Analysis of a Vesicular Glutamate Transporter (VGLUT2) Supports a Cell-leakage Mode in Addition to Vesicular Packaging

Abstract

VGLUT2 is one of three vesicular glutamate transporters that play crucial roles in glutamatergic excitatory neurotransmission. We explored the functional properties of the rat VGLUT2 by heterologous expression of VGLUT2 in Xenopus oocytes. Immunocytochemical analysis indicated that most VGLUT2 protein was expressed in intracellular compartments but that some expression occurred also on the plasma membrane. Functional analysis revealed VGLUT2 to be active in two independent modes, namely, uptake into intracellular organelles and efflux at the plasma membrane. VGLUT-specific transport was identified based on the strong preference for glutamate over aspartate--in contrast to plasma-membrane or mitochondrial glutamate transporters--and sensitivity to known VGLUT blockers. VGLUT2 expression in oocytes (1) stimulated the influx of L-[(3)H]glutamate, but not D-[(3)H]aspartate, into digitonin-permeabilized oocytes and (2) stimulated efflux of L-glutamate, but not L-aspartate, from intact oocytes preinjected with (3)H-labeled amino acids. In the latter assay, cellular efflux of glutamate (which was blocked by rose bengal and trypan blue) may be analogous to vesicular packaging of glutamate. Our data are consistent with VGLUT2-mediated H(+)/L-glutamate antiport, but not antiport with chloride. Expression of mammalian VGLUT1 and VGLUT3 also stimulated L-[(3)H]glutamate efflux from Xenopus oocytes, suggesting that this phenomenon is a general feature of vesicular glutamate transporters. Our findings support the idea that vesicular glutamate transporters, when transiently expressed on the neuronal plasma membrane, may mediate Ca(2+)-independent glutamate leakage in addition to their traditional role of packaging glutamate into synaptic vesicles for Ca(2+)-dependent exocytosis.

Protons Regulate Vesicular Glutamate Transporters through an Allosteric Mechanism

“…Low external pH is required to promote glutamate efflux across the plasma membrane of transfected HEK cells, as suggested previously although interpreted then to reflect H + exchange (Mackenzie et al, 2008). Our data now show that the requirement of VGLUTs for H + is allosteric.…”

Impact of vesicular glutamate leakage on synaptic transmission at the calyx of Held

“…; Schenck et al . ), whereas glutamate leakage is reportedly caused by the H + –glutamate antiport through VGLUTs (Mackenzie et al , ). The calyces of Held in rodents express both VGLUT1 and VGLUT2 (Billups, ; Blaesse et al .…”

VGLUTs in Peripheral Neurons and the Spinal Cord: Time for a Review

“…Thus, neuropeptides [147, 148], ATP [149], and even genetically expressed macromolecular tracers in DRG neurons [150] have been shown to undergo somatic release. Interestingly, recent studies in Xenopus oocytes transfected with VGLUT 2 suggest that this transporter can be found in two different states: (1) serving the traditional role of packaging glutamate into synaptic vesicles for Ca 2+ -dependent exocytosis, and (2) participating in Ca 2+ -independent leakage when present in the plasma membrane [151]. Moreover, this dual role appears to be also true for VGLUT 1 and VGLUT 3 [151].…”

“…Interestingly, recent studies in Xenopus oocytes transfected with VGLUT 2 suggest that this transporter can be found in two different states: (1) serving the traditional role of packaging glutamate into synaptic vesicles for Ca 2+ -dependent exocytosis, and (2) participating in Ca 2+ -independent leakage when present in the plasma membrane [151]. Moreover, this dual role appears to be also true for VGLUT 1 and VGLUT 3 [151]. Whether such states are also a feature in sensory neurons, and VGLUTs facilitated any form of somatic glutamatergic release remains to be established.…”