Hélène Varoqui

Cloning and Functional Identification of a Neuronal Glutamine Transporter

Glutamine is the preferred precursor for the neurotransmitter pool of glutamate, the major excitatory transmitter in the mammalian central nervous system. We have isolated a complementary DNA clone (designated GlnT) encoding a plasma membrane glutamine transporter from glutamatergic neurons in culture, and its properties have been examined using the T7 vaccinia system in fibroblasts. When GlnT is transfected into CV-1 cells, l-glutamine is the preferred substrate. Transport is Na+-dependent and inhibited by α-methylaminoisobutyric acid, a specific inhibitor of neutral amino acid transport system A. Kinetic analysis of glutamine uptake by GlnT is saturable, with a Michaelis constant (K m ) of 489 ± 88 μm at pH 7.4. Glutamine uptake mediated by GlnT is pH-sensitive with a 5-fold greater efficiency of uptake at pH 8.2 than at pH 6.6. Only the maximal velocity of transport increases without a significant change in K m . The distribution of GlnT mRNA and protein in the central nervous system is widespread and is expressed on neurons that use glutamate as their neurotransmitter. In cultured cerebellar granule cells, GlnT is expressed only on neurons and is absent from astrocytes. GlnT expression increases concomitantly with the morphologic and functional differentiation of these cells in vitro, consistent with its role of supplying glutamatergic neurons with their neurotransmitter precursor. GlnT is the first member of the system A family of neutral amino acid transporters with 11 putative membrane-spanning domains and is a potential target to modulate presynaptic glutamatergic function.

Presynaptic regulation of quantal size by the vesicular glutamate transporter VGLUT1.

A fundamental question in synaptic physiology is whether the unitary strength of a synapse can be regulated by presynaptic characteristics and, if so, what those characteristics might be. Here, we characterize a newly proposed mechanism for altering the strength of glutamatergic synapses based on the recently identified vesicular glutamate transporter VGLUT1. We provide direct evidence that filling in isolated synaptic vesicles is subject to a dynamic equilibrium that is determined by both the concentration of available glutamate and the number of vesicular transporters participating in loading. We observe that changing the number of vesicular transporters expressed at hippocampal excitatory synapses results in enhanced evoked and miniature responses and verify biophysically that these changes correspond to an increase in the amount of glutamate released per vesicle into the synaptic cleft. In addition, we find that this modulation of synaptic strength by vesicular transporter expression is endogenously regulated, both across development to coincide with a maturational increase in vesicle cycling and quantal amplitude and by excitatory and inhibitory receptor activation in mature neurons to provide an activity-dependent scaling of quantal size via a presynaptic mechanism. Together, these findings underscore that vesicular transporter expression is used endogenously to directly regulate the extent of glutamate release, providing a concise presynaptic mechanism for controlling the quantal efficacy of excitatory transmission during synaptic refinement and plasticity.

A Novel System A Isoform Mediating Na+/Neutral Amino Acid Cotransport

A cDNA clone encoding a plasma membrane alanine-preferring transporter (SAT2) has been isolated from glutamatergic neurons in culture and represents the second member of the system A family of neutral amino acid transporters. SAT2 displays a widespread distribution and is expressed in most tissues, including heart, adrenal gland, skeletal muscle, stomach, fat, brain, spinal cord, colon, and lung, with lower levels detected in spleen. No signal is detected in liver or testis. In the central nervous system, SAT2 is expressed in neurons. SAT2 is significantly up-regulated during differentiation of cerebellar granule cells and is absent from astrocytes in primary culture. The functional properties of SAT2, examined using transfected fibroblasts and in cRNA-injected voltage-clamped Xenopus oocytes, show that small aliphatic neutral amino acids are preferred substrates and that transport is voltage- and Na+-dependent (1:1 stoichiometry), pH-sensitive, and inhibited by α-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of system A. Kinetic analyses of alanine and MeAIB uptake by SAT2 are saturable, with Michaelis constants (K m ) of 200–500 μm. In addition to its ubiquitous role as a substrate for oxidative metabolism and a major vehicle of nitrogen transport, SAT2 may provide alanine to function as the amino group donor to α-ketoglutarate to provide an alternative source for neurotransmitter synthesis in glutamatergic neurons.

Homeostatic Scaling of Vesicular Glutamate and GABA Transporter Expression in Rat Neocortical Circuits

Homeostatic control of pyramidal neuron firing rate involves a functional balance of feedforward excitation and feedback inhibition in neocortical circuits. Here, we reveal a dynamic scaling in vesicular excitatory (vesicular glutamate transporters VGLUT1 and VGLUT2) and inhibitory (vesicular inhibitory amino acid transporter VIAAT) transporter mRNA and synaptic protein expression in rat neocortical neuronal cultures, using a well established in vitro protocol to induce homeostatic plasticity. During the second and third week of synaptic differentiation, the predominant vesicular transporters expressed in neocortical neurons, VGLUT1 and VIAAT, are both dramatically upregulated. In mature cultures, VGLUT1 and VIAAT exhibit bidirectional and opposite regulation by prolonged activity changes. Endogenous coregulation during development and homeostatic scaling of the expression of the transporters in functionally differentiated cultures may serve to control vesicular glutamate and GABA filling and adjust functional presynaptic excitatory/inhibitory balance. Unexpectedly, hyperexcitation in differentiated cultures triggers a striking increase in VGLUT2 mRNA and synaptic protein, whereas decreased excitation reduces levels. VGLUT2 mRNA and protein are expressed in subsets of VGLUT1-encoded neocortical neurons that we identify in primary cultures and in neocortex in situ and in vivo. After prolonged hyperexcitation, downregulation of VGLUT1/synaptophysin intensity ratios at most synapses is observed, whereas a subset of VGLUT1-containing boutons selectively increase the expression of VGLUT2. Bidirectional and opposite regulation of VGLUT1 and VGLUT2 by activity may serve as positive or negative feedback regulators for cortical synaptic transmission. Intracortical VGLUT1/VGLUT2 coexpressing neurons have the capacity to independently modulate the level of expression of either transporter at discrete synapses and therefore may serve as a plastic interface between subcortical thalamic input (VGLUT2) and cortical output (VGLUT1) neurons.

Functional properties and cellular distribution of the System A glutamine transporter SNAT1 support specialized roles in central neurons

Glutamine, the preferred precursor for neurotransmitter glutamate and GABA, is likely to be the principal substrate for the neuronal System A transporter SNAT1 in vivo. We explored the functional properties of SNAT1 (the product of the rat Slc38a1 gene) by measuring radiotracer uptake and currents associated with SNAT1 expression in Xenopus oocytes and determined the neuronal-phenotypic and cellular distribution of SNAT1 by confocal laser-scanning microscopy alongside other markers. We found that SNAT1 mediates transport of small, neutral, aliphatic amino acids including glutamine (K0.5 ≈ 0.3 mm), alanine, and the System A-specific analogue 2-(methylamino)isobutyrate. Amino acid transport is driven by the Na+ electrochemical gradient. The voltage-dependent binding of Na+ precedes that of the amino acid in a simultaneous transport mechanism. Li+ (but not H+) can substitute for Na+ but results in reduced Vmax. In the absence of amino acid, SNAT1 mediates Na+-dependent presteady-state currents (Qmax ≈ 9 nC) and a nonsaturable cation leak with selectivity Na+, Li+ » H+, K+. Simultaneous flux and current measurements indicate coupling stoichiometry of 1 Na+ per 1 amino acid. SNAT1 protein was detected in somata and proximal dendrites but not nerve terminals of glutamatergic and GABAergic neurons throughout the adult CNS. We did not detect SNAT1 expression in astrocytes but detected its expression on the luminal membranes of the ependyma. The functional properties and cellular distribution of SNAT1 support a primary role for SNAT1 in glutamine transport serving the glutamate/GABA-glutamine cycle in central neurons. Localization of SNAT1 to certain dopaminergic neurons of the substantia nigra and cholinergic motoneurons suggests that SNAT1 may play additional specialized roles, providing metabolic fuel (via α-ketoglutarate) or precursors (cysteine, glycine) for glutathione synthesis.

Active Transport of Acetylcholine by the Human Vesicular Acetylcholine Transporter

The characteristics of ATP-dependent transport of acetylcholine (ACh) in homogenates of pheochromocytoma (PC-12) cells stably transfected with the human vesicular acetylcholine transporter (VAChT) cDNA are described. The human VAChT protein was abundantly expressed in this line and appeared as a diffuse band with a molecular mass of ∼75 kDa on Western blots. Vesicular [3H]ACh accumulation increased ∼20 times over levels attained by the endogenous rat VAChT, expressed at low levels in control PC-12 cells. The transport of [3H]ACh by human VAChT was dependent upon the addition of exogenous ATP at 37°C. Uptake was abolished by low temperature (4°C), the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (2.5 µM) and bafilomycin A1 (1 µM), a specific inhibitor of the vesicular H+-ATPase. The kinetics of [3H]ACh uptake by human VAChT were saturable, exhibiting an apparent Km of 0.97 ± 0.1 mM and Vmax of 0.58 ± 0.04 nmol/min/mg. Maximal steady-state levels of vesicular [3H]ACh accumulation were directly proportional to the concentration of substrate present in the medium with saturation occurring at ∼4 mM. Uptake was stereospecifically inhibited by L-vesamicol with an IC50 of 14.7 ± 1.5 nM. The apparent affinity (Kd) of [3H]vesamicol for human VAChT was 4.1 ± 0.5 nM, and the Bmax was 8.9 ± 0.6 pmol/mg. The turnover (Vmax/Bmax) of the human VAChT was ∼65/min. This expression system should prove useful for the structure/function analysis of VAChT.

Activity-dependent regulation of vesicular glutamate and GABA transporters: A means to scale quantal size

The functional balance of glutamatergic and GABAergic signaling in neuronal cortical circuits is under homeostatic control. That is, prolonged alterations of global network activity leads to opposite changes in quantal amplitude at glutamatergic and GABAergic synapses. Such scaling of excitatory and inhibitory transmission within cortical circuits serves to restore and maintain a constant spontaneous firing rate of pyramidal neurons. Our recent work shows that this includes alterations in the levels of expression of vesicular glutamate (VGLUT1 and VGLUT2) and GABA (VIAAT) transporters. Other vesicle markers, such as synaptophysin or synapsin, are not regulated in this way. Endogenous regulation at the level of mRNA and synaptic protein controls the number of transporters per vesicle and hence, the level of vesicle filling with transmitter. Bidirectional and opposite activity-dependent regulation of VGLUT1 and VIAAT expression would serve to adjust the balance of glutamate and GABA release and therefore the level of postsynaptic receptor saturation. In some excitatory neurons and synapses, co-expression of VGLUT1 and VGLUT2 occurs. Bidirectional and opposite changes in the levels of two excitatory vesicular transporters would enable individual neocortical neurons to scale up or scale down the level of vesicular glutamate storage, and thus, the amount available for release at individual synapses. Regulated vesicular transmitter storage and release via selective changes in the level of expression of vesicular glutamate and GABA transporters indicates that homeostatic plasticity of synaptic strength at cortical synapses includes presynaptic elements.

Acidosis-Sensing Glutamine Pump SNAT2 Determines Amino Acid Levels and Mammalian Target of Rapamycin Signalling to Protein Synthesis in L6 Muscle Cells

Wasting of lean tissue as a consequence of metabolic acidosis is a serious problem in patients with chronic renal failure. A possible contributor is inhibition by low pH of the System A (SNAT2) transporter, which carries the amino acid L-glutamine (L-Gln) into muscle cells. The aim of this study was to determine the effect of selective SNAT2 inhibition on intracellular amino acid profiles and amino acid-dependent signaling through mammalian target of rapamycin in L6 skeletal muscle cells. Inhibition of SNAT2 with the selective competitive substrate methylaminoisobutyrate, metabolic acidosis (pH 7.1), or silencing SNAT2 expression with small interfering RNA all depleted intracellular L-Gln. SNAT2 inhibition also indirectly depleted other amino acids whose intracellular concentrations are maintained by the L-Gln gradient across the plasma membrane, notably the anabolic amino acid L-leucine. Consequently, SNAT2 inhibition strongly impaired signaling through mammalian target of rapamycin to ribosomal protein S6 kinase, ribosomal protein S6, and 4E-BP1, leading to impairment of protein synthesis comparable with that induced by rapamycin. It is concluded that even though SNAT2 is only one of several L-Gln transporters in muscle, it may determine intracellular anabolic amino acid levels, regulating the amino acid signaling that affects protein mass, nucleotide/nucleic acid metabolism, and cell growth.

Molecular analysis of vesicular amine transporter function and targeting to secretory organelles.

Vesicular transporters are responsible for the loading of neurotransmitters into specialized secretory organelles in neurons and neuroendocrine cells to make them available for regulated neurosecretion. The exocytotic release of neurotransmitter therefore depends on the functional activity of the vesicular transporters and their efficient sorting to these secretory organelles. Molecular analysis of vesicular transport proteins has revealed important information regarding structural domains responsible for their functional properties, including substrate specificity and trafficking to various classes of secretory vesicles. These studies have established the existence of an important functional relationship between transporter activity and presynaptic quantal neurosecretion.—Erickson, J. D., Varoqui, H. Molecular analysis of vesicular amine transporter function and targeting to secretory organelles. FASEB J. 14, 2450–2458 (2000)

SNAT2 Amino Acid Transporter Is Regulated by Amino Acids of the SLC6 γ-Aminobutyric Acid Transporter Subfamily in Neocortical Neurons and May Play No Role in Delivering Glutamine for Glutamatergic Transmission

System A transporters SNAT1 and SNAT2 mediate uptake of neutral α-amino acids (e.g. glutamine, alanine, and proline) and are expressed in central neurons. We tested the hypothesis that SNAT2 is required to support neurotransmitter glutamate synthesis by examining spontaneous excitatory activity after inducing or repressing SNAT2 expression for prolonged periods. We stimulated de novo synthesis of SNAT2 mRNA and increased SNAT2 mRNA stability and total SNAT2 protein and functional activity, whereas SNAT1 expression was unaffected. Increased endogenous SNAT2 expression did not affect spontaneous excitatory action-potential frequency over control. Long term glutamine exposure strongly repressed SNAT2 expression but increased excitatory action-potential frequency. Quantal size was not altered following SNAT2 induction or repression. These results suggest that spontaneous glutamatergic transmission in pyramidal neurons does not rely on SNAT2. To our surprise, repression of SNAT2 activity was not limited to System A substrates. Taurine, γ-aminobutyric acid, and β-alanine (substrates of the SLC6 γ-aminobutyric acid transporter family) repressed SNAT2 expression more potently (10×) than did System A substrates; however, the responses to System A substrates were more rapid. Since ATF4 (activating transcription factor 4) and CCAAT/enhancer-binding protein are known to bind to an amino acid response element within the SNAT2 promoter and mediate induction of SNAT2 in peripheral cell lines, we tested whether either factor was similarly induced by amino acid deprivation in neurons. We found that glutamine and taurine repressed the induction of both transcription factors. Our data revealed that SNAT2 expression is constitutively low in neurons under physiological conditions but potently induced, together with the taurine transporter TauT, in response to depletion of neutral amino acids.