Showing papers on "Electrochemical gradient published in 2001"

Structures and proton-pumping strategies of mitochondrial respiratory enzymes.

Abstract: Enzymes of the mitochondrial respiratory chain serve as proton pumps, using the energy made available from electron transfer reactions to transport protons across the inner mitochondrial membrane and create an electrochemical gradient used for the production of ATP. The ATP synthase enzyme is reversible and can also serve as a proton pump by coupling ATP hydrolysis to proton translocation. Each of the respiratory enzymes uses a different strategy for performing proton pumping. In this work, the strategies are described and the structural bases for the action of these proteins are discussed in light of recent crystal structures of several respiratory enzymes. The mechanisms and efficiency of proton translocation are also analyzed in terms of the thermodynamics of the substrate transformations catalyzed by these enzymes.

Natural-resistance-associated macrophage protein 1 is an H+/bivalent cation antiporter.

Abstract: In mammals, natural-resistance-associated macrophage protein 1 (Nramp1) regulates macrophage activation and is associated with infectious and autoimmune diseases. Nramp2 is associated with anaemia. Both belong to a highly conserved eukaryote/prokaryote protein family. We used Xenopus oocytes to demonstrate that, like Nramp2, Nramp1 is a bivalent cation (Fe2+, Zn2+ and Mn2+) transporter. Strikingly, however, where Nramp2 is a symporter of H+ and metal ions, Nramp1 is a highly pH-dependent antiporter that fluxes metal ions in either direction against a proton gradient. At pH 9.0, oocytes injected with cRNA from wild-type murine Nramp1 with a glycine residue at position 169 (Nramp1(G169); P=3.22x10(-6)) and human NRAMP1 (P=3.87x10(-5)) showed significantly enhanced uptake of radiolabelled Zn2+ compared with water-injected controls. At pH 5.5, Nramp1(G169) (P=1.34x10(-13)) and NRAMP1 (P=1.09x10(-6)) oocytes showed significant efflux of Zn2+. Zn2+ transport was abolished when the proton gradient was dissipated using carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Using pre-acidified oocytes, currents of 130+/-57 nA were evoked by 100 microM Zn2+ at pH 7.5, and 139+/-47 nA by 100 microM Fe2+ at pH 7.0, in Nramp1(G169) oocytes; currents of 254+/-49 nA and 242+/-26 nA were evoked, respectively, in NRAMP1 oocytes. Steady-state currents evoked by increasing concentrations of Zn2+ were saturable, with apparent affinity constants of approx. 614 nM for Nramp1(G169) and approx. 562 nM for NRAMP1 oocytes, and a curvilinear voltage dependence of transporter activity (i.e. the data points approximate to a curve that approaches a linear asymptote). In the present study we propose a new model for metal ion homoeostasis in macrophages. Under normal physiological conditions, Nramp2, localized to early endosomal membranes, delivers extracellularly acquired bivalent cations into the cytosol. Nramp1, localized to late endosomal/lysosomal membranes, delivers bivalent cations from the cytosol into this acidic compartment where they may directly affect antimicrobial activity.

Evidence for a role in growth and salt resistance of a plasma membrane H+-ATPase in the root endodermis

Abstract: The plasma membrane of plant cells is energized by an electrochemical gradient produced by P-type H+-ATPases (proton pumps). These pumps are encoded by at least 12 genes in Arabidopsis. Here we provide evidence that isoform AHA4 contributes to solute transport through the root endodermis. AHA4 is expressed most strongly in the root endodermis and flowers, as suggested by promoter-GUS reporter assays. A disruption of this pump (aha4-1) was identified as a T-DNA insertion in the middle of the gene (after VFP(574)). Truncated aha4-1 transcripts accumulate to approximately 50% of the level observed for AHA4 mRNA in wild-type plants. Plants homozygous for aha4-1 (-/-) show a subtle reduction in root and shoot growth compared with wild-type plants when grown under normal conditions. However, a mutant phenotype is very clear in plants grown under salt stress (e.g., 75 or 110 mM NaCl). In leaves of mutant plants subjected to Na stress, the ratio of Na to K increased 4-5-fold. Interestingly, the aha4-1 mutation appears to be semidominant and was only partially complemented by the introduction of additional wild-type copies of AHA4. These results are consistent with the hypothesis that aha4-1 may produce a dominant negative protein or RNA that partially disrupts the activity of other pumps or functions in the root endodermal tissue, thereby compromising the function of this cell layer in controlling ion homeostasis and nutrient transport.

Role of KCNE1-Dependent K+ Fluxes in Mouse Proximal Tubule

Abstract: . The electrochemical gradient for K + across the luminal membrane of the proximal tubule favors K + fluxes to the lumen. Here it was demonstrated by immunohistochemistry that KCNE1 and KCNQ1, which form together the slowly activated component of the delayed rectifying K + current in the heart, also colocalize in the luminal membrane of proximal tubule in mouse kidney. Micropuncture experiments revealed a reduced K + concentration in late proximal and early distal tubular fluid as well as a reduced K + delivery to these sites in KCNE1 knockout (-/-), compared with wild-type (+/+) mice. These observations would be consistent with KCNE1-dependent K + fluxes to the lumen in proximal tubule. Electrophysiological studies in isolated perfused proximal tubules indicated that this K + flux is essential to counteract membrane depolarization due to electrogenic Na + -coupled transport of glucose or amino acids. Clearance studies revealed an enhanced fractional urinary excretion of fluid, Na + , Cl - , and glucose in KCNE1 -/- compared with KCNE1 +/+ mice that may relate to an attenuated transport in proximal tubule and contribute to volume depletion in these mice, as indicated by higher hematocrit values.

Sulfide oxidation coupled to ATP synthesis in chicken liver mitochondria.

Abstract: Chicken liver mitochondria consumed O2 at an accelerated rate when supplied with low concentrations of hydrogen sulfide. Maximum respiration occurred in 10 μM sulfide, and continued more slowly up to concentrations as high as 60 μM. Sulfide oxidation was coupled to adenosine triphosphate (ATP) synthesis, as shown by firefly luciferase luminescence and by measurement of the mitochondrial membrane electrochemical gradient. Synthesis of ATP required low, steady-state concentrations of sulfide (<5 μM), which were maintained by use of a syringe pump. The ratio of consumed O2 to sulfide changed at low sulfide and O2 concentrations, indicating alternative metabolic reactions and products. In low concentrations of sulfide, presumably most similar to physiological, the O2/sulfide ratio was 0.75. This is the first report of sulfide oxidation linked to ATP synthesis in any organism not specifically adapted to a sulfide-rich environment. We suggest that this may be a widespread mitochondrial trait, and that it is consistent with the hypothesis that mitochondria originated from sulfide-oxidizing symbionts.

In vivo temperature dependence of cyclic and pseudocyclic electron transport in barley.

Abstract: The effect of temperature on the rate of electron transfer through photosystems I and II (PSI and PSII) was investigated in leaves of barley (Hordeum vulgare L.). Measurements of PSI and PSII photochemistry were made in 21% O2 and in 2% O2, to limit electron transport to O2 in the Mehler reaction. Measurements were made in the presence of saturating CO2 concentrations to suppress photorespiration. It was observed that the O2 dependency of PSII electron transport is highly temperature dependent. At 10 degrees C, the quantum yield of PSII (phi PSII) was insensitive to O2 concentration, indicating that there was no Mehler reaction operating. At high temperatures (> 25 degrees C) a substantial reduction in phi PSII was observed when the O2 concentration was reduced. However, under the same conditions, there was no effect of O2 concentration on the delta pH-dependent process of non-photochemical quenching. The rate of electron transport through PSI was also found to be independent of O2 concentration across the temperature range. We conclude that the Mehler reaction is not important in maintaining a thylakoid proton gradient that is capable of controlling PSII activity, and present evidence that cyclic electron transport around PSI acts to maintain membrane energisation at low temperature.

Identification of essential charged residues in transmembrane segments of the multidrug transporter MexB of Pseudomonas aeruginosa.

Abstract: The MexABM efflux pump exports structurally diverse xenobiotics, utilizing the proton electrochemical gradient to confer drug resistance on Pseudomonas aeruginosa. The MexB subunit traverses the inner membrane 12 times and has two, two, and one charged residues in putative transmembrane segments 2 (TMS-2), TMS-4, and TMS-10, respectively. All five residues were mutated, and MexB function was evaluated by determining the MICs of antibiotics and fluorescent dye efflux. Replacement of Lys342 with Ala, Arg, or Glu and Glu346 with Ala, Gln, or Asp in TMS-2 did not have a discernible effect. Ala, Asn, or Lys substitution for Asp407 in TMS-4, which is well conserved, led to loss of activity. Moreover, a mutant with Glu in place of Asp407 exhibited only marginal function, suggesting that the length of the side chain at this position is important. The only replacements for Asp408 in TMS-4 or Lys939 in TMS-10 that exhibited significant function were Glu and Arg, respectively, suggesting that the native charge at these positions is required. In addition, double neutral mutants or mutants in which the charged residues Asp407 and Lys939 or Asp408 and Lys939 were interchanged completely lost function. An Asp408→Glu/Lys939→Arg mutant retained significant activity, while an Asp407→Glu/Lys939→Arg mutant exhibited only marginal function. An Asp407→Glu/Asp408→Glu double mutant also lost activity, but significant function was restored by replacing Lys939 with Arg (Asp407→Glu/Asp408→Glu/Lys939→Arg). Taken as a whole, the findings indicate that Asp407, Asp408, and Lys939 are functionally important and raise the possibility that Asp407, Asp408, and Lys939 may form a charge network between TMS-4 and TMS-10 that is important for proton translocation and/or energy coupling.

Cytochrome b6f mutation specifically affects thermal dissipation of absorbed light energy in Arabidopsis

Abstract: Summary Light-induced lumenal acidification controls the efficiency of light harvesting by inducing thermal dissipation of excess absorbed light energy in photosystem II. We isolated an Arabidopsis mutant, pgr1 (proton gradient regulation), entirely lacking thermal dissipation, which was observed as little non-photochemical quenching of chlorophyll fluorescence. Map-based cloning showed that pgr1 had a point mutation in petC encoding the Rieske subunit of the cytochrome b6f complex. Although the electron transport rate was not affected at low light intensity, it was significantly restricted at high light intensity in pgr1, indicating that the lumenal acidification was not sufficient to induce thermal dissipation. This view was supported by (i) slow de-epoxidation of violaXanthin, which is closely related to lumenal acidification, and (ii) reduced 9-aminoacridine fluorescence quenching. Although lumenal acidification was insufficient to induce thermal dissipation, growth rate was not affected under low light growth conditions in pgr1. These results suggest that thermal dissipation is precisely regulated by lumenal pH to maintain maximum photosynthetic activity. We showed that pgr1 was sensitive to changes in light conditions, demonstrating that maximum activity of the cytochrome b6f complex is indispensable for short-term acclimation.

Ion fluxes considered in terms of membrane-surface electrical potentials

Abstract: Ions transported through plasma membranes encounter electrical charges, and associated electrical potentials, at the membrane surfaces. The ionic composition of the tissue-bathing medium influences both the surface charge density and the surface electrical potential. Changes in surface electrical potential may affect ion transport by altering two components of the chemical potential difference (Δµj ) of an ion through the membrane. First, the surface activity of the transported ion will change because of electrostatic attraction or repulsion. Second, the surface-to-surface transmembrane potential difference will change. (This is different from the bulk-phase-to-bulk-phase transmembrane potential difference measured with microelectrodes.) These changes in the components of the chemical potential may change the flux of an ion through the membrane even if the surface-to-surface Δµj (equal to the bulk-phase-to-bulk-phase Δµj ) remains constant. The Goldman-Hodgkin-Katz (GHK) flux equation does not take into account these surface-potential effects. The equation has been modified to incorporate surface potentials computed by a Gouy-Chapman-Stern model and surface ion activities computed by Nernst equations. The modified equation (despite several additional deficiencies of the GHK model) successfully predicts many transport phenomena not predicted by the standard GHK equation. Thus electrostatic effects may account for saturation, cis- and trans-inhibition, rectification, voltage gating, shifts in voltage optima, and other phenomena also attributable to other mechanisms.

Na+-to-sugar stoichiometry of SGLT3.

Abstract: Sodium-glucose cotransporters (SGLTs) mediate active transport of sugar across cell membranes coupled to Na+, by using the electrochemical gradient as a driving force. In the kidney, there is evide...

External alternative NADH:ubiquinone oxidoreductase redirected to the internal face of the mitochondrial inner membrane rescues complex I deficiency in Yarrowia lipolytica

Abstract: Alternative NADH:ubiquinone oxidoreductases are single subunit enzymes capable of transferring electrons from NADH to ubiquinone without contributing to the proton gradient across the respiratory membrane. The obligately aerobic yeast Yarrowia lipolytica has only one such enzyme, encoded by the NDH2 gene and located on the external face of the mitochondrial inner membrane. In sharp contrast to ndh2 deletions, deficiencies in nuclear genes for central subunits of proton pumping NADH:ubiquinone oxidoreductases (complex I) are lethal. We have redirected NDH2 to the internal face of the mitochondrial inner membrane by N-terminally attaching the mitochondrial targeting sequence of NUAM, the largest subunit of complex I. Lethality of complex I mutations was rescued by the internal, but not the external version of alternative NADH:ubiquinone oxidoreductase. Internal NDH2 also permitted growth in the presence of complex I inhibitors such as 2-decyl-4-quinazolinyl amine (DQA). Functional expression of NDH2 on both sides of the mitochondrial inner membrane indicates that alternative NADH:ubiquinone oxidoreductase requires no additional components for catalytic activity. Our findings also demonstrate that shuttle mechanisms for the transfer of redox equivalents from the matrix to the cytosolic side of the mitochondrial inner membrane are insufficient in Y. lipolytica.

The electric field generated by photosynthetic reaction center induces rapid reversed electron transfer in the bc1 complex.

Abstract: The cytochrome bc1 complex is the central enzyme of respiratory and photosynthetic electron-transfer chains. It couples the redox work of quinol oxidation and cytochrome reduction to the generation of a proton gradient needed for ATP synthesis. When the quinone processing Qi- and Qo-sites of the complex are inhibited by both antimycin and myxothiazol, the flash-induced kinetics of the b-heme chain, which transfers electrons between these sites, are also expected to be inhibited. However, we have observed in Rhodobacter sphaeroides chromatophores, that when a fraction of heme bH is reduced, flash excitation induces fast (half-time ∼0.1 ms) oxidation of heme bH, even in the presence of antimycin and myxothiazol. The sensitivity of this oxidation to ionophores and uncouplers, and the absence of any delay in the onset of this reaction, indicates that it is due to a reversal of electron transfer between bL and bH hemes, driven by the electrical field generated by the photosynthetic reaction center. In the pres...

Oscillations in proton transport revealed from simultaneous measurements of net current and net proton fluxes from isolated root protoplasts: MIFE meets patch-clamp

Abstract: Proton fluxes were measured non-invasively on patch-clamped protoplasts isolated from wheat roots using an external H + electrode to measure the electrochemical gradient in the external solution. Under voltage clamp in the whole-cell configuration, the H + fluxes across the plasma membrane could be measured as a function of voltage and time and correlated with the simultaneous measurements of membrane current. Protoplasts could exist in three states based on the current–voltage (I–V) curves and the flux–V curves. In the pump-state where the membrane voltage (Vm) was more negative than the electrochemical equilibrium potential for potassium (E K ), a net efflux of H + occurred that was voltage-dependent such that the efflux increased as Vm was clamped more positive. In the K-state, where Vm was close to E K , similar flux–V curves were observed. In the depolarised state where Vm was greater than E K the proton flux was characterised by a net influx of H + (H + -influx state) that reversed direction at more positive values of Vm. The inhibitory effect of DCCD and stimulatory effect of fusicoccin were used to correlate current and H + flux through the H + -ATPase for which there was reasonably good agreement within the limits of the flux measurements. Some protoplasts were kept in the whole-cell configuration for up to 3 h revealing slow sustained oscillations (period about 40 min) in H + flux that were in phase with oscillations in free-running Vm. These oscillations were also observed under voltage clamp, with membrane current in phase with H + flux, but which became damped out after a few cycles. The oscillations encompassed the pump-state, K + -state and H + -influx-state. The H +- flux–V curves and I–V curves were used to model the electrical characteristics of the plasma membrane with H + -ATPase, inward and outward K + rectifiers, a linear conductance, and a passive H + influx possibly through gated proton channels.

Extraction and separation of metal cations in solution by supported liquid membrane using lasalocid A as carrier

Abstract: Flat-sheet-supported liquid membranes incorporating lasalocid A (a natural ionophore), were previously shown to be permeable to metal cations (Cd 2 + and Zn 2 + ) against a proton gradient (ΔpH), which is the driving forceof the process [1]. This transport process has been extended to other metal species such as Pb 2 + , Na + , and Ag + and also to the case where two metal species compete for transport. A higher transport flux for Pb 2 + as compared to Cd 2 + and Zn 2 + is observed and partly explained by a higher rate of interfacial complexation owing to the smaller hydration shell of this species. This effect is confirmed by the data obtained with Ag + . However, the size of the metal cation in relation to its hydration shell does not appear as the major parameter to take into account for an estimate of the trans-membrane transport efficiency, as the Na + ions escape to this behavior, which has been considered until now as a general trend for metal cation ionophores.

Origin of Resting Membrane Potentials

Abstract: Most of the factors that determine or influence the resting E m of cells are discussed in this chapter. The structural and chemical composition of the cell membrane is briefly examined and correlated with the resistive and capacitative properties of the membrane. The factors that determine the intracellular ion concentrations in cells are examined. These factors include the Na + -K + -coupled pump , the Ca 2+ -Na + exchange reaction and the sarcolemmal Ca 2+ pump . The Na + -K + pump enzyme, Na + ,K + -ATPase, requires both Na + and K + for activity and transports three Na + ions outward and usually two K + ions inward per ATP hydrolyzed. Cardiac glycosides are specific blockers of this transport ATPase. The Na + -K + pump, if electroneutral, is not directly related to excitability, but only indirectly related by its role in maintaining the Na + and K + concentration gradients.

The cultured branchial epithelium of the rainbow trout as a model for diffusive fluxes of ammonia across the fish gill.

Abstract: A novel branchial epithelial preparation grown in L-15 medium in culture was used as a model system for understanding the diffusion of ammonia across the gills of the rainbow trout Oncorhynchus mykiss. The epithelium is known to contain both respiratory and mitochondria-rich cells in the approximate proportion in which they occur in vivo and to exhibit diffusive fluxes of Na+ and Cl- similar to in vivo values, but does not exhibit active apical-to-basolateral transport of Na+. Transepithelial resistance and paracellular permeability are also known to increase when the apical medium is changed from L-15 medium (symmetrical conditions) to fresh water (asymmetrical conditions). In the present study, net basolateral-to-apical ammonia fluxes increased as basolateral total ammonia concentration, basolateral-to-apical pH gradients and basolateral-to-apical P(NH(3)) gradients were experimentally increased and were greater under asymmetrical than under symmetrical conditions. The slope of the relationship between ammonia flux and P(NH(3)) gradient (i.e. NH(3) permeability) was the same under both conditions and similar to values for other epithelia. The higher fluxes under asymmetrical conditions were explained by an apparent diffusive flux of NH(4)+ that was linearly correlated with transepithelial conductance and was probably explained by the higher electrochemical gradient and higher paracellular permeability when fresh water was present on the apical surface. In this situation, NH(4)(+) diffusion was greater than NH(3) diffusion under conditions representative of in vivo values, but overall fluxes amounted to only approximately 20% of those in vivo. These results suggest that branchial ammonia excretion in the intact animal is unlikely to be explained by diffusion alone and, therefore, that carrier-mediated transport may play an important role.

Pancreatic ductal bicarbonate secretion: past, present and future.

Abstract: The pancreatic duct epithelium in the guinea-pig and many other species secretes HCO3 - at concentrations approaching 150 mM. This cannot be explained by conventional models based upon HCO3 - secretion via an anion exchanger at the luminal membrane because: 1) under these conditions, the Cl- and HCO3 - concentration gradients would favour HCO3 - reabsorption rather than secretion, and 2) the luminal anion exchanger appears to be inhibited by luminal HCO3 - concentrations of 125 mM or more. There may, however, be a sufficiently large electrochemical gradient to drive HCO3 - secretion across the luminal membrane via an anion conductance. In contrast to earlier studies on rat ducts, the membrane potential Em in guinea-pig duct cells does not depolarise appreciably upon stimulation with secretagogues but remains constant at about – 60 mV. Consequently, even with 125 mM or more HCO3 - in the lumen and an estimated 20 mM in the cytoplasm, the electrochemical gradient for HCO3 - will still favour secretion to the lumen. Under the same conditions, the intracellular Cl- concentration drops to very low levels (approximately 7 mM) presumably because, although Cl- may leave freely through the cystic fibrosis transmembrane conductance regulator (CFTR) channels in the luminal membrane, there is no major pathway for Cl- uptake across the basolateral membrane. Consequently a HCO3 - -rich secretion may arise as a result of the lack of competition from intracellular Cl- for efflux via the anion conductances at the luminal membrane. Whether CFTR, or another anion conductance, provides such a pathway for HCO3 - remains to be seen.

Effect of temperature on the luminescent characteristics in leaves of arabidopsis mutants with decreased unsaturation of the membrane lipids

Abstract: Summary. The luminescent characteristics of Arabidopsis wild type and two mutants, JB67 and LK3, deficient in lipid fatty acid desaturation are investigated as a function of temperature. The data reveal that lipid unsaturation exerts stronger effect on the delayed compared with on the prompt fluorescence. Some differences in the form of the induction curvcs of the delayed fluorescence and the kinetics of the delayed fluorescence dark relaxation between the wild type and the mutants were found. They are attributed to acceleration of the linear electron transfer, earlier activation of PSI and faster lightinduced generation of proton gradient in the mutants, particularly in LK3, as compared to the wild type. The temperature course of the fluorescence stationary level indicates higher thermal stability of the Arabidopsis mutants than that of the wild type. The splitting of the slow phase of the induction curves of DF into two maxima at low temperature in LK3 mutant suggests that the mutation renders structural rearrangements similar to those induced by heat treatment of the wild type. The importance of the membrane lipids unsaturation for the structural organization and photosynthetic activity of thylakoid membranes is discussed.

Coupled Interactions among Solute Diffusions, Membrane Surface Potentials, and Opposing Enzyme Reactions as a Mechanism for Active Transports Performed with Biomimetic Membranes

Abstract: The present theoretical analysis demonstrates that the membrane surface potential effect of a uniformly charged artificial membrane can be substituted by an electrical resistance term which balances the passive membrane resistance term. Depending on the sign of this electrical resistance term, the global membrane resistance for a charged solute may be either enhanced or lowered as compared to the passive membrane resistance term. By itself, such a membrane charge effect cannot lead to active transport phenomena. However, the combined effects between two opposite enzymic reactions acting on both parts of a uniformly charged porous membrane and in unstirred layers with the membrane surface potentials lead to the concept of biomimetic membranes able to specifically transport a molecule against its chemical gradient.

Method of improving biomass yield of lactic acid bacterial cultures

Abstract: A method of enhancing biomass yield of a lactic acid bacterial species cell culture, comprising cultivating the cells in a process comprising the steps of providing conditions that results in a reduced glycolytic flux and providing conditions that enable the cells to have, under aerobic conditions, a respiratory metabolism. The increased yield of biomass may be the result of an increased yield of ATP which can be obtained by activating the native ATP synthase activity of the H+-ATPase complex by lowering the ATP/ADP ratio, e.g. by carbon source limitation, and/or by increasing the proton gradient (membrane potential) of the cells, e.g. by enhancing or establishing an electron transport chain which can be achieved by enhancing expression of dehydrogenases or electron transport chain components, by adding to the medium a quinone or porphyrin compound or by enhancing the expression of the H+-ATPase activity.

A Na+-dependent D-mannose transporter in the apical membrane of chicken small intestine epithelial cells.

Abstract: The presence of a Na+/D-mannose cotransporter in brush-border membrane vesicles (BBMV) isolated from chicken small intestine was examined. In the presence of an electrochemical gradient for Na+, but not in its absence, D-mannose was accumulated transiently by the BBMV. D-Mannose uptake into the BBMV was energized by both the membrane potential and the chemical gradient for Na+. The relationship between D-mannose transport and external D-mannose concentration was described by an equation that represented the superposition of a saturable component (Michaelis-Menten constant Km 12.5 µM) and another component unsaturatable up to 80 µM D-mannose. D-Mannose uptake was inhibited by various substances in the following order of potency: D-mannose>>D-glucose>phlorizin>phloretin>D-fructose. For the uptake of α-methyl-glucopyranoside the order was D-glucose=phlorizin>>phloretin=D-fructose=D-mannose. The initial rate of D-mannose uptake increased as the extravesicular [Na+] increased, with a Hill coefficient of 1, suggesting that the Na+:D-mannose cotransport stoichiometry is 1:1. It is concluded that the intestinal apical membrane has a saturable, electrogenic and concentration- and Na+-dependent mannose transport mechanism that differs from the sodium-dependent glucose transporter SGLT1.

Ca2+ Sensitivity of Synaptic Vesicle Dopamine, γ-Aminobutyric Acid, and Glutamate Transport Systems

Abstract: 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 μM 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 μM Zn2+ that, like Ca2+, dissipates the proton concentration gradient by exchanging with H+. With respect to γ-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 γ-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.

Cl(-)-ATPases: Novel primary active transporters in biology.

Abstract: Five widely documented mechanisms of chloride transport across plasma membranes are anion-coupled antiport, sodium and hydrogen-coupled symport, Cl(-)channels, and an electrochemical coupling process. No genetic evidence has yet been provided for primary active chloride transport despite numerous reports of cellular Cl(-)-stimulated ATPases co-existing, in the same tissue, with uphill chloride transport that could not be accounted for by the five common chloride transport processes. Cl(-)-stimulated ATPase activity is a common property of practically all biological cells with the major location being of mitochondrial origin. It also appears that plasma membranes are sites of Cl(-)-stimulated ATPase activity. Recent studies of Cl(-)-stimulated ATPase activity and active chloride transport in the same membrane system, including liposomes, suggest a medication by the ATPase in net movement of chloride up its electrochemical gradient across plasma membranes. Further studies, especially from a molecular biological perspective, are required to confirm a direct transport role to plasma membrane-localized Cl(-)-stimulated ATPases. J. Exp. Zool. 289:215-223, 2001.