摘要
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,g-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 SHSY5Y 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 newavenue for understanding the molecular mechanism by which otoferlin functions in neurological pathways.
基金
This work was supported by funding from Canadian Institutes of Health Research and Natural Sciences and Engineering Research Council of Canada.Z.J.is a Canada Research Chair in Structural Biology.
