A non-equilibrium thermodynamics analysis of active transport within the framework of the chemiosotic theory.

The proton circuit devised by Mitchell in the chemiosmotic theory was subjected to analysis using the formalism of irreversible thermodynamics. The phenomenological coefficients and the degree of coupling relating co-permeant flows were derived from anion/H+, substrate/H+, cation/H+ and anion/anion biporter models. Linearity and equality of the cross-coefficients in Onsager relations were always satisfied. Macroscopic flows leading to charges splitting, such as oxido-reduction, hydro-dehydratation and transhydrogenase, are driven by a composite thermodynamic force which includes the proton-motive component. Multiple coupling occurs in the circuit when it is assumed that the net inward flux of protons becomes zero, i.e. when the circulation of protons reaches a stationary state. Under these conditions, oxidative phosphorylation, ATPase- or respiration-linked transhydrogenase and uptake of anion or cation against their electrochemical gradient may be predicted, in agreement with known experimental evidence.     

Cation-Anion Balance during Potassium and Sodium Absorption by Barley Roots

Steady-state rates of potassium ion and sodium ion absorption by excised barley roots accompanied by various anions were compared with the rates of anion absorption and the concomitant H⁺ and base release by the roots. The cation absorption rates were found to be independent of the identities, concentrations, and rates of absorption of the anions of the external solution, including bicarbonate. Absorption of the anion of the salt plus bicarbonate could not account for the cation absorption. H⁺ is released during cation absorption and base during anion absorption. The magnitude by which one or the other predominates depends on the relative rates of anion and cation absorption under various conditions of pH, cation and anion concentration, and inhibitor concentrations. The conclusion is that potassium and sodium ions are absorbed independently of the anions of the absorption solution in exchange for H⁺, while anions are exchanged for a base. The H⁺ release reflects a specificity between K⁺ and Na⁺ absorption such that it appears to be H⁺ exchanged in the specific rate-limiting reactions of the cation absorption.     

Pyridine nucleotide transhydrogenases of parasitic helminths.

Mitochondria from the parasitic helminth, Hymenolepis diminuta, catalyzed both NADPH:NAD⁺ and NADH:NADP⁺ transhydrogenase reactions which were demonstrable employing the appropriate acetylpyridine nucleotide derivative as the hydride ion acceptor. Thionicotinamide NAD⁺ would not serve as the oxidant in the former reaction. Under the assay conditions employed, neither reaction was energy linked, and the NADPH:NAD⁺ system was approximately five times more active than the NADH:NADP⁺ system. The NADH:NADP⁺ reaction was inhibited by phosphate and imidazole buffers, EDTA, and adenyl nucleotides, while the NADPH:NAD⁺ reaction was inhibited only slightly by imidazole and unaffected by EDTA and adenyl nucleotides. Enzyme coupling techniques revealed that both transhydrogenase systems functioned when the appropriate physiological pyridine nucleotide was the hydride ion acceptor. An NADH:NAD⁺ transhydrogenase system, which was unaffected by EDTA, or adenyl nucleotides, also was demonstrable in the mitochondria of H. diminuta. Saturation kinetics indicated that the NADH:NAD⁺ reaction was the product of an independent enzyme system. Mitochondria derived from another parasitic helminth, Ascaris lumbricoides, catalyzed only a single transhydrogenase reaction, i.e., the NADH:NAD⁺ activity. Transhydrogenase systems from both parasites were essentially membrane bound and localized on the inner mitochondrial membrane. Physiologically, the NADPH:NAD⁺ transhydrogenase of H. diminuta may serve to couple the intramitochondrial metabolism of malate (via an NADP linked “malic” enzyme) to the anaerobic NADH-dependent ATP-generating fumarate reductase system. In A. lumbricoides, where the intramitochondrial metabolism of malate depends on an NAD-linked “malic” enzyme which is localized primarily in the intermembrane space, the NADH:NAD⁺ transhydrogenase activity may serve physiologically in the translocation of hydride ions across the inner membrane to the anaerobic energy-generating fumarate reductase system.     

The possible role of redox-associated protons in growth of plant cells

The protons excreted by plant cells may arise by two different mechanisms: (1) by the action of the plasma membrane H⁺-ATPase and (2) by plasma membrane redox reactions. The exact proportion from each source is not known, but the plasma membrane H⁺-ATPase is, by far, the major contributor to proton efflux. There is still some question of whether the redox-associated protons produced by NADH oxidation on the inner side of the plasma membrane traverse the membrane in a 1 : 1 relationship with electrons generated in the redox reactions. Membrane depolarization observed in the presence of ferricyanide reduction by plasma membranes of whole cells or tissues or the lag period between ferricyanide reduction and medium acidification argue that only scalar protons may be involved. The other major argument against tight coupling between protons and electrons involves the concept of strong charge compensation. When ferricyanide is reduced to ferrocyanide on the outside of cells or tissues, an extra negative charge arises, which is compensated for by the release of H⁺ or K⁺, so that the total ratio of increased H⁺ plus K⁺ equals the electrons transferred by transmembrane electron transport. These are strong arguments against a tight coupling between electrons and protons excreted by the plasma membrane. On the other hand, there is no question that inhibitor studies provide evidence for two mechanisms of proton generation by plasma membranes. When the H⁺-ATPase activity is totally inhibited, the addition of ferricyanide induces a burst of extra proton excretion, orvice versa, when plasma membrane redox reactions are inhibited, the H⁺-ATPase can function normally. Since plasma membrane redox reactions and associated H⁺ excretion are related to growth, it is possible that in plants the ATPase-generated protons have a different function from redox-associated protons. The H⁺-ATPase-generated protons have been considered for many years to be necessary for cell wall expansion, allowing elongation to take place. A special function of the redox-generated protons may be in initiating proliferative cell growth, based on the presence of a hormone-stimulated NADH oxidase in membranes of soybean hypocotyls and stimulation of root growth by low concentrations of oxidants. Here we propose that this NADH oxidase and the redox protons released by its action control growth. The mechanism for this may be the evolution of protons into a special membrane domain, from which a signal to initiate cell proliferation may originate, independent of the action of the H⁺-ATPase-generated protons. It is also possible that both expansion and proliferative growth are controlled by redox-generated protons.     

A study on the mechanism of energy coupling in the redox chain

The present study demonstrates that the mitochondrial respiratory chain includes not three but four energy coupling sites, the fourth site being localized at the NADPH→NAD⁺ step.

1. The NADPH→NAD⁺-directed transhydrogenase reaction in sonicated beef heart submitochondrial particles energizes the particle membrane as judged by two membrane potential probes, i.e. uptake of a penetrating anion, phenyldicarbaundecaborane (PCB^?), and enhancement of anilinonaphthalene sulfonate (ANS^?) fluorescence.
2. The reverse reaction (NADH→NADP⁺) is accompanied by the oppositely directed anion movement, i.e. PCB^? efflux.
3. Being insensitive to rotenone, antimycin, cyanide, and oligomycin, both the influx and efflux of PCB^? coupled with transhydrogenase reaction can be prevented or reversed by uncouplers.
4. Equalization of concentrations of the transhydrogenase substrates and products also prevents (or reverses) the PCB^? influx coupled with oxidation of NADPH by NAD⁺, as well as the PCB^? efflux coupled with reduction of NADP⁺ by NADH.
5. The transhydrogenase-linked PCB^? uptake depends linearly on the energy yield of the oxidation reaction calculated according to formula $$\Delta G = RTln\frac{{[NADPH] x [NAD^ + ]}}{{[NADP^ + ] x [NADH]^ \cdot }}$$ No threshold value of Δ was found. Measurable PCB^? transport was still observed at Δ≤0.5 kcal/mole NADPH oxidized.
6. Partial uncoupling of transhydrogenase reaction and PCB^? transport, induced by low concentrations ofp-trifluoromethoxycarbonylcyanide phenylhydrazone (FCCP), dinitrophenol, or by removing coupling factor F₁, results in the decrease of the slope of the straight line showing the PCB^? uptake as a function of Δ. Oligomycin improves the coupling in F₁-deprived particles, the slope being increased. Rutamycin, dicyclohexylcarbodiimide (DCCD) and reconstitution of particles with F₁, also increase the coupling.
7. In phosphorylating particles oxidizing succinate by O₂, both the energy-dependent NADH→NADP⁺ hydrogen transfer and PCB^? influx possess equal sensitivity to FCCP, which is lower than the sensitivity of oxidative phosphorylation. Similarly, the decrease in the succinate oxidation rate induced by malonate arrests first phosphorylation and then under higher malonate concentration, PCB^? influx. The rate of NADPH→NAD⁺ transhydrogenase reaction was found to be lower than the threshold value of rate of succinate oxidation, still coupled with phosphorylation. Respectively, the values of PCB^? uptake under transhydrogenase reaction are lower than those inherent in phosphorylating oxidation of succinate.

The conclusion is made that the NADPH→NAD⁺-directed transhydrogenase reaction generates the membrane potential of the same polarity as respiration and ATP hydrolysis but of a lower magnitude (“plus” inside particles; the forward hydrogen transfer). The NADH→NADP⁺-directed transhydrogenase reaction forms the membrane potential of the opposite polarity (“minus” inside particles; the reverse hydrogen transfer). Under conditions used, the transhydrogenase-produced membrane potential proves to be too low to support ATP synthesis (and, most probably, the synthesis of any other high-energy compound) maintaining, nevertheless, some electrophoretic ion fluxes. A conclusion is made that transhydrogenase forms a membrane potential with no high-energy intermediates involved.     

Relationship of Cation Influxes and Effluxes in Yeast

The Na⁺ efflux from Na⁺-rich yeast cells into a cation-free medium is largely balanced by the excretion of organic anions. In the presence of Rb⁺, K⁺, or high levels of H⁺ (pH 3–4), the Na⁺ efflux is increased and the organic anion excretion is suppressed so that stoichiometric cation exchanges occur. H⁺ participates in the exchanges, moving into or out of the cells depending on the external pH and on the concentration of external Rb⁺(K⁺). The total cation efflux is dependent on the external Rb⁺ concentration in a "saturation" relationship, but the individual cations in the efflux stream are not. The discrimination factor in the efflux pathway between H⁺ and Na⁺ is very large (of the order of 10,000), and between Na⁺ and K⁺ considerable (of the order of 50). For the latter pair, the recycling of K⁺ from the cell wall space is an important factor in the discrimination. In addition, the Na⁺ efflux as a function of Na⁺ content follows a sigmoidal curve so that the discrimination factor is increased at high levels of cellular Na⁺. Although the influx and efflux pathways behave as a tightly coupled system, the mechanism of coupling is not entirely clear. A single system with different cation specificities and kinetic behaviors on the inside and outside faces of the membrane could account for the data.     

Amiloride-sensitive Na+/H+ exchange stimulated by cellular acidification or shrinkage in red blood cells of the frog,Rana temporaria

Simultaneous net uptake of Na⁺ and net extrusion of H⁺, both inhibited by amiloride, could be stimulated in red blood cells of the frog, Rana temporaria, either by intracellular acidification or cellular shrinkage. Net transports of Na⁺ and H⁺ were transient, dying out after 10–20 min (20°C) when stimulated by intracellular acidification but developing more slowly and proceeding for more than 60 min (20°C) when stimulated by cellular shrinkage. Evidence is presented suggesting a coupling between the transports of Na⁺ and H⁺ with an exchange ratio of 1:1 Na⁺/H⁺ exchange, stimulated by intracellular acidification, was able to readjust intracellular pH also when operating in parallel to a fully working anion exchanger in CO₂/HCO ₃ ^- -buffered media. Inhibition of anion exchange resulted in reduced cellular net uptake of Na⁺.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonate - DMSO dimethylsulphoxide - IU international unit - pH _e extracellular pH - pH _i intracellular pH - RBC red blood cell     

Na/H and k/h antiport in root membrane vesicles isolated from the halophyte atriplex and the glycophyte cotton

Proton fluxes have been followed into and out of membrane vesicles isolated from the roots of the halophyte Atriplex nummularia and the glycophyte Gossypium hirsutum, with the aid of the ΔpH probe [¹⁴C]methylamine. Evidence is presented for the operation of Na⁺/H⁺ and K⁺/H⁺ antiporters in the membranes of both plants. Cation supply after a pH gradient has been set up across the vesicle membrane (either as a result of providing ATP to the H⁺-ATPase or by imposing an artificial pH gradient) brings about dissipation of the ΔpH, but does not depolarize the membrane potential as observed in similar experiments, but in the absence of Cl⁻, using the ΔΨ probe SCN⁻. Cation/H⁺ exchange is thus indicated. This exchange is not due to nonspecific electric coupling, nor to competition for anionic adsorption sites on the membrane, nor to inhibition of the H⁺-ATPase; coupling of the opposed cation and H⁺ fluxes by a membrane component is the most likely explanation. Saturation kinetics have been observed for both Na⁺/H⁺ and K⁺/H⁺ antiport in Atriplex. Moreover, additive effects are obtained when Na⁺ is supplied together with saturating concentrations of K⁺, and vice versa, suggesting that separate antiporters for Na⁺ and for K⁺ may be operating. In the case of both Atriplex and Gossypium evidence was obtained suggesting the presence of antiporters in both plasmalemma and tonoplast.     

Ionic processes underlying the decrease in osmotic potential of Chara L-cell fragments in dilute electrolyte solutions

Movements of ions are considered to be governed by the electroneutrality rule. Therefore, a cation moving across the cell membrane into the cell either passively or actively should move together with its counterion, an anion, in equal amounts of charge or in exchange for another cation inside the cell. This means that the net influx of the cation in question should be affected by the permeability of its counterion and/or another cation inside the cell. To examine osmotic and ionic regulation in Chara cells, cell fragments of Chara having a lower osmotic pressure than normal (L-cell fragments) were prepared. The L-cell fragments were individually put into various dilute electrolyte solutions and their osmotic potentials were measured with a turgor balance. Concentrations of K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl^?, NO^?₃. and SO^2?₄. in the external electrolyte solutions in which L-cells had been incubated were also analysed by ion chromatography. The results showed that in 0.5 mM KCL + 0.1 mM CaCl₂ solution, Chara L-cell fragments absorbed K⁺ and Cl^? to maintain electroneutrality and then regained their osmotic potential very rapidly. When the anion was Cl, the cation absorbed at the highest rate was K⁺ On the other hand, when the cation was K, the anion absorbed at the highest rate was Cl, Other ions Ca²⁺, SO^2?₄ and NO^?₃ showed much less permeability than K⁺ and Cl ^?for the Chara plasma membrane. The conclusion from these findings was that due to the constraint of electroneutral transport, the uptake rate of a salt into L-cells is limited by the permeability of the least permeable ion.     

Relation of ATPases in rat renal brush-border membranes to ATP-driven H+ secretion

Summary In the presence of inhibitors for mitochondrial H⁺-ATPase, (Na⁺+K⁺)- and Ca²⁺-ATPases, and alkaline phosphatase, sealed brush-border membrane vesicles hydrolyse externally added ATP demonstrating the existence of ATPases at the outside of the membrane (ecto-ATPases). These ATPases accept several nucleotides, are stimulated by Ca²⁺ and Mg²⁺, and are inhibited by N,N-dicyclohexylcarbodiimide (DCCD), but not by N-ethylmaleimide (NEM). They occur in both brushborder and basolateral membranes. Opening of brush-border membrane vesicles with Triton X-100 exposes ATPases located at the inside (cytosolic side) of the membrane. These detergent-exposed ATPases prefer ATP, are activated by Mg²⁺ and Mn²⁺, but not by Ca²⁺, and are inhibited by DCCD as well as by NEM. They are present in brush-border, but not in basolateral membranes. As measured by an intravesicularly trapped pH indicator, ATP-loaded brush-border membrane vesicles extrude protons by a DCCD- and NEM-sensitive pump. ATP-driven H⁺ secretion is electrogenic and requires either exit of a permeant anion (Cl^–) or entry of a cation, e.g., Na⁺ via electrogenic Na⁺/d-glucose and Na⁺/l-phenylalanine uptake. In the presence of Na⁺, ATP-driven H⁺ efflux is stimulated by blocking the Na⁺/H⁺ exchanger with amiloride. These data prove the coexistence of Na⁺-coupled substrate transporters, Na⁺/H⁺ exchanger, and an ATP-driven H⁺ pump in brush-border membrane vesicles. Similar location and inhibitor sensitivity reveal the identity of ATP-driven H⁺ pumps with (a part of) the DCCD- and NEM-sensitive ATPases at the cytosolic side of the brush-border membrane.     

Studies on the mechanism of K+ transport in yeast.

The effect of uncouplers and diffusible acids on K⁺ transport was studied in yeast.Although the K⁺ transport system seems to depend on ATP to function, the effects of uncouplers are not due primarily to its action on the energy conserving systems of the cell.Other uncouplers with different structures to that of DNP showed also an inhibitory effect on K⁺ transport, which agrees with their reported ability to conduct protons through membranes.Uncouplers, besides inhibiting K⁺ uptake, produce an efflux of this cation; however, the rate of efflux produced is quantitatively important only when the cells have previously taken up the cation; there seems to exist a mechanism which prevents the loss of cations by yeast.In the absence of substrate, at pH 8.5, with 0.5 m KCl, TCS produces the efflux of H⁺, and when ⁸⁶Rb⁺ was used as a substitute for K⁺, an increase of the entrance of the cation could be detected in the presence of the uncoupler. It seems that the effect of the uncoupler depends on the direction of the combined H⁺ and K⁺ gradients, or the electrochemical potential of the cell.As reported by other authors, weak diffusible acids increase the uptake of K⁺ by yeast, and this effect is not due to changes in the metabolism, but to the magnitude of the entrance of the molecules to the yeast cell.It was found that the efflux of the acids (H₂CO₃), on the other hand, can produce an efflux of K⁺, which means that anions are important not only for the entrance of the cations, but for its permanence within the cell as well.The data seem to be in agreement with the hypothesis of the existence of a proton pump, responsible for the creation of an electrochemical potential, involved in K⁺ transport. At low pH, this pump seems to be activated by the transport of K⁺ into the cell.     

Induction of Na+ / K+ exchange in swollen heart mitochondria

Heart mitochondria swollen passively in nitrate salts contract in a respiration-dependent reaction which can be attributed to an endogenous cation/H⁺ exchange component (or components). The rate of contraction increases with increased extent of passive swelling in both Na⁺ and K⁺ salts. Since nearly constant internal cation concentrations are maintained during osmotic swelling, this result suggests that both Na⁺/H⁺ and K⁺/H⁺ exchange is enhanced by increased matrix volume. Endogenous Mg²⁺ is also lost with increased matrix volume, and this observation, in conjunction with other evidence available in the literature, suggests that monovalent cation/H⁺ exchanges may be regulated by divalent cations. Passive exchange of Na⁺/K⁺,⁴²K⁺/K⁺, and²⁴Na⁺/Na⁺ can be readily demonstrated in mitochondria swollen in nitrate. All these exchanges are low or not detectable in unswollen control mitochondria, and it appears that they are manifestations of the activated cation/H⁺ component (or components) functioning in the absence of pH.     

Influence of the level of nitrate nutrition on ion uptake and assimilation, organic Acid accumulation, and cation-anion balance in whole tomato plants

Tomato plants (Lycopersicon esculentum L. var. Ailsa Craig) were grown in water culture in nutrient solution in a series of 10 increasing levels of nitrate nutrition. Using whole plant data derived from analytical and yield data of individual plant parts, the fate of anion charge arising from increased NO₃ assimilation was followed in its distribution between organic anion accumulation in the plant and OH⁻ efflux into the nutrient solution as calculated by excess anion over cation uptake. With increasing NO₃ nutrition the bulk of the anion charge appeared as organic anion accumulation in the plants. OH⁻ efflux at a maximum accounted for only 20% of the anion charge shift. The major organic anion accumulated in response to nitrate assimilation was malate. The increase in organic anion accumulation was paralleled by an increase in cation concentration (K⁺, Ca²⁺, Mg²⁺, Na⁺). Total inorganic anion levels (NO₃⁻, SO₄²⁻, H₂PO₄⁻, Cl⁻) were relatively constant. The effect of increasing NO₃ nutrition in stimulating organic anion accumulation was much more pronounced in the tops than in the roots.     

Modification of bovine heart mitochondrial transhydrogenase with tetranitromethane

Modification of pyridine dinucleotide transhydrogenase with tetranitromethane resulted in inhibition of its activity. Development of a membrane potential in submitochondrial particles during the reduction of 3-acetylpyridine adenine dinucleotide (AcPyAD⁺) by NADPH decreased to nearly the same extent as the transhydrogenase rate on tetranitromethane treatment of the membrane. Kinetics of the inactivation of homogeneous transhydrogenase and the enzyme reconstituted into phosphatidylcholine liposomes indicate that a single essential residue was modified per active monomer. NADP⁺, NADPH and NADH gave substantial protection against tetranitromethane inactivation of both the nonenergy-linked and energy-linked transhydrogenase reactions of submitochondrial particles and the NADPH → AcPyAD⁺ reaction of reconstituted enzyme. NAD⁺ had no effect on inactivation. Tetranitromethane modification of reconstituted transhydrogenase resulted in a decrease in the rate of coupled H⁺ translocation that was comparable to the decrease in the rate of NADPH → AcPyAD⁺ transhydrogenation. It is concluded that tetranitromethane modification controls the H⁺ translocation process solely through its effect on catalytic activity, rather than through alteration of a separate H⁺-binding domain. Nitrotyrosine was not found in tetranitromethane-treated transhydrogenase. Both 5,5′-dithiobis(2-nitrobenzoate)-accessible and buried sulfhydryl groups were modified with tetranitromethane. NADH and NADPH prevented sulfhydryl reactivity toward tetranitromethane. These data indicate that the inhibition seen with tetranitromethane results from the modification of a cysteine residue.     

Active ion uptake and maintenance of cation-anion balance: A critical examination of their role in regulating rhizosphere pH

The processes responsible for maintenance of cation-anion balance in plants and their relation to active ion accumulation and changes in rhizosphere pH are outlined and discussed. The major processes involved are: (1) accumulation and degradation of organic acids which occur in the plant mainly as organic acid anions (and their transfer within the plant) and (2) extrusion of H⁺ or OH^– into the rhizosphere. The relative importance of the two processes is determined by the size of the excess anion or cation uptake. Indeed, plants typically absorb unequal quantities of nutritive cations (NH₄ ⁺+Ca²⁺+ Mg²⁺+K⁺+Na⁺) and anions (NO₃ ^–+Cl^–+SO₄ ^2–+H₂PO₄ ^–) and charge balance is maintained by excretion of an amount of H⁺ or OH^– which is stoichiometrically equal to the respective excess cation or anion uptake. The mechanisms and processes by which H⁺ and in particular OH^– ions are excreted in response to unequal cation-anion uptake are, however, poorly understood.The contemporary view is that primary active extrusion of H⁺, catalyzed by a membrane-located ATPase, is the major driving force for secondary transport of cations and anions across the plasma membrane. However, the fact that net OH^– extrusion often occurs (since excess anion absorption commonly takes place) implies there is a yet-to-be characterized OH^– ion efflux mechanism at the plasma membrane that is associated with anion uptake. There is, therefore, a need for future studies of the uptake mechanisms and stoichiometry of anion uptake; particularly that of NO₃ ^– which is often the predominant anion absorbed. Another related phenonenon which requires detailed study in terms of cation-anion balance is localized rhizosphere acidification which can occur in response to deficiencies of Fe and P.     

Characterization of native and reconstituted plasma membrane H+-ATPase from the plasma membrane ofBeta vulgaris

Summary Characteristics of the native and reconstituted H⁺-ATPase from the plasma membrane of red beet (Beta vulgaris L.) were examined. The partially purified, reconstituted H⁺-ATPase retained characteristics similar to those of the native plasma membrane H⁺-ATPase following reconstitution into proteoliposomes. ATPase activity and H⁺ transport of both enzymes were inhibited by vanadate, DCCD, DES and mersalyl. Slight inhibition of ATPase activity associated with native plasma membranes by oligomycin, azide, molybdate or NO ₃ ^– was eliminated during solubilization and reconstitution, indicating the loss of contaminating ATPase activities. Both native and reconstituted ATPase activities and H⁺ transport showed a pH optimum of 6.5, required a divalent cation (Co²⁺>Mg²⁺>Mn²⁺>Zn²⁺>Ca²⁺), and preferred ATP as substrate. The Mg:ATP kinetics of the two ATPase activities were similar, showing simple Michaelis-Menten kinetics. Saturation occurred between 3 and 5mM Mg: ATP, with aK _m of 0.33 and 0.46mM Mg: ATP for the native and reconstituted enzymes, respectively. The temperature optimum for the ATPase was shifted from 45 to 35°C following reconstitution. Both native and reconstituted H⁺-ATPases were stimulated by monovalent ions. Native plasma membrane H⁺-ATPase showed an order of cation preference of K⁺>NH ₄ ⁺ >Rb⁺>Na⁺>Cs⁺>Li⁺>choline⁺. This basic order was unchanged following reconstitution, with K⁺, NH ₄ ⁺ , Rb⁺ and Cs⁺ being the preferred cations. Both enzymes were also stimulated by anions although to a lesser degree. The order of anion preference differed between the two enzymes. Salt stimulation of ATPase activity was enhanced greatly following reconstitution. Stimulation by KCl was 26% for native ATPase activity, increasing to 228% for reconstituted ATPase activity. In terms of H⁺ transport, both enzymes required a cation such as K⁺ for maximal transport activity, but were stimulated preferentially by Cl^– even in the presence of valinomycin. This suggests that the stimulatory effect of anions on enzyme activity is not simply as a permeant anion, dissipating a positive interior membrane potential, but may involve a direct anion activation of the plasma membrane H⁺-ATPase.     

Two Open States with Progressive Proton Selectivities in the Branched Channelrhodopsin-2 Photocycle

Channelrhodopsins are light-gated ion channels that mediate vision in phototactic green algae like Chlamydomonas. In neurosciences, channelrhodopsins are widely used to light-trigger action potentials in transfected cells. All known channelrhodopsins preferentially conduct H⁺. Previous studies have indicated the existence of an early and a late conducting state within the channelrhodopsin photocycle. Here, we show that for channelrhodopsin-2 expressed in Xenopus oocytes and HEK cells, the two open states have different ion selectivities that cause changes in the channelrhodopsin-2 reversal voltage during a light pulse. An enzyme kinetic algorithm was applied to convert the reversal voltages in various ionic conditions to conductance ratios for H⁺ and divalent cations (Ca²⁺ and/or Mg²⁺), as compared to monovalent cations (Na⁺ and/or K⁺). Compared to monovalent cation conductance, the H⁺ conductance, α, is ∼3 × 10⁶ and the divalent cation conductance, β, is ∼0.01 in the early conducting state. In the stationary mixture of the early and late states, α is larger and β smaller, both by a factor of ∼2. The results suggest that the ionic basis of light perception in Chlamydomonas is relatively nonspecific in the beginning of a light pulse but becomes more selective for protons during longer light exposures.     

Leaching of solutes by the action of acidic rain: a comparison of efflux from twigs and single needles of Picea abies (L. Karst.)

Abstract. Leaching of inorganic cations (K⁺, Mg²⁺) and in some cases of inorganic anions and sugars from detached twigs and single needles of spruce Picea abies L. Karst.) in the presence of acid rain (H₂SO₄, 1 mol m^?3) or salt solutions (Na₂ SO₄, 1 mol m^?3) was examined under laboratory conditions. Cation leaching (as percentage of the total water soluble ion content of the tissue per hour) was: K⁺: 0.01-0.02%; Mg²⁺: 0.005-0.01%; Ca²⁺: 0.1-0.2%. Leaching rates of anions were even lower than that and concentrations in the leachate were often below the detection limit of anion chromatography. Spraying with H₂SO₄ (pH 2.95, 1 mol m^?3) increased leaching only transiently. Similar effects were found when Na₂SO₄ was used instead of H₂SO₄. The transiently enhanced leaching was apparently due to H⁺/cation or cation/cation exchange at the twig or leaf surfaces. Feeding of K⁺ or Al³⁺ through the stems increased leaching of all cations within a few hours, again demonstrating rapid ion exchange in the apoplast. Leaching of potassium and magnesium from single needles occurred at similar relative rates as from twigs. Loss of Ca²⁺ ions, however, was even smaller from needles than from twigs. Apparently, a large part of the Ca²⁺ lost from twigs originated from the bark and not from the needles. Efflux of ions from longitudinal needle sections was about 1000 times taster than the rates obtained with intact needles, indicating that the cuticle was the main barrier Preventing solute loss. In relation to the total amount of mineral nutrients in trees, leaching is considered to be too small to be the primary cause of damage to trees stressed by acid rain, as has been suggested in the literature.     

K+ and Cl− Conductance of Arabidopsis thaliana Plasma Membrane at Depolarized Voltages

The ion currents, activated by depolarizations, across the plasma membrane of Arabidopsis thaliana cultured cells were studied by means of the patch-clamp technique in the whole-cell configuration. The electrical conductance of the membrane could be shifted from a cation to an anion conducting state by changes in the [K⁺]: [Cl⁻] ratio in the external medium. For ratios between 1:1 and 1:5 the currents were due to K⁺ efflux and for a ratio of 1:10 to Cl⁻ influx. In the cation conducting state the permeability ratio of K⁺ over NH⁺₄ and the alkali metal ions was: K⁺ ≅ NH>Na⁺ ≅ Li⁺ >Cs⁺. In the anion conducting state the permeability of NO⁻₃ was the same as that of Cl⁻. These channels were activated by depolarizations in the range of physiological potentials (-70/-80mV) and, either by mediating the efflux of cations or the influx of anions, they could function to re-hyperpolarize the membrane potential after depolarizations due to the influx of cations or of solutes cotransported with protons and/or to the inhibition of electrogenic pumps.     

Ion modulation of membrane permeability: Effect of cations on intact cells and on cells and phospholipid bilayers treated with pore-forming agents

Summary Leakage of ions (Na⁺, K⁺) and phosphorylated metabolites (phosphorylcholine, 2-deoxyglucose 6-phosphate) through membrane lesions in intact cells or in cells modified by pore-forming agent has been studied. Leakage from intact cells isinduced by protons and by divalent cations such as Cu²⁺, Cd²⁺ or Zn²⁺. Leakage from agent-modified cells—or across phospholipid bilayers modified by agent—isprevented by low concentrations of the same cations and by higher concentrations of Ca²⁺, Mn²⁺ or Ba²⁺; Mg²⁺, dimethonium, spermine, or spermidine are virtually ineffective. The relative efficacy of a particular cation (e.g. Ca²⁺) depends more on cell type than on the nature of the pore-forming agent. The predominant effect is on binding of cation to specific sites, not on surface charge. Surface charge, on the other hand, does affect leakage from agent-modified cells in that suspension in nonionic media reduces leakage, which can be restored by increasing the ionic strength: univalent (Na⁺, K⁺, Rb⁺, NH ₄ ⁺ ) and divalent (Mg²⁺, dimethonium) cations are equally effective; addition of protons or divalent cations such as Zn²⁺ to this system inhibits leakage. From this and other evidence here presented it is concluded that leakage across membranes is modulated by the presence of endogenous anionic components: when these are in the ionized state, leakage is favored; when unionized (as a result of protonation) or chelated (by binding to divalent cation), leakage is prevented. It is suggested that such groups are exposed at the extracellular face of the plasma membrane.