The quantal release of glutamate depends on its transport into synaptic vesicles. Recent work has shown that a protein previously implicated in the uptake of inorganic phosphate across the plasma membrane catalyzes glutamate uptake by synaptic vesicles. However, only a subset of glutamate neurons expresses this vesicular glutamate transporter (VGLUT1). We now report that excitatory neurons lacking VGLUT1 express a closely related protein that has also been implicated in phosphate transport. Like VGLUT1, this protein localizes to synaptic vesicles and functions as a vesicular glutamate transporter (VGLUT2). The complementary expression of VGLUT1 and 2 defines two distinct classes of excitatory synapse.

Synaptic transmission of most vertebrate synapses is thought to be terminated by rapid transport of the neurotransmitter into presynaptic nerve terminals or neuroglia. L-Glutamate is the major excitatory transmitter in brain and its transport represents the mechanism by which it is removed from the synaptic cleft and kept below toxic levels. Here we use an antibody against a glial L-glutamate transporter from rat brain to isolate a complementary DNA clone encoding this transporter. Expression of this cDNA in transfected HeLa cells indicates that L-glutamate accumulation requires external sodium and internal potassium and transport shows the expected stereospecificity. The cDNA sequence predicts a protein of 573 amino acids with 8-9 putative transmembrane alpha-helices. Database searches indicate that this protein is not homologous to any identified protein of mammalian origin, including the recently described superfamily of neurotransmitter transporters. This protein therefore seems to be a member of a new family of transport molecules.

Glutamate, the major excitatory neurotransmitter in brain, is almost exclusively intracellular due to the action of the glutamate transporters in the plasma membranes. To study the localization and properties of these proteins, we have raised antibodies specifically recognizing parts of the sequences of two cloned rat glutamate transporters, GLT-1 (Pines et al., 1992) and GLAST (Storck et al., 1992). On immunoblots the antibodies against GLT-1 label a broad heterogeneous band with maximum density at around 73 kDa, while the antibody against GLAST labels a similarly broad band at around 66 kDa in the cerebellum and a few kilodaltons lower in other brain regions. GLT-1 is expressed at the highest concentrations in the hippocampus, lateral septum, cerebral cortex, and striatum, while GLAST is preferentially expressed in the molecular layer of the cerebellum. However, both transporters are present throughout the brain, and have roughly parallel distributions in the cerebral hemispheres and brainstem. Preembedding light and electron microscopical immunocytochemistry shows that both GLT-1 and GLAST are restricted to astrocytes, which appear to express both proteins concomitantly, but in different proportions in different parts of the brain. Nerve terminal labeling was not observed. Both the amino and carboxyl terminals of GLT- 1 and GLAST are located intracellularly, indicating an even number of transmembrane segments. Antibodies against a synthetic peptide corresponding to amino acid residues 2–11 of the proposed sequence of GLT-1 recognize the native rat brain GLT-1 protein, confirming that the translation initiation site is at the first ATG.

Quantal release of the principal excitatory neurotransmitter glutamate requires a mechanism for its transport into secretory vesicles. Within the brain, the complementary expression of vesicular glutamate transporters (VGLUTs) 1 and 2 accounts for the release of glutamate by all known excitatory neurons. We now report the identification of VGLUT3 and its expression by many cells generally considered to release a classical transmitter with properties very different from glutamate. Remarkably, subpopulations of inhibitory neurons as well as cholinergic interneurons, monoamine neurons, and glia express VGLUT3. The dendritic expression of VGLUT3 by particular neurons also indicates the potential for retrograde synaptic signaling. The distribution and subcellular location of VGLUT3 thus suggest novel modes of signaling by glutamate.