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Cav1.3 Ca2+ 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 Cav1.3 voltage-dependent facilitation (VDF) in transfected HEK293T cells in a manner that depends on the identity of the auxiliary Ca2+ channel β subunit.Cav1.3 VDF is impaired, and synchronous exocytosis and the Ca2+ efficiency of exocytosis are reduced, in IHCs from deaf-circler mice expressing a mutant form of harmonin (dfcr) that cannot interact with Cav1.3.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.Cav1.3 channels mediate Ca2+ 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 Cav1.3 α1 subunit (α11.3) and controls cell surface Cav1.3 levels by promoting ubiquitin-dependent proteosomal degradation. However, PDZ-domain-containing proteins have diverse functions and regulate other Cav1.3 properties, which could collectively influence presynaptic transmitter release. Here, we report that harmonin binding to the α11.3 distal C-terminus (dCT) enhances voltage-dependent facilitation (VDF) of Cav1.3 currents both in transfected HEK293T cells and in mouse inner hair cells. In HEK293T cells, this effect of harmonin was greater for Cav1.3 channels containing the auxiliary Cavβ1 than with the β2 auxiliary subunit. Cav1.3 channels lacking the α11.3 dCT were insensitive to harmonin modulation. Moreover, the ‘deaf-circler’dfcr mutant form of harmonin, which does not interact with the α11.3 dCT, did not promote VDF. In mature IHCs from mice expressing the dfcr harmonin mutant, Cav1.3 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 Ca2+ influx for triggering exocytosis. Collectively, our results indicate a multifaceted presynaptic role of harmonin in IHCs in regulating Cav1.3 Ca2+ channels and exocytosis.