Proton Concentration Gradient Research Articles

Modeling Mitochondrial ROS: A Great Balancing Act

‘‘So be surewhen you step, Step with care and great tact. And remember that life’s A Great Balancing Act. Just never forget to be dexterous and deft, and never mix up your right foot with your left.’’ —Dr. Seuss, Oh, The Places You’ll Go! Random House, 1990. The benefits and risks of a cell’s use of O2 can be a tricky balance. Consumption of O2 by mitochondria greatly enhances the efficiency of aerobic metabolism, allowing generation of 15-fold more ATP than is possible by anaerobic glycolysis alone. Most O2 consumption occurs in the electron transport chain. Under normal conditions, the electron transport chain takes high-energy electrons from NADH or FADH2 to generate a proton concentration gradient and membrane potential gradient between the inner mitochondrial space and the mitochondrial matrix. O2 plays a key role by capturing four low energy electrons coming out of the electron transport chain, forming H2O in the process. The proton gradient is used to power ATP synthase, which harnesses the energy from protons flowing back into the mitochondrial matrix to phosphorylate ADP, yielding ATP. Yet if a highenergy electron leaks from the electron transport chain too early, it may be captured by O2 to form the reactive oxygen species (ROS) known as superoxide (O2� ). Superoxide can be quite damaging because it has an extremely high affinity for electrons, ripping them away from nearby proteins, lipids, and nucleic acids via oxidation. Indeed, ROS species play a key role in a wide range of pathologies suchasatherosclerosis(1),Alzheimer’s disease (2), and cancer (3). Therefore the cell needs to balance the benefits of an efficient aerobic metabolism with the risks of generating toxic ROS. Although the interplay between ATP synthesis and ROS generation has been studied intensively, there have emerged apparent inconsistencies between explanations of the conditions and mechanisms responsible for mitochondrial ROS production from isolated mitochondria and intact cells. As an attempt to resolve these discrepancies, Aon et al. (4) showed that ROS overflow is minimal at intermediate redox states, increasing in either highly reduced/high mitochondrial membrane

The inevitable journey to being.

Life is evolutionarily the most complex of the emergent symmetry-breaking, macroscopically organized dynamic structures in the Universe. Members of this cascading series of disequilibria-converting systems, or engines in Cottrell's terminology, become ever more complicated-more chemical and less physical-as each engine extracts, exploits and generates ever lower grades of energy and resources in the service of entropy generation. Each one of these engines emerges spontaneously from order created by a particular mother engine or engines, as the disequilibrated potential daughter is driven beyond a critical point. Exothermic serpentinization of ocean crust is life's mother engine. It drives alkaline hydrothermal convection and thereby the spontaneous production of precipitated submarine hydrothermal mounds. Here, the two chemical disequilibria directly causative in the emergence of life spontaneously arose across the mineral precipitate membranes separating the acidulous, nitrate-bearing CO2-rich, Hadean sea from the alkaline and CH4/H2-rich serpentinization-generated effluents. Essential redox gradients-involving hydrothermal CH4 and H2 as electron donors, CO2 and nitrate, nitrite, and ferric iron from the ambient ocean as acceptors-were imposed which functioned as the original 'carbon-fixing engine'. At the same time, a post-critical-point (milli)voltage pH potential (proton concentration gradient) drove the condensation of orthophosphate to produce a high energy currency: 'the pyrophosphatase engine'.

Asymmetric Ion Transport through Ion-Channel-Mimetic Solid-State Nanopores

Both scientists and engineers are interested in the design and fabrication of synthetic nanofluidic architectures that mimic the gating functions of biological ion channels. The effort to build such structures requires interdisciplinary efforts at the intersection of chemistry, materials science, and nanotechnology. Biological ion channels and synthetic nanofluidic devices have some structural and chemical similarities, and therefore, they share some common features in regulating the traverse ionic flow. In the past decade, researchers have identified two asymmetric ion transport phenomena in synthetic nanofluidic structures, the rectified ionic current and the net diffusion current. The rectified ionic current is a diode-like current-voltage response that occurs when switching the voltage bias. This phenomenon indicates a preferential direction of transport in the nanofluidic system. The net diffusion current occurs as a direct product of charge selectivity and is generated from the asymmetric diffusion through charged nanofluidic channels. These new ion transport phenomena and the elaborate structures that occur in biology have inspired us to build functional nanofluidic devices for both fundamental research and practical applications. In this Account, we review our recent progress in the design and fabrication of biomimetic solid-state nanofluidic devices with asymmetric ion transport behavior. We demonstrate the origin of the rectified ionic current and the net diffusion current. We also identify several influential factors and discuss how to build these asymmetric features into nanofluidic systems by controlling (1) nanopore geometry, (2) surface charge distribution, (3) chemical composition, (4) channel wall wettability, (5) environmental pH, (6) electrolyte concentration gradient, and (7) ion mobility. In the case of the first four features, we build these asymmetric features directly into the nanofluidic structures. With the final three, we construct different environmental conditions in the electrolyte solutions on either side of the nanochannel. The novel and well-controlled nanofluidic phenomena have become the foundation for many promising applications, and we have highlighted several representative examples. Inspired by the electrogenic cell of the electric eel, we have demonstrated a proof-of-concept nanofluidic reverse electrodialysis system (NREDS) that converts salinity gradient energy into electricity by means of net diffusion current. We have also constructed chirality analysis systems into nanofluidic architectures and monitored these sensing events as the change in the degree of ionic current rectification. Moreover, we have developed a biohybrid nanosystem, in which we reconstituted the F0F1-ATPase on a liposome-coated, solid-state nanoporous membrane. By applying a transmembrane proton concentration gradient, the biohybrid nanodevice can synthesize ATP in vitro. These findings have improved our understanding of the asymmetric ion transport phenomena in synthetic nanofluidic systems and offer innovative insights into the design of functional nanofluidic devices.

Chemical Bias Coupled Photoelectrochemical Zero Bias Hydrogen Generation Utilizing Self-Assembled TiO2 Nanoarchitecture Electrode

ABSTRACTPhotoelectrochemical zero bias hydrogen generation has been achieved with self-assembled nanoporous anatase type TiO2 (SANAT) photoelectrode and chemical bias. The SANAT fabricated using halogen free conventional electrolytes by anodization exhibits more than 2 times superior performance to rutile single crystal electrodes in photoelectrolysis of water. The chemical bias assisted cell consists of two separate compartments connected by a liquid junction. The SANAT anode is immersed in alkaline electrolyte, Pt cathode is in acidic electrolyte. The use of electrolytes of two different pH values produces a chemical bias of 0.059 ∆pH V due to the proton concentration gradient. Under zero bias condition, photocurrent sufficient for photolysis of water was observed. Hydrogen evolution was visible at counter electrode without the application of any external voltage. We call this system fuel type photoelectrochemical zero bias hydrogen generation or water splitting.

Tracing the Trail of Protons through Complex I of the Mitochondrial Respiratory Chain

Mitochondria are the structures that produce the bulk part of the cellular energy currency ATP, which drives numerous energy requiring processes in the cell. This process involves a series of large enzyme complexes—the respiratory chain—that couples the transfer of electrons to the creation of a concentration gradient of protons across the inner mitochondrial membrane, which drives ATP synthesis. Complex I (or NADH-quinone oxidoreductase) is the largest and by far the most complicated of the respiratory chain enzyme complexes. The molecular mechanism whereby it couples electron transfer to proton extrusion has remained mysterious until very recently. Low-resolution X-ray structures of complex I have, surprisingly, suggested that electron transfer in the hydrophilic arm, protruding into the mitochondrial matrix, causes movement of a coupling rod that influences three putative proton pumps within the hydrophobic arm embedded in the inner mitochondrial membrane. In this Primer, we will briefly introduce the recent progress made in this area and highlight the road ahead that likely will unravel the detailed molecular mechanisms of complex I function.

STIM1 and STIM2 Are Located in the Acidic Ca2+ Stores and Associates with Orai1 upon Depletion of the Acidic Stores in Human Platelets

Mammalian cells accumulate Ca2+ into agonist-sensitive acidic organelles, vesicles that possess a vacuolar proton-ATPase. Acidic Ca2+ stores include secretory granules and lysosome-related organelles. Current evidence clearly indicates that acidic Ca2+ stores participate in cell signaling and function, including the activation of store-operated Ca2+ entry in human platelets upon depletion of the acidic stores, although the mechanism underlying the activation of store-operated Ca2+ entry controlled by the acidic stores remains unclear. STIM1 has been presented as the endoplasmic reticulum Ca2+ sensor, but its role sensing intraluminal Ca2+ concentration in the acidic stores has not been investigated. Here we report that STIM1 and STIM2 are expressed in the lysosome-related organelles and dense granules in human platelets isolated by immunomagnetic sorting. Depletion of the acidic Ca2+ stores using the specific vacuolar proton-ATPase inhibitor, bafilomycin A1, enhanced the association between STIM1 and STIM2 as well as between these proteins and the plasma membrane channel Orai1. Depletion of the acidic Ca2+ stores also induces time-dependent co-immunoprecipitation of STIM1 with the TRPC proteins hTRPC1 and hTRPC6, as well as between Orai1 and both TRPC proteins. In addition, bafilomycin A1 enhanced the association between STIM2 and SERCA3. These findings demonstrate the location of STIM1 and STIM2 in the acidic Ca2+ stores and their association with Ca2+ channels and ATPases upon acidic stores discharge.

Application of cyclic voltammetry to investigate enhanced catalytic current generation by biofilm-modified anodes of Geobacter sulfurreducens strain DL1vs. variant strain KN400

A biofilm of Geobacter sulfurreducens will grow on an anode surface and catalyze the generation of an electrical current by oxidizing acetate and utilizing the anode as its metabolic terminal electron acceptor. Here we report qualitative analysis of cyclic voltammetry of anodes modified with biofilms of G. sulfurreducens strains DL1 and KN400 to predict possible rate-limiting steps in current generation. Strain KN400 generates approximately 2 to 8-fold greater current than strain DL1 depending upon the electrode material, enabling comparative electrochemical analysis to study the mechanism of current generation. This analysis is based on our recently reported electrochemical model for biofilm-catalyzed current generation expanded here to a five step model; Step 1 is mass transport of acetate, carbon dioxide and protons into and out of the biofilm, Step 2 is microbial turnover of acetate to carbon dioxide and protons, Step 3 is the non-concerted, 1-electron reduction of 8 equivalents of electron transfer (ET) mediator, Step 4 is extracellular electron transfer (EET) through the biofilm to the electrode surface, and Step 5 is the reversible oxidation of reduced mediator by the electrode. Five idealized voltammetric current vs. potential dependencies (voltammograms) are derived, one for when each step in the model is assumed to limit catalytic current. Comparison to experimental voltammetry of DL1 and KN400 biofilm-modified anodes suggests that for both strains, the microbial oxidation of acetate (Step 2) is fast compared to microbial reduction of ET mediator (Step 3), and either Step 3 or EET through the biofilm (Step 4) limits catalytic current generation. The possible limitation of catalytic current by Step 4 is consistent with proton concentration gradients observed within these biofilms and finite thicknesses achieved by these biofilms. The model presented here has been universally designed for application to biofilms other than G. sulfurreducens and could serve as a platform for future quantitative voltammetric analysis of non-corrosive anode and cathode reactions catalyzed by microorganisms.

Computational modeling of a direct propane fuel cell

The first two dimensional mathematical model of a complete direct propane fuel cell (DPFC) is described. The governing equations were solved using FreeFem software that uses finite element methods. Robin boundary conditions were used to couple the anode, membrane, and cathode sub-domains successfully. The model showed that a polytetrafluoroethylene membrane having its pores filled with zirconium phosphate (ZrP–PTFE), in a DPFC at 150°C performed much the same as other electrolytes; Nafion, aqueous H3PO4, and H2SO4 doped polybenzimidazole, when they were used in DPFCs. One advantage of a ZrP–PTFE at 150°C is that it operates without liquid phase water. As a result corrosion will be much less severe and it may be possible for non-precious metal catalysts to be used. Computational results showed that the thickness of the catalyst layer could be increased sufficiently so that the pressure drop between the reactant and product channels of the interdigitated flow fields is small. By increasing the width of the land and therefore the reactant's contact time with the catalyst it was possible to approach 100% propane conversion. Therefore fuel cell operation with a minimum concentration of propane in the product stream should be possible. Finally computations of the electrical potential in the ZrP phase, the electron flux in the Pt/C phase, and the overpotential in both the anode and cathode catalyst layers showed that serious errors in the model occurred because proton diffusion, caused by the proton concentration gradient, was neglected in the equation for the conservation of protons.

Essentials for ATP Synthesis by F1F0 ATP Synthases

The majority of cellular energy in the form of adenosine triphosphate (ATP) is synthesized by the ubiquitous F(1)F(0) ATP synthase. Power for ATP synthesis derives from an electrochemical proton (or Na(+)) gradient, which drives rotation of membranous F(0) motor components. Efficient rotation not only requires a significant driving force (DeltamuH(+)), consisting of membrane potential (Deltapsi) and proton concentration gradient (DeltapH), but also a high proton concentration at the source P side. In vivo this is maintained by dynamic proton movements across and along the surface of the membrane. The torque-generating unit consists of the interface of the rotating c ring and the stator a subunit. Ion translocation through this unit involves a sophisticated interplay between the c-ring binding sites, the stator arginine, and the coupling ions on both sides of the membrane. c-ring rotation is transmitted to the eccentric shaft gamma-subunit to elicit conformational changes in the catalytic sites of F(1), leading to ATP synthesis.

Precise detection of pH inside large unilamellar vesicles using membrane-impermeable dendritic porphyrin-based nanoprobes

Accurate real-time measurements of proton concentration gradients are pivotal to mechanistic studies of proton translocation by membrane-bound enzymes. Here we report a detailed characterization of the pH-sensitive fluorescent nanoprobe Glu 3, which is well suited for pH measurements in microcompartmentalized biological systems. The probe is a polyglutamic porphyrin dendrimer in which multiple carboxylate termini ensure its high water solubility and prevent its diffusion across phospholipid membranes. The probe’s p K is in the physiological pH range, and its protonation can be followed ratiometrically by absorbance or fluorescence in the ultraviolet-visible spectral region. The usefulness of the probe was enhanced by using a semiautomatic titration system coupled to a charge-coupled device (CCD) spectrometer, enabling fast and accurate titrations and full spectral coverage of the system at millisecond time resolution. The probe’s p K was measured in bulk solutions as well as inside large unilamellar vesicles in the presence of physiologically relevant ions. Glu 3 was found to be completely membrane impermeable, and its distinct spectroscopic features permit pH measurements inside closed membrane vesicles, enabling quantitative mechanistic studies of membrane-spanning proteins. Performance of the probe was demonstrated by monitoring the rate of proton leakage through the phospholipid bilayer in large vesicles with and without the uncoupler gramicidin present. Overall, as a probe for biological proton translocation measurements, Glu 3 was found to be superior to the commercially available pH indicators.

Mitochondrial Lactate Oxidation Complex and an Adaptive Role for Lactate Production

The intracellular lactate shuttle (ILS) hypothesis holds that lactate produced as the result of glycolysis and glycogenolysis in the cytosol is balanced by oxidative removal in mitochondria of the same cell. Also, the ILS is a necessary component of the previously described cell-cell lactate shuttle (CCLS), because lactate supplied from the interstitium and vasculature can be taken up and used in highly oxidative cells (red skeletal and cardiac myocytes, hepatocytes, and neurons). This ILS emphasizes the role of mitochondrial redox in creating the proton and lactate anion concentration gradients necessary for the oxidative disposal of lactate in the mitochondrial reticulum during exercise and other conditions. The hypothesis was initially supported by direct measurement of lactate oxidation in isolated mitochondria as well as findings of the existence of mitochondrial monocarboxylate transporters (mMCT) and lactate dehydrogenase (mLDH). Subsequently, the presence of a mitochondrial lactate oxidation complex (composed of mMCT1, CD147 (basigin), mLDH, and cytochrome oxidase (COX)) was discovered, which lends support to the presence of the ILS. Most recently, efforts have been made to evaluate the role of lactate as a cell-signaling molecule (i.e., a "lactormone") that is involved in the adaptive response to exercise. Lactate is capable of upregulating MCT1 and COX gene and protein expression. Current findings allow us to understand how lactate production during exercise represents a physiological signal for the activation of a vast transcription network affecting MCT1 protein expression and mitochondrial biogenesis, thereby explaining how training increases the capacity for lactate clearance via oxidation.

Controlled fluid transport using ATP-powered protein pumps

Plants have the ability to move fluids using the chemical energy available with bio-fuels.The energy released by the cleavage of a terminal phosphate ion during the hydrolysis of abio-fuel assists the transport of ions and fluids in cellular homeostasis. The device discussedin this paper uses protein pumps cultured from plant cells to move fluid across a membranebarrier for controllable fluid transport. This paper demonstrates the ability to reconstitutea protein pump extracted from a plant cell on a supported bilayer lipid membrane (BLM)and use the pump to transport fluid expending adenosine triphoshate (ATP). TheAtSUT4 protein used in this demonstration is cultured from Arabidopsis thaliana.This protein transporter moves a proton and a sucrose molecule in the presenceof an applied proton gradient or by using the energy released from adenosinetriphosphate’s hydrolysis reaction. The BLM supporting the AtSUT4 is formed from1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-L-Serine] (sodium salt) (POPS),1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine (POPE) lipids supported on aporous lead silicate glass plate. The BLM is formed with the transporter and theATP-phosphohydrolase (red beet ATPase) enzyme, and the ATP required for thereaction is added as a magnesium salt on one side of the membrane. The ATPhydrolysis reaction provides the required energy for transporting a proton–sucrosemolecule through the protein pump. It is observed that there is no fluid transportin the absence of the enzyme and the amount of fluid transported through themembrane is dependent on the amount of enzyme reconstituted in the BLM for a fixedsucrose concentration. This demonstrates the dependence of the fluid flux on theATP hydrolysis reaction catalyzed by the ATP-ase enzyme. The dependenceof fluid flux on the amount of ATP-ase provides convincing evidence that thebiochemical reaction is producing the fluid transport. The fluid flux resulting from theATP-powered transport is observed to be higher than the rates observed witha proton concentration gradient driven transport reported in our earlier work.

The Extracellular pH Dependency of Transport Activity by Human Oligopeptide Transporter 1 (hPEPT1) Expressed Stably in Chinese Hamster Ovary (CHO) Cells: A Reason for the Bell-Shaped Activity versus pH

Human oligopeptide transporter (hPEPT1) translocates di/tri-peptide by coupling to movement of proton down the electrochemical gradient. This transporter has the characteristics that the pH-profile of neutral dipeptide transport shows a bell-shaped curve with an optimal pH of 5.5. In the present study, we examined the reason for the decrease in the acidic region with hPEPT1-transfected CHO cells stably oeverexpressing hPEPT1 (CHO/hPEPT1). The pH profile of the transport activity vs. pH was measured in the presence of nigericin/monensin. Under this condition, the inwardly directed proton concentration gradient was dissipated while the membrane potential remained. As pH increased the activity increased, and the Henderson-Hasselbalch equation with a single pKa was fitted well to the activity curve. The pKa value was estimated to be 6.7+/-0.2. This value strongly suggests that there is a key amino acid residue, which is involved in pH regulation of transport activity. To identify the key amino acid residue, we examined the effects of various chemical modifications on pH-profile of the transport activity. Modification of carboxyl groups or hydroxyl groups had no significant influence on the pH-profile, whereas a chemical modification of histidine residue with diethylpyrocarbonate (DEPC) completely abolished the transport activity in CHO/hPEPT1 cells. On the other hand, this abolishment was almost prevented by the presence of 10 mM Gly-Sar. This protection was observed only in the presence of the substrate of hPEPT1, indicating that the histidine residue is located at the substrate recognition site. The pH-profile of the transport activity in CHO/hPEPT1 cells treated with DEPC in the presence of 10 mM Gly-Sar also showed a bell-shape similar to that in non-treated CHO/hPEPT1 cells. These data stressed that the histidine residue located at or near the substrate binding site is involved in the pH regulation of transport activity.

The mechanism of photosynthetic water splitting

Oxygenic photosynthesis, which provides the biosphere with most of its chemical energy, uses water as its source of electrons. Water is photochemically oxidized by the protein complex photosystem II (PSII), which is found, along with other proteins of the photosynthetic light reactions, in the thylakoid membranes of cyanobacteria and of green plant chloroplasts. Water splitting is catalyzed by the oxygen-evolving complex (OEC) of PSII, producing dioxygen gas, protons and electrons. O(2) is released into the atmosphere, sustaining all aerobic life on earth; product protons are released into the thylakoid lumen, augmenting a proton concentration gradient across the membrane; and photo-energized electrons pass to the rest of the electron-transfer pathway. The OEC contains four manganese ions, one calcium ion and (almost certainly) a chloride ion, but its precise structure and catalytic mechanism remain unclear. In this paper, we develop a chemically complete structure of the OEC and its environment by using molecular mechanics calculations to extend and slightly adjust the recently-obtained X-ray crystallographic model with reference to this structure and to some important recent experimental results.

Catalytic Micropumps: Microscopic Convective Fluid Flow and Pattern Formation

As innovations continue to be made in the fields of microfluidics and the colloidal assembly, new strategies for moving particles and fluids may be needed. Heterogeneous catalysis provides means of locally converting the stored chemical energy of fuels to mechanical energy. We report an ambient temperature stationary "pump" that generates a proton concentration gradient through the bipolar electrochemical decomposition of hydrogen peroxide on patterned silver-gold surfaces. The resulting electric field drives convective fluid flow and pattern formation of colloidal tracer particles at the microscopic level by a combination of electroosmotic and electrophoretic forces.

Photoresponsive Liquid Membrane Transport of Alkali Metal Ions Using Crowned Spirobenzopyrans

Several azacrown ether derivatives, which are monoaza-12-crown-4, -15-crown-5, and -18-crown-6 and diaza-12-crown-4 and -18-crown-6, bearing one or two spirobenzopyran(s), which we call crowned spirobenzopyran or crowned bis(spirobenzopyran), were synthesized and were used as carriers for liquid membrane transport of alkali metal ions. The passive alkali metal transports through liquid membranes containing crowned spirobenzopyrans were carried out under dark, and UV- and visible-light irradiation conditions. The metal ion transport was accelerated and retarded by UV- and visible-light irradiation, respectively. On the other hand, the photoresponse of the metal ion selectivity in membrane transport by crowned spirobenzopyrans was different, depending on the kind of crown ether units. Especially, diaza-12-crown-4-bis(spirobenzopyran) exhibited an excellently selective and effective transporting ability for Li(+). The uphill transports of Li(+) through a liquid membrane containing monoaza-12-crown-4-spirobenzopyran or diaza-12-crown-4-bis(spirobenzopyran) were realized under the conditions where the same aqueous solution was used as the source and receiving phases with UV and visible lights being irradiated onto the boundary phases between the source and membrane phases and between the receiving and membrane phases, respectively. The uphill transport of Li(+) from the source to receiving phases through a liquid membrane containing a crowned spirobenzopyran was also attained by the proton-concentration gradient between the source and receiving phases under dark conditions, and the transporting ability was remarkably increased by photoirradiation.

Telemetric electrochemical sensor

A telemetric system was designed and constructed to sense pH and ethanol variation in aqueous solutions. The measured signals were transferred by software digitally and transmitted wirelessly by the telemeter, personal digital assistant (PDA), through the General Packet Radio Service (GPRS) protocol. The pH sensing electrode was designed to measure a chemical potential induced by a proton concentration gradient on the electrode’s surface which exhibits internal Donnon diffusion behavior, and a linear relationship between the electrical potential and pH was found. The result shows that the wireless sensing system allowed not only long-term usage and long-distance transmission but also with high accuracy (e.g. S.D. less than ±2%). The telemetric system can also be modified to measure ethanol concentration in aqueous solution amperometrically. It was found that the sensitivity of that ex situ measurements matched those of in field measurements with negligible deviation, less than 4%.

Ca2+ sensitivity of synaptic vesicle dopamine, gamma-aminobutyric acid, and glutamate transport systems.

The effect of Ca2+ on the uptake of neurotransmitters by synaptic vesicles was investigated in a synaptic vesicle enriched fraction isolated from sheep brain cortex. We observed that dopamine uptake, which is driven at expenses of the proton concentration gradient generated across the membrane by the H+-ATPase activity, is strongly inhibited (70%) by 500 microM Ca2+. Conversely, glutamate uptake, which essentially requires the electrical potential in the presence of low Cl- concentrations, is not affected by Ca2+, even when the proton concentration gradient greatly contributes for the proton electrochemical gradient. These observations were checked by adding Ca2+ to dopamine or glutamate loaded vesicles, which promoted dopamine release, whereas glutamate remained inside the vesicles. Furthermore, similar effects were obtained by adding 150 microM Zn2+ that, like Ca2+, dissipates the proton concentration gradient by exchanging with H+. With respect to gamma-aminobutyric acid transport, which utilizes either the proton concentration gradient or the electrical potential as energy sources, we observed that Ca2+ or Zn2+ do not induce great alterations in the gamma-aminobutyric acid accumulation by synaptic vesicles. These results clarify the nature of the energy source for accumulation of main neurotransmitters and suggest that stressing concentrations of Ca2+ or Zn2+ inhibit the proton concentration gradient-dependent neurotransmitter accumulation by inducing H+ pump uncoupling rather than by interacting with the neurotransmitter transporter molecules.

Kinetic model of ATP synthase: pH dependence of the rate of ATP synthesis

Recently, a novel molecular mechanism of torque generation in the F 0 portion of ATP synthase was proposed [Rohatgi, Saha and Nath (1998) Curr. Sci. 75, 716–718]. In this mechanism, rotation of the c-subunit was conceived to take place in 12 discrete steps of 30° each due to the binding and unbinding of protons to/from the leading and trailing Asp-61 residues of the c-subunit, respectively. Based on this molecular mechanism, a kinetic scheme has been developed in this work. The scheme considers proton transport driven by a concentration gradient of protons across the proton half-channels, and the rotation of the c-subunit by changes in the electrical potential only. This kinetic scheme has been analyzed mathematically and an expression has been obtained to explain the pH dependence of the rate of ATP synthesis by ATP synthase under steady state operating conditions. For a single set of three enzymological kinetic parameters, this expression predicts the rates of ATP synthesis which agree well with the experimental data over a wide range of pH in and pH out. A logical consequence of our analysis is that ΔpH and Δ ψ are kinetically inequivalent driving forces for ATP synthesis.

Crucial role of the membrane potential for ATP synthesis by F(1)F(o) ATP synthases.

ATP, the universal carrier of cell energy, is manufactured from ADP and phosphate by the enzyme ATP synthase using the free energy of an electrochemical gradient of protons (or Na(+)). The proton-motive force consists of two components, the transmembrane proton concentration gradient (delta pH) and the membrane potential. The two components were considered to be not only thermodynamically but also kinetically equivalent, since the chloroplast ATP synthase appeared to operate on delta pH only. Recent experiments demonstrate, however, that the chloroplast ATP synthase, like those of mitochondria and bacteria, requires a membrane potential for ATP synthesis. Hence, the membrane potential and proton gradient are not equivalent under normal operating conditions far from equilibrium. These conclusions are corroborated by the finding that only the membrane potential induces a rotary torque that drives the counter-rotation of the a and c subunits in the F(o) motor of Propionigenium modestum ATP synthase.