scholarly journals Glutamate uptake into synaptic vesicles of bovine cerebral cortex and electrochemical potential difference of proton across the membrane

1989 ◽  
Vol 258 (2) ◽  
pp. 499-504 ◽  
Author(s):  
J Shioi ◽  
S Naito ◽  
T Ueda
Keyword(s):  
Cerebral Cortex ◽  
Synaptic Vesicles ◽  
Driving Force ◽  
Significant Contribution ◽  
Atp Hydrolysis ◽  
Glutamate Uptake ◽  
Protonmotive Force ◽  
Chromaffin Granule ◽  
Carbonyl Cyanide ◽  
Disulphonic Acid

Measurements have been made of the ATP-dependent membrane potential (delta psi) and pH gradient (delta pH) across the membranes of the synaptic vesicles purified from bovine cerebral cortex, using the voltage-sensitive dye bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxanol and the delta pH-sensitive fluorescent dye 9-aminoacridine respectively. A pre-existing small delta pH (inside acidic) was detected in the synaptic vesicles, but no additional significant contribution by MgATP to delta pH was observed. In contrast, delta psi (inside positive) increased substantially upon addition of MgATP. This ATP-dependent delta psi was reduced by thiocyanate anion (SCN-), a delta psi dissipator, or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a protonmotive-force dissipator. Correspondingly, a substantially larger glutamate uptake occurred in the presence of MgATP, which was inhibited by SCN- and FCCP. A nonhydrolysable analogue of ATP, adenosine 5′-[beta gamma-methylene]triphosphate, did not substitute for ATP in either delta psi generation or glutamate uptake. The results support the hypothesis that a H+-pumping ATPase generates a protonmotive force in the synaptic vesicles at the expense of ATP hydrolysis, and the protonmotive force thus formed provides a driving force for the vesicular glutamate uptake. The delta psi generation by ATP hydrolysis was not affected by orthovanadate, ouabain or oligomycin, but was inhibited by N-ethylmaleimide, quercetin, trimethyltin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid. These results indicate that the H+-pumping ATPase in the synaptic vesicle is similar to that in the chromaffin granule, platelet granule and lysosome.

scholarly journals Artificially imposed electrical potentials drive l-glutamate uptake into synaptic vesicles of bovine cerebral cortex

1990 ◽  
Vol 267 (1) ◽  
pp. 63-68 ◽  
Author(s):  
J Shioi ◽  
T Ueda
Keyword(s):  
Cerebral Cortex ◽  
Synaptic Vesicles ◽  
Glutamate Uptake ◽  
Electrical Potential ◽  
Potential Gradient ◽  
Protonmotive Force ◽  
Uptake System ◽  
Vesicle Membrane ◽  
Excitatory Neurotransmitter ◽  
Electrical Potentials

L-Glutamate is a major excitatory neurotransmitter in the central nervous system. MgATP-dependent glutamate uptake and H(+)-pumping ATPase activity were reported in highly purified synaptic vesicles [Naito & Ueda (1983) J. Biol. Chem. 258, 696-699; Shioi, Naito & Ueda (1989) Biochem. J. 258, 499-504], and it is hypothesized that an electrochemical H+ gradient across the vesicle membrane, the so-called protonmotive force, elicits the neurotransmitter uptake. An inside-positive diffusion potential across the vesicle membrane was established with valinomycin plus Rb+. This artificial electrical potential promoted the uptake of glutamate, but not aspartate, in the synaptic vesicles prepared from bovine cerebral cortex. The uptake was inhibited by the protonmotive-force dissipators carbonyl cyanide p-trifluoro-methoxyphenylhydrazone or nigericin, and was enhanced by concomitant imposition of a pH jump (alkalinization) in the external medium. Subcellular and subvesicular distributions showed the uptake system to be predominantly associated with small synaptic vesicles. The results support the hypothesis that glutamate uptake into synaptic vesicles is coupled with a H+ efflux down the electrochemical potential gradient, which is generated by H(+)-pumping ATPase.

scholarly journals Proton translocation of the bovine chromaffin-granule membrane

1978 ◽  
Vol 170 (3) ◽  
pp. 661-672 ◽  
Author(s):  
J H Phillips ◽  
V P Allison
Keyword(s):  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Potential Gradient ◽  
Protonmotive Force ◽  
Chromaffin Granule ◽  
Anion Transporter ◽  
Direct Measurements ◽  
Granule Membrane ◽  
Potential Gradients ◽  
Proton Movement

Bovine chromaffin granules were lysed and their membranes resealed to give osmotically sensitive ‘ghosts’. These swell in the presence of salts and MgATP. It is shown that this is due to proton entry accompanied by anions. The rate of swelling depends on the anion present, but swelling is not limited to media containing permeant anions. It is quite marked in solutions of sulphates, phosphates and acetates. It is not uncoupler-sensitive, suggesting that at least one component of swelling is due to coupled proton and anion entry (non-electrogenic proton translocation). Direct measurements of transmembrane pH and potential gradients generated in the presence of MgATP shows that these are rapidly established in sucrose media, and are rather little affected by the presence of salts. They contribute roughly equally to the total protonmotive force. The potential gradient is establihsed very rapidly, but the pH gradient is generated over several minutes. The gradients are not completely dissipated by uncoupler, and it is shown that, in media containing sulphate but no permeant anion, sulphate can be taken up by the ‘ghosts’. There thus appear to be two mechanisms of proton translocation across the membrane, both dependent on ATP hydrolysis: an electrogenic transfer of protons, and proton movement linked to an anion transporter of broad specificity.

scholarly journals Transport of γ-aminobutyrate and l-glutamate into synaptic vesicles. Effect of different inhibitors on the vesicular uptake of neurotransmitters and on the Mg2+-ATPase

1991 ◽  
Vol 276 (2) ◽  
pp. 363-367 ◽  
Author(s):  
E M Fykse ◽  
F Fonnum
Keyword(s):  
Benzoic Acid ◽  
Rat Brain ◽  
Synaptic Vesicles ◽  
Glutamate Uptake ◽  
Gaba Uptake ◽  
Anion Channels ◽  
Low Concentrations ◽  
Lipophilic Anion ◽  
Vesicular Uptake ◽  
Disulphonic Acid

The uptakes of gamma-aminobutyrate (GABA) and L-glutamate into synaptic vesicles isolated from rat brain were compared with respect to the effects of 4-acetamido-4′-isothiocyanostilbene-2,2′- disulphonic acid (SITS), 4,4′-di-isothiocyanostilbene-2,2′-disulphonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (N144), agents known to block anion channels. The uptake of glutamate was inhibited by low micromolar concentrations of SITS, DIDS and N144. GABA uptake was much less sensitive to these agents than was glutamate uptake. SITS and N144 inhibited the vacuolar H(+)-ATPase of synaptic vesicles to a smaller extent than the glutamate uptake. The uptake of GABA was not affected by the permeant anions Cl- and Br-, whereas the uptake of glutamate was highly stimulated by low concentrations of these ions. The uptakes of both glutamate and GABA were inhibited by similar, but not identical, concentrations of the lipophilic anion SCN-.

scholarly journals The effect of chloroform on mitochondrial energy transduction

1996 ◽  
Vol 320 (3) ◽  
pp. 837-845 ◽  
Author(s):  
Lee-Feng CHIEN ◽  
Martin D. BRAND
Keyword(s):  
Electron Transport ◽  
Atp Hydrolysis ◽  
Electron Flow ◽  
Protonmotive Force ◽  
Rate Dependent ◽  
Carbonyl Cyanide ◽  
Apparent Decrease ◽  
Electron Transport Complexes ◽  
The Relationship ◽  
Flow Pathway

The effect of chloroform on mitochondrial respiration with succinate was investigated by applying the method of Brand, Chien and Diolez [(1994) Biochem. J. 297, 27–29] to examine whether chloroform causes redox slip (fewer protons pumped per electron transferred) during mitochondrial electron transport. N,N,N´,N´-Tetramethyl-p-phenylenediamine (TMPD), which lowers H+/O (the number of protons pumped to the external medium by the electron transport complexes per oxygen atom consumed) by altering the electron flow pathway, was investigated for comparison. Non-phosphorylating mitochondria that had been treated with 350 µM TMPD or 30 mM chloroform were titrated with malonate in the presence of submaximal concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Linear relations between CCCP-induced extra respiration and protonmotive force were obtained. These results showed that there was no measurable protonmotive force-dependent or rate-dependent slip in mitochondria treated with either TMPD or chloroform. However, both TMPD and chloroform seemed to decrease H+/O in a manner independent of protonmotive force and rate. The relationship between non-phosphorylating respiration and protonmotive force was simulated in mitochondria of which 25% of the total population were assumed to have been broken. The simulation showed that the apparent decrease in H+/O on the addition of TMPD or chloroform to mitochondria could be in principle accounted for by breakage. Assays of mitochondrial breakage (ATP hydrolysis in the presence of atractyloside and oxidation of exogenous NADH) showed that chloroform broke mitochondria but TMPD did not. We conclude that chloroform changes the measured H+/O as an artifact by causing mitochondrial breakage and does not cause measurable redox slip, whereas TMPD genuinely lowers H+/O.

Effects of methylmalonic and propionic acids on glutamate uptake by synaptosomes and synaptic vesicles and on glutamate release by synaptosomes from cerebral cortex of rats

2001 ◽  
Vol 920 (1-2) ◽  
pp. 194-201 ◽  
Author(s):  
Ana Maria Brusque ◽  
Liane Nanci Rotta ◽  
Rejane Giacomelli Tavares ◽  
Tatiana Emanuelli ◽  
Carolina Vargas Schwarzbold ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Synaptic Vesicles ◽  
Glutamate Uptake ◽  
Glutamate Release

Synaptic vesicles immunoisolated from rat cerebral cortex contain high levels of glutamate

Neuron ◽  
1989 ◽  
Vol 3 (6) ◽  
pp. 715-720 ◽  
Author(s):  
Peter M. Burger ◽  
Ehrenfried Mehl ◽  
Patricia L. Cameron ◽  
Peter R. Maycox ◽  
Marion Baumert ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Synaptic Vesicles ◽  
Rat Cerebral Cortex

scholarly journals Thermodynamic control of electron flux through mitochondrial cytochrome bc1 complex

1985 ◽  
Vol 225 (2) ◽  
pp. 399-405 ◽  
Author(s):  
G C Brown ◽  
M D Brand
Keyword(s):  
Liver Mitochondria ◽  
Cytochrome Bc1 ◽  
Rat Liver Mitochondria ◽  
Protonmotive Force ◽  
Bc1 Complex ◽  
Thermodynamic Control ◽  
Cytochrome Bc1 Complex ◽  
Redox States ◽  
Force Ratio ◽  
Carbonyl Cyanide

The redox states of exogenously added ubiquinone-2 and cytochrome c, and the protonmotive force (delta p) of rat liver mitochondria were measured as the respiration rate was titrated with the uncoupler carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone. The force ratio delta Eh/delta p across the bc1 complex was close to 1:1 in State 4, indicating an H+/e- stoichiometry of 1:1 for the cytochrome bc1 complex, excluding protons moved by pool ubiquinone. Assuming a constant stoichiometry the rate of electron transport increased linearly with the disequilibrium (delta Eh - delta p) across the complex.

scholarly journals Activity and cytosolic Na+ regulates synaptic vesicle endocytosis

2019 ◽  
Author(s):  
Yun Zhu ◽  
Dainan Li ◽  
Hai Huang
Keyword(s):  
Synaptic Transmission ◽  
High Frequency ◽  
Synaptic Vesicles ◽  
Glutamate Uptake ◽  
High Frequencies ◽  
Vesicle Recycling ◽  
Two Photon ◽  
Readily Releasable Pool ◽  
Content Change ◽  
Intracellular Ca

ABSTRACTRetrieval of synaptic vesicles via endocytosis is essential for maintaining sustained synaptic transmission, especially for neurons that fire action potentials at high frequencies. However, how activity regulates synaptic vesicles recycling is largely unknown. Here we report that Na+ substantially accumulated in the mouse calyx of Held terminals during repetitive high-frequency spiking. Elevated presynaptic Na+ accelerated both slow and rapid forms of endocytosis and facilitated endocytosis overshoot but did not affect the readily releasable pool size, Ca2+ influx, or exocytosis. To examine whether this facilitation of endocytosis is related to the Na+-dependent vesicular content change, we dialyzed increasing concentrations of glutamate into the presynaptic cytosol or blocked the vesicular glutamate uptake with bafilomycin and found the rate of endocytosis was not affected by regulating the glutamate content in the presynaptic terminal. Endocytosis is critically dependent on intracellular Ca2+, and the activity of Na+/Ca2+ exchanger (NCX) may be altered when the Na+ gradient is changed. However, neither NCX blocker nor change of extracellular Na+ concentration affected the endocytosis rate. Moreover, two-photon Ca2+ imaging showed that presynaptic Na+ did not affect the action potential-evoked intracellular Ca2+ transient and decay. Therefore, we revealed a novel mechanism of cytosolic Na+ in accelerating vesicle endocytosis. During high-frequency synaptic transmission, when large amounts of synaptic vesicles are fused, Na+ accumulated in terminals, facilitated vesicle recycling and sustained reliable synaptic transmission.

scholarly journals A Mechanistic Perspective on PEX1 and PEX6, Two AAA+ Proteins of the Peroxisomal Protein Import Machinery

2019 ◽  
Vol 20 (21) ◽  
pp. 5246 ◽  
Author(s):  
Ana G. Pedrosa ◽  
Tânia Francisco ◽  
Maria J. Ferreira ◽  
Tony A. Rodrigues ◽  
Aurora Barros-Barbosa ◽  
...  
Keyword(s):  
Self Assembly ◽  
Driving Force ◽  
Matrix Protein ◽  
Protein Import ◽  
Atp Hydrolysis ◽  
Transport Proteins ◽  
Gtp Hydrolysis ◽  
Assembly Mechanism ◽  
Dependent Protein ◽  
Organelle Membrane

In contrast to many protein translocases that use ATP or GTP hydrolysis as the driving force to transport proteins across biological membranes, the peroxisomal matrix protein import machinery relies on a regulated self-assembly mechanism for this purpose and uses ATP hydrolysis only to reset its components. The ATP-dependent protein complex in charge of resetting this machinery—the Receptor Export Module (REM)—comprises two members of the “ATPases Associated with diverse cellular Activities” (AAA+) family, PEX1 and PEX6, and a membrane protein that anchors the ATPases to the organelle membrane. In recent years, a large amount of data on the structure/function of the REM complex has become available. Here, we discuss the main findings and their mechanistic implications.

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