Hair cells (HCs) mediate auditory and vestibular sensation through synaptic transmission. Here, I identify asteroid (ast), an ENU-induced deafness mutant, as encoding vesicular glutamate transporter 3 (Vglut3). ast⁻/⁻ hair cells show normal mechanotransduction and morphology, but a splicing defect excluding exon 2 of vglut3 correlates with vestibular dysfunction. Morpholino knockdown phenocopies a weaker balance defect. While synaptic ribbons appear normal, ribbon-associated vesicles are reduced, suggesting a role for Vglut3 in synaptic vesicle biogenesis or trafficking. These findings identify the hair cell glutamate transporter and establish asteroid as a model for hair cell synaptic biology.
