Analysis of a vesicular glutamate transporter (VGLUT2) supports a cell-leakage mode in addition to vesicular packaging.
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.
Mackenzie
Bryan
B
Department of Molecular & Cellular Physiology, University of Cincinnati College of Medicine, PO Box 670576, Cincinnati, OH 45267-0576, USA. bryan.mackenzie@uc.edu
Illing
Anthony C
AC
Morris
Marie E K
ME
Varoqui
Hélène
H
Erickson
Jeffrey D
JD
D000818:Animals; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014316:Tritium; D050599:Vesicular Glutamate Transport Protein 2 / Q000378:metabolism*; D014981:Xenopus
D011994:Recombinant Proteins; C493475:Slc17a6 protein, rat; D050599:Vesicular Glutamate Transport Protein 2; D014316:Tritium