Introduction l Glutamate is a major
Introduction l-Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system (CNS) that contributes not only to fast synaptic neurotransmission, but also to complex physiological processes like memory, learning, plasticity, and neuronal cell death , . Glutamate is synthesized in the cytoplasm and stored in synaptic vesicles by an uptake system that depends on the proton electrochemical gradient, the vesicular glutamate transporters (VGLUTs). Following its exocytotic release, glutamate activates ionotropic glutamate receptors for fast excitatory neurotransmission and metabotropic receptors for slower modulatory affects on transmission. To terminate the action of glutamate and maintain its extracellular concentration below excitotoxic levels, Na+-dependent high affinity glutamate transporters (excitatory amino 98014 transporters: EAATs) located on the plasma membrane of neurons and glial cells rapidly remove glutamate from the extracellular space (for reviews, see , , ). Glutamate is thought to be released not only synaptically but also extrasynaptically by exocytosis , cystine-glutamate antiporter  and volume-regulated anion channels . However, most of the glutamate is released synaptically and transits through the glutamate–glutamine cycle before being repackaged into synaptic vesicles , . Glutamate taken up into glial cells is metabolized to glutamine, which is then transported back into neurons, converted to glutamate and sequestered into synaptic vesicles by the VGLUTs (Fig. 1). Glutamate transported by EAATs residing on postsynaptic GABAergic neurons serves as a precursor for the synthesis of GABA and recently has been shown to impact vesicular GABA content and inhibitory synaptic strength in the hippocampus . In early studies of sodium-dependent glutamate transport, radiolabeled neurotransmitters and heterogeneous preparations such as brain slices and synaptosomes were utilized to identify pharmacologically distinct subtypes of EAATs, which differed in their substrate specificity and sensitivity to inhibitors , , , . In 1992, the genes encoding the three plasma membrane glutamate transporters were identified and designated GLAST, GLT-1 and EAAC1 , , . GLAST is expressed in glial cells. GLT-1 is primarily expressed in glial cells and EAAC1 is a neuronal transporter. In 1994, three human glutamate transporters, EAAT1, EAAT2 and EAAT3 corresponding to GLAST, GLT-1 and EAAC1, were identified . Two additional mammalian subtypes were identified subsequently, EAAT4 which is abundant in cerebellar Purkinje cells and EAAT5 which is expressed in the retina , . Studies using isolated synaptic vesicles and radiolabeled neurotransmitters also determined the unique characteristics of the VGLUTs. First, VGLUTs exhibit a substrate specificity that differs from the EAATs. Second, VGLUTs require a H+ electrochemical gradient across the vesicle membrane that is generated by the vacuolar-type proton ATPase. Third, the uptake activity of VGLUTs is stimulated by physiological concentrations of Cl− (1–5 mM) . In 2000, two different groups, the Edwards Laboratory at UCSF and the Jahn laboratory at the Max-Planck Institute reported the molecular identity of a vesicular glutamate transporter (VGLUT1). Both groups reported that the brain-specific Na+-dependent inorganic phosphate cotransporter (BNPI), which had originally been reported as a carrier of inorganic phosphate, acted as a vesicular glutamate transporter , . Because BNPI shares 82% amino acid homology with differentiation–association Na+-dependent inorganic phosphate cotransporter (DNPI), DNPI was postulated to also act as a VGLUT and after functional characterization was designated VGLUT2 , , , . Interestingly, VGLUT1 and VGLUT2 are sufficient to confer a glutamatergic phenotype when heterologously expressed in hippocampal GABAergic neurons. Recently, a third vesicular glutamate transporter (VGLUT3) was cloned and characterized , , , . The three VGLUTs share more than 70% amino acid homology and have mostly non-overlapping distributions throughout the brain , , , , .