Harmonin enhances voltage‐dependent facilitation of Ca v 1.3 channels and synchronous exocytosis in mouse inner hair cells

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.