Electrochemical Potential Of Na Research Articles

The Ammonia Transport, Retention and Futile Cycling Problem in Cyanobacteria

Ammonia is the preferred nitrogen source for many algae including the cyanobacterium Synechococcus elongatis (Synechococcus R-2; PCC 7942). Modelling ammonia uptake by cells is not straightforward because it exists in solution as NH(3) and NH (4) (+) . NH(3) is readily diffusible not only via the lipid bilayer but also through aquaporins and other more specific porins. On the other hand, NH (4) (+) requires cationic transporters to cross a membrane. Significant intracellular ammonia pools (≈1-10mol m(-3)) are essential for the synthesis of amino acids from ammonia. The most common model envisaged for how cells take up ammonia and use it as a nitrogen source is the "pump-leak model" where uptake occurs through a simple diffusion of NH(3) or through an energy-driven NH (4) (+) pump balancing a leak of NH(3) out of the cell. The flaw in such models is that cells maintain intracellular pools of ammonia much higher than predicted by such models. With caution, [(14)C]-methylamine can be used as an analogue tracer for ammonia and has been used to test various models of ammonia transport and metabolism. In this study, simple "proton trapping" accumulation by the diffusion of uncharged CH(3)NH(2) has been compared to systems where CH(3)NH (3) (+) is taken up through channels, driven by the membrane potential (ΔU (i,o)) or the electrochemical potential for Na(+) (ΔμNa (i,o) (+) ). No model can be reconciled with experimental data unless the permeability of CH(3)NH(2) across the cell membrane is asymmetric: permeability into the cell is very high through gated porins, whereas permeability out of the cell is very low (≈40nm s(-1)) and independent of the extracellular pH. The best model is a Na (in) (+) /CH(3)NH (3) (+) (in) co-porter driven by ΔμNa (i,o) (+) balancing synthesis of methylglutamine and a slow leak governed by Ficks law, and so there is significant futile cycling of methylamine across the cell membrane to maintain intracellular methylamine pools high enough for fixation by glutamine synthetase. The modified pump-leak model with asymmetric permeability of the uncharged form is a viable model for understanding ammonia uptake and retention in plants, free-living microbes and organisms in symbiotic relationships.

Aeromonas hydrophila motY is essential for polar flagellum function, and requires coordinate expression of motX and Pom proteins

By the analysis of the Aeromonas hydrophila ATCC7966(T) genome we identified A. hydrophila AH-3 MotY. A. hydrophila MotY, like MotX, is essential for the polar flagellum function energized by an electrochemical potential of Na(+) as coupling ion, but is not involved in lateral flagella function energized by the proton motive force. Thus, the A. hydrophila polar flagellum stator is a complex integrated by two essential proteins, MotX and MotY, which interact with one of two redundant pairs of proteins, PomAB and PomA(2)B(2). In an A. hydrophila motX mutant, polar flagellum motility is restored by motX complementation, but the ability of the A. hydrophila motY mutant to swim is not restored by introduction of the wild-type motY alone. However, its polar flagellum motility is restored when motX and -Y are expressed together from the same plasmid promoter. Finally, even though both the redundant A. hydrophila polar flagellum stators, PomAB and PomA(2)B(2), are energized by the Na(+) ion, they cannot be exchanged. Furthermore, Vibrio parahaemolyticus PomAB and Pseudomonas aeruginosa MotAB or MotCD are unable to restore swimming motility in A. hydrophila polar flagellum stator mutants.

The Involvement of Protein-Protein Interactions in the Mechanism of the Na+,K+-ATPase

The Na+,K+-ATPase (or sodium pump) was the first ion pump to be discovered (Skou, 1957) and it is one of the most fundametally important enzymes of animal physiology. The electrochemical potential for Na+, which the enzyme maintains, is used as the driving force for numerous secondary transport systems, e.g. voltage-sensitive Na+ channels in nerve. ATP provides the energy source to drive ion pumping. However, it also plays a crucial allosteric role, accelerating significantly the enzyme's rate determining E2-E1 transition and the associated release of K+ ions to the cytoplasm. Based on the results of stopped-flow kinetic experiments and recently published crystal structural data for the related enzyme, the sarcoplasmic reticulum Ca2+-ATPase, it is suggested that the allosteric role of ATP in the mechanism of the Na+,K+-ATPase can be explained by an ATP-induced closing of the cytoplasmic domains of the enzyme which relieves steric hindrance arising from interactions between neighbouring pump molecules within the native membrane environment and hence an acceleration of the E2-E1 conformational change (Clarke, 2009). In the presence of millimolar concentrations of ATP, therefore, it is proposed that the enzyme functions as a monomer (alpha-beta protomer), whereas at low ATP concentrations it functions as a dimer ((alpha-beta)2 diprotomer) or higher aggregate. The physiological advantage of protein-protein interactions is still unclear, but a possibility is that they may lead to an enhancement of the enzyme's ATP affinity and allow it to continue functioning even under hypoxic conditions.Skou JC. (1957) Biochimica et Biophysica Acta, 23: 394-401.Clarke RJ (2009) European Biophysics Journal, in press.

Na+-dependent HCO3-transport in the cyanobacteriumSynechocystisPCC6803

The effect of Na+on HCO3-transport, inorganic carbon (Ci) accumulation, and photosynthesis was investigated in the unicellular cyanobacterium Synechocystis sp. PCC6803 using the silicone fluid filtering centrifugation technique. Unlike other cyanobacteria, Synechocystis cells grown at low Ciin standing culture had little capacity for Na+-independent HCO3-transport, when assayed at pH 9.6. However, 25 mM NaCl, but not KCl, strongly promoted HCO3-transport and accumulation. Kinetic analysis indicated that the HCO3-concentration required for one half the maximum rate of transport, K0.5(HCO3-), decreased in the presence of Na+while the maximum rate of transport, VMAX, increased by up to 15-fold. Na+-dependent HCO3-transport occurred against an electrochemical potential of up to 24 kJ ·mol-1, indicating the involvement of carrier-mediated active transport. Li+(1-3 mM) partially substituted for Na+in that K0.5( HCO3-) values were similar (38 vs. 50 µM), but VMAXwas reduced by twofold. At higher concentrations, Li+counteracted the effects of Na+. Monensin reversibly inhibited Na+-dependent HCO3-transport and acted by reducing VMAXwithout affecting K0.5(HCO3-). Monensin inhibition suggested that the electrochemical potential for Na+may play a role in Na+-dependent HCO3-transport, possibly through an involvement in intracellular pH regulation during transport. Na+also stimulated photosynthetic C fixation and O2evolution and these effects were correlated with the Na+-dependent increase in intracellular Ciaccumulation. The Na+-requirement for photosynthesis could be relieved by the provision of CA to the cell suspension, in agreement with the proposal that Na+is required for transport and not directly involved in the photosynthetic process.Key words: active transport, CO2-concentrating mechanism, cyanobacteria, Na+-dependent HCO3-transport, photosynthesis, Synechocystis PCC6803.

Solubilization and reconstitution of high-and low-affinity Na +-dependent neutral l-α-amino acid transporters from rabbit small intestine

High- and low-affinity Na +-dependent neutral l-α-amino acid transporters were solubilized with 0.25% octaethylene glycol dodecyl ether (C 12E 8) after removal of the proteins from the brush-border membrane vesicles with 2% CHAPS and 4 M urea. When the CHAPS-insoluble protein was treated with papain before its solubilization with C 12E 8, a substantial amount of protein was removed without any decrease of the transport activities. The solubilized transporters were reconstituted into proteo-liposomes after removal of C 12E 8 with Bio-Beads SM2. Several parameters proved to be important for optimal reconstitution efficiency: (a) the type of detergent, and (b) the phospholipid/protein and detergent/protein ratio during reconstitution, and (c) the salt concentration during reconstitution. Reconstituted proteoliposomes showed rapid uptake of neutral l-α-amino acids but not imino acid, basic or acidic amino acids driven by an electrochemical potential of Na + (out > in). The uptakes under low-and high-substrate condition were further augmented by an artificial membrane potential introduced by K + diffusion via valinomycin (negative interior). Kinetic analysis revealed that both the brush-border membranes and the solubilized fraction involved two carrier-mediated pathways for alanine transport. The kinetic parameters were determined by curve fitting with a computer to be K n  0.28 mM (0.21 mM) and K t2  43.2 mM (28.4 mM), respectively (those with brush-border membrane vesicles in parentheses). Studies on the specific activities for transport of individual amino acids under low or high substrate concentration and the cross-inhibitory effects of various amino acids on alanine uptake (low concentration) revealed that these transporters possess broad specificity for neutral l-α-amino acids.

A novel mechanism of cation/substrate contransport: Na +/H +/adenosine cotransport in Vibrio parahaemolyticus

Adenosine is actively transported with Na+ in Vibrio parahaemolyticus (Sakai, Y., Tsuda, M., Tsuchiya, T. (1987) Biochim, Biophys. Acta 893, 43-48). The proton conductor carbonylcyanide m-chlorophenylhydrazone, CCCP, strongly inhibited active transport of adenosine at pH 8.5 as well as at pH 7.0. This seemed peculiar because the driving force, an electrochemical potential of Na+, is established by the Na(+)-extruding respiratory chain at pH 8.5 in this organism, although it is established by the function of the Na+/H+ antiporter at pH 7.0. This suggested that H+ might be involved in the adenosine transport. We detected H+ uptake induced by adenosine influx in V. parahaemolyticus cells in the presence of Na+, but not in its absence, suggesting the occurrence of Na+/H+/adenosine cotransport. We isolated formycin A-resistant mutants which showed defective adenosine transport. The mutation resulted in simultaneous losses of Na+ uptake and H+ uptake induced by adenosine. In revertants from these mutants the Na+ uptake and H+ uptake were restored simultaneously. The frequencies of reversion were in the order of 10(-7), indicating that the mutations were single mutations; namely that Na+/adenosine cotransport and H+/adenosine cotransport took place via the same carrier. Thus, we conclude that adenosine is transported by the novel mechanism of Na+/H+/adenosine cotransport in V. parahaemolyticus.

In vitro protein translocation into inverted membrane vesicles prepared from Vibrio alginolyticus is stimulated by the electrochemical potential of Na + in the presence of Escherichia coli SecA

A protein translocation system was reconstituted from inverted membrane vesicles prepared from Na + pump-possessing Vibrio alginolyticus and purified Escherichia coli SecA. The translocation required ATP and was stimulated by the functioning of the Na + pump, suggesting that the electrochemical potential of Na + but not that of H +, is important for protein translocation in Vibrio.

Partial purification of alanine carrier from rabbit small intestine brush border membrane and its functional reconstitution into proteoliposomes

An alanine transport carrier was partially purified from brush border membranes of rabbit small intestine. The alanine carrier activity was not solubilized with 0.4% deoxycholate but recovered in the detergent-insoluble fraction. The detergent-insoluble proteins were reconstituted into proteoliposomes with soybean phospholipids. The reconstituted proteoliposomes were capable of uptake of alanine driven by an electrochemical potential of Na+. The initial rate of alanine uptake into the proteoliposomes was 90 pmoles/mg protein/sec, which was 15-fold higher than that observed with the native membrane vesicles. The uptake of alanine was effectively suppressed by various neutral amino acids but not by either cationic or anionic amino acids.

Roles of the respiratory Na+ pump in bioenergetics of Vibrio alginolyticus.

Bioenergetic characteristics of Na+ pump-defective mutants of a marine bacterium Vibrio alginolyticus were compared with those of the wild type and revertant. Generation of membrane potential and motility at pH 8.5 in the mutants were completely inhibited by a proton conductor, carbonylcyanide m-chlorophenylhydrazone, whereas those in the wild type or revertant were resistant to the inhibitor. Motility and amino acid transport were driven by the electrochemical potential of Na+ not only in the wild type or revertant but also in the mutants. In the absence of the proton conductor, motility and amino acid transport of the mutants did not significantly differ from those of the wild type or revertant even at pH 8.5, where the Na+ pump has maximum activity. Therefore, the electrochemical potential of Na+ in the mutants seemed to be maintained at a normal level by a respiration-dependent H+ pump and Na+/H+ antiporter. On the other hand, growth of the mutants became defective as the medium pH increased, especially on minimal medium. These results indicate that the Na+ pump is an important energy-generating mechanism when nutrients are limited at alkaline pH.

Sucrose uptake is driven by the Na+ electrochemical potential in the marine bacterium Vibrio alginolyticus.

Na+ was found to be essential for the accumulation of sucrose by Vibrio alginolyticus. Sucrose uptake was completely inhibited by the addition of proton conductor at neutral pH, but not at alkaline pH, where the primary electrogenic Na+ pump generates the Na+ electrochemical gradient. We therefore conclude that sucrose transport is driven by the electrochemical potential of Na+ in this organism.

Role of ATP on the initial rate of amino acid uptake in ehrlich ascites cells

Incubation with graded doses of rotenone can bring about a graded lowering of the cellular ATP level. Using cells with varying ATP levels, it can be shown that the initial uptake of [ 14C]glycine, before the cellular concentration exceeds that of the medium, is decreased as ATP decreases. Alterations in cellular cations cannot account for the difference in glycine influx. Prolonged exposure of cells to a lowered ATP content increases the exodus of cellular glycine. Valinomycin reduces the steady-state level of glycine uptake, but its effects can to a major extent be overcome by the addition of glucose. At high extracellular K + (70 mM) neither the sum of the Na ++K + gradients, nor the electrochemical potential of Na + provides sufficient energy to account for glycine and 2-aminoisobutyrate accumulation if a 1:1 coupling between Na + and the amino acid occurs.