Intravesicular pH changes in submitochondrial particles induced by monovalent cations: relationship to the Na+/H+ and K+/H+ antiporters

The fluorescence of internalized fluorescein isothiocyanate dextran has been used to monitor the intravesicular pH of submitochondrial particles (SMP). Respiring SMP maintain a steady-state delta pH (interior acid) that results from the inwardly directed H+ flux of respiration and an opposing passive H+ leak. Addition of K+, Na+, or Li+ to SMP results in a shift to a more alkaline interior pH (pHi) in both respiring and nonrespiring SMP. The K+-dependent change in pHi, like the K+/H+ antiport in intact mitochondria, is inhibited by quinine and by dicyclohexylcarbodiimide. The Na+-dependent reaction is only partially inhibited by these reagents. Both the Na+- and the K+-dependent pH changes are sensitive to amiloride derivatives. The Km for both Na+ and K+ is near 20 mM whereas that for Li+ is closer to 10 mM. The K+/H+ exchange reaction is only slightly inhibited by added Mg2+, but abolished when A23187 is added with Mg2+. The passive exchange is optimal at pHi 6.5 with either Na+ or K+, and cannot be detected above pHi of 7.2. Both the Na+/H+ and the K+/H+ exchange reactions are optimal at an external pH of 7.8 in respiring SMP (pHi 7.1). Valinomycin stimulates the K+-dependent pH change in nonrespiring SMP, as does nigericin. It is concluded that SMP show K+/H+ antiport activity with properties distinct from those of Na+/H+ antiport. However, the properties of the K+/H+ exchange do not correspond in all respects to those of the antiport in intact mitochondria. Donnan equilibria and parallel uniport pathways for H+ and cations appear to contribute to cation-dependent pH changes in SMP.     

Regulation of mitochondrial K+/H+ antiport activity by hydrogen ions

The effect of matrix pH (pHi) on the activity of the mitochondrial K+/H+ antiport has been studied using the fluorescence of 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) to monitor pHi and passive swelling in K+ acetate to follow antiport activity. Heart mitochondria suspended in hypotonic K+ acetate in the absence of respiration show an initial delta pH of -0.4 (interior acid) that decays slowly. Addition of A23187 to deplete matrix Mg2+ results in a further acid shift in pHi followed by equilibration of delta pH. This equilibration appears to depend on K+/H+ antiport and is slow at acid pHi but very rapid when the matrix is alkaline. Swelling of Mg(2+)-depleted mitochondria in K+ acetate is multiphasic with a slow initial rate, a period of maximum swelling, and a final period in which the rate declines. At constant external pH (pH0), the initial rate of swelling is faster with increasing pHi and the time to the onset of the maximum swelling rate decreases. The maximum swelling rate is initiated at pHi 7.4 when pH0 is 7.8 and at pHi 7.1 when pH0 is 7.4. The maximum rate of swelling increases linearly with increasing pH0 in the range from 7.0 to 8.2. This rate also shows a linear relationship to the value of pHi at the time the maximum rate is attained. Dixon plots of the reciprocal of the maximum swelling rate vs [H+]0 suggest that external [H+] is a noncompetitive inhibitor of K+ entry on the antiport. It is concluded that K+/H+ antiport in Mg(2+)-depleted heart mitochondria can be regulated by matrix [H+] (see Beavis, A. D., and Garlid, K. D. (1990) J. Biol. Chem. 265, 2538-2545), but that this antiport is also sensitive to external [H+] or to delta pH when it acts in the direction of K+ uptake.     

Matrix magnesium and the permeability of heart mitochondria to potassium ion

Isolated beef heart mitochondria were treated with A23187 in the presence of different concentrations of Mg2+ or EDTA to establish varying levels of total mitochondrial Mg2+. The Mg2+ content was related to the rate of passive swelling of the mitochondria in potassium acetate and other potassium salts in which swelling is presumed to depend on K+ entry via an endogenous K+/H+ antiport. Swelling in these salts does not commence until Mg2+ has been depleted from an initial value of 36 nmol X mg-1 of protein to 8 nmol/mg-1, or less. Below this level, swelling increases linearly with decreasing Mg2+ to a maximum rate at 2 nmol of Mg2+ X mg-1. Rotenone-treated heart mitochondria suspended in 75 mM potassium acetate at pH 7.80 show no delta pH by 5,5-dimethyl-2,4-oxazolidinedione distribution. Distribution of methylamine also shows essentially no delta pH under these conditions when allowance is made for binding of [14C]methylamine by mitochondrial membranes under these conditions. Addition of A23187 results in a small and transient delta pH (delta pH less than 0.14, acid interior) as measured by methylamine distribution. Estimation of the maximum matrix free Mg2+ concentration from the maximum delta pH observed and the external free Mg2+ concentration at equilibrium with A23187 shows that swelling is not initiated until matrix free Mg2+ is decreased to below 150 microM. An independent estimate of free Mg2+ using a null-point procedure gives a lower, but quite similar value (50 microM) for maximum matrix free Mg2+ when swelling commences. The large depletion of total and free Mg2+ that is required to activate swelling in potassium acetate (and presumably K+/H+ antiport activity) does not appear to be compatible with previous indications that free Mg2+ acts as a "carrier brake" to regulate K+ extrusion from the mitochondrion on such an antiport (Garlid, K. D. (1980) J. Biol. Chem. 255, 11273-11279). The removal of a tightly bound component of mitochondrial Mg2+ is closely related to increased K+ permeability and increased passive swelling in potassium salts. This Mg2+ appears to play a role in the maintenance of mitochondrial membrane structure and integrity.     

The respiration of brain mitochondria and its regulation by monovalent cation transport.

The Na+ and K+ permeability properties of rat brain mitochondria were determined to explain the influences of these cations upon respiration. A new procedure for isolating exceptionally intact mitochondria with minimal contamination by synaptosomes was developed for this purpose. Respiration was uncoupled by Na+ and less so by K+. Uncoupling was maximal in the presence of EDTA plus Pi and was decreased by Mg2+. Maximal uncoupler-stimulated respiration rates were inhibited by Na+ but largely unaffected by K+. The inhibition by Na+ was relatively insensitive to Mg2+. Membrane Na+ and K+ conductances as well as neutral exchanges (Na+/H+ and K+/H+ antiport activities) were determined by swelling measurements and correlated with metabolic effects of the cations. Cation conductance, i.e. electrophoretic Na+ or K+ permeation, was increased by EDTA (Na+ greater than K+) and decreased by Mg2+. Magnesium preferentially suppressed Na+ conductance so as to reverse the cation selectivity (K+ greater than Na+). Neutral cation/H+ exchange rates (Na+ greater than K+) were not influenced by chelator or Mg2+. The extent of cation-dependent uncoupling of respiration correlated best with the inner membrane conductance of the ion according to an empirical relationship derived with the model K+ conductor valinomycin. The metabolic influences of Na+ and K+ can be explained in terms of coupled flow of these ions with protons and their effect upon the H+ electrochemical gradient although alternative possibilities are discussed. These in vitro studies are compared to previous observations in situ to assess their physiological significance.     

Kinetics of inhibition and binding of dicyclohexylcarbodiimide to the 82,000-dalton mitochondrial K+/H+ antiporter

Inhibition of K+/H+ antiport by N,N'-dicyclohexylcarbodiimide in Mg2+ depleted mitochondria follows first order kinetics, exhibiting a half-time of 13 min when mitochondria are incubated with 50 nmol/mg inhibitor at 0 degrees C. 14C radiolabeled N,N'-dicyclohexylcarbodiimide binds to the 82,000-dalton protein, and the second order rate constant for binding is found to be approximately the same as the second order rate constant for inhibition. These findings provide additional confirmation of the identification of this porter with the 82,000-dalton protein and permit us to estimate that rat liver mitochondria contain about 8 pmol/mg of K+/H+ antiporter with a turnover number of 700 s-1. The K+/H+ antiporter of rat liver mitochondria is protected from N,N'-dicyclohexylcarbodiimide inhibition and binding by quinine and by endogenous Mg2+. An 82,000-dalton, [14C]N,N'-dicyclohexylcarbodiimide-binding protein is also observed in rat liver submitochondrial particles, establishing this as an integral protein of the inner membrane. Submitochondrial particles, presumed to be inverted in membrane orientation, are protected from radiolabeling by external Mg2+, supporting the contention that the Mg2+ binding site is localized to the matrix side of the K+/H+ antiporter.     

Activation of potassium ion transport in mitochondria by cadmium ion

Low levels of Cd2+ (1-5 microM) produce rapid swelling of mitochondria, which is respiration-dependent and uncoupler-sensitive. No cation requirement is apparent, since the swelling occurs in a medium containing only sucrose and the respiratory substrate. The swelling is inhibited by ruthenium red, suggesting that this effect of Cd2+ requires its entry into mitochondria. In medium containing 9 mM K+, addition of Cd2+ along with ruthenium red increases the rate of K+ influx threefold. In the presence of K+, Rb+ or Li+, but not of Na+, addition of Cd2+ produces first efflux of H+ into the medium followed by discharge of the pH gradient or uncoupling. Only the latter effect is inhibited by ruthenium red, showing that the efflux and influx of H+ are independent reactions. The H+ efflux appears to be an antiport response to the induced K+ entry. Its activation by Cd2+ is similar to the known effect of p-chloromercuriphenyl sulfonate. The H+ influx or uncoupling appears to result from binding of Cd2+ to some matrix-facing membrane site, perhaps the dithiol group on coupling factor B, and may relate to apparent permeability changes associated Cd2+-induced swelling.     

Na+/Ca2+ antiport in cultured arterial smooth muscle cells. Inhibition by magnesium and other divalent cations

Cultured smooth muscle cells from rat aorta were loaded with Na+, and Na+/Ca2+ antiport was assayed by measuring the initial rates of 45Ca2+ influx and 22Na+ efflux, which were inhibitable by 2',4'-dimethylbenzamil. The replacement of extracellular Na+ with other monovalent ions (K+, Li+, choline, or N-methyl-D-glucamine) was essential for obtaining significant antiport activity. Mg2+ competitively inhibited 45Ca2+ influx via the antiporter (Ki = 93 +/- 7 microM). External Ca2+ or Sr2+ stimulated 22Na+ efflux as would be expected for antiport activity. Mg2+ did not stimulate 22Na+ efflux, which indicates that Mg2+ is probably not transported by the antiporter under the conditions of these experiments. Mg2+ inhibited Ca2+-stimulated 22Na+ efflux as expected from the 45Ca2+ influx data. The replacement of external N-methyl-D-glucamine with K+, but not other monovalent ions (choline, Li+), decreased the potency of Mg2+ as an inhibitor of Na+/Ca2+ antiport 6.7-fold. Other divalent cations (Co2+, Mn2+, Cd2+, Ba2+) also inhibited Na+/Ca2+ antiport activity, and high external potassium decreased the potency of each by 4.3-8.6-fold. The order of effectiveness of the divalent cations as inhibitors of Na+/Ca2+ antiport (Cd2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+) correlated with the closeness of the crystal ionic radius to that of Ca2+.     

Volume-regulating behavior of human platelets

Human platelets exposed to hypotonic media undergo an initial swelling followed by shrinking (regulatory volume decrease [RVD]). If the RVD is blocked, the degree of swelling is in accord with osmotic behavior. The cells could swell at least threefold without significant lysis. Two methods were used to follow the volume changes, electronic sizing and turbidimetry. Changes in shape produced only limited contribution to the measurements. The RVD was very rapid, essentially complete in 2 to 8 minutes, with a rate proportional to the degree of initial cell swelling. RVD involved a loss of KCl via volume-activated conductive permeability pathways for K+ and anions, presumably Cl-. In media containing greater than 50 mM KCl, the shrinking was inhibited and with higher concentrations was reversed (secondary swelling), suggesting that it is driven by the net gradient of K+ plus Cl-. The K+ pathway was specific for Rb+ and K+ compared to Li+ and Na+. The Cl- pathway accepted NO-3 and SCN- but not citrate or SO4(2-). In isotonic medium, the permeability of platelets to Cl- appeared to be low compared to that of K+. After hypotonic swelling both permeabilities were increased, but the Cl- permeability exceeded that of K+. The Cl- conductive pathway remained open as long as the cells were swollen. RVD was incomplete unless amiloride, an inhibitor of Na+/H+ exchange, was present or unless Na+ was replaced by an impermeant cation. In addition, acidification of the cytoplasm occurred upon cell swelling. This reduction in pHi appeared to activate Na+/H+ exchange, with a resultant uptake of Na+ and reduction in the rate and amount of shrinking. Like other cells, platelets responded to hypertonic shrinking with activation of Na+/H+ exchange, but regulatory volume increase was not detectable.     

Evidence for the allosteric regulation of the mitochondrial K+/H+ antiporter by matrix protons

It is well accepted that the mitochondrial K+/H+ antiporter is regulated by matrix Mg2+; however, this is not the only factor controlling its activity. The precise conditions used to deplete divalent cations have profound effects on the subsequent activity of the antiporter in a KOAc assay medium. Examination of the proton fluxes during both pretreatment and subsequent assay of K+/H+ antiport reveals that differences in K+/H+ antiport activity correlate very well with differences in matrix pH. Thus, inhibition of the K+/H+ antiporter following depletion of Mg2+ appears to result from inhibition by matrix protons. To test this hypothesis, we have examined the effect of modulating matrix pH in three different ways on the activity of the K+/H+ antiporter: 1) lowering the pH of the K+ pretreatment medium to 6.7 leads to inactivation of the K+/H+ antiporter; 2) adding NH4+ to the assay medium eliminates the lag in activity induced by depleting Mg2+ in a pretreatment medium containing NH4+; 3) permitting mitochondria to respire in a tetraethylammonium(+)-containing pretreatment medium activates the K+/H+ antiporter. Each one of these procedures leads to a change in matrix pH and an effect on K+/H+ antiport which appears to require regulation of the K+/H+ antiporter by matrix protons. This finding is not only physiologically significant but also provides a useful definition of conditions required for unmasking the K+/H+ antiporter in a reproducible manner.     

Kinetic properties of the Na+/H+ antiport of heart mitochondria

The fluorescence of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) has been used to follow the Na+/H+ antiport activity of isolated heart mitochondria as a Na+-dependent extrusion of matrix H+. The antiport activity measured in this way shows a hyperbolic dependence on external Na+ or Li+ concentration when the external pH (pHo) is 7.2 or higher. The apparent Km for Na+ decreases with increasing pHo to a limit of 4.6 mM. The Ki for external H+ as a competitive inhibitor of Na+/H+ antiport averages 3.0 nM (pHo 8.6). The Vmax at 24 degrees C is 160 ng ion of H+ min-1 (mg of protein)-1 and does not vary with pHo. Li+ reacts with the antiporter with higher affinity, but much lower Vmax, and is a competitive inhibitor of Na+/H+ antiport. The rate of Na+/H+ antiport is optimal when the pHi is near 7.2. When pHo is maintained constant, Na+-dependent extrusion of matrix H+ shows a hyperbolic dependence on [H+]i with an apparent Km corresponding to a pHi of 6.8. The Na+/H+ antiport is inhibited by benzamil and by 5-N-substituted amiloride analogues with I50 values in the range from 50 to 100 microM. The pH profile for this inhibition seems consistent with the availability of a matrix binding site for the amiloride analogues. The mitochondrial Na+/H+ antiport resembles the antiport found in the plasma membrane of mammalian cells in that Na+, Li+, and external H+ appear to compete for a common external binding site and both exchanges are inhibited by amiloride analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypotonic swelling activates the Na*/H* exchange in rat brain synaptosomes

The influence of hypotonic swelling and hypertonic shrinking on cytosolic pH in synaptosomes was investigated. It was shown that decreasing the osmolarity of incubation medium to 230 mOsm leads to alkalization and increasing the osmolarity of incubation medium to 810 mOsm leads to acidification. Alkalization was inhibited by amiloride, indicating the involvement of the Na+/H+ exchanger. The acidification of cytosol upon hypertonic shrinking was insensitive, to amiloride and the inhibitor of Na+, K+, Cl- cotransport bumetanide. Thus, the Na+/H+ exchange in synaptosomes is activated by hypotonic swelling but not hypertonic shrinking, in contrast with erythrocytes and lymphocytes, which have been investigated earlier.     

Mg2+ efflux is accomplished by an amiloride-sensitive Na+/Mg2+ antiport

Mg2+ efflux from Mg2+-preloaded chicken erythrocytes is caused by an electroneutral Na+/Mg2+ antiport. It depends specifically on extracellular Na+, according to Michaelis-Menten kinetics (Km = 25 mM), and is reversibly noncompetitively inhibited by amiloride (Ki = 0.59 mM). In contrast to Na+/H+ antiport, Li+, Ca2+ and N-ethylmaleimide do not interfere with Na+/Mg2+ antiport. The Na+/Mg2+ antiport is driven by the intracellular/extracellular Mg2+ gradient.     

Valinomycin-induced chloride permeability in isolated rat liver mitochondria

1. Ionophore-induced osmotic swelling was used to study Cl- transport in isolated rat liver mitochondria. 2. Energy-dependent, neutral ionophore-induced swelling in Cl- salts at pH 7.2 required K+ and was preceded by a brief lag phase that was absent in chlorotributyltin-induced swelling. 3. Treatments that stimulated or inhibited mitochondrial K+/H+ exchange had qualitatively similar effects on both valinomycin-induced swelling and the associated lag phase. 4. The results suggest that valinomycin-induced Cl- permeability results from an interaction between the K+/H+ antiporter and neutral ionophore K+ complexes.     

Cu2+-induced permeability of the Escherichia coli cytoplasmic membrane]

Cu(2+)-induced permeability of cytoplasmic membranes of Escherichia coli for different cations and neutral molecules of saccharose was estimated by studying their effect on cell plasmolysis during uncharged exchange of cytoplasmic K+ ions by periplasmic space cations. The addition of copper resulted in the exchange of K+ ions by periplasmic Na+, Tris+, streptomycin2+, Cu2+, Ca2+, Mg2+, Cd2+, and Mn2+. It is concluded that Cu(2+)-induced conducting pathways in bacterial membranes are hydrophilic channels with a radius of approximately 0.5 nm and a nonselective permeability for different cations.     

Red cell volume regulation: the pivotal role of ionic strength in controlling swelling-dependent transport systems.

A volume increase of trout erythrocytes can be induced either by beta-adrenergic stimulation of a Na+/H+ antiport in an isotonic medium (isotonic swelling) or by suspending red cells in an hypotonic medium (hypotonic swelling). In both cases cells regulate their volume by a loss of osmolytes via specific pathways. After hypotonic swelling several volume-dependent pathways were activated allowing K+, Na+, taurine and choline to diffuse. All these pathways were fully inhibited by furosemide and inhibitors of the anion exchanger (DIDS, niflumic acid), and the K+ loss was mediated essentially via a 'Cl(-)-independent' pathway. After isotonic swelling, the taurine, choline and Na+ pathways were practically not activated and the K+ loss was strictly 'Cl(-)-dependent'. Thus cellular swelling is a prerequisite for activation of these pathways but, for a given volume increase, the degree of activation and the degree of anion-dependence of the K+ pathway depend on the nature of the stimulus, whether hormonal or by reduction of osmolality. It appears that the pattern of the response induced by hormonal stimulation is not triggered by either cellular cAMP (since it can be reproduced in the absence of hormone by isotonic swelling in an ammonium-containing saline) or by the tonicity of the medium in which swelling occurs since after swelling in an isotonic medium containing urea, the cells adopt the regulatory pattern normally observed after hypotonic swelling. We demonstrated that the stimulus is the change in cellular ionic strength induced by swelling: when ionic strength drops, the cells adopt the hypotonic swelling pattern; when ionic strength increases, the isotonic swelling pattern is activated. To explain this modulating effect of ionic strength a speculative model is proposed, which also allows the integration of two further sets of experimental results: (i) all the volume-activated transport systems are blocked by inhibitors of the anion exchanger and (ii) a Cl(-)-dependent, DIDS-sensitive K+ pathway can be activated in static volume trout red cells (i.e., in the absence of volume increase) by the conformational change of hemoglobin induced by the binding of O2 or CO to the heme.     

K+/H+ exchange in yeast mitochondria: sensitivity to inhibitors, solubilization and reconstitution of the activity in proteoliposomes.

The K+/H+ exchange activity of the inner mitochondrial membrane was investigated in the yeast Saccharomyces cerevisiae. Swelling experiments in potassium acetate indicated that the K+/H+ exchange was active without any additional treatment after the mitochondria isolation, such as a Mg2+ depletion. As in mammalian mitochondria, the activity of yeast mitochondria was stimulated by increasing pH and was inhibited by the amphiphilic amines quinine and propranolol and by the carboxyl reagent dicyclohexylcarbodiimide. However, the activity was poorly inhibited by Mg2+ and consequently was only slightly stimulated by the Mg2+/H+ exchanger A23187. On the other hand, Zn2+ was very efficient for inhibiting the exchange and consequently the activity was strongly stimulated by the permeant metal-chelator o-phenanthroline. The [86Rb]Rb+ accumulation in mitochondria and mitoplasts was only partially inhibited by quinine and propranolol suggesting that part of the accumulation monitored under these conditions was due to cation leak through the inner membrane together with adsorption on the membrane. The DCCD-sensitive activity could be reconstituted from mitochondria and from mitoplasts solubilized with Triton X-100; this activity, measured by [86Rb]Rb+ accumulation, was quinine- and propranolol-sensitive. A spectrophotometric method, based on the capacity of negatively charged proteoliposomes to swell, was then developed in order to continuously follow the reconstituted activity.     

Solubilization and reconstitution of the gastric H,K-ATPase

Proteoliposomes containing the hog gastric H+,K+-ATPase were prepared from cholate and n-octyl glucoside extracts of native microsomes. Experiments were presented which show reconstitution-dependent selective purification of a 94-kDa peptide capable of Rb+/Rb+ exchange and active H+ transport. The absence of selective enrichment of residual protein contamination in this material suggests but does not prove that those transport reactions are attributable only to the 94-kDa peptide. Transport demonstrated inhibitor sensitivity and cation specificity comparable to the microsomal gastric ATPase. In K2SO4 media the H+ transport reaction was protonophore insensitive and correlated with MgATP-dependent 86Rb+ extrusion. This and other evidence suggested that active transport occurs via electroneutral H+in for K+out exchange. 86Rb+ exchange (uptake) in the proteoliposomes demonstrated both saturable and nonsaturable components. At a K0.5 = 1.5 mM, saturable 86Rb+ uptake accounted for about 90% of Rb+ influx. The vanadate-sensitive cation exchange indicated that the ATPase was reconstituted asymmetrically into the proteoliposomes (70% cis-/30% trans-vanadate site). 86Rb+ exchange was inhibited by ATP and stimulated about 2-fold by low Mg2+ and 5 mM phosphate. These ligand effects and the demonstration of comparable rates of passive exchange and active Rb+ efflux suggest that passive K+ exchange is not severely limited by a K+-occluded enzyme form in the H,K-ATPase. A model compatible with this hypothesis is suggested.     

Gliotoxin induces Mg2+ efflux from intact brain mitochondria

Gliotoxin (GT) is a hydrophobic fungal metabolite of the epipolythiodioxopiperazine group which reacts with membrane thiols. When added to a suspension of energized brain mitochondria, it induces matrix swelling of low amplitude, collapse of membrane potential (DeltaPsi), and efflux of endogenous cations such as Ca2+ and Mg2+, typical events of mitochondrial permeability transition (MPT) induction. These effects are due to opening of the membrane transition pore. The addition of cyclosporin A (CsA) or ADP slightly reduces membrane potential collapse, matrix swelling and Ca2+ efflux; Mg2+ efflux is not affected at all. The presence of exogenous Mg2+ or spermine completely preserve mitochondria against DeltaPsi collapse, matrix swelling and Ca2+ release. Instead, Mg2+ efflux is only slightly affected by spermine. Our results demonstrate that, besides inducing MPT, gliotoxin activates a specific Mg2+ efflux system from brain mitochondria.     

Cation exchanges of yeast in the absence of magnesium.

Environmental Mg2+ was found to influence the K+/Na+ exchange rate of metabolizing yeast. Addition of EDTA increased the exchange rate and Mg2+ reversed the effect of EDTA. Yeast starved in the absence of Mg2+ exchanged cellular K+ or Na+ for external H+ when maintained at acidic pH. The exchange rate depended on cellular pH and showed the same kinetics for both K+ and Na+. At acidic pH, the presence of external cations neither inhibited H+ absorption nor changed the cation/H+ 1 : 1 stoichiometry. At neutral pH, external cations inhibited H+ influx but did not change the cation efflux. The K+/Na+ exchange is discussed as electrically coupled and the K+/H+ and Na+/H+ exchanges as electroneutral antiports.     

H+/ion antiport as the principal mechanism of transport systems in the vacuolar membrane of the yeast Saccharomyces carlsbergensis

The secondary transport systems of the yeast vacuolar membrane have been investigated by the method of radioactive isotopes [( 14C]arginine); activation of H+-ATPase by cations (Cat+), when the enzyme is under H+ control and measurement of changes in the proton gradient (delta pH) and membrane potential (Em) due to the supposed substrates of the transporters. The main mechanism of cation transport across the yeast tonoplast is probably H+/Cat+ antiport. The apparent Km of antiporters for Ca2+, Mg2+, Mn2+, Zn2+ and Pi are 0.06, 0.3, 0.8, 0.055-0.17 and 1.5 mM, respectively.