Harmonin enhances voltage‐dependent facilitation of Ca_v1.3 channels and synchronous exocytosis in mouse inner hair cells

Abstract

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.

Clarin-1 gene transfer rescues auditory synaptopathy in model of Usher syndrome

“…Consistent with these results, IHCs lacking harmonin in dfcr mice also have greater Ca 2+ currents, with a negative shift in voltage-dependence, and a clustering defect of Ca V 1.3 Ca 2+ channels (27,28). Based on the observed interactions of clarin-1 ( Figure 11E), we suggest that clarin-1, harmonin, and Ca V 1.3 channels, via their Ca 2+ channel subunit Ca V β 2 , form a molecular platform associated with F-actin in the active zones of the IHC.…”

Hair cells use active zones with different voltage dependence of Ca 2+ influx to decompose sounds into complementary neural codes

“…5H), and normal sound-pressure dependence of firing (Fig. 5I) (44,45). Here, we revisited the regulation of IHC AZs by harmonin in deaf-circler mice (dfcr) (46), reasoning that site-specific expression of harmonin might contribute to the heterogeneity of maximal Ca 2+ influx and voltage dependence of activation.…”

Usher proteins in inner ear structure and function

“…1). In this minireview, we limit our focus to the roles of USH proteins in the hair cell stereocilia, even though some of these proteins are also important for signal transmission at the hair cell synapses (13,14).…”