Glutamate uptake system in the presynaptic vesicle: Glutamic acid analogs as inhibitors and alternate substrates (original) (raw)
Related papers
Neurochemical Research, 2008
Noncyclic fluorine-substituted and cyclic analogs of glutamic acid were tested for their ability to inhibit glutamate uptake in isolated bovine presynaptic vesicles, in order to assess the specific structural requirements of the glutamate translocation system in the vesicle membrane. Cyclic analogs that permitted close interaction between the positive and negative charges of the glutamate molecule were effective inhibitors; maximum inhibitory potency was observed with L-trans-1-aminocyclopentane-1,3-dicarboxylic acid (L-t-ACPD), while D-t-ACPD was less active. Analogs with a larger or smaller ring (as in trans-1-aminocyclohexane-1,3-dicarboxylic acid or trans-1aminocyclobutane-1,3-dicarboxylic acid) were also inhibitory, but somewhat less so. trans-ACPD was also taken up by the vesicles with a time course and ATP dependence similar to uptake of glutamate, and this uptake was inhibited by glutamate. The K m value for t-ACPD uptake was similar to its K i for inhibition of glutamate uptake, while its rate of uptake was lower than that of glutamate. Fluorine-substituted noncyclic analogs with substitutions at the 4-carbon were less effective than glutamic acid itself, although 4,4-difluoroglutamic acid was equal in activity to the unsubstituted compound. Inhibition by these derivatives appeared to be competitive in nature, and they probably were also transported by the vesicle uptake system.
Journal of Neurochemistry, 1989
Rat brain synaptic vesicles exhibit ATP-dependent uptake of y-[3H]amino-n-butyric acid ([3H]GABA) and L-[3H]glutamate. After hypotonic shock, the highest specific activities of uptake of both L-glutamate and GABA were recovered in the 0.4 M fraction of a sucrose gradient. The uptakes of L-glutamate and GABA were inhibited by similar, but not identical, concentrations of the mitochondria1 uncoupler carbonyl cyanide m-chlorophenylhydrazone and the ionophores nigericin and gramicidin, but they were not inhibited by the K' carrier valinomycin. N,N'-Dicyclohexylcarbodiimide and N-ethylmaleimide, Mg2+-ATPase inhibitors, inhibited the GABA and L-glutamate uptakes similarly. Low concentrations of C1-stimulated the vesicular uptake of L-glutamate but not that of GABA. The uptakes of both L-glutamate and GABA were inhibited by high concentrations of CI-. These results indicate that the vesicular GABA and L-glutamate uptakes are driven by an electrochemical proton gradient generated by a similar Mg2+-ATPase. The vesicular uptake mechanisms are discussed in relation to other vesicle uptake systems. Key Words: Synaptic vesicles-Vesicular uptake-MgZ+-ATPase-Proton gradient-Inhibitors. Fykse E. M. et al. Comparison of the properties of y-aminobutyric acid and L-glutamate uptake into synaptic vesicles isolated from rat brain.
Characterization of Glutamate Uptake into Synaptic Vesicles
Journal of Neurochemistry, 1985
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 ATPdependent 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. Key Words: Glutamate-Synaptic vesicle-Uptake-Mg-ATPase-Excitatory neurotransmitter. Naito S. and Ueda T. Characterization of glutamate uptake into synaptic vesicles.
Synaptic Vesicular Glutamate Uptake: Modulation by a Synaptosomal Cytosolic Factor
Journal of Neurochemistry, 1990
We have demonstrated previously that L-glutamate is taken up into isolated synaptic vesicles in an ATP-dependent manner, supporting the neurotransmitter role of this acidic amino acid. We now report that a nerve terminal cytosolic factor inhibits the ATP-dependent vesicular uptake of glutamate in a dose-dependent manner. This factor appears to be a protein with a molecular weight >100,000, as estimated by size exclusion chromatography, and is precipitated by ammonium sulfate (40% saturation). The inhibitory factor is inactivated by heating to 100°C. Proteolytic digestion of the ammonium sulfate fraction by trypsin or chymotrypsin did not reduce, but rather increased slightly, the inhibition of glutamate uptake. Unlike the native factor, the digest retained inhibitory activity after heating, suggesting that proteolytic digestion may generate active fragments. The inhibition of ATP-dependent vesicular glutamate uptake is not species-specific, as the factor obtained from both rat and bovine brains produced an equal degree of inhibition of glutamate uptake into vesicles of each species. These observations raise the possibility that vesicular uptake of glutamate may be regulated by an endogenous factor in vivo.
Neurochemical Research
would represent an efficient mechanism for swift glutamate loading into synaptic vesicles, supporting maintenance of normal synaptic transmission. Keywords Synaptic vesicles • Aspartate aminotransferase • Glutamate • VGLUT • Local synthesis • Efficient mechanism Abbreviations AAT Aspartate aminotransferase ACPD 1-Aminocyclopentane-1,3-dicarboxylic acid α-KGA α-Ketoglutarate FCCP Carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone GAPDH Glyceraldehyde-3-phophate dehydrogenase GABA γ-Aminobutyric acid GDH Glutamate dehydrogenase Gln Glutamine GOT Glutamate oxaloacetate aminotransferase HPLC High pressure liquid chromatography hsp Heat shock protein SV Synaptic vesicle TCA Tricarboxylic acid VGLUT Vesicular glutamate transporter
Uptake of L-[3H] glutamic acid by crude and purified synaptic vesicles from rat brain
Biochemical and Biophysical Research Communications, 1982
Rat brain synaptic vesicles suspended in a yiun crnnprised of potassium tartrate displayed saturable accumulation of L[ H] glutamic acid at 37" (Km 2.0 x 10JtM; 311*13 pmol/mg protein), 60 min. which was stable for periods up to The accumulation was temperature sensitive and partially ATP-dependent, uptake levels being reduced to 18.7tO.8 ~pnol/xg protein at 4", and to 14124 pnol/nq protein in the absence of ATP. Fractionation of a crude vesicle preparation on a discontinuous sucrose gradient demonstrated the accumulation to be specifically associated with the synaptic vesicle fraction.
An anion binding site that regulates the glutamate transporter of synaptic vesicles
1993
Glutamate, the major excitatory neurotransmitter of the mammalian central nervous system, is stored in synaptic vesicles and released by exocytosis upon depolarization of the presynaptic nerve terminal. Synaptic vesicles possess an active glutamate-specific transporter that is driven by an electrochemical proton gradient across the vesicle membrane and requires chloride for maximal activity. In this study, we have characterized the role of chloride in vesicular glutamate transport using 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a potent inhibitor of anion translocators. DIDS inhibited glutamate uptake with an ICao of 0.7 I.IM or less. In contrast, all energy gradient parameters (membrane potential, pH gradient, and ATPase activity) required at least &fold higher concentration of DIDS for inhibition. Furthermore, high concentrations of chloride but not of glutamate or other anions prevented DIDS inhibition of glutamate uptake. In contrast to uptake, glutamate efflux from glutamate-loaded vesicles was independent of chloride over a wide concentration range. However, efflux was still susceptible to DIDS inhibition. DIDS inhibition was prevented by excess chloride. We conclude that the vesicular glutamate transporter possesses a DIDSsensitive chloride binding site on the cytoplasmic side, distinct from the substrate binding site, which regulates transport activity.
Neuroscience Letters, 1991
Synaptic vesicle fractions have been isolated from cerebral cortex, subcortical telencephalon, whole brain and spinal cord by density gradient centrifugation. The Mg 2+ ATP-dependent vesicular uptake and the Na+-dependent synaptosomal uptake of glycine, GABA and L-glutamate has been compared in the different regions. All these regions contain GABA as inhibitory neurotransmitter, whereas glycine only plays a dominant role as such in the spinal cord. The ratio between GABA and glycine uptake in the different vesicle fractions was similar, and the ratios differed greatly from the ratios in the synaptosomal uptake. In contrast, the ratio between glutamate and GABA uptake in vesicles from different regions differed, and these ratios corresponded to the ratios in the synaptosomal uptake. These results indicate that glycine is taken up into synaptic vesicles from non-glycine terminals, and we suggest that GABA and glycine can be taken up into the same vesicle population.
Neurochemistry International, 1983
Purified synaptic vesicles were isolated from hog cerebral cortex by a rapid procedure consisting of homogenization of cerebral cortex slices in iso-osmotic sucrose, differential centrifugation and sucrose densitygradient centrifugation. The purity of the vesicles was evaluated both biochemically and morphologically. The vesicles contained high amounts of 7-aminobutyrate (GABA) and acetylcholine at specific concentrations of 390 nmol/mg protein and 7.2 nmol/mg protein respectively. Glutamate decarboxylase, the enzyme which catalyses GABA formation, binds to the synaptic vesicles in a calcium-dependent manner. The percentage of glutamate decarboxylase bound to the vesicles increases from about 5!'i; without calcium, reaching a plateau of about 60% at 4 mM Ca-'-. Magnesium in concentrations 0.2 l0 mM has no significant effect on glutamate decarboxylase binding. Also in phospholipid vesicles (small unilamellar phosphatidylserine pfiosphatidylcholine, 2:1 liposomes} Ca 2 +. but not Mg 2 +, induced the binding of glutamate decarboxylase, reaching a plateau of 50% at 2 mM Ca 2+. Both in synaptic vesicles and in phospholipid vesicles the calcium-dependent glutamate decarboxylase binding seems to be specific, and not caused by unspecific association of proteins, since the specific binding (bound enzyme activity/mg bound protein) increases 3-fold from 0 to 4 mM Ca 2+. The functional role of this binding was studied in GAD containing vesicles by measuring the relationship between the accumulation of [3H]GABA, newly synthetized from [3H]glutamate, and the uptake of added [14C]GABA. No significant uptake of [14C]GABA was found under the experimental conditions used, whereas large amounts of [3H]GABA were found within the vesicles. It appears that the [3H]GABA accumulation process is functionally linked to [3H]GABA synthesis and is mediated by the membrane-bound glutamate decarboxylase. This synthesis-coupled uptake of GABA into synaptic vesicles possibly serves to bring about a plasticity effect in previously stimulated GABAergic nerve endings.
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.