Stimulation of K+ flux into mitochondria by phenylarsine oxide

The dithiol-reactive reagent phenylarsine oxide causes a pH-dependent stimulation of unidirectional K⁺ flux into respiring rat liver mitochondria. This stimulation is diminished by subsequent addition of either the dithiol 2,3-dimercaptopropanol or the monothiol 2-mercaptoethanol. In contrast, uncoupling by phenylarsine oxide is reversed by 2,3-dimercaptopropanol but not by 2-mercaptoethanol. The data suggest separate sites of interaction of phenylarsine oxide with mechanisms of K⁺ entry and ATP synthesis. Stimulatory effects of mersalyl and phenylarsine oxide on K⁺ influx are not additive. Thus PheASO and mersalyl may affect K⁺ influx at a common site. Pretreatment of the mitochondria with DCCD, which inhibits K⁺ influx, fails to alter sensitivity to PheAsO or mersalyl. Thus the DCCD binding site associated with the K⁺ influx mechanism appears to be separate from and independent of the sulfhydryl group(s) which mediate stimulation of K⁺ influx by PheAsO and mersalyl.PheAsO, like mersalyl, also increases the rate of unidirectional K⁺ efflux from respiring mitochondria. The combined presence of PheAsO plus mersalyl causes a greater stimulation of K⁺ efflux than is observed with either reagent alone.Abbreviations used: BAL, British AntilLewisite or 2,3-dimercaptopropanol; DCCD, dicyclohexylcarbodiimide; DBCT, dibutylchloromethyltin chloride; 2-ME, 2-mercaptoethanol; PheAsO, phenylarsine oxide.     

Ba2+ uptake and the inhibition by Ba2+ of K+ flux into rat liver mitochondria

Summary Rapid uptake of Ba²⁺ by respiring rat liver mitochondria is accompanied by a transient stimulation of respiration. Following accumulation of Ba²⁺, e.g. at a concentration of 120 nmol per mg protein, the mitochondria exhibit reduced rates of state 3 and uncoupler-stimulated respiration. ADP-stimulated respiration is inhibited at a lower concentration of Ba²⁺ than is required to affect uncoupler-stimulated respiration, suggesting a distinct effect of Ba²⁺ on mechanisms involved in synthesis of ATP. Ba²⁺, which has an ionic radius similar to that of K⁺, inhibits unidirectional K⁺ flux into respiring rat liver mitochondria. This effect on K⁺ influx is observable at concentrations of Ba²⁺, e.g. 23 to 37 nmol per mg protein, which cause no significant change in state 4 or uncoupler-stimulated respiration. The accumulated Ba²⁺ decreases the measuredV _max of K⁺ influx, while having little effect on the apparentK _m for K⁺. The inhibition of K⁺ influx by Ba²⁺ is seen in the presence and absence of mersalyl, an activator of K⁺ influx. In contrast, under the conditions studied, Ba²⁺ has no apparent effet on the rate of unidirectional K⁺ efflux. These data are consistent with the idea that K⁺ may enter and leave mitochondria via spearate mechanisms.     

Kinetics of Mg2+ flux into rat liver mitochondria.

Unidirectional fluxes of Mg2+ across the limiting membranes of rat liver mitochondria have been measured in the presence of the respiratory substrate succinate by means of the radioisotope 28Mg. Rates of both influx and efflux of Mg2+ are decreased when respiration is inhibited. A linear dependence of the reciprocal of the Mg2+ influx rate on the reciprocal of the Mg2+ concentration is observed. The apparent Km for Mg2+ averages about 0.7 mM. N-Ethyl-maleimide, an inhibitor of transmembrane phosphate-hydroxyl exchanges, enhances the observed pH dependence of Mg2+, influx. In the presence of MalNEt, the apparent Vmax of Mg2+ influx is greater at pH 8 than at pH 7, and there is a linear dependence of the Mg2+ influx rate on the external OH- concentration. The K+ analogue Tl+ inhibits Mg2+ influx, while La3+, an inhibitor of mitochondrial Ca2+ transport, has no effect on Mg2+ influx. Mg2+ competitively inhibits the flux of K+ into rat liver mitochondria. The mechanism(s) mediating mitochondrial Mg2+ and K+ fluxes appear to be similar in their energy dependence, pH dependence, sensitivity to Tl+, and insensitivity to La3+.     

Effects of quinine on K+ transport in heart mitochondria

Quinine inhibits the respiration-dependent extrusion of K⁺ from Mg²⁺-depleted heart mitochondria and the passive osmotic swelling of these mitochondria in K⁺ and Na⁺ acetate at alkaline pH. These observations concur with those of Nakashima and Garlid (J. Biol. Chem. 257, 9252, 1982) using rat liver mitochondria. Quinine also inhibits the respiration-dependent contraction of heart mitochondria swollen passively in Na⁺ or K⁺ nitrate and the increment of elevated respiration associated with the extrusion of ions from these mitochondria. Quinine, at concentrations up to 0.5 mM, inhibits the respiration-dependent⁴²K⁺/K⁺ exchange seen in the presence of mersalyl, but higher levels of the drug produce increased membrane permeability and net K⁺ loss from the matrix. These results are all consistent with an inhibition of the putative mitochondrial K⁺/H⁺ antiport by quinine. However, quinine has other effects on the mitochondrial membrane, and possible alternatives to this interpretation are discussed.     

Effects of K+ and other monovalent cations on yeast mitochondria.

In mitochondria from Saccharomyces cerevisiae and in the presence of ethanol or NADH, K+ or Na+ increased the rate of O2 uptake in states 3 and uncoupled as well as in sonicated mitochondria. The respiratory control, the ADP:O ratio and the synthesis of ATP also increased. ATP hydrolysis by sonicated mitochondria increased depending on the cation added as follows: K+ = NH4+ = Rb+ Na+ Li+. This correlated with the ionic radii of the cations. Monovalent cations increased the activity of: 1) F1F0ATPase which was sensitive to cation size and 2) complex I of the respiratory chain, which seemed to regulate the rate of oxidative phosphorylation, but did not discriminate between K+ or Na+.     

Energy-dependence exchange of K+ in heart mitochondria. K+ efflux.

The efflux ⁴²K⁺ from isolated beef heart mitochondria under conditions of near steadystate K⁺ is increased by repiration and is sensitive to uncouplers and to exogenous Mg² The respiration-dependent efflux is strongly activated by inorganic phosphate in the presence of external K⁺, but not Na⁺, and is inhibited by oxidative phosphorylation. Low concentrations of mersalyl also activate respiration-dependent efflux of ⁴²K⁺ in the absence of net alteration in matrix K⁺. Acetate in the presence of mersalyl brings about net accumulation of K⁺ with retention of internal ⁴²K⁺. The results are consistent with a model in which nearly constant matrix K⁺ is maintained by the regulated interplay between a K⁺ uniport (which is responsive to membrane potential and which is the pathway for K⁺ influx) and a $\text{K}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchanger (which responds to the transmembrane pH differential and which is the pathway for net K⁺ efflux).     

Energy-dependent exchange of K+ in heart mitochondria. K+ influx.

The tuberculostatic and carcinogenic drug isonicotinic acid hydrazide (“isoniazid”) is oxidized to pyridine-4-carboxaldehyde by the horseradish peroxidase/Mn²⁺/O₂ system. Eosin and related sensitizers greatly accelerate the reaction and generate light detectable with the liquid scintillation counter or with the photon counter. If the isoniazid concentration is raised, the rate and extent of O₂ uptake are increased, but above a certain concentration of isoniazid, emission is reduced and even suppressed. The strong quencher of triplet eosin, potassium ferricyanide, abolished both effects of eosin, that is, catalysis and light emission. Superoxide dismutase at high concentrations partially suppressed the emission and almost totally removed the catalytic effect. It is tentatively proposed that the isoniazid/peroxidase/Mn²⁺/O₂ system efficiently produces the aldehyde in the triplet state, which in turn transfers energy to eosin. Because of the presence of oxygen, only a small yield of triplet eosin is obtained and only a small fraction of these triplet eosin molecules is able to react with isoniazid. Nevertheless, it will contribute efficiently to a cyclic process of oxidation of the isoniazid.     

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.     

Mitochondrial K + transport: effect of N-ethyl maleimide on 42 K flux

The energy-linked flux of K⁺ into rat liver mitochondria is found to be stimulated by the sulfhydryl reagent, N-ethyl maleimide. The stimulation of K⁺ influx by N-ethyl maleimide is observed only at alkaline external pH. N-ethyl maleimide also stimulates efflux of K⁺ from the mitochondria. The stimulation by N-ethyl maleimide of K⁺ influx, but not K⁺ efflux, is dependent on the availability of metabolic energy. It is suggested that the effect of N-ethyl maleimide on K⁺ influx may be secondarily the result of an inhibition of phosphate-hydroxyl exchange. The dependence of energy-linked K⁺ influx on the external pH may be interpreted as evidence for a role of OH^? as a counterion accompanying K⁺ through the mitochondrial pump mechanism.     

Kinetics of ATP-dependent Mg2+ flux in mitochondria.

ATP-dependent Mg2+ accumulation in isolated mitochondria occurs predominantly in the matrix and inner membrane compartments. In mitochondria contaminated with lysosomes, the time course and magnitude of ATP-dependent Mg2+ accumulation are influenced by various cytoplasmic substances, besides substrates of the citric acid cycle. Removal of lysosomes by treatment of the mitochondrial preparation with low concentrations of digitonin, which does not damage the mitoplast, eliminates the modifying influence of cytoplasmic components on Mg2+ flux. In lysosome-free mitochondria, the kinetics of Mg2+ flux is dependent only on the concentration of ATP, of Mg2+, and on the availability of site specific reducing substrates of the electron transport system. Oligomycin at concentrations sufficient to inhibit phosphorylation coupled electron transport and ATP synthesis does not modify Mg2+ flux, which is dependent on added ATP. Site specific inhibitors of the electron transport system inhibit the augmenting effect of oxidizable substrates on Mg2+ uptake, even when electron transfer is inhibited by oligomycin. Atractyloside, by inhibiting the action of externally added ATP, diminishes Mg2+ flux. Ruthenium red is a powerful inhibitor of ATP dependent Mg2+ flux. Uncouplers not only inhibit Mg2+ uptake, but induce Mg2+ efflux. From the time course of Mg2+ flux, a first-order rate constant of egress of Mg2+ and other kinetic constants were calculated and a kinetic model was derived which describes the bi-directional movement of Mg 2+ in mitoplasts.     

Effect of antibodies against mitochondrial K+-transporting protein on K+ transport in rat liver mitochondria

The K+ transport in rat liver mitochondria was studied by the immunochemical method. Antibodies to mitochondrial K+-transporting protein with molecular weight 60 kDa were obtained and used as possible inhibitor or K+ transport. Antibodies depressed the DNP-induced K+ efflux and energy-dependent swelling by 50% without causing changes in respiration and oxidative phosphorylation.     

Channel-mediated K+ flux in barley aleurone protoplasts

Gibberellic acid (GA₃) stimulates K⁺ efflux from the barley (Hordeum vulgare L. cv. Himalaya) aleurone. We investigated the mechanism of K⁺ flux across the plasma membrane of aleurone protoplasts using patch-clamp techniques. Potassium-ion currents, measured over the entire surface of the protoplast plasma membrane, were induced when the electrochemical gradient for K⁺ was inward (into the cytoplasm). The magnitude and voltage-dependence of this inward current were the same in protoplasts treated with GA₃ and in control protoplasts (no GA₃). Inward currents activated by negative shifts in the membrane potential (E_M) from the Nernst potential for K⁺ (E_K) showed membrane conductance to be a function of the electrochemical gradient (i.e. E_M-E_K). Single-channel influx currents of K⁺ were recorded in small patches of the plasma membrane. These channels had a single-channel conductance of 5–10 pS with 100 mM K⁺ on the inside and 10 mM K⁺ on the outside of the plasma membrane. Single-channel currents, like whole-cell currents, were the same in protoplasts treated with GA₃ and control protoplasts. Voltage-gated efflux currents were found only in protoplasts tha thad been incubated without GA₃. We conclude that K⁺ influx in the aleurone is mediated by channels and these membrane proteins are not greatly effected by GA₃.Abbreviations and symbols F_K Nernst potential for K⁺ - E_M membrane potential - E_rev reversal potential - GA₃ gibberellic acid - K_i concentration of K⁺ inside the cell - K_o concentration of K⁺ outside the cell - R gas constant - S conductance (siemens) - T temperature (^oK) - _i ionic activity coefficient for internal (cytoplasmic) solution - _o ionic activity coefficient for external medium     

K+/H+ antiport in heart mitochondria

Heart mitochondria depleted of endogenous divalent cations by treatment with A23187 and EDTA swell in (a) K+ acetate or (b) K+ nitrate when an uncoupler is present. These mitochondria also exchange matrix 42K+ with external K+, Na+, or Li+ in a reaction that does not require respiration and is insensitive to uncouplers. Untreated control mitochondria do not swell in either medium nor do they show the passive cation exchange. Both the swelling and the exchange reactions are inhibited by Mg2+ and by quinine and other lipophilic amines. Swelling and exchange are both strongly activated at alkaline pH, and the exchange reaction is also increased markedly by hypotonic conditions. All of these properties correspond to those reported for a respiration-dependent extrusion of K+ from Mg2+-depleted mitochondria, a reaction attributed to a latent Mg2+- and H+-sensitive K+/H+ antiport. The swelling reactions are strongly inhibited by dicyclohexylcarbodiimide reacted under hypotonic conditions, but the exchange reaction is not sensitive to this reagent. Heart mitochondria depleted of Mg2+ show marked increases in their permeability to H+, to anions, and possibly to cations, and the permeability to each of these components is further increased at alkaline pH. This generalized increase in membrane permeability makes it likely that K+/H+ antiport is not the only pathway available for K+ movement in these mitochondria. It is concluded that the swelling, 42K+ exchange, and K+ extrusion data are all consistent with the presence of the putative K+/H+ antiport but that definitive evidence for the participation of such a component in these reactions is still lacking.     

Effect of quinine on mitochondrial K+ and Mg++ flux

Quinine decreases rates of unidirectional K+ flux into and out of respiring rat liver mitochondria. K+ efflux is more sensitive to quinine than K+ influx. The data are consistent with the proposal that two separate mechanisms may mediate K+ influx, only one of which is sensitive to quinine. Effects on K+ flux of the stereoisomer quinidine are similar to effects of quinine. The smaller quinuclidine causes at most a slight inhibition of K+ efflux under the same conditions. Mg++ flux exhibits a pattern of inhibition by quinine similar to that of K+ flux. Mg++ efflux is more sensitive to quinine than is Mg++ influx. These and earlier findings indicate marked similarities between liver mitochondrial transport mechanisms for K+ and Mg++.     

Effect of beta-adrenoceptor blockade on H+ and K+ flux in exercising humans

The effect of beta-adrenoceptor blockade (beta B) on muscle release and uptake of H+ and K+ in humans during maximal exercise has been investigated. Eight volunteers cycled intermittently at power outputs corresponding to 100% of maximal O2 uptake. Prior to exercise either propranolol (beta B) or saline (control) was infused into the femoral vein. Arterial and femoral venous blood samples were drawn at rest, during exercise, and during 30-min recovery. Peak arterial blood values for K+, lactic acid (LA), and base deficit (BD) (mean +/- SE) were respectively 5.5 +/- 0.1, 9.5 +/- 0.6, and 11.7 +/- 0.9 mmol/l during beta B and 5.1 +/- 0.1, 8.3 +/- 0.6, and 10.3 +/- 1.0 for control (P less than 0.05). The release of K+ from the working leg did not differ between treatments during exercise, but K+ uptake during late recovery (5-30 min) was slightly lower during beta B. Thus the higher arterial K+ levels during exercise (beta B) cannot be attributed to greater release by active muscle but are likely due to decreased K+ uptake by noncontracting muscle. Arterial-femoral venous differences for LA and BD did not differ significantly between treatments. Additionally LA exchange across the leg was similar to H+ exchange (arterial-femoral venous differences for BD) under all conditions. During early recovery (1-5 min), regardless of experimental treatment, BD levels iin arterial blood were higher than LA (P less than 0.05). These elevated BD levels may be due to unequal removal rates between LA and H+ equivalents by nonexercised tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sensitivity of mitochondrial Mg++ flux to reagents which affect K+ flux

Effects on Mg⁺⁺ transport in rat liver mitochondria of three reagents earlier shown to affect mitochondrial K⁺ transport have been examined. The sulfhydryl reactive reagent phenylarsine oxide, which activates K⁺ flux into respiring mitochondria, also stimulates Mg⁺⁺ influx. The K⁺ analog Ba⁺⁺, when taken up into the mitochondrial matrix, inhibits influx of both K⁺ and Mg⁺⁺. The effect on Mg⁺⁺ influx is seen only if Mg⁺⁺, which blocks Ba⁺⁺ accumulation, is added after a preincubation with Ba⁺⁺. Thus the inhibition of Mg⁺⁺ influx appears to require interaction of Ba⁺⁺ at the matrix side of the inner mitochondrial membrane. Added Ba⁺⁺ also diminishes observed rates of Mg⁺⁺ efflux but not K⁺ efflux. This difference may relate to a higher concentration of Ba⁺⁺ remaining in the medium in the presence of Mg⁺⁺ under the conditions of our experiments. Pretreatment of mitochondria with dicyclohexylcarbodiimide (DCCD), under conditions which result in an increase in the apparentK _m for K⁺ of the K⁺ influx mechanism, results in inhibition of Mg⁺⁺ influx from media containing approximately 0.2 mM Mg⁺⁺. The inhibitory effect of DCCD on Mg⁺⁺ influx is not seen at higher external Mg⁺⁺ (0.8 mM). This dependence on cation concentration is similar to the dependence on K⁺ concentration of the inhibitory effect of DCCD on K⁺ influx. Although mitochondrial Mg⁺⁺ and K⁺ transport mechanisms exhibit similar reagent sensitivities, whether Mg⁺⁺ and K⁺ share common transport catalysts remains to be established.Abbreviations used: DCCD, dicyclohexylcarbodiimide; PheAsO, phenylarsine oxide.