Vesicular glutamate transporter contains two independent transport machineries

Vesicular glutamate transporters (VGLUTs) are responsible for the vesicular storage of l-glutamate and play an essential role in glutamatergic signal transmission in the central nervous system. The molecular mechanism of the transport remains unknown. Here, we established a novel in vitro assay procedure, which includes purification of wild and mutant VGLUT2 and their reconstitution with purified bacterial F(o)F(1)-ATPase (F-ATPase) into liposomes. Upon the addition of ATP, the proteoliposomes facilitated l-glutamate uptake in a membrane potential (DeltaPsi)-dependent fashion. The ATP-dependent l-glutamate uptake exhibited an absolute requirement for approximately 4 mm Cl(-), was sensitive to Evans blue, but was insensitive to d,l-aspartate. VGLUT2s with mutations in the transmembrane-located residues Arg(184), His(128), and Glu(191) showed a dramatic loss in l-glutamate transport activity, whereas Na(+)-dependent inorganic phosphate (P(i)) uptake remained comparable to that of the wild type. Furthermore, P(i) transport did not require Cl(-) and was not inhibited by Evans blue. Thus, VGLUT2 appears to possess two intrinsic transport machineries that are independent of each other: a DeltaPsi-dependent l-glutamate uptake and a Na(+)-dependent P(i) uptake.     

Vesicular neurotransmitter transporter trafficking in vivo: Moving from cells to flies

During exocytosis, classical and amino acid neurotransmitters are released from the lumen of synaptic vesicles to allow signaling at the synapse. The storage of neurotransmitters in synaptic vesicles and other types of secretory vesicles requires the activity of specific vesicular transporters. Glutamate and monoamines such as dopamine are packaged by VGLUTs and VMATs respectively. Changes in the localization of either protein have the potential to up- or down regulate neurotransmitter release, and some of the mechanisms for sorting these proteins to secretory vesicles have been investigated in cultured cells in vitro. We have used Drosophila molecular genetic techniques to study vesicular transporter trafficking in an intact organism and have identified a motif required for localizing Drosophila VMAT (DVMAT) to synaptic vesicles in vivo. In contrast to DVMAT, large deletions of Drosophila VGLUT (DVGLUT) show relatively modest deficits in localizing to synaptic vesicles, suggesting that DVMAT and DVGLUT may undergo different modes of trafficking at the synapse. Further in vivo studies of DVMAT trafficking mutants will allow us to determine how changes in the localization of vesicular transporters affect the nervous system as a whole and complex behaviors mediated by aminergic circuits.     

Vesicular glutamate transporter 1 immunoreactivity in extrinsic and intrinsic innervation of the rat esophagus

Encouraged by the recent finding of vesicular glutamate transporter 2 (VGLUT2) immunoreactivity (-ir) in intraganglionic laminar endings (IGLEs) of the rat esophagus, we investigated also the distribution and co-localization patterns of VGLUT1. Confocal imaging revealed substantial co-localization of VGLUT1-ir with selective markers of IGLEs, i.e., calretinin and VGLUT2, indicating that IGLEs contain both VGLUT1 and VGLUT2 within their synaptic vesicles. Besides IGLEs, we found VGLUT1-ir in both cholinergic and nitrergic myenteric neuronal cell bodies, in fibers of the muscularis mucosae, and in esophageal motor endplates. Skeletal neuromuscular junctions, in contrast, showed no VGLUT1-ir. We also tested for probable co-localization of VGLUT1-ir with markers of extrinsic and intrinsic esophageal innervation and glia. Within the myenteric neuropil we found, besides co-localization of VGLUT1 and substance P, no further co-localization of VGLUT1-ir with any of these markers. In the muscularis mucosae some VGLUT1-ir fibers were shown to contain neuronal nitric oxide synthase (nNOS)-ir. VGLUT1-ir in esophageal motor endplates was partly co-localized with vesicular acetylcholine transporter (VAChT)/choline acetyltransferase (ChAT)-ir, but VGLUT1-ir was also demonstrated in separately terminating fibers at motor endplates co-localized neither with ChAT/VAChT-ir nor with nNOS-ir, suggesting a hitherto unknown glutamatergic enteric co-innervation. Thus, VGLUT1-ir was found in extrinsic as well as intrinsic innervation of the rat esophagus.     

The glutamate/GABA-glutamine cycle: aspects of transport, neurotransmitter homeostasis and ammonia transfer

Neurons are metabolically handicapped in the sense that they are not able to perform de novo synthesis of neurotransmitter glutamate and gamma-aminobutyric acid (GABA) from glucose. A metabolite shuttle known as the glutamate/GABA-glutamine cycle describes the release of neurotransmitter glutamate or GABA from neurons and subsequent uptake into astrocytes. In return, astrocytes release glutamine to be taken up into neurons for use as neurotransmitter precursor. In this review, the basic properties of the glutamate/GABA-glutamine cycle will be discussed, including aspects of transport and metabolism. Discussions of stoichiometry, the relative role of glutamate vs. GABA and pathological conditions affecting the glutamate/GABA-glutamine cycling are presented. Furthermore, a section is devoted to the accompanying ammonia homeostasis of the glutamate/GABA-glutamine cycle, examining the possible means of intercellular transfer of ammonia produced in neurons (when glutamine is deamidated to glutamate) and utilized in astrocytes (for amidation of glutamate) when the glutamate/GABA-glutamine cycle is operating. A main objective of this review is to endorse the view that the glutamate/GABA-glutamine cycle must be seen as a bi-directional transfer of not only carbon units but also nitrogen units.     

Vesicular glutamate transporter 2-immunoreactive afferent nerve terminals in the carotid body of the rat

The carotid body is a peripheral chemoreceptor that detects decreases in arterial pO₂ and subsequently activates the carotid sinus nerve. The hypoxia-evoked activity of the carotid sinus nerve has been suggested to be modulated by glutamate. In the present study, we investigate the immunohistochemical localization of vesicular glutamate transporters in the carotid body of the rat. Vesicular glutamate transporter 2 (VGLUT2) labeling was closely associated with glomus cells immunoreactive to tyrosine hydroxylase but was not in the cytoplasm of these cells. The VGLUT2 immunoreactivity was observed within nerve endings that were immunoreactive to P2X3 and densely localized inside P2X3-immunoreactive axon terminals. These results suggest that VGLUT2 is localized in the afferent nerve terminals of the carotid body. Glutamate may be released from afferent nerve terminals to modulate the chemosensory activity of the carotid body.     

Inhibition of glutamine transport depletes glutamate and GABA neurotransmitter pools: further evidence for metabolic compartmentation

The role of glutamine and alanine transport in the recycling of neurotransmitter glutamate was investigated in Guinea pig brain cortical tissue slices and prisms, and in cultured neuroblastoma and astrocyte cell lines. The ability of exogenous (2 mm) glutamine to displace 13C label supplied as [3-13C]pyruvate, [2-13C]acetate, l-[3-13C]lactate, or d-[1-13C]glucose was investigated using NMR spectroscopy. Glutamine transport was inhibited in slices under quiescent or depolarising conditions using histidine, which shares most transport routes with glutamine, or 2-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of the neuronal system A. Glutamine mainly entered a large, slow turnover pool, probably located in neurons, which did not interact with the glutamate/glutamine neurotransmitter cycle. This uptake was inhibited by MeAIB. When [1-13C]glucose was used as substrate, glutamate/glutamine cycle turnover was inhibited by histidine but not MeAIB, suggesting that neuronal system A may not play a prominent role in neurotransmitter cycling. When transport was blocked by histidine under depolarising conditions, neurotransmitter pools were depleted, showing that glutamine transport is essential for maintenance of glutamate, GABA and alanine pools. Alanine labelling and release were decreased by histidine, showing that alanine was released from neurons and returned to astrocytes. The resultant implications for metabolic compartmentation and regulation of metabolism by transport processes are discussed.     

Post-translational regulation of an Aplysia glutamate transporter during long-term facilitation

Regulation of glutamate transporters accompanies plasticity of some glutamatergic synapses. The regulation of glutamate uptake at the Aplysia sensorimotor synapse during long-term facilitation (LTF) was investigated. Previously, increases in levels of ApGT1 ( Aplysia glutamate transporter 1) in synaptic membranes were found to be related to long-term increases in glutamate uptake. In this study, we found that regulation of ApGT1 during LTF appears to occur post-translationally. Serotonin (5-HT) a transmitter that induces LTF did not increase synthesis of ApGT1. A pool of ApGT1 appears to exist in sensory neuron somata, which is transported to the terminals by axonal transport. Blocking the rough endoplasmic reticulum-Golgi-trans-Golgi network (TGN) pathway with Brefeldin A prevented the 5-HT-induced increase of ApGT1 in terminals. Also, 5-HT produced changes in post-translational modifications of ApGT1 as well as changes in the levels of an ApGT1-co-precipitating protein. These results suggest that regulation of trafficking of ApGT1 from the vesicular trafficking system (rough endoplasmic reticulum-Golgi-TGN) in the sensory neuron somata to the terminals by post-translational modifications and protein interactions appears to be the mechanism underlying the increase in ApGT1, and thus, glutamate uptake during memory formation.     

Vesicular glutamate transporter modulates sex differences in dopamine neuron vulnerability to age‐related neurodegeneration

Age is the greatest risk factor for Parkinson''s disease (PD) which causes progressive loss of dopamine (DA) neurons, with males at greater risk than females. Intriguingly, some DA neurons are more resilient to degeneration than others. Increasing evidence suggests that vesicular glutamate transporter (VGLUT) expression in DA neurons plays a role in this selective vulnerability. We investigated the role of DA neuron VGLUT in sex‐ and age‐related differences in DA neuron vulnerability using the genetically tractable Drosophila model. We found sex differences in age‐related DA neurodegeneration and its associated locomotor behavior, where males exhibit significantly greater decreases in both DA neuron number and locomotion during aging compared with females. We discovered that dynamic changes in DA neuron VGLUT expression mediate these age‐ and sex‐related differences, as a potential compensatory mechanism for diminished DA neurotransmission during aging. Importantly, female Drosophila possess higher levels of VGLUT expression in DA neurons compared with males, and this finding is conserved across flies, rodents, and humans. Moreover, we showed that diminishing VGLUT expression in DA neurons eliminates females'' greater resilience to DA neuron loss across aging. This offers a new mechanism for sex differences in selective DA neuron vulnerability to age‐related DA neurodegeneration. Finally, in mice, we showed that the ability of DA neurons to achieve optimal control over VGLUT expression is essential for DA neuron survival. These findings lay the groundwork for the manipulation of DA neuron VGLUT expression as a novel therapeutic strategy to boost DA neuron resilience to age‐ and PD‐related neurodegeneration.     

The anion conductance of the glutamate transporter EAAC1 depends on the direction of glutamate transport

The steady-state and pre-steady-state kinetics of glutamate transport by the neuronal glutamate transporter EAAC1 were determined under conditions of outward glutamate transport and compared to those found for the inward transport mode. In both transport modes, the glutamate-induced current is composed of two components, the coupled transport current and the uncoupled anion current, and inhibited by a specific non-transportable inhibitor. Furthermore, the glutamate-independent leak current is observed in both transport modes. Upon a glutamate concentration jump outward transport currents show a distinct transient phase that deactivates within 15 ms. The results demonstrate that the general properties of EAAC1 are symmetric, but the rates of substrate transport and anion flux are asymmetric with respect to the orientation of the substrate binding site in the membrane. Therefore, the EAAC1 anion conductance differs from normal ligand-gated ion channels in that it can be activated by glutamate and Na(+) from both sides of the membrane.     

Vesicular glutamate transport at a central synapse limits the acuity of visual perception in zebrafish

The neural circuitry that constrains visual acuity in the CNS has not been experimentally identified. We show here that zebrafish blumenkohl (blu) mutants are impaired in resolving rapid movements and fine spatial detail. The blu gene encodes a vesicular glutamate transporter expressed by retinal ganglion cells. Mutant retinotectal synapses release less glutamate, per vesicle and per terminal, and fatigue more quickly than wild-type in response to high-frequency stimulation. In addition, mutant axons arborize more extensively, thus increasing the number of synaptic terminals and effectively normalizing the combined input to postsynaptic cells in the tectum. This presumably homeostatic response results in larger receptive fields of tectal cells and a degradation of the retinotopic map. As predicted, mutants have a selective deficit in the capture of small prey objects, a behavior dependent on the tectum. Our studies successfully link the disruption of a synaptic protein to complex changes in neural circuitry and behavior.     

Vesicular transport and kidney development.

Vesicular transport processes play crucial roles in the biogenesis of cellular membranes and in the polarized transport functions of epithelial cells. During the 1990's we have witnessed major progress in elucidation of the machineries responsible for the intracellular membrane trafficking. The components of these machineries are abundant in tissues with a high content of epithelial cells, such as the kidney. However, the developmental role of the membrane trafficking apparatus in higher eukaryotes has been addressed hardly at all. We summarize here data on the presence and the functional role of vesicle transport proteins in the kidney, and describe work addressing the developmentally regulated expression and localization of three molecules suggested to be involved in polarized trafficking in kidney epithelia, Rab17, syntaxin 3, and Munc-18-2. The results show that specialized transport machinery is induced during differentiation of renal epithelia. However, the expression levels of the components under study are highest in the mature structures, indicating that the proteins are predominantly required for the function of mature epithelia and possibly for the maintenance of the polarized phenotype of specific epithelial cells. The proteins are, however, detected at low levels already in earlier, differentiating structures, and could thus also be involved in the differentiation of kidney epithelia.     

N-methyl-D-aspartate receptor-dependent regulation of the glutamate transporter excitatory amino acid carrier 1

The neuronal transporter excitatory amino acid carrier 1 (EAAC1) is enriched in perisynaptic regions, where it may regulate synaptic spillover of glutamate. In this study we examined potential interactions between EAAC1 and ionotropic glutamate receptors. N-Methyl-D-aspartate (NMDA) receptor subunits NR1, NR2A, and NR2B, but not the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluR2, were co-immunoprecipitated with EAAC1 from neuron-enriched hippocampal cultures. A similar interaction was observed in C6 glioma and human embryonic kidney cells after co-transfection with Myc epitope-tagged EAAC1 and NMDA receptor subunits. Co-transfection of C6 glioma with the combination of NR1 and NR2 subunits dramatically increased (approximately 3-fold) the amount of Myc-EAAC1 that can be labeled with a membrane-impermeable biotinylating reagent. In hippocampal cultures, brief (5 min), robust (100 microM NMDA, 10 microM glycine) activation of the NMDA receptor decreased biotinylated EAAC1 to approximately 50% of control levels. This effect was inhibited by an NMDA receptor antagonist, intracellular or extracellular calcium chelators, or hypertonic sucrose. Glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid with cyclothiazide, and thapsigargin mimicked the effects of NMDA. These studies suggest that NMDA receptors interact with EAAC1, facilitate cell surface expression of EAAC1 under basal conditions, and control internalization of EAAC1 upon activation. This NMDA receptor-dependent regulation of EAAC1 provides a novel mechanism that may shape excitatory signaling during synaptic plasticity and/or excitotoxicity.     

Altered astrocyte glutamate transporter regulation of hypothalamic neurosecretory neurons in heart failure rats

Neurohumoral activation, which includes augmented plasma levels of the neurohormone vasopressin (VP), is a common finding in heart failure (HF) that contributes to morbidity and mortality in this disease. While an increased activation of magnocellular neurosecretory cells (MNCs) and enhanced glutamate function in HF is well documented, the precise underlying mechanisms remain to be elucidated. Here, we combined electrophysiology and protein measurements to determine whether altered glial glutamate transporter function and/or expression occurs in the hypothalamic supraoptic nucleus (SON) during HF. Patch-clamp recordings obtained from MNCs in brain slices show that pharmacological blockade of astrocyte glutamate transporter 1 (GLT1) function [500 μM dihydrokainate (DHK)], resulted in a persistent N-methyl-D-aspartate receptor (NMDAR)-mediated inward current (tonic I(NMDA)) in sham rats, an effect that was significantly smaller in MNCs from HF rats. In addition, we found a diminished GLT1 protein content in plasma membrane (but not cytosolic) fractions of SON punches in HF rats. Conversely, astrocyte GLAST expression was significantly higher in the SON of HF rats, while nonselective blockade of glutamate transport activity (100 μM TBOA) evoked an enhanced tonic I(NMDA) activation in HF rats. Steady-state activation of NMDARs by extracellular glutamate levels was diminished during HF. Taken together, these results support a shift in the relative expression and function of two major glial glutamate transporters (from GLT1 to GLAST predominance) during HF. This shift may act as a compensatory mechanism to preserve an adequate basal glutamate uptake level in the face of an enhanced glutamatergic afferent activity in HF rats.     

The discovery of slowness: low-capacity transport and slow anion channel gating by the glutamate transporter EAAT5

Excitatory amino acid transporters (EAATs) control the glutamate concentration in the synaptic cleft by glial and neuronal glutamate uptake. Uphill glutamate transport is achieved by the co-/countertransport of Na⁺ and other ions down their concentration gradients. Glutamate transporters also display an anion conductance that is activated by the binding of Na⁺ and glutamate but is not thermodynamically coupled to the transport process. Of the five known glutamate transporter subtypes, the retina-specific subtype EAAT5 has the largest conductance relative to glutamate uptake activity. Our results suggest that EAAT5 behaves as a slow-gated anion channel with little glutamate transport activity. At steady state, EAAT5 was activated by glutamate, with a K_m= 61 ± 11 μM. Binding of Na⁺ to the empty transporter is associated with a K_m = 229 ± 37 mM, and binding to the glutamate-bound form is associated with a K_m = 76 ± 40 mM. Using laser-pulse photolysis of caged glutamate, we determined the pre-steady-state kinetics of the glutamate-induced anion current of EAAT5. This was characterized by two exponential components with time constants of 30 ± 1 ms and 200 ± 15 ms, which is an order of magnitude slower than those observed in other glutamate transporters. A voltage-jump analysis of the anion currents indicates that the slow activation behavior is caused by two slow, rate-limiting steps in the transport cycle, Na⁺ binding to the empty transporter, and translocation of the fully loaded transporter. We propose a kinetic transport scheme that includes these two slow steps and can account for the experimentally observed data. Overall, our results suggest that EAAT5 may not act as a classical high-capacity glutamate transporter in the retina; rather, it may function as a slow-gated glutamate receptor and/or glutamate buffering system.