Action of local anaesthetics on passive and energy-linked ion translocation in the inner mitochondrial membrane

The effect of dibucaine on passive and respiration-driven ion translocation and oxidative phosphorylation in submitochondrial particles from beef-heart has been studied.Dibucaine inhibited the nigericin-mediated H⁺/K⁺ exchange diffusion and the electrogenic, valinomycin-mediated K⁺ translocation in submitochondrial particles.The local anaesthetic exerted a direct stimulatory effect on the respiration-driven proton uptake and on the passive proton-diffusion reactions. The increase of the respiration-linked proton turnover caused by dibucaine was accompanied by uncoupling of oxidative phosphorylation. It is concluded that spontaneous noncoupled as well as ionophoremediated K⁺ translocation in mitochondria occurs across phospholipid bilayer regions of the membrane whilst other components of the membrane would be specifically involved in active and passive proton translocation across the membrane.The results indicate that polar groups of membrane phospholipids play an important role in energy conservation and transfer in the mitochondrial membrane.     

Monovalent ion and calcium ion fluxes in sarcoplasmic reticulum

Summary The ion permeability of sarcoplasmic reticulum vesicles from skeletal and heart muscle has been characterized by radioisotope flux, osmotic and membrane potential measurements, and by incorporating vesicles into planar phospholipid bilayers. The sarcoplasmic reticulum membrane is uniquely permeable to various biologically relevant monovalent ions. At least two and possibly three separate passive permeation systems for monovalent ions have been identified: 1) a K⁺, Na⁺ channel, 2) an anion channel, and 3) a H⁺ (OH^–) permeable pathway which may or may not be synonymous with the anion channel. A possible physiological function of these monovalent ion permeation systems is to permit rapid movement of K⁺, Na⁺, H⁺ and Cl across the membrane to counter electrogenic Ca²⁺ fluxes during Ca²⁺ release and uptake by sacroplasmic reticulum.     

The ionic relations of Porphyra purpurea(Roth) C.Ag. (Rhodophyta, Bangiales)

Abstract Radioisotope equilibration techniques have been used to determine the intracellular concentration of K⁺, Na⁺ and Cl_?, together with the unidirectional ion fluxes across the plasmalemma of Porphyra purpurea. Influx and efflux of ⁴²K⁺, ²⁴Na⁺ and ³⁶C1^? are biphasic, the rapid, initial uptake and loss of tracer from individual thalli being attributable to desorption from extracellular regions. Cellular fluxes are slower and monophasic, cells discriminating in favour of K⁺ and Cl^? and against Na⁺. A comparison between the equilibrium potential of individual ion species and the measured membrane potential demonstrates that there is an active component of K⁺ and Cl^? influx and Na⁺ efflux. ‘Active’ uptake and ‘passive’ loss of K⁺ and Cl^? are reduced when plants are kept in darkness, suggesting that a fraction of the transport of K⁺ and Cl^? may be due to ‘exchange diffusion’ (K⁺/K⁺ and Cl^?/Cl^?antiport).     

K+/H+ antiport in mitochondria

Mitochondria contain a latent K⁺/H⁺ antiporter that is activated by Mg²⁺-depletion and shows optimal activity in alkaline, hypotonic suspending media. This K⁺/H⁺ antiport activity appears responsible for a respiration-dependent extrusion of endogenous K⁺, for passive swelling in K⁺ acetate and other media, for a passive exchange of matrix⁴²K⁺ against external K⁺, Na⁺, or Li⁺, and for the respiration-dependent ion extrusion and osmotic contraction of mitochondria swollen passively in K⁺ nitrate. K⁺/H⁺ antiport is inhibited by quinine and by dicyclohexylcarbodiimide when this reagent is reacted with Mg²⁺-depleted mitochondria. There is good suggestive evidence that the K⁺/H⁺ antiport may serve as the endogenous K⁺-extruding device of the mitochondrion. There is also considerable experimental support for the concept that the K⁺/H⁺ antiport is regulated to prevent futile influx-efflux cycling of K⁺. However, it is not yet clear whether such regulation depends on matrix free Mg²⁺, on membrane conformational changes, or other as yet unknown factors.     

Mechanism of respiration-driven proton translocation in the inner mitochondrial membrane. Kinetics of proton translocation and role of cations

The kinetics and mechanism of passive and active proton translocation in submitochondrial vesicles, obtained by sonication of beef heart mitochondria, have been studied.Analysis of the anaerobic release of the protons taken up by submitochondrial particles in the respiring steady state shows that proton diffusion consists of two parallel, apparent first-order processes: a fast reaction which, on the basis of its kinetic properties and response to cations and various effectors, is considered to consist of a proton/monovalent cation exchange; and a slow process which, on analogous grounds, is considered as a single electrogenic flux.The study of the various parameters of the respiration-linked active proton translocation and of the accompanying migration of permeant anions and K⁺ led to the following conclusions: (i) The oxidoreduction-linked proton translocation is electrogenic. (ii) Cation counterflow is not a necessary factor in the respiration-driven proton translocation. (iii) The membrane potential developed by active proton translocation exerts a coupling with respect to permeant cations and anions. (iv) The respiration-driven proton translocation is secondarily coupled, through the Δμ_H component of the electrochemical proton gradient and at the level of a proton-cation exchange system of the membrane, to the flow of K⁺ and Na⁺.     

Relationship between coupled Na+−K+−Cl− transport and water absorption across the seawater eel intestine

Summary Simultaneous measurements of net ion and water fluxes were made in the stripped intestine of the seawater eel, and the relationship between Na⁺, K⁺, Cl^– and water transport were examined in the presence of mucosal KCl and serosal NaCl Ringer (standard condition). When Cl^– was removed from both sides of the intestine, net K⁺ flux from mucosa to serosa was reduced, accompanied by complete blockage of water absorption. Since it has been shown that net Cl^– and water fluxes depend on K⁺ transport under the standard condition (Ando 1983), the interdependence of K⁺ and Cl^– transport suggests the existence of a coupled KCl transport system, while the parallelism between the net Cl^– and water fluxes suggests that water absorption is linked to the coupled KCl transport. The coupled KCl and water transport were inhibited by treatment with ouabain or with Na⁺-free Ringer solutions, suggesting the existence of a Na⁺-dependent KCl transport system and linkage of water absorption to the coupled Na⁺–K⁺–Cl^– transport. Since ouabain blocked the active Na⁺–K⁺–Cl^– transport almost completely, the permeability coefficients for K⁺ and Na⁺ through the paracellular shunt pathway were estimated as P_K=0.076 and P_Na=0.058 cm/h, and P_Cl was calculated as 0.005 cm/h. Although Na⁺-independent K⁺ and Cl^tt- fluxes were observed again in the present study, these fluxes were not inhibited by CN^–, ouabain or diuretics, and evoked even after blocking the Na⁺–K⁺–Cl^– transport completely with ouabain. These results indicate that the Na⁺-independent K⁺ and Cl^– fluxes are distinct from the active Na⁺–K⁺–Cl^– transport and are not themselves active.     

Ion fluxes and phytochrome protons in mung bean hypocotyl segments: I. Fluxes of potassium

K⁺ [⁸⁶Rb⁺] uptake by Phaseolus aureus Roxb. hypocotyl segments cut immediately below the hook is inhibited by the active form of phytochrome (Pfr). Short load-short wash experiments indicate that the inhibition of uptake occurs across the plasmalemma. A maximal inhibition of short term uptake occurs in 10 to 50 millimolar KCI. Low temperature had only a small effect on influx and the inhibition of influx from 50 millimolar KCI. A consideration of the electrochemical gradient for K⁺ suggests that passive K⁺ fluxes may predominate under these conditions. Red light induces small depolarizations of membrane potential in subhook cells. Far red light antagonizes this effect. Pfr inhibits efflux of K⁺[⁸⁶Rb⁺] from subhook segments. This effect is also relatively insensitive to low temperature. This inhibition of efflux may reflect inhibition of a K⁺ -K⁺ exchange process, or reduced passive permeability of the plasmalemma to K⁺. In contrast, Pfr enhances short term uptake of K⁺[⁸⁶Rb⁺] in apical hypocotyl hook segments of Phaseolus aureus Roxb. Short load-short wash experiments indicate that fluxes across the plasmalemma are modified by Pfr. A maximal enhancement of short term influx occurs in 50 millimolar KCI. Influx and the red light enhancement of influx from 50 millimolar KCI are relatively insensitive to low temperature. Pfr also enhances efflux of K⁺[⁸⁶Rb⁺] from preloaded apical hook segments. This increased influx may reflect enhancement of a K⁺ -K⁺ exchange process or increased passive permeability of the plasmalemma to K⁺.     

Fluxes and compartmentation of potassium and chloride in the green alga Mougeotia

Summary Some ionic relations of the filamentous green alga Mougeotia sp. have been analyzed under different light conditions. Data from influx and efflux measurements using ⁸⁶Rb⁺ and ³⁶Cl^- fit the model of three cellular compartments (cell wall, cytoplasm, vacuole) in series. This result is remarkable, since in a Mougeotia cell at least two thirds of the cytoplasmic compartment are occupied by the cell-filling, flat and nearly rectangular chloroplast which is axially oriented. The chloroplast is concluded to be part of the cytoplasmic flux compartment.Photosynthetically saturating irradiances of continuous white light enhance the active and passive fluxes of K⁺ and Cl^- at the plasmalemma by a factor of 3. Photosystem II is responsible for the light-dependent increase of the uptake of Cl^- (³⁶Cl^-) whereas the uptake of K⁺ (⁸⁶Rb⁺) depends additionally on energy from photosystem I.Ion flux measurements performed after irradiations with red and far-red, respectively, show that the fluxes of K⁺ and Cl^- across the plasmalemma are not affected by the state of phytochrome.     

High affinity k uptake in maize roots: a lack of coupling with h efflux

We report here on the putative coupling between a high affinity K⁺ uptake system which operates at low external K⁺ concentrations (K_m = 10-20 micromolar), and H⁺ efflux in roots of intact, low-salt-grown maize plants. An experimental approach combining electrophysiological measurements, quantification of unidirectional K⁺(⁸⁶Rb⁺) influx, and the simultaneous measurement of net K⁺ and H⁺ fluxes associated with individual cells at the root surface with K⁺- and H⁺-selective microelectrodes was utilized. A microelectrode system described previously (IA Newman, LV Kochian, MA Grusak, and WJ Lucas [1987] Plant Physiol 84: 1177-1184) was used to quantify net ion fluxes from the measurement of electrochemical potential gradients for K⁺ and H⁺ ions within the unstirred layer at the root surface. No evidence for coupling between K⁺ uptake and H⁺ efflux could be found based on: (a) extremely variable K⁺:H⁺ flux stoichiometries, with K⁺ uptake often well in excess of H⁺ efflux; (b) dramatic time-dependent variability in H⁺ extrusion when both fluxes were measured at a particular location along the root over time; and (c) a lack of pH sensitivity by the high affinity K⁺ uptake system (to changes in external pH) when net K⁺ uptake, unidirectional K⁺(⁸⁶Rb⁺) influx, and K⁺-induced depolarizations of the membrane potential were determined in uptake solutions buffered at pH values from pH 4 to 8. Based on the results presented here, we propose that high affinity active K⁺ absorption into maize root cells is not mediated by a K⁺/H⁺ exchange mechanism. Instead, it is either due to the operation of a K⁺-H⁺ cotransport system, as has been hypothesized for Neurospora, or based on the striking lack of sensitivity to changes in extracellular pH, uptake could be mediated by a K⁺-ATPase as reported for Escherichia coli and Saccharomyces.     

H fluxes in excised samanea motor tissue : I. Promotion by light

Previous investigators revealed that white light-promoted leaflet opening in Samanea saman (Jacq) Merrill depends upon K⁺ uptake by extensor cells and efflux from flexor cells of the pulvinus, while dark-promoted closure depends upon K⁺ fluxes in the opposite directions. We now monitored H⁺ fluxes during pulvinar movement to test a model proposing coupled H⁺/K⁺ fluxes. H⁺ fluxes were monitored by measuring changes in the pH of a weakly buffered solution (initial pH = 5.5) bathing excised strips of extensor or flexor tissue. White light at hour 3 of the usual dark period promoted pulvinar opening, H⁺ efflux from extensor cells and uptake by flexor cells, while darkness at hours 2 to 4 of the usual light period promoted pulvinar closure, H⁺ uptake by extensor cells and efflux from flexor cells. The following conditions altered H⁺ fluxes during dark-promoted closure. (a) Light reversed the directions of the fluxes in both extensor and flexor cells. (b) Anoxia increased the rate of H⁺ uptake by extensor cells and promoted H⁺ uptake (rather than efflux) by flexor cells, consistent with an outwardly directed H⁺ pump. KCN showed similar effects initially, but they were transient. (c) Increase in external pH from 5.5 to 6.7 promoted H⁺ efflux (rather than uptake) by extensor cells and increased the rate of H⁺ efflux from flexor cells, presumably by decreasing the rate of inward diffusion. (d) Change in external K⁺ did not alter H⁺ fluxes by extensor cells, but removal of external K⁺ decreased the rate of H⁺ efflux from flexor cells by 70%. These observations support a model for coupled H⁺/K⁺ fluxes in pulvinar cells during light-and dark-promoted leaflet movements.     

Adenine nucleotide translocase as a site of regulation by ADP of the rat liver mitochondria permeability to H+ and K+ lons

In the presence of oligomycin ADP inhibits the osmotic swelling of the nonenergized rat liver mitochondria in the NH₄NO₃ medium. With the energized mitochondria ADP enhances contraction of the mitochondria swollen in the NH₄NO₃ medium. Carboxyatractyloside and atractyloside abolish or prevent the effects of ADP. The direct measurements of the proton conductance of rat liver mitochondria shows that the inhibitory action of ADP + oligomycin on the H⁺ permeability does not depend on the energization of mitochondria. In these experiments the local anesthetic nupercaine and ADP additively inhibit the inner membrane conductance for protons, but carboxyatractyloside abolishes only the effect of ADP. In the presence of oligomycin ADP also inhibits the osmotic swelling of the nonenergized liver mitochondria in the KNO₃ medium, and the energy-dependent swelling of rat liver mitochondria in the medium with K⁺ ions and P_i. The inhibition by ADP of the membrane passive permeability for K⁺ is also sensitive to carboxyatractyloside. It is concluded that rat liver mitochondria possess an ADP-regulated channel for H⁺ and K⁺. The properties of this pathway for protons and potassium ions favor the idea that ADP regulates the mitochondrial permeability via adenine nucleotide translocase. It is assumed that the adenine nucleotides carrier should operate according to the “gated pore” mechanism.     

Effect of Insulin on Potassium Flux and Water and Electrolyte Content of Muscles from Normal and from Hypophysectomized Rats

It was reported previously that insulin hyperpolarized rat skeletal muscle and decreased K⁺ flux in both directions. The observations on K⁺ flux are now extended to take advantage of the greater sensitivity to insulin of hyperphysectomized rats. Insulin caused a shift of water from extracellular to intracellular space if glucose was present, but not in its absence. Insulin caused net gain of muscle fiber K⁺, though not necessarily an increase in K⁺ concentration in fiber water. It probably also decreased intrafiber Na⁺ and Cl^-. Insulin decreased K⁺ efflux. The effect was dose-dependent. Muscles from hypophysectomized rats were more sensitive to the action of insulin on K⁺ flux than were those from normal rats. The effect was demonstrable within the time resolution of the system, suggesting that insulin's action is on cell surfaces. K⁺ influx was also decreased by insulin. Bookkeeping suggests that some K⁺ influx be called active. Insulin seemed to decrease active K⁺ influx and passive K⁺ efflux. It is not resolved whether insulin has a true dual effect or whether it acts only on passive fluxes in both directions (the apparent action on active K⁺ influx being an artefact of incomplete definition of passive flux) or whether a single alteration in the membrane may affect both active and passive fluxes.     

Active and passive transport of potassium in cells of excised pea epicotyls

The Ussing-Theorell equation, which provides a fundamental test for the independent passive movement of ions under conditions of nonequilibrium, has been used to assess the active and passive components of K⁺ uptake by segments of pea epicotyl (Pisum sativum L. cultivar Alaska), incubated for 24 hours in both 1-fold and 10-fold concentrations of a complete nutrient solution. Measurements of the rates at which ⁴²K diffused out of the segments provided data from which were estimated the K⁺ content of, and the fluxes to and from, the nonfree space compartments, interpreted as being cytoplasm and vacuole. For this analysis the serial model of MacRobbie and Dainty and Pitman for the spatial arrangement of cell compartments was used. On the basis of these values, and measurements of electrical potential across the cell membranes, the vacuolar K⁺ concentration was found to be fairly close to that expected as a result of passive diffusion between the cytoplasm and vacuole provided that no potential exists across the tonoplast. Cytoplasmic K⁺ concentration, however, was much too high in both treatments to be accounted for in passive terms. It was concluded, therefore, that, on the basis of the model, the high ratio of influx to efflux was maintained in the cells by an active K⁺ pump located at the plasmalemma. There is some reason to question the applicability of this model for flux analysis to the conditions of high net influx as encountered here; nonetheless, it provides a first approach to an over-all flux analysis in pea stem tissue.     

Effect of cholesterol content on some physical and functional properties of mitochondria isolated from adult rat liver,fetal liver,cholesterol-enriched liver and hepatomas AH-130, 3924A and 5123

The choleterol to phospholipid ratio in mitochodria from hepatomas AH-130, 3924A and 5123 is higher than in the particles isolated from adult or fetal rat livers. Nearly all the cholesterol of hepatoma mitochonda is located in membranes. As in liver mitochondria, in the particles isolated from hepatoma AH-130 there is more cholesterol in the outer than in the inner membrane.In mitochondria from cholesterol-enriched liver and hepatomas, there occur a decrease in extent of hypoosmotic and phosphate-induced sweeling and decrease of conformational linked energy states. The phenomenon is more marked in particles which exhibit higher cholesterol to phospholopid ratios. A statistically significant negative correlation exists between the cholesterol to phospholipid ratio and extent of volume or conformation changes. No significant modifications of these parameters were found in fetal liver mitochondria.Cholesterol content does not influence K⁺ uptake by cholesterol-enriched or hepatoma mitochondria. Nor does cholesterol content affect the respiratory increment related to this uptake. As a consequence of K⁺ uptake, total mitochodrial water exchangeable with tritiated water rises 20% while sucrose-impermeable water rises 42–48% in both adult rat liver and hepatoma AH-130 mitochondria. Absorbance changes linked to ion uptake do not correspond merely to variations in mitochobdrial water content. Water content. Water is apparently not influenced by the cholesterol to phospholipid ratio. However, the ratio is signifacantly correlated to both extent and initial rate of absorbance decrease of mitochondrial suspension during K⁺ uptake. The higher the ratio, the lower the extent and and initial rate of absorbance decrease.     

Ion transport by heart mitochondria: XXVII. The relation of mercurial-dependent adenosine triphosphatase activity to ion movements

The properties of a mercurial-dependent adenosine triphosphatase activity have been examined in isolated beef heart mitochondria. The reaction differs from that induced by uncouplers in that it is associated with extensive ion uptake and osmotic swelling, is highly specific for K⁺ over Na⁺, and is enhanced by respiration. Evidence is presented which suggests that the following events can account for the observations: (1) The mercurial blocks the phosphate transporter so that phosphate hydrolyzed from ATP is trapped in the matrix. (2) This interior negative potential causes cations to move inward and swelling results. (3) Permeability to K⁺ but not to Na⁺ is enhanced greatly by the reaction of the mercurial with the membrane. The inward movement of K⁺ closely resembles that produced by valinomycin, in that it is accompanied by proton ejection into the medium and it rapidly establishes a condition in which ion gradients cannot be maintained. This marked increase in permeability may be related to the pH gradient and is manifest as additional passive swelling in the absence of sucrose and passive contraction when sucrose is present. A comparison of the kinetics of swelling and of ATP hydrolysis shows that the elevated rates of ATPase are correlated with this condition of high permeability. When a corresponding condition of high permeability to Na⁺ is established by treatment with gramicidin or EDTA, the mercurial-dependent ATPase is nearly as rapid in Na⁺ as in the K⁺ medium. It appears, therefore, that the K⁺ specificity resides at the level of membrane permeability and is not a feature of the ATPase reaction per se. (4) Respiration appears to affect the ATPase reaction by virtue of its ability to extrude ions from the matrix in the presence of the mercurial. p-Chloromercuriphenyl sulfonate causes a switch from respiration-dependent ion accumulation to respiration-dependent ion extrusion to occur. A model to explain these reactions is presented.     

Voltage-dependent Inwardly Rectifying Potassium Conductance in the Outer Membrane of Neuronal Mitochondria

Potassium fluxes integrate mitochondria into cellular activities, controlling their volume homeostasis and structural integrity in many pathophysiological mechanisms. The outer mitochondrial membrane (OMM) is thought to play a passive role in this process because K⁺ is believed to equilibrate freely between the cytosol and mitochondrial intermembrane space. By patch clamping mitochondria isolated from the central nervous systems of adult mitoCFP transgenic mice, we discovered the existence of I_OMMKi, a novel voltage-dependent inwardly rectifying K⁺ conductance located in the OMM. I_OMMKi is regulated by osmolarity, potentiated by cAMP, and activated at physiological negative potentials, allowing K⁺ to enter the mitochondrial intermembrane space in a controlled regulated fashion. The identification of I_OMMKi in the OMM supports the notion that a membrane potential could exist across this membrane in vivo and suggests that the OMM possesses regulated pathways for K⁺ uptake.     

Computer simulations of neuron-glia interactions mediated by ion flux

Extracellular potassium concentration, [K⁺]_o, and intracellular calcium, [Ca²⁺]_i, rise during neuron excitation, seizures and spreading depression. Astrocytes probably restrain the rise of K⁺ in a way that is only partly understood. To examine the effect of glial K⁺ uptake, we used a model neuron equipped with Na⁺, K⁺, Ca²⁺ and Cl⁻ conductances, ion pumps and ion exchangers, surrounded by interstitial space and glia. The glial membrane was either “passive”, incorporating only leak channels and an ion exchange pump, or it had rectifying K⁺ channels. We computed ion fluxes, concentration changes and osmotic volume changes. Increase of [K⁺]_o stimulated the glial uptake by the glial 3Na/2K ion pump. The [K⁺]_o flux through glial leak and rectifier channels was outward as long as the driving potential was outwardly directed, but it turned inward when rising [K⁺]_o/[K⁺]_i ratio reversed the driving potential. Adjustments of glial membrane parameters influenced the neuronal firing patterns, the length of paroxysmal afterdischarge and the ignition point of spreading depression. We conclude that voltage gated K⁺ currents can boost the effectiveness of the glial “potassium buffer” and that this buffer function is important even at moderate or low levels of excitation, but especially so in pathological states.     

Glucose Accumulation Can Account for the Initial Water Flux Triggered by Na+/Glucose Cotransport

Over the last decade, several cotransport studies have led to the proposal of secondary active transport of water, challenging the dogma that all water transport is passive. The major observation leading to this interpretation was that a Na⁺ influx failed to reproduce the large and rapid cell swelling induced by Na⁺/solute cotransport. We have investigated this phenomenon by comparing a Na⁺/glucose (hSGLT1) induced water flux to water fluxes triggered either by a cationic inward current (using ROMK2 K⁺ channels) or by a glucose influx (using GLUT2, a passive glucose transporter). These proteins were overexpressed in Xenopus oocytes and assayed through volumetric measurements combined with double-electrode electrophysiology or radioactive uptake measurements. The osmotic gradients driving the observed water fluxes were estimated by comparison with the swelling induced by osmotic shocks of known amplitude. We found that, for equivalent cation or glucose uptakes, the combination of substrate accumulations observed with ROMK2 and GLUT2 are sufficient to provide the osmotic gradient necessary to account for a passive water flux through SGLT1. Despite the fact that the Na⁺/glucose stoichiometry of SGLT1 is 2:1, glucose accumulation accounts for two-thirds of the osmotic gradient responsible for the water flux observed at t = 30 s. It is concluded that the different accumulation processes for neutral versus charged solutes can quantitatively account for the fast water flux associated with Na⁺/glucose cotransport activation without having to propose the presence of secondary active water transport.     

Electrochemical gradients and k and cl fluxes in excised corn roots

The compartmental analysis method was used to estimate the K⁺ and Cl⁻ fluxes for cells of excised roots of Zea mays L. cv. Golden Bantam. When the measured fluxes are compared to those calculated with the Ussing-Teorell flux-ratio equation, an active inward transport of Cl⁻ across the plasmalemma is indicated; the plasmalemma K⁺ fluxes are not far different from those predicted for passive diffusion, although an active inward transport cannot be precluded. Whether fluxes across the tonoplast are active or passive depends upon the vacuolar potential which is unknown. Assuming no electropotential gradient, the tracer flux ratios are fairly close to those predicted for passive movement. However, if the vacuole is positive by about 10 millivolts relative to the cytoplasm, the data suggest active inward transport for K⁺ and outward transport for Cl⁻.     

Ion transport processes in corn mitochondria : I. Effect of the local anesthetic dibucaine

The local anesthetic dibucaine inhibited respiration-dependent contraction mediated by the K⁺/H⁺ antiport system of isolated corn mitochondria. Respiration declined concurrently. Nigericin, an exogenous K⁺/H⁺ exchanger, restored ion efflux in dibucaine-blocked corn mitochondria. It was concluded that dibucaine inhibited ion efflux via blockage of the K⁺/H⁺ antiport. Further experiments determined that dibucaine also inhibited proton influx facilitated by protonophores and by the ATPase complex during state III respiration. These results are discussed in relation to the mechanism by which dibucaine inhibits proton translocation across the inner mitochondrial membrane.