Glutamine uptake by System A transporters maintains neurotransmitter GABA synthesis and inhibitory synaptic transmission (original) (raw)
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Proceedings of the National Academy of Sciences, 2003
L-Glutamic acid decarboxylase (GAD) exists as both membraneassociated and soluble forms in the mammalian brain. Here, we propose that there is a functional and structural coupling between the synthesis of ␥-aminobutyric acid (GABA) by membraneassociated GAD and its packaging into synaptic vesicles (SVs) by vesicular GABA transporter (VGAT). This notion is supported by the following observations. First, newly synthesized [ 3 H]GABA from [ 3 H]L-glutamate by membrane-associated GAD is taken up preferentially over preexisting GABA by using immunoaffinity-purified GABAergic SVs. Second, the activity of SV-associated GAD and VGAT seems to be coupled because inhibition of GAD also decreases VGAT activity. Third, VGAT and SV-associated Ca 2؉ ͞ calmodulin-dependent kinase II have been found to form a protein complex with GAD. A model is also proposed to link the neuronal stimulation to enhanced synthesis and packaging of GABA into SVs.
The Glutamine Transporter Slc38a1 Regulates GABAergic Neurotransmission and Synaptic Plasticity
Cerebral Cortex, 2019
GABA signaling sustains fundamental brain functions, from nervous system development to the synchronization of population activity and synaptic plasticity. Despite these pivotal features, molecular determinants underscoring the rapid and cell-autonomous replenishment of the vesicular neurotransmitter GABA and its impact on synaptic plasticity remain elusive. Here, we show that genetic disruption of the glutamine transporter Slc38a1 in mice hampers GABA synthesis, modifies synaptic vesicle morphology in GABAergic presynapses and impairs critical period plasticity. We demonstrate that Slc38a1-mediated glutamine transport regulates vesicular GABA content, induces high-frequency membrane oscillations and shapes cortical processing and plasticity. Taken together, this work shows that Slc38a1 is not merely a transporter accumulating glutamine for metabolic purposes, but a key component regulating several neuronal functions.
Neurochemical Research, 2014
The operation of a glutamine-glutamate/ GABA cycle in the brain consisting of the transfer of glutamine from astrocytes to neurons and neurotransmitter glutamate or GABA from neurons to astrocytes is a wellknown concept. In neurons, glutamine is not only used for energy production and protein synthesis, as in other cells, but is also an essential precursor for biosynthesis of amino acid neurotransmitters. An excellent tool for the study of glutamine transfer from astrocytes to neurons is [ 14 C]acetate or [ 13 C]acetate and the glial specific enzyme inhibitors, i.e. the glutamine synthetase inhibitor methionine sulfoximine and the tricarboxylic acid cycle (aconitase) inhibitors fluoro-acetate and -citrate. Acetate is metabolized exclusively by glial cells, and [ 13 C]acetate is thus capable when used in combination with magnetic resonance spectroscopy or mass spectrometry, to provide information about glutamine transfer. The present review will give information about glutamine trafficking and the tools used to map it as exemplified by discussions of published work employing brain cell cultures as well as intact animals. It will be documented that considerably more glutamine is transferred from astrocytes to glutamatergic than to GABAergic neurons. However, glutamine does have an important role in GABAergic neurons despite their capability of re-utilizing their neurotransmitter by re-uptake.
A Role for GAT-1 in Presynaptic GABA Homeostasis?
Frontiers in Cellular Neuroscience, 2011
former preferentially synthesizes GABA for vesicular release, the latter for cytoplasmic stores (Soghomonian and Martin, 1998). Indeed, ratio of GAD 65 to GAD 67 is higher in synaptic vesicle fractions than in cytosol (Solimena et al., 1993). GAD 65 may be anchored to synaptic vesicles by forming a complex that includes the vesicular GABA transporter VGAT, an integral membrane protein of synaptic vesicles responsible for their filling (McIntire et al., 1997). This may provide a structural and functional coupling between synthesis and vesicular packaging of GABA (Hsu et al., 2000; Jin et al., 2003). Interestingly, [ 3 H]GABA newly synthesized from [ 3 H]Glu by synaptic vesicle-associated GAD is taken up preferentially into vesicles over cytosolic GABA (Jin et al., 2003). Minor sources of GABA, such as putrescine, spermine, spermidine, and ornithine, offer a negligible contribution to releasable GABA. Glu used for GABA synthesis may originate from diverse sources. Glu-GABA/glutamine cycle Glutamate derived from glutamine (Gln) is an important GABA precursor (Bradford et al., 1983; Sonnewald et al., 1993). Released GABA is taken up by astrocytic transporters (i.e., GAT-3; Minelli et al., 1996), and catabolized to the tricarboxylic acid (TCA) cycle intermediate succinate by GABA transaminase and succinate semialdehyde dehydrogenase; the resulting α-ketoglutarate is then transformed to Glu which is converted to Gln by Gln synthetase (Martinez-Hernandez et al., 1977). Gln is then extruded from astrocytes by SNAT3, a system N transporter (Chaudry et al., 1999, 2002; Boulland et al., 2002), and taken up by axon terminals. Gln influx into neurons is thought to be mediated by SNAT1 and/or SNAT2, two system A transporters (Fricke et al., 2007); this view is compatible with expression of SNAT1 and SNAT2 in most GABAergic cells (Melone et al., 2004, 2006; Conti and Melone, 2006). In neurons, Gln is converted to Glu by phosphate-activated glutaminase (PAG; Kvamme et al., 2001).
Journal of Neurochemistry, 2008
Adequate neurotransmitter synthesis and subsequent uptake and storage in synaptic vesicles are important mechanisms for excitatory and inhibitory neurotransmission, mediated by stimulus-induced exocytosis of the amino acids glutamate and GABA, respectively. Glutamate, which serves as an energy substrate, a building block of proteins, an excitatory neurotransmitter and as the immediate precursor for the inhibitory neurotransmitter GABA (Fonnum 1984), is synthesized directly from the mitochondrial precursor a-ketoglutarate or from glutamine by phosphate-activated glutaminase (PAG; EC 3.5.1.2), followed by release to the cytosol (Fonnum 1993). In GABAergic neurons, cytosolic glutamate is further metabolized to GABA by glutamic acid decarboxylase (GAD; EC 4.1.1.15) (Martin and Rimvall 1993). Cytosolic glutamate and GABA are then sequestered into synaptic vesicles by distinct vesicular transporters specific for glutamate (VGLUT1-3) and GABA (VGAT) in glutamatergic and GABAergic neurons, respectively (McIntire et al. 1997; Fremeau et al. 2004b). The lethal phenotype of animals with a complete absence of vesicular transporters for glutamate, GABA or monoamines demonstrates the major importance of vesicular
Glutamate, aspartate, and γ-aminobutyrate transport by membrane vesicles prepared from rat brain
Archives of Biochemistry and Biophysics, 1981
To prepare membrane vesicles, nerve terminal preparations (synaptosomes) isolated from rat cerebral cortex were first subjected to hypotonic lysis. After collecting the membranes contained in this fraction by centrifugation, membrane vesicles were then reconstituted during incubation in a potassium salt solution at 37°C. The transport of glutamate, aspartate, or y-aminobutyrie acid (GABA) was measured by transferring vesicles to 10 vol of 0.1 M NaCl solution containing the radioactive substrate. Transport was temperature dependent and exhibited saturation kinetics with an apparent K, of 2.5 brM. The rates and extent of L-glutamate and L-aspartate uptake were equivalent and were greater than those for GABA. Valinomycin increased the rate of uptake of each of these substances suggesting a role for an electrogenic component in transport. Consonant with this notion, external K+ and Rb+ decreased uptake of all three compounds. External thiocyanate also increases the rate of glutamate, aspartate, and GABA transport. Uptake of these neuroactive amino acids was absolutely dependent on external Na+; no other monovalent cation tested substitutes for it. Gramicidin D and nigericin inhibit glutamate transport by abolishing both the Na+ and Kf gradients. Monensin inhibits uptake by selectively dissipating the Na+ gradient. For both glutamate and GABA transport, the Na+ and K+ gradients are synergistic and not additive.
Glutamine efflux from astrocytes is mediated by multiple pathways
Journal of Neurochemistry, 2003
The neurotransmitter glutamate, once released into the synaptic cleft, is largely recycled by the glutamate-glutamine cycle, which involves uptake into astrocytes, conversion into glutamine and subsequent release of glutamine from astrocytes as a precursor for neuroneal glutamate synthesis. We analysed glutamine efflux from cultured astrocytes by pre-loading cells with labelled glutamine for 30 min and subsequently measured glutamine efflux for 30 min. Efflux of preloaded glutamine was rapid and almost complete after 30 min with a first order rate of 0.11 ± 0.01/min. Efflux was 50% reduced when cells were depleted of intracellular Na + . Increasing intracellular Na + concentration had a small stimulatory effect on glutamine efflux, indicating the participation of a Na + -dependent transport mechanism. About 50% of the basal efflux could not be inhibited by depletion of the intracellular Na + , suggesting the presence of an additional Na + -independent transport mechanism. Glutamine efflux was stimulated two-to threefold by addition of extracellular neutral amino acids, such as alanine or leucine. The stimulatory effects of alanine and leucine had a Na + -dependent and a Na + -independent component, suggesting the presence of two antiport mechanisms one involving Na + . When compared to the expression of glutamine transporter mRNAs in cultured astrocytes it appeared likely that glutamine efflux was mediated by SN1, LAT2, ASCT2 and an additional, yet unidentified, transporter that mediates about 40% of the basal efflux.