Glutamate Transporters EAAT4 and EAAT5 Are Expressed in Vestibular Hair Cells and Calyx Endings

Abstract

Glutamate is the neurotransmitter released from hair cells. Its clearance from the synaptic cleft can shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear and in other sensory organs where there is a continually high rate of neurotransmitter release. In the case of most cochlear and type II vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glutamate transporter. A similar mechanism cannot work in vestibular type I hair cells as the presence of calyx endings separates supporting cells from hair-cell synapses. Because of this arrangement, it has been conjectured that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrate that a glutamate-activated anion current, attributable to a high-affinity glutamate transporter and blocked by DL-TBOA, is expressed in type I, but not in type II hair cells. Molecular investigations reveal that EAAT4 and EAAT5, two glutamate transporters that could underlie the anion current, are expressed in both type I and type II hair cells and in calyx endings. EAAT4 has been thought to be expressed almost exclusively in the cerebellum and EAAT5 in the retina. Our results show that these two transporters have a wider distribution in mice. This is the first demonstration of the presence of transporters in hair cells and provides one of the few examples of EAATs in presynaptic elements.

Distribution of Na,K-ATPase α Subunits in Rat Vestibular Sensory Epithelia

“…A previous study of EAAT5 expression in mouse vestibular hair cells and calyx endings reported expression of EAAT5 in type I and type II hair cells as well as the calyx inner face (Dalet et al 2012). Using one of the EAAT5 antibodies used in that study, we also observed immunoreactivity in the calyx inner face, but, in contrast to the previous report, labeling of the calyx outer face was observed, and labeling of type II hair cells was not observed.…”

“…Firstly, the envelopment of the type I hair cell by the afferent calyx precludes glutamate uptake by supporting cells and necessitates mechanisms of glutamate clearance localized directly to the type I hair cell and/or calyx terminal. Indeed, recent work provides evidence that the glutamate transporter EAAT5 is expressed in calyx endings (Dalet et al 2012). Like other glutamate transporters, EAAT5 relies at least indirectly on NKAs to generate the ion gradients necessary for the Na + -dependent transport of glutamate (Beart and O'Shea 2007).…”

“…6A-C), as well as the glutamate transporter EAAT5 (Fig. 6D-F), recently localized to the calyx afferent terminal (Dalet et al 2012). We specifically examined afferent calyces in single optical sections from the striolar region of the utricle immunolabeled with a monoclonal antibody against NKAα3 (green, Fig.…”

Evidence that protons act as neurotransmitters at vestibular hair cell–calyx afferent synapses

“…2), and because puncta of high ΔpH were observed at locations distant from sites of quantal release (Fig. 4), we can rule out synaptic vesicles as the primary source of cleft acidification (24,25). It is more likely that protons were extruded into the cleft during hair cell depolarization through mechanisms such as Na + /H + exchange or electrogenic Na + /HCO 3 cotransport.…”

Deletion of Shank1 has minimal effects on the molecular composition and function of glutamatergic afferent postsynapses in the mouse inner ear

“…In fact, a handful of proteins necessary for glutamateric signaling that are relatively selectively expressed in the inner ear have been identified. For example, VLUT3, a vesicular glutamate transporter (Seal et al, 2008), otoferlin, a Ca 2+ sensor necessary for glutamate release from hair cells (Roux et al, 2006), and EAAT5, a glutamate transporter expressed in the vestibular sensory epithelia (Dalet et al, 2012) show more or less restricted expression to the inner ear (or ribbon synapses). Therefore, future work should investigate the expression of novel Shank isoforms in the inner ear.…”