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

Abstract

Ca(v)1.3 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 Ca(v)1.3 (1) subunit (alpha(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 alpha(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 beta(1) than with the beta(2) auxiliary subunit. Ca(v)1.3 channels lacking the alpha(1)1.3 dCT were insensitive to harmonin modulation. Moreover, the deaf-circler'dfcr mutant form of harmonin, which does not interact with the alpha(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 Ca2+ influx for triggering exocytosis. Collectively, our results indicate a multifaceted presynaptic role of harmonin in IHCs in regulating Ca(v)1.3 Ca2+ channels and exocytosis.