Characterization of Glutamate Uptake into Synaptic Vesicles

Recent evidence indicates that L-glutamate is taken up into synaptic vesicles in an ATP-dependent manner, supporting the notion that synaptic vesicles may be involved in glutamate synaptic transmission. In this study, we further characterized the ATP-dependent vesicular uptake of glutamate. Evidence is provided that a Mg-ATPase, not Ca-ATPase, is responsible for the ATP hydrolysis coupled to the glutamate uptake. The ATP-dependent glutamate uptake was inhibited by agents known to dissipate the electrochemical proton gradient across the membrane of chromaffin granules. Hence, it is suggested that the vesicular uptake of glutamate is driven by electrochemical proton gradients generated by the Mg-ATPase. Of particular interest is the finding that the ATP-dependent glutamate uptake is markedly stimulated by chloride over a physiologically relevant, millimolar concentration range, suggesting an important role of intranerve terminal chloride in the accumulation of glutamate in synaptic vesicles. The vesicular glutamate translocator is highly specific for L-glutamate, and failed to interact with aspartate, its related agents, and most of the glutamate analogs tested. It is proposed that this vesicular translocator plays a crucial role in determining the fate of glutamate as a neurotransmitter.     

Large Structural Change in Isolated Synaptic Vesicles upon Loading with Neurotransmitter

The size of a synaptic vesicle (SV) is generally thought to be determined by the amount of lipid and membrane protein it contains. Once formed, it is thought to remain constant in size. Using fluorescence correlation spectroscopy and cryogenic electron microscopy, we show that glutamatergic vesicles reversibly increase their size upon filling with glutamate. The increase (∼25% in diameter) corresponds to an increase in surface area of ∼50% and in volume of ∼100%. This large size increase implies a large structural change in the SV upon loading with neurotransmitters. Vesicles lacking SV protein 2A (SV2A) did not manifest a change in size after loading with glutamate, indicating that SV2A is required for this phenomenon.     

Glutamate Uptake into Synaptic Vesicles: Competitive Inhibition by Bromocriptine

The ATP-dependent uptake of L-glutamate into synaptic vesicles has been well characterized, implicating a key role for synaptic vesicles in glutamatergic neurotransmission. In the present study, we provide evidence that vesicular glutamate uptake is selectively inhibited by the peptide-containing halogenated ergot bromocriptine. It is the most potent inhibitor of the agents tested: the IC50 was determined to be 22 microM. The uptake was also inhibited by other ergopeptines such as ergotamine and ergocristine, but with less potency. Ergots devoid of the peptide moiety, however, such as ergonovine, lergotrile, and methysergide, had little or no effect. Although bromocriptine is known to elicit dopaminergic and serotonergic effects, its inhibitory effect on vesicular glutamate uptake was not mimicked by agents known to interact with dopamine and serotonin receptors. Kinetic data suggest that bromocriptine competes with glutamate for the glutamate binding site on the glutamate translocator. It is proposed that this inhibitor could be useful as a prototype probe in identifying and characterizing the vesicular glutamate translocator, as well as in developing a more specific inhibitor of the transport system.     

ATP-Dependent Glutamate Uptake into Synaptic Vesicles from Cerebellar Mutant Mice

The ATP-dependent glutamate uptake system in synaptic vesicles prepared from mouse cerebellum was characterized, and the levels of glutamate uptake were investigated in the cerebellar mutant mice, staggerer and weaver, whose main defect is the loss of cerebellar granule cells, and the nervous mutant, whose main defect is the loss of Purkinje cells. The ATP-dependent glutamate uptake is stimulated by low concentrations of chloride, is insensitive to aspartate, and is inhibited by agents known to dissipate the electrochemical proton gradient. These properties are similar to those of the glutamate uptake system observed in the highly purified synaptic vesicles prepared from bovine cortex. The ATP-dependent glutamate uptake system is reduced by 68% in the staggerer and 57-67% in the weaver mutant; these reductions parallel the substantial loss of granule cells in those mutants. In contrast, the cerebellar levels of glutamate uptake are not altered significantly in the nervous mutant, which has lost Purkinje cells, but not granule cells. In view of evidence that granule cells are glutamatergic neurons and Purkinje cells are GABAergic neurons, these observations support the notion that the ATP-dependent glutamate uptake system is present in synaptic vesicles of glutamatergic neurons.     

Enhanced Glutamate Uptake into Synaptic Vesicles Fueled by Vesicle-generated ATP from Phosphoenolpyruvate and ADP

Glycolytic ATP synthesis by synaptic vesicles provides an efficient mechanism for fueling vesicular loading of the neurotransmitter glutamate. This is achieved in part by vesicle-bound pyruvate kinase. However, we have found that vesicular glutamate uptake, in the presence of the pyruvate kinase substrates ADP and phosphoenolpyruvate (PEP), substantially exceeds that caused by exogenous ATP. We propose that this much enhanced uptake is in part due to extra ATP produced via a mechanism involving a novel enzyme, PEP-dependent ADP synthase. We discuss implications for this enzyme in energy homeostasis and pathophysiology, as well as in efficient synaptic glutamate transmission.     

Inhibition of Glutamate Uptake and Proton Pumping in Synaptic Vesicles by S-Nitrosylation

Abstract: Nitric oxide (NO; including NO^•, NO⁺, and NO⁻) was found to inhibit glutamate uptake by isolated synaptic vesicles of rat brain. This was observed when two unrelated NO donors, S -nitrosogluthathione and S -nitroso- N -acetylpenicillamine, were used. The primary target of NO is the H⁺-ATPase found in the synaptic vesicles, which leads to dissipation of the electrochemical proton gradient and inhibition of glutamate uptake. Oxyhemoglobin (12 µ M ) and, to a much lesser extent, methemoglobin protected the vacuolar H⁺-ATPase from inhibition. Inhibition of H⁺ pumping by NO was reversed by addition of 0.5 m M dithiothreitol. The results indicate that the vacuolar H⁺-ATPase from synaptic vesicles is inhibited by NO by a mechanism that involves S -nitrosylation of critical sulfhydryl groups in the enzyme. The interaction of NO with synaptic vesicles might be of importance for the understanding of the multiple effects of NO in neurotransmission.     

A Biochemical and Functional Protein Complex Involving Dopamine Synthesis and Transport into Synaptic Vesicles

Synaptic transmission depends on neurotransmitter pools stored within vesicles that undergo regulated exocytosis. In the brain, the vesicular monoamine transporter-2 (VMAT₂) is responsible for the loading of dopamine (DA) and other monoamines into synaptic vesicles. Prior to storage within vesicles, DA synthesis occurs at the synaptic terminal in a two-step enzymatic process. First, the rate-limiting enzyme tyrosine hydroxylase (TH) converts tyrosine to di-OH-phenylalanine. Aromatic amino acid decarboxylase (AADC) then converts di-OH-phenylalanine into DA. Here, we provide evidence that VMAT₂ physically and functionally interacts with the enzymes responsible for DA synthesis. In rat striata, TH and AADC co-immunoprecipitate with VMAT₂, whereas in PC 12 cells, TH co-immunoprecipitates with the closely related VMAT₁ and with overexpressed VMAT₂. GST pull-down assays further identified three cytosolic domains of VMAT₂ involved in the interaction with TH and AADC. Furthermore, in vitro binding assays demonstrated that TH directly interacts with VMAT₂. Additionally, using fractionation and immunoisolation approaches, we demonstrate that TH and AADC associate with VMAT₂-containing synaptic vesicles from rat brain. These vesicles exhibited specific TH activity. Finally, the coupling between synthesis and transport of DA into vesicles was impaired in the presence of fragments involved in the VMAT₂/TH/AADC interaction. Taken together, our results indicate that DA synthesis can occur at the synaptic vesicle membrane, where it is physically and functionally coupled to VMAT₂-mediated transport into vesicles.     

Quinolinic Acid-induced Seizures Stimulate Glutamate Uptake into Synaptic Vesicles from Rat Brain: Effects Prevented by Guanine-based Purines

Glutamate uptake into synaptic vesicles is a vital step for glutamatergic neurotransmission. Quinolinic acid (QA) is an endogenous glutamate analog that may be involved in the etiology of epilepsy and is related to disturbances on glutamate release and uptake. Guanine-based purines (GBPs) guanosine 5′-monophosphate (GMP and guanosine) have been shown to exert anticonvulsant effects against QA-induced seizures. The aims of this study were to investigate the effects of in vivo administration of several convulsant agents on glutamate uptake into synaptic vesicles and investigate the role of MK-801, guanosine or GMP (anticonvulsants) on glutamate uptake into synaptic vesicles from rats presenting QA-induced seizures. Animals were treated with vehicle (saline 0.9%), QA 239.2 nmoles, kainate 30 mg/kg, picrotoxin 6 mg/kg, PTZ (pentylenetetrazole) 60 mg/kg, caffeine 150 mg/kg or MES (maximal transcorneal electroshock) 80 mA. All convulsant agents induced seizures in 80–100% of animals, but only QA stimulated glutamate uptake into synaptic vesicle. Guanosine or GMP prevented seizures induced by QA (up to 52% of protection), an effect similar to the NMDA antagonist MK-801 (60% of protection). Both GBPs and MK-801 prevented QA-induced glutamate uptake stimulation. This study provided additional evidence on the role of QA and GBPs on glutamatergic system in rat brain, and point to new perspectives on seizures treatment.     

Uptake of l-Glutamate into Rat Brain Synaptic Vesicles: Effect of Inhibitors that Bind Specifically to the Glutamate Transporter

Abstract: In this study we have described a series of new and potent inhibitors of the vesicular uptake of glutamate. The two most efficient inhibitors were the dyes Evans blue and Chicago Skye Blue 6B, which are structurally related to glutamate and were competitive inhibitors in the nanomolar range. The anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (SITS) and the diuretics furosemide and bumetanide are inhibitors of chloride transport in other organs but were competitive inhibitors of glutamate and noncompetitive with respect to chloride ions. Evans blue, Chicago Skye Blue 6B, SITS, furosemide, and bumetanide are all large organic acids with two centers of negative charge and an electron-donating group at close vicinity of the negative charge at physiological pH. The inhibition of the glutamate uptake with these inhibitors was noncompetitive with respect to Cl⁻. The inhibitors, therefore, probably interact directly with the glutamate carrier. Bafilomycin A₁, which is a specific vacuolar ATPase inhibitor, was used as a control and inhibited the vesicular dopamine, glutamate, and GABA uptake to the same extent. None of the inhibitors had any effect on the plasma membrane carrier, which is therefore clearly different from the vesicular carrier.     

Characterization of Nucleotide Transport into Rat Brain Synaptic Vesicles

ATP transport to synaptic vesicles from rat brain has been studied using the fluorescent substrate analogue 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP). The increase in intravesicular concentration was time dependent for the first 30 min, epsilon-ATP being the most abundant nucleotide. The complexity of the saturation curve indicates the existence of kinetic and allosteric cooperativity in the nucleotide transport, which exhibits various affinity states with K0.5 values of 0.39 +/- 0.06 and 3.8 +/- 0.1 mM with epsilon-ATP as substrate. The Vmax values obtained were 13.5 +/- 1.4 pmol x min(-1) x mg of protein(-1) for the first curve and 28.3 +/- 1.6 pmol x min(-1) x mg of protein(-1) considering both components. This kinetic behavior can be explained on the basis of a mnemonic model. The nonhydrolyzable adenine nucleotide analogues adenosine 5'-O-3-(thiotriphosphate), adenosine 5'-O-2-(thiodiphosphate), and adenosine 5'-(beta,gamma-imino)triphosphate and the diadenosine polyphosphates P1,P3-di(adenosine)triphosphate, P1,P4-di(adenosine)tetraphosphate, and P1,P5-di(adenosine)pentaphosphate inhibited the nucleotide transport. The mitochondrial ATP/ADP exchange inhibitor atractyloside, N-ethylmaleimide, and polysulfonic aromatic compounds such as Evans blue and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid also inhibit epsilon-ATP vesicular transport.     

CtBP1-Mediated Membrane Fission Contributes to Effective Recycling of Synaptic Vesicles

1. Download : Download high-res image (168KB)
2. Download : Download full-size image

     

Uptake of Glycine into Synaptic Vesicles Isolated from Rat Spinal Cord

Glycine was taken up by a synaptic vesicle fraction from spinal cord in a Mg-ATP-dependent manner. The accumulation of glycine was inhibited by carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and nigericin, agents known to destroy the proton gradient across the vesicle membrane. Vesicular uptake of glycine was clearly different from synaptosomal uptake, with respect to both the affinity constant and the effect of Na+, ATP, CCCP, and temperature. Oligomycin and strychnine did not inhibit the vesicular uptake, showing that neither mitochondrial H(+)-ATPase nor binding to strychnine-sensitive glycine receptors was involved. It is suggested that the vesicular uptake of glycine is driven by a proton gradient generated by a Mg2(+)-ATPase. A low concentration of Cl- had little effect on the uptake of glycine, whereas the uptake of glutamate in the same experiment was highly stimulated. High concentrations of gamma-amino-n-butyric acid and beta-alanine inhibited vesicular glycine uptake, but glutamate did not. Accumulation of glycine was found to be fourfold higher in a spinal cord synaptic vesicle fraction than in a vesicle fraction from cerebral cortex.     

Mg-ATPase and Torpedo Cholinergic Synaptic Vesicles

The reported presence of Mg-ATPase activity in cholinergic synaptic vesicles from the electric organ of Torpedo marmorata was reinvestigated in view of possible contamination of vesicles by other subcellular fractions. After dilution in concentrated sucrose, the vesicular fraction isolated on a sedimentation sucrose gradient was purified further on a flotation density gradient. It appears that this treatment allows separation of the vesicles according to their content. The two vesicular content markers, acetylcholine and ATP, are recovered as sharp coincident peaks at a density close to 0.48 M sucrose. Empty vesicles are identified in denser regions by the protein pattern on gel electrophoresis which is identical to the pattern obtained for filled vesicles. Refractionation of vesicles depleted of their acetylcholine content by valinomycin leads to an extreme picture, with a massive shift of the vesicles toward denser regions. We have then shown that a ouabain-insensitive Mg-ATPase is indeed associated with the vesicle membrane, but the activity is fully apparent only when vesicles are permeabilized either as the result of the fractionation procedure or after detergent treatment. The relative insensitivity of the Mg-ATPase associated with the synaptic vesicles to oligomycin, N,N'-dicyclohexylcarbodiimide, and azide indicates that this enzyme differs from the classic F1F0 mitochondrial enzyme. The most striking finding is the sensitivity to vanadate of the vesicular Mg-ATPase, which suggests the involvement of a phosphorylated intermediate. On the basis of both the difference in inhibitor sensitivity between untreated and detergent-treated vesicles and of the pronase experiments, the possibility that the enzyme has an inward orientation is discussed.     

Axonal Transport of Synaptic Vesicles and Muscarinic Receptors: Effect of Protein Synthesis Inhibitors

The effect of cycloheximide, a protein synthesis inhibitor, was studied on the axonal transport of noradrenergic synaptic vesicles and presynaptic muscarinic receptors, identified by in vitro binding of [3H]dihydrotetrabenazine and [3H]quinuclidinylbenzilate, respectively, in rat sciatic nerve. Cycloheximide (1.5 mg/kg) administered subcutaneously 2 h before ligation decreased by approximately 50% the accumulation of vesicles and receptors in the proximal segment above the ligature placed on the nerve; its action was detectable after a lag period of 10 h and disappeared 96 h after administration. Double ligatures were placed on the nerve at various time intervals between the first (distal) and the second (proximal) ligature, and the accumulation of vesicles and receptors proximal to the second ligature was measured; the first ligature diminished the accumulation above the second ligature. At an interval of 96 h between the first and the second ligature, cycloheximide completely prevented the accumulation of vesicles and receptors proximal to the second ligature. The effects of double ligatures and the response to cycloheximide treatment can best be explained on the assumption that an important proportion of synaptic vesicles and presynaptic receptors is being recycled in the nerve cell bodies after retrograde transport.     

Biochemical Characterization of the Coating Mechanism of the Endosomal Donor Compartment of Synaptic Vesicles

The heterotetrameric adaptor protein complex, AP-3, sorts proteins to both the endosome/lysosome and the synaptic vesicles. We have characterized the recruitment of pure AP-3 complex and ADP-ribosylation factor (ARF) onto the endosomal donor compartments that give rise to synaptic vesicles. We demonstrated that endosomes become heavier in a sucrose gradient after incubation with rat brain cytosol and a nonhydrolyzable GTP analog, GTPgammaS. This process requires a small GTPase, ARF-1. Furthermore, the endosomal coating is specific for AP-3 but not the AP-2 complex. This process requires only two soluble proteins AP-3 and ARF, with the recruitment of AP-3 being saturable at about 30 nM. These results establish that the synaptic vesicle's donor membrane is coated with AP-3 before vesiculation, in a coat-protein-specific and dose-dependent fashion.     

Neurotransmitter Receptor Trafficking and the Regulation of Synaptic Strength

Modulation of the strength of synapses is thought to be one of the mechanisms that underlies learning and memory and is also likely to be important in processes of neuropathology and drug tolerance. This review focuses on the emerging role of postsynaptic neurotransmitter receptor trafficking as an essential mechanism underlying the dynamic regulation of synaptic strength.     

Membrane Composition of Adrenergic Large and Small Dense Core Vesicles and of Synaptic Vesicles: Consequences for Their Biogenesis

The membrane proteins of adrenergic large dense core vesicles, in particular those of chromaffin granules, have been characterized in detail. With the exception of the nucleotide carrier all major peptides have been cloned. There has been a controversy whether these vesicles contain antigens like synaptophysin, synaptotagmin and VAMP or synaptobrevin found in high concentration in synaptic vesicles. One can now conclude that large dense core vesicles also contain these peptides although in lower concentrations. The biosynthesis of large dense core vesicles is analogous to that of other peptide secreting vesicles of the regulated pathway. One cannot yet definitely define the biosynthesis of small dense core vesicles which apparently have a very similar membrane composition to that of large dense core vesicles. They may form directly from large dense core vesicles when their membranes have been retrieved after exocytosis. These membranes may become sorted in an endosomal compartment where peptides may be deleted or added. Such an addition could be derived from synaptophysin-rich vesicles present in adrenergic axons. However small dense core vesicle peptides may also be transported axonally independent of large dense core vesicles. For proving one of these possibilities some crucial experiments have been suggested.     

ATP-Stimulated Ca2+ Transport into Cholinergic Torpedo Synaptic Vesicles

Abstract: Activation of the Ca²⁺/Mg²⁺ ATPase associated with highly purified Torpedo synaptic vesicles results in ⁴⁵Ca²⁺ uptake. The accumulated ⁴⁵Ca²⁺ is released by hypoosmotic buffer and by the Ca²⁺ ionophore A23187. Density-gradient centrifugation and permeation chromatography reveal that vesicular acetylcholine and the membrane-bound ⁴⁵Ca²⁺ co-migrate, thus implying that ⁴⁵Ca²⁺ is transported into cholinergic vesicles. ATP-dependent ⁴⁵Ca²⁺ uptake follows saturation kinetics, with K_mCa²⁺= 50 μM, K_mATP= 5 μM, and V_max= 3 ± 0.3 nmol Ca²⁺/mg protein/min. Treatment of the vesicles with mersalyl, dicyclohexyl-carbodiimide, and quercetin leads to inactivation of the Ca²⁺/Mg²⁺ ATPase and to comparable inhibition of ⁴⁵Ca²⁺ transport. Ruthenium red and ouabain have no effect on either of these activities. Nigericin in the presence of external K⁺ is a potent inhibitor of ⁴⁵Ca²⁺ translocation, whereas gramicidin activates transport. The proton translocator carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP) and FCCP + the ionophore valinomycin partially inhibit ⁴⁵Ca²⁺ transport. By contrast, the above ionophores do not affect Ca²⁺/Mg²⁺ ATPase activity. Tentative mechanisms for ATP-dependent Ca²⁺ transport into cholinergic synaptic vesicles and the physiological significance of this process are discussed.     

Ganglioside Composition of Synaptic Vesicles from Torpedo Electric Organ

We have studied the ganglioside content and pattern of synaptic vesicles isolated from the electric organs of two species of Torpedinidae, Torpedo californica and Torpedo marmorata. The ganglioside concentrations were high relative to protein content (77 and 58 micrograms of N-acetylneuraminic acid/mg of protein, respectively), owing to the low protein-to-lipid ratio; however, they were also appreciable in relation to phospholipid (15.6 and 10.0 micrograms of N-acetylneuraminic acid/mg of phospholipid). The fact that a membrane fraction that separated from synaptic vesicles of T. californica on a controlled-pore glass-bead column and constituted the main potential source of contamination in this preparation had a lower ganglioside content and a different TLC pattern than synaptic vesicles indicated the relatively high purity of the latter. Most of the gangliosides from synaptic vesicles of both species migrated on TLC in the vicinity of standards with three or more sialic acids. Synaptosomes from T. marmorata had a higher lipid N-acetylneuraminic acid/phospholipid ratio and a different TLC pattern than synaptic vesicles. Considering these results and other data appearing recently in the literature, we suggest that reexamination of synaptic vesicles from mammalian brain for the possible presence of gangliosides is warranted.     

Cholinergic Synaptic Vesicles Contain a V-Type and a P-Type ATPase

Fifty to eighty-five percent of the ATPase activity in different preparations of cholinergic synaptic vesicles isolated from Torpedo electric organ was half-inhibited by 7 microM vanadate. This activity is due to a recently purified phosphointermediate, or P-type, ATPase, Acetylcholine (ACh) active transport by the vesicles was stimulated about 35% by vanadate, demonstrating that the P-type enzyme is not the proton pump responsible for ACh active transport. Nearly all of the vesicle ATPase activity was inhibited by N-ethylmaleimide. The P-type ATPase could be protected from N-ethylmaleimide inactivation by vanadate, and subsequently reactivated by complexation of vanadate with deferoxamine. The inactivation-protection pattern suggests the presence of a vanadate-insensitive, N-ethylmaleimide-sensitive ATPase consistent with a vacuolar, or V-type, activity expected to drive ACh active transport. ACh active transport was half-inhibited by 5 microM N-ethylmaleimide, even in the presence of vanadate. The presence of a V-type ATPase was confirmed by Western blots using antisera raised against three separate subunits of chromaffin granule vacuolar ATPase I. Both ATPase activities, the P-type polypeptides, and the 38-kilodalton polypeptide of the V-type ATPase precisely copurify with the synaptic vesicles. Solubilization of synaptic vesicles in octaethyleneglycol dodecyl ether detergent results in several-fold stimulation of the P-type activity and inactivation of the V-type activity, thus explaining why the V-type activity was not detected previously during purification of the P-type ATPase. It is concluded that cholinergic vesicles contain a P-type ATPase of unknown function and a V-type ATPase which is the proton pump.