moreover, the 'deaf-circler' dfcr mutant form of harmonin, which does not interact with the α 1 1.3 dCT, did not promote VDF. In mature IHCs from mice expressing the dfcr harmonin mutant, Ca v 1.3 VDF was less than in control IHCs. This difference was not observed between control and dfcr IHCs prior to hearing onset. Membrane capacitance recordings from dfcr IHCs revealed a role for harmonin in synchronous exocytosis and in increasing the efficiency of Ca 2+ influx for triggering exocytosis. Collectively, our results indicate a multifaceted presynaptic role of harmonin in IHCs in regulating Ca v 1.3 Ca 2+ channels and exocytosis.F. D. Gregory and T. Pangršič contributed equally to this work.

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