Function coupling of otoferlin with GAD65 acts to modulate GABAergic activity.

Otoferlin， an integral membrane protein implicated in a late stage of exocytosis， has been reported to play a critical role in hearing although the underlying mechanisms remain elusive. However， its widespread tissue distribution infers a more ubiquitous role in synaptic vesicle trafficking. Glutamate， an excitatory neurotransmitter， is converted to its inhibitory counterpart， γ-aminobutyric acid (GABA)， by L-glutamic acid decarboxylase (GAD)， which exists in soluble (GAD67) and membrane-bound (GAD65) forms. For the first time， we have revealed a close association between otoferlin and GAD65 in both HEK293 and neuronal cells， including SH-SY5Y neuroblastoma and primary rat hippocampus cells， showing a direct interaction between GAD65 and otoferlin's C2 domains. In primary rat hippocampus cells， otoferlin and GAD65 co-localized in a punctate pattern within the cell body， as well as in the axon along the path of vesicular traffic. Significantly， GABA is virtually abolished in otoferlin-knockdown neuronal cells whereas otoferlin overexpression markedly increases endogenous GABA. GABA attenuation in otoferlin-knockdown primary cells is correlated with diminished L-type calcium current. This previously unknown and close correlation demonstrates that otoferlin， through GAD65， modulates GABAergic activity. The discovery of otoferlin-GAD65 functional coupling provides a new avenue for understanding the molecular mechanism by which otoferlin functions in neurological pathways.