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.     

Modulation of Na+/H+ exchange activity by Cl-

Na+/H+ exchanger (NHE) activity is exquisitely dependent on the intra- and extracellular concentrations of Na+ and H+. In addition, Cl- ions have been suggested to modulate NHE activity, but little is known about the underlying mechanism, and the Cl- sensitivity of the individual isoforms has not been established. To explore their Cl- sensitivity, types 1, 2, and 3 Na+/H+ exchangers (NHE1, NHE2, and NHE3) were heterologously expressed in antiport-deficient cells. Bilateral replacement of Cl- with nitrate or thiocyanate inhibited the activity of all isoforms. Cl- depletion did not affect cell volume or the cellular ATP content, which could have indirectly altered NHE activity. The number of plasmalemmal exchangers was unaffected by Cl- removal, implying that inhibition was due to a decrease in the intrinsic activity of individual exchangers. Analysis of truncated mutants of NHE1 revealed that the anion sensitivity resides, at least in part, in the COOH-terminal domain of the exchanger. Moreover, readdition of Cl- into the extracellular medium failed to restore normal transport, suggesting that intracellular Cl- is critical for activity. Thus interaction of intracellular Cl- with the COOH terminus of NHE1 or with an associated protein is essential for optimal activity.     

Electroneutral K+/H+ exchange in mitochondrial membrane vesicles involves Yol027/Letm1 proteins

YOL027c in yeast and LETM1 in humans encode integral proteins of the inner mitochondrial membrane. They have been implicated in mitochondrial K+ homeostasis and volume control. To further characterize their role, we made use of submitochondrial particles (SMPs) with entrapped K+- and H+-sensitive fluorescent dyes PBFI and BCECF, respectively, to study the kinetics of K+ and H+ transport across the yeast inner mitochondrial membrane. Wild-type SMPs exhibited rapid, reciprocal translocations of K+ and H+ driven by concentration gradients of either of them. K+ and H+ translocations have stoichiometries similar to those mediated by the exogenous K+/H+ exchanger nigericin, and they are shown to be essentially electroneutral and obligatorily coupled. Moreover, [K+] gradients move H+ against its concentration gradient, and vice-versa. These features, as well as the sensitivity of K+ and H+ fluxes to quinine and Mg2+, qualify these activities as K+/H+ exchange reactions. Both activities are abolished when the yeast Yol027p protein is absent (yol027Delta mutant SMPs), indicating that it has an essential role in this reaction. The replacement of the yeast Yol027p by the human Letm1 protein restores K+/H+ exchange activity confirming functional homology of the yeast and human proteins. Considering their newly identified function, we propose to refer to the yeast YOL027c gene and the human LETM1 gene as yMKH1 and hMKH1, respectively.     

Mechanism of osmotic activation of Na+/H+ exchange in rat thymic lymphocytes

The activity of the Na+/H+ exchange system of rat thymic lymphocytes was determined by means of intracellular (pHi) and extracellular pH (pH0) measurements. In isotonic media, the antiport is virtually quiescent at physiological pHi (7.0-7.1), but is greatly activated by cytoplasmic acidification. At normal pHi, the antiport can also be activated by osmotic shrinking. Osmotic activation occurs after a delay of 20-30 s and is reversed several minutes after iso-osmolarity is restored. The mechanism of activation was analyzed by comparing the kinetic parameters of transport in resting (isotonic) and hyperosmotically stressed cells. The affinities of the external substrate site for Na+ and H+ are not altered in shrunken cells. In contrast, the Hi+ sensitivity of the antiport (which is largely dictated by an allosteric modifier site) was increased, which accounted for the activation. The concentration of free cytoplasmic Ca2+ [( Ca2+]i) increased after osmotic shrinking. This increase was dependent on the presence of extracellular Ca2+ and Na+ and was blocked by inhibitors of Na+/H+ exchange, which suggests that it is a consequence, rather than the cause, of the activation of the antiport. It is concluded that the shift in the pHi dependence of the modifier site of the Na+/H+ antiport is the primary event underlying the regulatory volume increase that follows osmotic shrinkage.     

Reconstitution of the mitochondrial non-selective Na+/H+ (K+/H+) antiporter into proteoliposomes

Mitochondria contain two Na+/H+ antiporters, one of which transports K+ as well as Na+. The physiological role of this non-selective Na+/H+ (K+/H+) antiporter is to provide mitochondrial volume homeostasis. The properties of this carrier have been well documented in intact mitochondria, and it has been identified as an 82,000-dalton inner membrane protein. The present studies were designed to solubilize and reconstitute this antiporter in order to permit its isolation and molecular characterization. Proteins from mitoplasts made from rat liver mitochondria were extracted with Triton X-100 in the presence of cardiolipin and reconstituted into phospholipid vesicles. The reconstituted proteoliposomes exhibited electroneutral 86Rb+ transport which was reversibly inhibited by Mg2+ and quinine with K0.5 values of approximately 150 and 300 microM, respectively. Incubation of reconstituted vesicles with dicyclohexylcarbodiimide resulted in irreversible inhibition of 86Rb+ uptake into proteoliposomes. Incubation of vesicles with [14C]dicyclohexylcarbodiimide resulted in labeling of an 82,000-dalton protein. These properties, which are also characteristic of the native Na+/H+ (K+/H+) antiporter, lead us to conclude that this mitochondrial carrier has been reconstituted into proteoliposomes with its known native properties intact.     

Nigericin-induced Na+/H+ and K+/H+ exchange in synaptosomes: Effect on [3H]GABA release

The effect of the putative K⁺/H⁺ ionophore, nigericin on the internal Na⁺ concentration ([Na _i ]), the internal pH (pH _i ), the internal Ca²⁺ concentration ([Ca _i ]) and the baseline release of the neurotransmitter, GABA was investigated in Na⁺-binding benzofuran isophtalate acetoxymethyl ester (SBFIAM), 2′,7′-bis(carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM), fura-2 and [³H]GABA loaded synaptosomes, respectively. In the presence of Na⁺ at a physiological concentration (147 mM), nigericin (0.5 μM) elevates [Na _i ] from 20 to 50 mM, increases thepH _i , 0.16 pH units, elevates four fold the [Ca _i ] at expense of external Ca²⁺ and markedly increases (more than five fold) the release of [³H]GABA. In the absence of a Na⁺ concentration gradient (i.e. when the external Na⁺ concentration equals the [Na _i ]), the same concentration (0.5 μM) of nigericin causes the opposite effect on thepH _i (acidifies the synaptosomal interior), does not modify the [Na _i ] and is practically unable to elevate the [Ca _i ] or to increase [³H]GABA release. Only with higher concentrations of nigericin than 0.5 μM the ionophore is able to elevate the [Ca _i ] and to increase the release of [³H]GABA under the conditions in which the net Na⁺ movements are eliminated. These results clearly show that under physiological conditions (147 mM external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore, and all its effects are triggered by the entrance of Na⁺ in exchange for H⁺ through the ionophore itself. Nigericin behaves as a K⁺/H⁺ ionophore in synaptosomes just when the net Na⁺ movements are eliminated (i.e. under conditions in which the external and the internal Na⁺ concentrations are equal). In summary care must be taken when using the putative K⁺/H⁺ ionophore nigericin as an experimental tool in synaptosomes, as under standard conditions (i.e. in the presence of high external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore.     

Amiloride inhibits phorbol ester-stimulated Na+/H+ exchange and protein kinase C. An amiloride analog selectively inhibits Na+/H+ exchange

The human leukemic cell line, HL-60, differentiates in response to tumor-promoting phorbol esters. Recently, we have reported that one of the first events evoked by phorbol esters in HL-60 cells is the stimulation of Na+-dependent H+ efflux. In efforts to determine whether stimulation of Na+/H+ exchange by phorbol esters is coupled to induction of cellular differentiation, we found that 1) amiloride, a frequently used inhibitor of Na+/H+ exchange, rapidly inhibits phorbol ester-stimulated protein phosphorylation in vivo and protein kinase C-mediated phosphorylation in vitro, both with potency similar to that with which amiloride inhibits Na+/H+ exchange; 2) an amiloride analog, dimethylamiloride, is a far more potent inhibitor of Na+/H+ exchange than is amiloride, while being no more potent than amiloride in inhibiting phorbol ester/protein kinase C-mediated phosphorylation; and 3) at concentrations sufficient to completely inhibit Na+/H+ exchange, amiloride blocked phorbol ester-induced adhesion of HL-60 cells (adhesion being a property indicative of the differentiated state), but dimethylamiloride (as well as ethylisopropylamiloride, another very potent amiloride analog) did not. Thus, dimethylamiloride represents a potential tool for distinguishing protein kinase C-coupled from Na+/H+ exchange-coupled events in phorbol ester-stimulated cells.     

Human cytomegalovirus infection enhances osmotic stimulation of Na+/H+ exchange in human fibroblasts

Infection withhuman cytomegalovirus (HCMV) causes an enlargement (cytomegaly) ofhuman fibroblasts (MRC-5). As a first step toward determining whethersolute uptake, mediated in part by Na⁺/H⁺exchange, is responsible for the development of cytomegaly, we studiedthe effects of HCMV infection on intracellular pH(pH_i) regulation (nominalCO₂/concn = 0) by comparing cytomegalic cells with mock-infected cells.Seventy-two hours after HCMV infection of MRC-5 cells we observed thefollowing changes relative to mock-infected cells: restingpH_i is 0.1-0.2 pH unit morealkaline; the intrinsic buffering power of the cytoplasm was reduced by~40-50%; acid-loadingH⁺-equivalent fluxes were reduced;and there were alterations of Na⁺/H⁺exchanger (NHE) properties, including an alkaline shift of the pH_i dependence of activity, areduction of the apparent affinity for extracellularNa⁺, and an increase of theapparent maximum velocity and a large increase in stimulation by ahyperosmotic challenge. These results indicate that HCMV infectionexerts a profound effect on functional properties of the NHE, onacid-loading mechanisms, and on intrinsic cellular buffering power.These effects are consistent with a role for the NHE in the developmentof cytomegaly.

     

High capacity Na+/H+ exchange activity in mineralizing osteoblasts

Osteoblasts synthesize bone in polarized groups of cells sealed by tight junctions. Large amounts of acid are produced as bone mineral is precipitated. We addressed the mechanism by which cells manage this acid load by measuring intracellular pH (pHi) in non‐transformed osteoblasts in response to weak acid or bicarbonate loading. Basal pHi in mineralizing osteoblasts was ～7.3 and decreased by ～1.4 units upon replacing extracellular Na⁺ with N‐methyl‐D ‐glucamine. Loading with 40 mM acetic or propionic acids, in normal extracellular Na⁺, caused only mild cytosolic acidification. In contrast, in Na⁺‐free solutions, weak acids reduced pHi dramatically. After Na⁺ reintroduction, pHi recovered rapidly, in keeping with Na⁺/H⁺ exchanger (NHE) activity. Sodium‐dependent pHi recovery from weak acid loading was inhibited by amiloride with the Ki consistent with NHEs. NHE1 and NHE6 were expressed strongly, and expression was upregulated highly, by mineralization, in human osteoblasts. Antibody labeling of mouse bone showed NHE1 on basolateral surfaces of all osteoblasts. NHE6 occurred on basolateral surfaces of osteoblasts mainly in areas of mineralization. Conversely, elevated HCO alkalinized osteoblasts, and pH recovered in medium containing Cl^?, with or without Na⁺, in keeping with Na⁺‐independent Cl^?/HCO exchange. The exchanger AE2 also occurred on the basolateral surface of osteoblasts, consistent with Cl^?/HCO exchange for elimination of metabolic carbonate. Overexpression of NHE6 or knockdown of NHE1 in MG63 human osteosarcoma cells confirmed roles of NHE1 and NHE6 in maintaining pHi. We conclude that in mineralizing osteoblasts, slightly basic basal pHi is maintained, and external acid load is dissipated, by high‐capacity Na⁺/H⁺ exchange via NHE1 and NHE6. J. Cell. Physiol. 226: 1702–1712, 2011. © 2010 Wiley‐Liss, Inc.     

Na+/H+ exchange activity in the plasma membrane of Arabidopsis

In plants, Na+/H+ exchangers in the plasma membrane are critical for growth in high levels of salt, removing toxic Na+ from the cytoplasm by transport out of the cell. The molecular identity of a plasma membrane Na+/H+ exchanger in Arabidopsis (SOS1) has recently been determined. In this study, immunological analysis provided evidence that SOS1 localizes to the plasma membrane of leaves and roots. To characterize the transport activity of this protein, purified plasma membrane vesicles were isolated from leaves of Arabidopsis. Na+/H+ exchange activity, monitored as the ability of Na to dissipate an established pH gradient, was absent in plants grown without salt. However, exchange activity was induced when plants were grown in 250 mm NaCl and increased with prolonged salt exposure up to 8 d. H+-coupled exchange was specific for Na, because chloride salts of other monovalent cations did not dissipate the pH gradient. Na+/H+ exchange activity was dependent on Na (substrate) concentration, and kinetic analysis indicated that the affinity (apparent Km) of the transporter for Na+ is 22.8 mm. Data from two experimental approaches supports electroneutral exchange (one Na+ exchanged for one proton): (a) no change in membrane potential was measured during the exchange reaction, and (b) Na+/H+ exchange was unaffected by the presence or absence of a membrane potential. Results from this research provide a framework for future studies into the regulation of the plant plasma membrane Na+/H+ exchanger and its relative contribution to the maintenance of cellular Na+ homeostasis during plant growth in salt.     

Epidermal growth factor up-regulates intestinal Na+/H+ exchange activity.

The present studies were designed to examine the regulation of Na+/H+ exchange activity by epidermal growth factor (EGF) in an in vitro system. Na+/H+ exchange activity was determined in brush-border membranes isolated from rat jejunal enterocytes incubated with epidermal growth factor and a number of second messengers. EGF at physiological concentrations stimulated Na+/H+ exchange activity without affecting vesicle size. The stimulation of Na+/H+ activity was the result of increasing Vmax of Na+/H+ (6.0 +/- 0.4 compared with 3.3 +/- 0.27 nmol/mg protein/5 sec, P < 0.01). Km values of the Na+/H+ exchanger in brush-border membrane from cells stimulated with EGF and controls were similar (16.0 +/- 3.0 vs 13.0 +/- 3.0, respectively). Na+/H+ activity was inhibited by phorbol esters, calmodulin, and cyclic AMP. The effects of EGF, calmodulin, cyclic AMP, and phorbol esters were dependent on ATP, because depleting the cells from ATP masked the effects on Na+/H+ exchange activity. The results suggest that EGF stimulates Na+/H+ exchange activity in the enterocytes. This stimulation is most likely not via activation of the phosphatidylinositol pathway.     

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.     

Route,mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps

A single Na⁺/K⁺-ATPase pumps three Na⁺ outwards and two K⁺ inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na⁺ than K⁺ generates outward current across the cell membrane. Less well understood is the ability of Na⁺/K⁺ pumps to generate an inward current of protons. Originally noted in pumps deprived of external K⁺ and Na⁺ ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K⁺ and Na⁺ concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na⁺ release from phosphorylated Na⁺/K⁺ pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na⁺/K⁺ pumps that enables proton import is not required for completion of the 3 Na⁺/2 K⁺ transport cycle. However, the back-step occurs readily during Na⁺/K⁺ transport when external K⁺ ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na⁺-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na⁺ and K⁺ ions that passes through binding site II. The inferred occurrence of Na⁺/K⁺ exchange and H⁺ import during the same conformational cycle of a single molecule identifies the Na⁺/K⁺ pump as a hybrid transporter. Whether Na⁺/K⁺ pump–mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.     

Reconstitution of the beef heart and rat liver mitochondrial K+/H+ (Na+/H+) antiporter. Quantitation of K+ transport with the novel fluorescent probe, PBFI

New indicators for fluorescent measurement of Na+ and K+ ions should prove particularly useful for studies of reconstituted carriers of these ions. We show that PBFI, a K(+)-specific probe, provides a convenient and sensitive assay for the study of K+ uptake mediated by the reconstituted mitochondrial K+/H+ (Na+/H+) antiporter. Fluorescent measurements have enabled us for the first time to establish reconstitution of the K+/H+ (Na+/H+) antiporter from beef heart as well as from rat liver mitochondria. This technique has also enabled us to establish that dicyclohexylcarbodiimide is capable of complete inhibition of K+/H+ antiport in the reconstituted system, in accord with findings in intact mitochondria. PBFI fluorescence, which measures net K+ uptake, was essential for this corroboration, since dicyclohexylcarbodiimide is not capable of complete inhibition of 42K+/K+ or 86Rb+/Rb+ exchange, presumably because it acts selectively on proton transport within the carrier.     

Activation of Na+/H+ and K+/H+ exchange by calyculin A in Amphiuma tridactylum red blood cells: implications for the control of volume-induced ion flux activity

Alteration in cell volume of vertebrates results in activation of volume-sensitive ion flux pathways. Fine control of the activity of these pathways enables cells to regulate volume following osmotic perturbation. Protein phosphorylation and dephosphorylation have been reported to play a crucial role in the control of volume-sensitive ion flux pathways. Exposing Amphiuma tridactylu red blood cells (RBCs) to phorbol esters in isotonic medium results in a simultaneous, dose-dependent activation of both Na(+)/H(+) and K(+)/H(+) exchangers. We tested the hypothesis that in Amphiuma RBCs, both shrinkage-induced Na(+)/H(+) exchange and swelling-induced K(+)/H(+) exchange are activated by phosphorylation-dependent reactions. To this end, we assessed the effect of calyculin A, a phosphatase inhibitor, on the activity of the aforementioned exchangers. We found that exposure of Amphiuma RBCs to calyculin-A in isotonic media results in simultaneous, 1-2 orders of magnitude increase in the activity of both K(+)/H(+) and Na(+)/H(+) exchangers. We also demonstrate that, in isotonic media, calyculin A-dependent increases in net Na(+) uptake and K(+) loss are a direct result of phosphatase inhibition and are not dependent on changes in cell volume. Whereas calyculin A exposure in the absence of volume changes results in stimulation of both the Na(+)/H(+) and K(+)/H(+) exchangers, superimposing cell swelling or shrinkage and calyculin A treatment results in selective activation of K(+)/H(+) or Na(+)/H(+) exchange, respectively. We conclude that kinase-dependent reactions are responsible for Na(+)/H(+) and K(+)/H(+) exchange activity, whereas undefined volume-dependent reactions confer specificity and coordinated control.     

The K+/site and H+/site stoichiometry of mitochondrial electron transport.

Electrode measurements of the average number of H+ ejected and K+ taken up (in the presence of valinomycin) per pair of electrons passing the energy-conserving sites of the respiratory chain of rat liver and rat heart mitochondria have given identical values of the H+/site and 5+/site ratios very close to 4 in the presence of N-ethylmaleimide, an inhibitor of interfering respiration-coupled uptake of H+ + H2PO4-. The K+/site uptake ratio of 4 not only shows that inward movement of K+ provides quantitative charge-compensation for the 4 H+ ejected, but also confirms that 4 charges are separated per pair of electrons per site. When N-ethylmaleimide is omitted, the H+/site ejection ratio is depressed, because of the interfering secondary uptake of H/+ with H2PO4- on the phosphate carrier, but the K+/site uptake ratio remains at 4.0. Addition of phosphate or acetate, which can carry H+ into respiring mitochondria, further depresses the H+/site ratio, but does not affect the K+/site ratio, which remains at 4.0. These and other considerations thus confirm our earlier stoichiometric measurements that the average H+/site ratio is 4.0 and also show that the K+/site uptake ratio can be used as a measure of the intrinsic H+/site ratio, regardless of the presence of phosphate in the medium and without the necessity of adding N-ethylmaleimide or other inhibitors of H+ + H2PO4- transport.     

ATP/ADP exchange activity of gastric (H+ +K+)-ATPase

The ATP/ADP exchange is shown to be a partial reaction of the (H+ +K+)-ATPase by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to P1, P5-di(adenosine-5') pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg2+ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration (K0.5=116 microM) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg2+ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K+ as a function of pH and Mg2+.     

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.