Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Active accumulation of neurotransmitters by synaptic vesicles is an essential component of the synaptic transmission cycle. Isolated vesicles show energy-dependent uptake of several transmitters by processes which are apparently mediated by a proton electrochemical potential across the vesicle membrane. Although this energy gradient is probably generated by a proton ATPase, the functional separation of ATP cleavage and transmitter uptake activity has only been shown clearly for monoamine transport. We report here that the light-driven proton pump, bacteriorhodopsin, can replace the endogenous proton ATPase in proteoliposomes reconstituted from vesicular detergent extracts. The system shows light-dependent uptake of glutamate with properties very similar to those observed in intact vesicles, e.g. chloride dependence or stimulation by NH4+. Our experiments show that the proton pump and the glutamate transporter are separate entities and provide a powerful tool for further characterization of the glutamate carrier.
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PMID:Bacteriorhodopsin drives the glutamate transporter of synaptic vesicles after co-reconstitution. 197 Feb 94

Plasma membrane potential generated by Na+, K(+)-ATPase provides the driving force for high-affinity, Na(+)-dependent uptake of glutamate into the cytoplasm of glutamatergic nerve endings and glial cells. Ca2(+)-calmodulin-dependent ATPase in the plasma membrane and Ca2(+)-ATPase in the endoplasmic reticulum influence the intracellular [Ca2+] and, therefore, the exocytotic release of neurotransmitter glutamate. The membrane potential across the membrane of the synaptic vesicles, generated by a H(+)-ATPase, provides the driving force for synaptic vesicular uptake of glutamate as well as that of GABA and glycine. Hypoxia and ischemia lead to release of glutamate, perhaps in consequence of an increased endogenous pool of glutamate and/or lack of substrate (ATP) for the ATPases. This release, rather than being exocytotic, is believed to result mainly from a reversal of the Na(+)-dependent high-affinity glutamate transporter in the plasma membrane.
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PMID:Interrelationship between glutamate and membrane-bound ATPases in nerve cells. 198 May 85

Pinealocytes, endocrine cells that synthesize and secrete melatonin, possess a large number of synaptic-like microvesicles (MVs) containing the L-glutamate transporter (Moriyama, Y., and Yamamoto, A. (1995) FEBS Lett., 367, 233-236). In this study, the L-glutamate transporter in MVs isolated from bovine pineal glands was characterized as to its driving force, requirement of anions, and substrate specificity. Upon the addition of ATP, the MVs accumulated L-glutamate. The uptake was significantly dependent on the extravesicular Cl- concentration, being negligible in the absence of Cl- and maximum at 2-5 mM and decreasing gradually at 20-100 mM. The membrane potential (inside positive) was maximum at 0-10 mM Cl- and then decreased gradually depending on the Cl- concentration, whereas a pH gradient was practically absent without Cl- and increased gradually up to 100 mM Cl-. Ammonium acetate or nigericin plus K+, a dissipator of a pH gradient, had little effect on or was slightly stimulatory toward the uptake, whereas valinomycin plus K+ inhibited both formation of the membrane potential and the glutamate uptake to similar extents. The ATP- and Cl(-)-dependent glutamate uptake was inhibited by fluoride, iodide, or thiocyanate, without vacuolar H(+)-ATPase being affected. An anion channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, similarly inhibited the glutamate uptake in a Cl- protectable manner. Furthermore, ATP- and glutamate-dependent acidification of MVs was observed when 4 mM Cl- was present. Among more than 50 kinds of glutamate analogues tested, only a few compounds, including 1-aminocyclohexane-trans-1,3-dicarboxylic acid, caused similar acidification. A good correlation was observed between the acidification and the inhibition of glutamate uptake by glutamate analogues. These results indicated that 1) the major driving force of the glutamate uptake is the membrane potential, 2) Cl- regulates the glutamate uptake, probably via anion-binding site(s) on the transporter, and 3) the transporter shows strict substrate specificity. Hence, the overall properties of the vesicular glutamate transporter in the MVs well matched those of the synaptic vesicle glutamate transporter. We concluded that the vesicular glutamate transporter, being similar if not identical to the neuronal counterpart, operates in endocrine cells.
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PMID:Vesicular L-glutamate transporter in microvesicles from bovine pineal glands. Driving force, mechanism of chloride anion activation, and substrate specificity. 767 14

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 IC50 of 0.7 microM or less. In contrast, all energy gradient parameters (membrane potential, pH gradient, and ATPase activity) required at least 5-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 DIDS-sensitive chloride binding site on the cytoplasmic side, distinct from the substrate binding site, which regulates transport activity.
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PMID:An anion binding site that regulates the glutamate transporter of synaptic vesicles. 822 29

Astrocyte end-feet surround intraparenchymal microvessels and represent therefore the first cellular barrier for glucose entering the brain. As such, they are a likely site of prevalent glucose uptake. Astrocytic processes are also wrapped around synaptic contacts, implying that they are ideally positioned to sense and be functionally coupled to increased synaptic activity. We have recently demonstrated that glutamate, the main excitatory neurotransmitter, stimulates in a concentration-dependent manner 2-DG uptake and phosphorylation by astrocytes in primary culture. The effect is not receptor-mediated but rather proceeds via one of the recently cloned glutamate transporter. The mechanism involves an activation of the Na+/K+ ATPase. Concomitant to the stimulation of glucose uptake, glutamate causes a concentration-dependent increase in lactate efflux. These observations suggest that glutamate uptake is coupled to aerobic glycolysis in astrocytes. In addition, since glutamate release occurs following the modality-specific activation of a brain region, the glutamate-evoked uptake of glucose into astrocytes provides a simple mechanism to couple neuronal activity to energy metabolism. These data also suggest that modality-specific activation visualized using 2DG-based autoradiography or PET may primarily reflect glutamate-mediated uptake of 2DG into astrocytes.
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PMID:Cellular mechanisms of brain energy metabolism. Relevance to functional brain imaging and to neurodegenerative disorders. 862 17

Glutamate uptake into synaptic vesicles is driven by an electrochemical proton gradient formed across the membrane by a vacuolar H+-ATPase. Chloride has a biphasic effect on glutamate transport, which it activates at low concentrations (2-8 mM) and inhibits at high concentrations (>20 mM). Stimulation with 4 mM chloride was due to an increase in the Vmax of transport, whereas inhibition by high chloride concentrations was related to an increase in Km to glutamate. Both stimulation and inhibition by Cl- were observed in the presence of A23187 or (NH4)2SO4, two substances that dissipate the proton gradient (deltapH). With the use of these agents, we show that the transmembrane potential regulates the apparent affinity for glutamate, whereas the deltapH antagonizes the effect of high chloride concentrations and is important for retaining glutamate inside the vesicles. Selective dissipation of deltapH in the presence of chloride led to a significant glutamate efflux from the vesicles and promoted a decrease in the velocity of glutamate uptake. The H+-ATPase activity was stimulated when the deltapH component was dissipated. Glutamate efflux induced by chloride was saturable, and half-maximal effect was attained in the presence of 30 mM Cl-. The results indicate that: (i) both transmembrane potential and deltapH modulate the glutamate uptake at different levels and (ii) chloride affects glutamate transport by two different mechanisms. One is related to a change of the proportions between the transmembrane potential and the deltapH components of the electrochemical proton gradient, and the other involves a direct interaction of the anion with the glutamate transporter.
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PMID:Regulation of glutamate transport into synaptic vesicles by chloride and proton gradient. 866 10

The postsynaptic actions of glutamate are rapidly terminated by high affinity glutamate uptake into glial cells. In this study we demonstrate the stimulation of both glutamate uptake and Na,K-ATPase activity in rat astrocyte cultures in response to sublethal ischemia-like insults. Primary cultures of neonatal rat cortical astrocytes were subjected to hypoxia, or to serum- and glucose-free medium, or to both conditions (ischemia). Cell death was assessed by propidium iodide staining of cell nuclei. To measure sodium pump activity and glutamate uptake, 3H-glutamate and 86Rb were both simultaneously added to the cell culture in the presence or absence of 2 mM ouabain. Na,K-ATPase activity was defined as ouabain-sensitive 86Rb uptake. Concomitant transient increases (2-3 times above control levels) of both Na,K-ATPase and glutamate transporter activities were observed in astrocytes after 4-24 h of hypoxia, 4 h of glucose deprivation, and 2-4 h of ischemia. A 24 h ischemia caused a profound loss of both activities in parallel with significant cell death. The addition of 5 mM glucose to the cells after 4 h ischemia prevented the loss of both sodium pump activity and glutamate uptake and rescued astrocytes from death observed at the end of 24 h ischemia. Reoxygenation after the 4 h ischemic event caused the selective inhibition of Na,K-ATPase activity. The observed increases in Na,K-ATPase activity and glutamate uptake in cultured astrocytes subjected to sublethal ischemia-like insults may model an important functional response of astrocytes in vivo by which they attempt to maintain ion and glutamate homeostasis under restricted energy and oxygen supply.
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PMID:Stimulation of glutamate uptake and Na,K-ATPase activity in rat astrocytes exposed to ischemia-like insults. 903 29

In mammalian cells, the uptake of amino acids is mediated by specialized, energy-dependent and passive transporters with overlapping substrate specificities. Most energy-dependent transporters are coupled either to the cotransport of Na+ or Cl- or to the countertransport of K+. Passive transporters are either facilitated transporters or channels. As a prelude to the molecular characterization of the different classes of transporters, we have isolated transporter cDNAs by expression-cloning with Xenopus laevis oocytes and we have characterized the cloned transporters functionally by uptake studies into oocytes using radiolabelled substrates and by electrophysiology to determine substrate-evoked currents. Mammalian transporters investigated include the dibasic and neutral amino acid transport protein D2/NBAT (system b0+) and the Na(+)- and K(+)-dependent neuronal and epithelial high-affinity glutamate transporter EAAC1 (system XAG-). A detailed characterization of these proteins has provided new information on transport characteristics and mechanisms for coupling to different inorganic ions. This work has furthermore advanced our understanding of the roles these transporters play in amino acid homeostasis and in various pathologies. For example, in the central nervous system, glutamate transporters are critically important in maintaining the extracellular glutamate concentration below neurotoxic levels, and defects of the human D2 gene have been shown to account for the formation of kidney stones in patients with cystinuria. Using similar approaches, we are investigating the molecular characteristics of K(+)-coupled amino acid transporters in the larval lepidopteran insect midgut. In the larval midgut, K+ is actively secreted into the lumen through the concerted action of an apical H+ V-ATPase and an apical K+/2H+ antiporter, thereby providing the driving force for absorption of amino acids. In vivo, the uptake occurs at extremely high pH (pH 10) and is driven by a large potential difference (approximately -200 mV). Studies with brush-border membrane vesicles have shown that there are several transport systems in the larval intestine with distinct amino acid and cation specificities. In addition to K+, Na+ can also be coupled to amino acid uptake at lower pH, but the Na+/K+ ratio of the hemolymph is so low that K+ is probably the major coupling ion in vivo. The neutral amino acid transport system of larval midgut has been studied most extensively. Apart from its cation selectivity, it appears to be related to the amino acid transport system B previously characterized in vertebrate epithelial cells. Both systems have a broad substrate range which excludes 2-(methylamino)-isobutyric acid, an amino acid analog accepted by the mammalian Na(+)-coupled system A. In order to gain insights into the K(+)-coupling mechanism and into amino acid and K+ homeostasis in insects, current studies are designed to delineate the molecular characteristics of these insect transporters. Recent data showed that injection of mRNA prepared from the midgut of Manduca sexta into Xenopus laevis oocytes induced a 1.5- to 2.5-fold stimulation of the Na(+)-dependent uptake of both leucine and phenylalanine (0.2 mmoll-1, pH 8). The molecular cloning of these transporters is now in progress. Knowledge of their unique molecular properties could be exploited in the future to control disease vectors and insect pests.
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PMID:Molecular characteristics of mammalian and insect amino acid transporters: implications for amino acid homeostasis. 905 Feb 35

Pinealocytes, the neuroendocrine cells that produce melatonin, accumulate glutamate in microvesicles through a specific vesicular transporter energetically coupled with vacuolar-type proton ATPase. The glutamate is secreted into the extracellular space through microvesicle-mediated exocytosis and then stimulates neighboring pinealocytes, resulting in inhibition of norepinephrine-dependent melatonin synthesis. In this study, we identified and characterized the plasma membrane-type glutamate transporter in rat pinealocytes. The [3H]glutamate uptake by cultured pinealocytes was driven by extracellular Na+, saturated with the [3H]glutamate concentration used, and significantly inhibited by L-glutamate, L-aspartate, beta-threo-hydroxyaspartate, pyrrolidine dicarboxylate, and L-cysteine sulfinate, substrates or inhibitors of the plasma membrane glutamate transporter. Consistently, the clearance of extracellular glutamate, as measured by HPLC, was also dependent on Na+ and inhibited by beta-threo-hydroxyaspartate and L-cysteine sulfinate. Immunological studies with site-specific antibodies against three isoforms of the Na+-dependent glutamate transporter (GLT-1, GLAST, and EAAC1) revealed the expression of only the GLT-1 type transporter in pineal glands. Expression of the GLT-1 type transporter in pineal glands was further demonstrated by means of reverse transcription-polymerase chain reaction with specific DNA probes. Immunohistochemical analysis indicated that the immunological counterpart(s) of the GLT-1 is localized in pinealocytes. These results suggested that the GLT-1-type Na+-dependent transporter is expressed and functions as a reuptake system for glutamate in rat pinealocytes. The physiological role of the transporter in the termination of the glutamate signal in the pineal gland is discussed.
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PMID:Functional expression of a GLT-1 type Na+-dependent glutamate transporter in rat pinealocytes. 932 78

Synapse loss, deposits of amyloid beta-peptide (Abeta), impaired energy metabolism, and cognitive deficits are defining features of Alzheimer's disease (AD). Estrogen replacement therapy reduces the risk of developing AD in postmenopausal women. Because synapses are likely sites for initiation of neurodegenerative cascades in AD, we tested the hypothesis that estrogens act directly on synapses to suppress oxidative impairment of membrane transport systems. Exposure of rat cortical synaptosomes to Abeta25-35 (Abeta) and FeSO4 induced membrane lipid peroxidation and impaired the function of the plasma membrane Na+/K+-ATPase, glutamate transporter, and glucose transporter. Pretreatment of synaptosomes with 17beta-estradiol or estriol largely prevented impairment of Na+/K+-ATPase activity, glutamate transport, and glucose transport; other steroids were relatively ineffective. 17Beta-estradiol suppressed membrane lipid peroxidation induced by Abeta and FeSO4, but did not prevent impairment of membrane transport systems by 4-hydroxynonenal (a toxic lipid peroxidation product), suggesting that an antioxidant property of 17beta-estradiol was responsible for its protective effects. By suppressing membrane lipid peroxidation in synaptic membranes, estrogens may prevent impairment of transport systems that maintain ion homeostasis and energy metabolism, and thereby forestall excitotoxic synaptic degeneration and neuronal loss in disorders such as AD and ischemic stroke.
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PMID:17Beta-estradiol attenuates oxidative impairment of synaptic Na+/K+-ATPase activity, glucose transport, and glutamate transport induced by amyloid beta-peptide and iron. 940 14


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