Cholesterol Stimulation of Penetration of Unilamellar Liposomes by Hydrophobic Compounds.

The incorporation of cholesterol into unilamellar liposomes greatly increased the transmembranous movement of hydrophobic ionophores such as nigericin. In reconstituted liposomes containing rhodopsin as the only protein, the presence of cholesterol lowers by 10-fold or more the amount of negericin required to eliminate the light-driven proton gradient. These effects are seen both above and below the transition temperature of the phospholipid used for reconstitution. Cholesterol similarly increases the ability of A-23187, 1799, or NH4SCN to collapse the proton gradient of bacteriorhodopsin vesicles. Cholesterol also lowers the concentration of nigericin or valinomycin required for a rapid translocation of Rb+ into protein-free liposomes. It also lowers the concentration of A-23187 required for the release of Ca45 trapped in protein-free liposomes. In contrast to these observations and in confirmation of previous findings, we observed that cholesterol decreased the permeability of liposomes for glucose. Thus the effects of cholesterol on the permeability of the membrane vary with the chemical nature of the permeating compounds. We have also confirmed that in multilamellar liposomes cholesterol decreases the permeability of Rb+ in the presence of valinomycin. It therefore appears that the effect of cholesterol changes with the size and structural features of the model membranes.     

Use of 1-anilino-8-naphthalene-sulfonate as a probe of gastric vesicle transport

Summary The interaction of 1-anilino-8-naphthalene-sulfonate (ANS) with vesicles derived from hog fundic mucosa was studied in the presence of valinomycin and with the addition of ATP. Evidence was found for two classes of sites, those rapidly accessible to ANS with aK _D of 7.5 m and those slowly accessible, but rapidly accessed in the presence of valinomycin with aK _D of 2.5 m. ATP transiently increases the quantum yield of the latter ANS binding sites only in the presence of valinomycin, but does not alter the number ofK _D of those sites. The time course of this increase correlates with H⁺ uptake and Rb⁺ extrusion by those vesicles and H⁺ carriers such as tetrachlorsalicylanilide or nigericin abolish the ATP response. With ATP addition in the presence of SC¹⁴N and valinomycin there is transient uptake of SCN^–. It is concluded that ANS is acting as a probe of a structural change dependent on a potential and H⁺ gradient.     

Valinomycin-induced potassium and rubidium permeability of intact cells of Acholeplasma laidlawii B

The valinomycin-induced K⁺ and Rb⁺ permeability in cells of Acholeplasma laidlawii B differing in fatty acid and cholesterol content was studied using three different techniques: (i) by following the swelling of the cells in potassium acetate optically; (iii) by recording the efflux of K⁺ using a potassium-selective glass electrode; and (ii) by measuring the efflux of Rb⁺ (after preincubation of the cells with ⁸⁶Rb⁺) with a filter technique.If unsaturation of the membrane lipids was increased, the permeability was found to increase. Cholesterol appeared to cause a slight decrease in permeability.The valinomycin-induced efflux of K⁺ is gradually reduced when the temperature is lowered and becomes zero below the gel-liquid crystalline phase transition.     

Proton transport by gastric membrane vesicles

A highly purified membrane fraction was derived from hog gastric mucosa by a combination of differential and density gradient centrifugation and free flow electrophoresis. This final fraction was 35-fold enriched with respect to cation activated ouabain-insensitive ATPase. Antibody against this fraction was shown to be bound to the luminal surface of the gastric glands. The addition of ATP to this fraction or the density gradient fraction resulted in H⁺ uptake into an osmotically sensitive space. The apparent K_m for ATP was 1.7 · 10^?4 M in the absence of a K⁺ gradient similar to that found for ATPase activity. The reaction is specific for ATP and requires cation in the sequence K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺ and is inhibited by ATPase inhibitors such as N,N′-dicylclohexylcarbodiimide. Maximal H⁺ uptake occurs with an outward K⁺ gradient but the minimal apparent K_A is found in the absence of a K⁺ gradient. The pH optimum for H⁺ uptake is between 5.8 and 6.2 which corresponds to the pH range for phosphorylation of the enzyme, but is considerably less than the pH maximum of the K⁺ dependent dephosphorylation. In the presence of an inward K^? gradient, protonophores such as tetrachlorsalicylanilide only partially abolish the H⁺ gradient but valinomycin dissipates 75% of the gradient, and nigericin abolishes the gradient. The vesicles therefore have a low K⁺ conductance but a measurable H⁺ conductance, hence a K⁺ gradient can produce an H⁺ gradient in the presence of valinomycin. The uptake and spontaneous leak of H⁺ are temperature sensitive skin with a similar transition temperature. Ultraviolet irradiation inactivates ATPase and proton transport at the same rate, approximately at twice the rate of p-nitrophenylphosphatase inactivation. It is concluded that H⁺ uptake by these vesicles is probably due to a dimeric (H⁺ + K⁺)-ATPase and is probably non-electrogenic.     

Action of protein synthesis inhibitors in blocking electrogenic h efflux from corn roots

The block in the electrogenic H⁺ efflux produced by protein synthesis inhibitors in corn root tissue can be released or by-passed by addition of fusicoccin or nigericin. The inhibition also lowers cell potential, and the release repolarizes. Associated with the inhibition of H⁺ efflux is inhibition of K⁺ influx and the growth of the root tip; fusicoccin partially relieves these inhibitions, but nigericin does not. The inhibition of H⁺ efflux which arises from blocking the proton channel of the ATPase by oligomycin or N,N′-dicyclohexylcarbodiimide can also be partially relieved by fusicoccin, but not by nigericin; the inhibition produced by diethylstilbestrol is not relieved by fusicoccin.     

Factors affecting the development of the capacity for ATP formation in isolated chloroplasts

Full development of the capacity for ATP formation in isolated thylakoid membranes coincides with the beginning of illumination. Indeed, the yield of ATP per ms of illumination is about twice as great during the first 15 ms of high-intensity illumination as it is thereafter. The presence of valinomycin and K⁺ prevents the formation of a membrane potential (as indicated by the obliteration of most of the change in absorbance at 518 nm) and at the same time delays the formation of the capacity for ATP synthesis for many milliseconds. Presumably, phosphorylation is initially dependent on a rapidly formed membrane potential, whereas after a delay a ΔpH sufficient to drive ATP formation forms. The actual duration of this delay depends on the phosphoryl group transfer potential (i.e., ΔG_ATP) of the ATP-synthesizing reaction. If the delay in the presence of valinomycin and K⁺ represents the time required to develop a ΔpH capable of driving phosphorylation by itself, then the effect of ΔG_ATP on the duration of the delay suggests that the onset of phosphorylation is determined by the magnitude of the electrochemical potential of protons and not by factors affecting the activation of the coupling factor enzyme. The initial ATP formation, which is almost entirely dependent on the electrical potential, should not be affected by the electrically neutral exchange of cations catalyzed by nigericin. When the external pH is 7.0 this seems to be true, since the ATP synthesis which is initially sensitive to valinomycin and K⁺ is largely insensitive to nigericin and K⁺. However, when the external pH is 8.0 the response to nigericin is exactly the opposite and the ATP formation which is sensitive to valinomycin is also abolished by nigericin. These data suggest that there may be either an energetic requirement for both a ΔpH and membrane potential at alkaline pH or a non-energetic requirement for a minimum proton activity in the initiation of phosphorylation.     

Energized transport of potassium ions in the absence of valinomycin by cytochrome c oxidase-reconstituted vesicles

Valinomycin-independent energized uptake of K⁺ was observed in cytochrome c oxidase reconstituted proteoliposome. The rate of K⁺ influx was proportoinal to the magnitude of electron flux. The energized uptake of K⁺ was abolished by p-trifluoromethoxycarbonylcyanide phenylhydrazone or by nigericin. Using the safranine fluorescence technique, it was demonstrated that even in the absence of valinomycin, liposomes and proteoliposomes reconstituted with cytochrome c oxidase are able to discriminate between Na⁺ and K⁺ and show a preference for K⁺ in the presence of excess Na⁺.     

Measurement and significance of the membrane potential in Methanoba cterium bryantii

The membrane potential (Δψ) of Methanobacterium bryantii was 133–142 mV as measured from the distribution of ⁸⁶Rb⁺ in valinomycin-treated cells, and was considerably higher than that obtained using triphenylmethylphosphonium in the presence of tetraphenylboron. The Δψ measured using the Rb⁺/valinomycin method was sensitive to certain ionophores including gramicidin, nigericin, carbonyl cyanide m-chlorophenylhydrazone and 3,3′,4′,5-tetrachlorosalicylanilide. It was also dissipated by 1 mM tetraphenylphosphonium and was abolished in heat-treated or permeabilized cells. The Δψ could be varied by adjusting the extracellular potassium concentration in valinomycin-treated cells. Monensin-treated cells possessed a significantly increased Δψ, as monitored by the Rb⁺ / valinomycin method. Tetraphenylphosphonium cation (1 mM) abolished methane synthesis, intracellular ATP and Δψ, supporting a role for Δψ in ATP and CH₄ synthesis. However, lower concentrations of the lipophilic cation (50 μM) greatly elevated both the intracellular ATP concentration and Δψ but decreased the rate of CH₄ synthesis by almost 50%. Thus, tetraphenylphosphonium cation exerts a primary inhibitory effect on CH₄ synthesis which cannot be attributed to the loss of Δψ or ATP.     

Driving forces in hepatocellular uptake of phalloidin and cholate

Active uptake of phalloidin and cholate in isolated rat liver cells depends upon both Na⁺ gradient and membrane potential. Omission of Na⁺ or inhibition of the (Na⁺ + K⁺)-ATPase diminished both phalloidin and cholate uptake. Dissipation of the sodium, potassium or proton gradient by monensin, nigericin, gramicidin and valinomycin blocked phalloidin uptake and also caused reduction of cholate transport. Chelation of Ca²⁺ and Mg²⁺ by EGTA or incubation of liver cells with NH₄Cl neither influenced phalloidin nor cholate uptake. Hyperpolarization of liver cells by the lipophilic anions NO₃⁻ or SCN⁻ enhanced phalloidin but reduced cholate uptake. Depolarization induced by a reversed K⁺ gradient reduced both kinds of transport. The results indicate that sodium ions and the membrane potential are driving forces for phalloidin and cholate uptake in hepatocytes.     

Light-dependent rubidium transport in intact Halobacterium halobium cells

The uptake of rubidium in intact Halobacterium halobium cells was followed, and found to be light-dependent. The exchange process is slow, the steady-state rate of ⁸⁶Rb⁺/Rb⁺ exchange being given by k = 6.3 · 10^?4 min^?1. Starved cells exhibited a faster rate than unstarved cells. The influx of ⁸⁶Rb⁺ was almost completely blocked in the presence of proton conductors (CCCP, FCCP, and SF 6847), and was sensitive to the presence of the permeant cation TPMP⁺. Valinomycin very slightly increased the rate of uptake, while 1 · 10^?6 M nigericin showed significant inhibition. On the other hand, release of ⁸⁶Rb⁺ was not light-dependent, although still affected by uncouplers, TPMP⁺, and nigericin. These experimental observations may be explained in terms of a passive flux driven by an electrical potential difference, and influenced by positive isotope interaction within the membrane. In carefully matched influx-efflux studies, the extent of the positive isotope interaction was measured. Using the formal treatment of Kedem and Essig, the ratio (exchange resistance)/(resistance to net flow) for ⁸⁶Rb⁺ was found to be 1.7.     

Effect of 2,4-dinitrophenol and ionophores on growth and methanogenesis inMethanobacterium thermoautotrophicum

2,4-Dinitrophenol and gramicidin D completely inhibited growth and methanogenesis inMethanobacterium thermoautotrophicum. At low K⁺ concentrations valinomycin inhibited growth and methanogenesis relatively slightly, at high K⁺ concentrations (0.1m KCl) growth was inhibited completely and methanogenesis by about 50%. Monensin and nigericin inhibited growth completely; methanogenesis was inhibited like with valinomycin at high K⁺ concentrations. The results can be interpreted in terms of Mitchell’s chemiosmotic theory as follows. The protonmotive force inM. thermoautotrophicum is the basic source of energy for endergonic processes. Dissipation of the electrical component of protonmotive force may probably be compensated by an increased generation of the proton gradient. However, the osmotic component is essential for growth ofM. thermoautotrophicum.     

Functional reconstitution of the gamma-aminobutyric acid transporter from synaptic vesicles using artificial ion gradients.

The gamma-aminobutyric acid transporter of rat brain synaptic vesicles was reconstituted in proteoliposomes, and its activity was studied in response to artificially created membrane potentials or proton gradients. Changes of the membrane potential were monitored using the dyes oxonol VI and 3,3'-diisopropylthiodicarbocyanine iodide, and changes of the H+ gradient were followed using acridine orange. An inside positive membrane potential was generated by the creation of an inwardly directed K+ gradient and the subsequent addition of valinomycin. Under these conditions, valinomycin evoked uptake of [3H]GABA which was saturable. Similarly, [3H]glutamate uptake was stimulated by valinomycin, indicating that both transporters can be driven by the membrane potential. Proton gradients were generated by the incubation of K(+)-loaded proteoliposomes in a buffer free of K+ or Na+ ions and the subsequent addition of nigericin. Proton gradients were also generated via the endogenous H+ ATPase by incubation of K(+)-loaded proteoliposomes in equimolar K+ buffer in the presence of valinomycin. These proton gradients evoked nonspecific, nonsaturable uptake of GABA and beta-alanine but not of glycine in proteoliposomes as well as protein-free liposomes. Therefore, transporter activity was monitored using glycine as an alternative substrate. Proton gradients generated by both methods elicited saturable glycine uptake in proteoliposomes. Together, our data confirm that the vesicular GABA transporter can be energized by both the membrane potential and the pH gradient and show that transport can be achieved by artificial gradients independently of the endogenous proton ATPase.     

Energy coupling to nitrate uptake into the denitrifying cells of Paracoccus denitrificans

This study deals with the effects of the agents that dissipate the individual components of the proton motive force (short-chain fatty acids, nigericin, and valinomycin) upon the methyl viologen-coupled nitrate reductase activity in intact cells. Substitution of butyrate or acetate for chloride in Tris-buffered assay media resulted in a marked inhibition at pH 7. In a Tris-chloride buffer of neutral pH, the reaction was almost fully inhibitable by nigericin. Alkalinisation increased the IC₅₀ value for nigericin and decreased the maximal inhibition attained. Both types of inhibitions could be reversed by the permeabilisation of cells or by the addition of nitrite, and that caused by nigericin disappeared at high extracellular concentrations of potassium. These data indicate that nitrate transport step relies heavily on the pH gradient at neutral pH. Since the affinity of cells for nitrate was strongly diminished by imposing an inside-positive potassium (or lithium) diffusion potential at alkaline external pH, a potential dependent step may be of significance in the transporter cycle under these conditions. Experiments with sodium-depleted media provided no hints for Na⁺ as a possible H⁺ substitute.     

Nigericin forms highly stable complexes with lithium and cesium

Nigericin is a monocarboxylic polyether molecule described as a mobile K⁺ ionophore unable to transport Li⁺ and Cs⁺ across natural or artificial membranes. This paper shows that the ion carrier molecule forms complexes of equivalent energy demands with Li⁺, Cs⁺, Na⁺, Rb⁺, and K⁺. This is in accordance with the similar values of the complex stability constants obtained from nigericin with the five alkali metal cations assayed. On the other hand, nigericinalkali metal cation binding isotherms show faster rates for Li⁺ and Cs⁺ than for Na⁺, K⁺, and Rb⁺, in conditions where the carboxylic proton does not dissociate. Furthermore, proton NMR spectra of nigericin-Li⁺ and nigericin-Cs⁺ complexes show wide broadenings, suggesting strong cation interaction with the ionophore; in contrast, the complexes with Na⁺, K⁺, and Rb⁺ show only clear-cut chemical shifts. These latter results support the view that nigericin forms highly stable complexes with Li⁺ and Cs⁺ and contribute to the explanation for the inability of this ionophore to transport the former cations in conditions where it catalyzes a fast transport of K⁺>Rb⁺>Na⁺.Part of the results of this paper were presented at the 14th International Congress of Biochemistry in Prague, Czechoslovakia.     

Photophosphorylation as a function of illumination time. I. Effects of permeant cations and permeant anions

(1) Very brief periods of illumination do not initiate photophosphorylation in isolated chloroplast lamellae. The time of illumination required before any phosphorylation can be detected is inversely proportional to the light intensity. At very high intensities, phosphorylation is initiated after illumination for about 4 ms.(2) There is no similar delay in the initiation of electron transport. The rate of electron transport is very high at first but declines at about the time the capacity for ATP synthesis develops. When the chloroplasts are uncoupled with gramicidin the high initial rate persists.(3) Various ions which permeate the thylakoid membrane (K⁺ or Rb⁺ in the presence of valinomycin, SCN^?, I^?, or ClO₄^?) markedly increase the time of illumination required to initiate phosphorylation. Potassium ions in the presence of valinomycin increase the delay to a maximum of about 50 ms whereas thiocyanate ions increase the delay to a maximum of about 25 ms. The effects of K⁺ with valinomycin and the effect of SCN^? are not additive. Permeant ions and combinations of permeant ions have little or no effect on phosphorylation during continuous illumination.(4) The reason for the threshold in the light requirement and the reason for the effect of permeant ions thereon are both obscure. However, it could be argued that the energy for phosphorylation initially resides in an electric potential gradient which is abolished by migration of ions in the field, leaving a more slowly developing proton concentration gradient as the main driving force for phosphorylation during continuous illumination. If so, the threshold in the presence of permeant ions should depend on internal hydrogen ion buffering.     

The effect of trypsin treatment on the incorporation and energy-transducing properties of bacteriorhodopsin in liposomes

Bacteriorhodopsin and trypsin-modified bacteriorhodopsin have been reconstituted into liposomes by means of a low pH-sonication procedure. The incorporation of bacteriorhodopsin in these proteoliposomes is predominantly in the same direction as in vivo and the direction of proton pumping is from inside to outside the liposomes. The direction of proton translocation and electrical potential generation was studied as a function of the reconstitution pH. Light-dependent proton extrusion and generation of a Δp, interior negative and alkaline was observed at a reconstitution pH below 3.0 using bacteriorhodopsin, and at a pH below 3.5 using trypsin-modified bacteriorhodopsin. The shift in inflection point is explained in terms of differences between bacteriorhodopsin and trypsin-modified bacteriorhodopsin in a specific protein-phospholipid interaction which depends on the surface charge density of the cytoplasmic side of bacteriorhodopsin. The magnitude of the protonmotive force (Δp) generated by trypsin-modified bacteriorhodopsin in liposomes was quantitated. Illumination of the proteoliposomes resulted in the generation of a high Δp (135 mV, inside negative and alkaline), with a major contribution of the pH gradient. The ionophores nigericin and valinomycin induced, respectively, a compensatory interconversion of ΔpH into Δψ and vice versa. If no endogenous proton permeability of the membrane would exist, a protonmotive force could be generated of − 143 mV as electrical potential alone or − 162 mV as pH gradient alone.     

Potassium-stimulated ATPase activity and hydrogen transport in gastric microsomal vesicles

The Mg²⁺-dependent, K⁺-stimulated ATPase of microsomes from pig gastric mucosa has been studied in relation to observed active H⁺ transport into vesicular space. Uptake of fluorescent dyes (acridine orange and 9-aminoacridine) was used to monitor the generated pH gradient. Freeze-fracture electron microscopy showed that the vesicular gastric microsomes have an asymmetric distribution of intramembraneous particles (P-face was particulate; E-face was relatively smooth).Valinomycin stimulated both dye uptake and K⁺-ATPase (valinomycin-stimulated K⁺-ATPase); stimulation by valinomycin was due to increased K⁺ entry to some intravesicular activating site, which in turn depends upon the accompanying anion. Using the valinomycin-stimulated K⁺-ATPase and H⁺ accumulation as an index, the sequence for anion permeation was NO₃^? > Br^? > Cl^? > I^? > acetate ≈ isethionate. When permeability to both K⁺ and H⁺ was increased (e.g using valinomycin plus a protonophore or nigericin), stimulation of K⁺-ATPase was much less dependent on the anion and the observed dissipation of the vesicular pH gradient was consistent with an ‘uncoupling’ of ATP hydrolysis from H⁺ accumulation.Thiocyanate interacts with valinomycin inhibiting the typical action of the K⁺ ionophore. But stimulation of ATPase activity was seen by adding 10 mM SCN^? to membranes preincubated with valinomycin. From the relative activation of the valinomycin-stimulated K⁺-ATPase, it appears that SCN^? is a very     

Amiloride sensitivity of proton-conductive pathways in gastric and intestinal apical membrane vesicles

Summary Passive proton permeability of gastrointestinal apical membrane vesicles was determined. The nature of the pathways for proton permeation was investigated using amiloride. The rate of proton permeation (k _H ⁺ was determined by addition of vesicles (pH _i = 6.5) to a pH 8.0 solution containing acridine orange. The rate of recovery of acridine orange fluorescence after quenching by the acidic vesicles ranged from 4 × 10^–3 (gastric parietal cell stimulation-associated vesicles; SAV) and 5 × 10^–3 (duodenal brush-border membrane vesicles; dBBMV) to 11 × 10+^–3 sec^–1 (ileal BBMV; iBBMV). Amiloride, 0.03 and 0.1 mm, significantly reduced the rate of proton permeation in dBBMV and iBBMV, but not gastric SAV. The decreases in k _H ⁺ were proportionately greater in iBBMV as compared with dBBMV. The presence of Na⁺/H⁺ exchange was demonstrated in both dBBMV and iBBMV by proton-driven (pH _i < pH _o ) ²²Na⁺ uptake. Evidence was also sought for the conductive nature of pathways for proton permeation. Intravesicular acidification, again determined by quenching of acridine orange fluorescence, was observed during imposition of K⁺-diffusion potential ([K⁺] _i [K⁺ _o ). In dBBMV and iBBMV, intravesicular acidification was enhanced in the presence of the K⁺-ionophore valinomycin, indicating that the native K⁺ permeability is rate limiting. In the presence of valinomycin, the K⁺-diffusion potential drove BBMV intravesicular acidification to levels close to the electrochemical potential. In gastric SAV, acidification was not limited by the K⁺ permeability. Valinomycin was without effect, but the K⁺/H⁺ ionophore nigericin enhanced acidification in gastric SAV, illustrating the low proton permeability of these membranes. Amiloride, 0.03–1 mm, resulted in concentration-dependent reductions of K⁺-diffusion potential-driven acidification in dBBMV and iBBMV but not in gastric SAV. These data demonstrate that proton permeation in the three membrane types is rheogenic. The sensitivity of the proton-conductive pathways in intestinal BBMV to high concentrations of amiloride correlated with the presence of the Na⁺/H⁺ antiport and indicates that this transmembrane protein may represent a pathway for proton permeation.We thank Ruth Briggs for assistance with the Na/H exchange experiments. This work was supported by a grant from the Medical Research Council (G8418056CA).     

Changes in the Light Scattering by Symbiosomes from Vicia faba Root Nodules as Indicator of Ion and Metabolite Transport across the Peribacteroid Membrane

Passive transport of ions and metabolites across the peribacteroid membrane (PBM) was investigated on symbiosome preparations isolated from the broad bean (Vicia faba L.) root nodules and suspended in a potassium-free medium. Optical density of the symbiosome suspension at 546 nm was monitored as an indicator of light-scattering changes. Depolarization of the PBM with tetraphenylphosphonium cation (TPP⁺) caused an increase in light scattering of symbiosome suspension. This effect was enhanced after adding a K⁺ ionophore valinomycin to the incubation medium. A similar effect was observed after supplementing the symbiosome suspension with nigericin, a K⁺/H⁺ antiporter. Similar experiments on bacteroid suspensions prepared from isolated symbiosomes did not reveal any appreciable changes in light scattering in the presence of the same membrane-active substances. The light scattering by symbiosome suspensions decreased after adding malate or succinate, while the subsequent addition of centimolar concentrations of K⁺ substantially accelerated this process. Light scattering by the symbiosome suspension was insensitive to the addition of glutamate, a substance normally impermeant through the PBM of legume root nodules. These results suggest that the changes in light scattering by symbiosomes reflect the osmotically induced changes of symbiosome volume. These volume changes were assigned to alteration of the peribacteroid space (PBS). The incubation of symbiosomes in a potassium-free medium acidified their the PBS; this acidification was accelerated by valinomycin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), and nigericin, and it was abolished in the presence of comparatively high concentrations of K⁺ in the incubation medium. The results indicate a relatively high permeability of the PBM to K⁺ ions.     

Inhibition and stimulation of respiration-linked Mg2+ efflux in rat heart mitochondria

Respiration-driven Mg²⁺ efflux from rat heart mitochondria has been studied in different conditions. Almost total release of Mg²⁺ from the mitochondria occurs upon addition of a proton/bivalent cation exchanger, A23187. The content of Mg²⁺ remaining in mitochondria after A23187 treatment is the same if part of the mitochondrial Mg²⁺ has already been extruded through the energy-linked mechanism. Some inhibition of Mg²⁺ efflux is observed in the presence of high concentrations of La³⁺ (100 µM). A proton/monovalent cation exchanger, nigericin, completely prevents Mg²⁺ efflux, whereas a cation conductor, valinomycin, considerably stimulates it. The results indicate that the main part of mitochondrial Mg²⁺ is present in a membrane-bounded compartment, probably in the matrix space. The driving force of the Mg²⁺ efflux appears to be the proton gradient (pH) created by mitochondrial respiration.