Monovalent Cation Transport Research Articles

Ion-transport chain of cytochrome oxidase: the two chain-direct coupling principle of energy coupling.

Cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) couples the aerobic oxidation of ferrocytochrome c to the cyclical transport of monovalent cations or to the active transport of monovalent and divalent cations. This transport capability is mediated by an intracomplex ion-transport chain of two protein-bound molecules of cardiolipin per molecule of cytochrome oxidase. Cardiolipin in a two-phase system shows the identical ionophoric pattern as does the cytochrome oxidase coupled system. A molecular model of the cardiolipin chain suggests the possibility of a cage-like structure through which cations can be transferred from phosphate group to phosphate group. The ion-transport chain and the electron-transport chain are anchored to the same set of subunits (I+IV); the close proximity of the two chains argues for the direct coupling of electron and cation flow. The ion-transport chain of cytochrome oxidase provides an introduction to the molecular mechanisms by which ions are moved across membranes in energy-coupling systems.

Energized transport of cations by cytochrome oxidase

Summary Cytochrome oxidase, when incorporated into a phospholipid liposome, can mediate the energy-dependent transport of monovalent and divalent cations and amino acids. Cation uptake is accompanied by anion uptake. Little specificity for the transportable cation seems to exist, and under appropriate conditions all cations tested may be equally transported.

Properties of the interaction of the sodium channel with permeant monovalent cations.

The use of sea anemone toxin, veratridine and scorpion toxin which specifically interact with the gating system of the sodium channel and maintain the channel in an open conformation has permitted a study of the mechanism of transport of monovalent cations through the selectivity filter of this channel. The initial rate of 22Na+ influx through the tetrodotoxin-sensitive Na+ channels of excitable cells is dependent upon the external concentrations of Na+ and Na+-substitutes with the following properties. (a) It is saturable at high Na+ concentrations and increases with the external Na+ concentration in a cooperative manner (nH = 1.6). (b) At low external Na+ concentrations (1 mM), it is activated and then inhibited by increasing external concentrations of monovalent cations such as Li+, guanidinium, hydrazinium, hydroxylamine and K+. The activating effect of these cations disappears at higher external Na+ concentrations (10 mM). The experimental data are consistent with a model involving at least two allosteric cation-binding sites per Na+ channel. The binding of monovalent cations to Na+ sites is characterized by a high positive homotropic cooperativity. Most of the work describes the properties of the Na+ channel in neuroblastoma cells. The mechanism has also been shown to be valid for excitable cells of other types and origins.

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

The Na + and K + permeability properties of rat brain mitochondria were determined to explain the influences of these cations upon respiration. A new procedure for isolating exceptionally intact mitochondria with minimal contamination by synaptosomes was developed for this purpose. Respiration was uncoupled by Na + and less so by K +. Uncoupling was maximal in the presence of EDTA plus P i and was decreased by Mg 2+. Maximal uncoupler-stimulated respiration rates were inhibited by Na + but largely unaffected by K +. The inhibition by Na + was relatively insensitive to Mg 2+. Membrane Na + and K + conductances as well as neutral exchanges (Na +/H + and K +/H + antiport activities) were determined by swelling measurements and correlated with metabolic effects of the cations. Cation conductance, i.e. electrophoretic Na + or K + permeation, was increased by EDTA (Na + > K +) and decreased by Mg 2+. Magnesium preferentially suppressed Na + conductance so as to reverse the cation selectivity (K + > Na +). Neutral cation/H + exchange rates (Na + > K +) were not influenced by chelator or Mg 2+. The extent of cation-dependent uncoupling of respiration correlated best with the inner membrane conductance of the ion according to an empirical relationship derived with the model K + conductor valinomycin. The metabolic influences of Na + and K + can be explained in terms of coupled flow of these ions with protons and their effect upon the H + electrochemical gradient although alternative possibilities are discussed. These in vitro studies are compared to previous observations in situ to assess their physiological significance.

Monovalent cation transport in myotonic dystrophy: NaK pump ratio in erythrocytes

Myotonic dystrophy is a dominantly-inherited disorder which affects skeletal muscle in combination with several other systems. Because of abnormalities in red blood cells, a universal membrane defect has been proposed as the primary disturbance. Erythrocyte cation pump ratios have also been reported to be abnormal. Hyperinsulinemia and glucose intolerance are present in a large number of patients. Since dramatic effects of insulin on membrane cation transport have been shown in several tissues, notably skeletal muscle, we wished first to confirm reports of altered pump ratio in these patients and then to evaluate the effects of insulin on cation fluxes. However, in our experiments myotonic dystrophy patients had normal pump ratios when compared with disease controls.

Kinetics of Ca 2+ and Sr 2+ uptake by yeast. Effects of pH, cations and phosphate

The uptake of Ca 2+ and Sr 2+ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics. The rate of Ca 2+ and Sr 2+ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentrations of Ca 2+ and Sr 2+ at low pH may be explained by a decrease of the surface potential. The inhibition of Ca 2+ and Sr 2+ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane. Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.

Effects of inotropic and arrhythmogenic digoxin doses and of digoxin-specific antibody on myocardial monovalent cation transport in the dog.

The effects of digoxin on monovalent cation active transport were determined in cardiac tissue obtained from dogs given inotropic, toxic, or lethal doses of digoxin. In hemodynamically monitored dogs, active uptake of the K+ analogue Rb+ was determined in vitro in a control myocardial biopsy, and then in serial biopsies from the same dog after the infusion of [3H]digoxin in doses sufficient to cause a sustained positive inotropic effect in the absence of toxicity, and finally after additional doses to induce overt toxicity. Nontoxic digoxin doses producing a mean increase of 20% in left ventricular (LV) dP/dt significantly reduced Rb+ active transport by 25% below control values. At the onset of digoxin-induced arrhythmias, maximal LV dP/dt was 53% above control whereas active Rb+ transport was reduced by 60% below baseline values (P less than 0.001). Control dogs given vehicle alone showed no significant change in contractility or in monovalent cation active transport. In another group of dogs given a lethal dose of digoxin, Rb+ active transport was reduced 59% below control levels at the onset of overt toxicity and was further reduced 80% below control at the time of onset of a fatal rhythm disturbance. When dogs were given high affinity digoxin-specific IgG or Fab fragments at the onset of overt toxicity, toxicity was rapidly reversed, and monovalent cation active transport increased to 51% of control at the time of restoration of sinus rhythm. Twenty-four hours after antibody reversal of arrhythmias, monovalent cation transport values approximated normal control levels. These data provide quantitative estimates of the extent of inhibition of monovalent cation transport by digoxin at inotropic, toxic, and lethal endpoints. Similar degrees of transport inhibition were present at the time of onset of digoxin-induced arrhythmias and at the time or arrhythmia reversal by digoxin-specific antibodies.

Effect of primycin on monovalent cation transport of erythrocyte membrane and lipid bilayer.

The effects of primycin were investigated on the alkali-cation transport of human erythrocytes and on the electric conduction of bimolecular lipid membranes. In the concentration range of 3.10(-6) approximately 10(-5) M primycin increased the permeability of erythrocytes to alkali-cations according to the sequences Cs+ greater than Rb+ approximately K+ greater than Na+, while the conductance of the negatively charged phosphatidylserine bimolecular lipid membrane increased by 2 approximately 3 orders of magnitude. The resistance-lowering effect of primycin strongly depended on the cationic species applied and a selectivity order Na+ greater than K+ greater than Rb+ greater than Cs+ was found. A possible mechanism of the primycin-membrane interaction is suggested on the basis of experimental data.

Increased ouabain-sensitive glycolysis of lymphocytes treated with phytohemagglutinin: relationship to potassium transport.

AbstractAn early increase in lymphocyte plasma membrane K+ transport is essential for PHA stimulated lymphocytes to divide. Little is known about the specific source and amount of energy required to support the increased transport by activated lymphocytes. Since ouabain, a cardiac glycoside, specifically inhibits the transport ATPase, we have measured the decrement in glycolysis and tricarboxylic acid cycle activity when untreated and PHA treated lymphocytes were exposed to ouabain. This metabolic decrement represents the portion of metabolism associated with monovalent cation transport and closely related processes. Since TCA cycle activity accounted for only 0.2% of glucose consumption, aerobic glycolysis was the major source of energy, i.e., ATP, for increased transport. Approximately one‐third of the total lactate production in both control and PHA stimulated lymphocytes was ouabain‐sensitive. Ouabain sensitive lactate production in control, 105 μmol/1010 cells/hour, increased 1.8‐fold to 193 μmol/1010 cells/hour after PHA treatment. Active K+ influx in similar cell populations increased from 40 μmol/1010 cells/hour to 74 μmol/1010 cells/hour (1.9‐fold) after PHA treatment. The increment in ouabain‐sensitive energy production and K+ transport were closely correlated and, therefore, 0.38 moles of K+ are transported for each mole of ATP generated in both control and PHA treated cells. The increased requirement for transport related energy is provided by increasing the ouabain‐sensitive ATP production rather than altering the efficiency of ATP transduction.

Monovalent cation transport in irreversibly sickled cells.

Using discontinuous density gradients of Stractan II, we have separated sickle cell blood into discrete subpopulations of reticulocytes, mature discoid cells, and irreversibly sickled cells (ISCs). We have measured active and passive fluxes of monovalent cations in mature discoid cells, ISCs, and normal control cells, also separated upon density gradients. These measurements revealed a decreased active cation transport in ISC-rich populations. However, parallel measurements of Na, K-ATPase activity showed normal ouabain-sensitive ATPase activity in ISCs. Passive permeability to external Rb was also normal in ISCs. The observation of depressed pump activity in intact ISCs, contrasted with normal ATPase activity in ISC membranes, suggests the presence of factors in the intact cell which inhibit the active transport of Na and K in ISCs.

Potassium transport system of Rhodopseudomonas capsulata.

Rhodopseudomonas capsulata required potassium (or rubidium or cesium as analogs of potassium) for growth. These cations were actively accumulated by the cells by a process following Michaelis-Menten saturation kinetics. The monovalent cation transport system had Km's of 0.2 mM K+, 0.5 mM Rb+, and 2.6 mM Cs+. The rates of uptake of substrates by the potassium transport system varied with the age of the culture, although the affinity constant for the substrates remained constant. The maximal velocity of uptake of K+ was lower in aerobically grown cells than in photosynthetically grown cells, although the Km's for K+ and for Rb+ were about the same.

Effect of Rb+ Substituted for K+ on HeLa Cells: Cellular Content and Membrane Transport of Monovalent Cations, and Cell Growth

The usefulness of Rb+ was investigated as an analog of K+ in membrane transport and in supporting the growth of HeLa cells. On replacement of extracellular K+ by Rb+, intracellular Rb+ content increased linearly at 15 min. K+ content declined in contrast with Rb+, but Na+ content was unaffected. In Rb+-preloaded cells, Rb+ and K+ were exchanged after replacement of extracellular Rb+ with K. A kinetic analysis of intracellular Rb+ content showed that most K+ was exchangeable with Rb+, but a small part of K+ was not. Both Rb+ and K+ influxes were equally inhibited by ouabain or furosemide. Rb+ efflux was repressed by furosemide, whereas K+ efflux wasnot influenced. When 90 % of K+ was replaced by Rb+ in serum-free chemically defined medium, cell growth and protein synthesis were inhibited. After total replacement of extracellular K+ by Rb+, cell growth ceased and cellular protein content decreased, while a small part of intracellular K+ remained unexchanged. HeLa cells seemed to accumulate Rb+ in preference to K+.

In vitro studies of skeletal muscle membranes. Effects of denervation on the macromolecular components of cation transport in red and white skeletal muscle.

The effects of denervation on the macromolecular components of active monovalent cation transport in skeletal muscle have been studied using purified sarcolemma membranes. A comparison of membrane activities of fast-twitch, slow-twitch, and mixed-fiber muscles was made to determine what role, if any, the motor nerve has in regulating this important aspect of muscle metabolism. A dramatic increase in the basal sarcolemmal Mg++ ATPase activity (three- to fourfold) was found for both major muscle types. An increase in the ouabain-inhibitable (Na+ + K+)-stimulated enzyme was also found, but the effect was substantially less (1.5- to twofold). [3H]-ouabain binding, as an index of glycoside receptor sites, also increased (two- to threefold) midway in the course of denervation. On the other hand, the phosphorylated intermediate activity, a functional component of the transport system, clearly decreased over the same time course and remained below control values for the remainder of the course. This resulted in a two- to threefold increase in the turnover number, suggesting that active transport of cations should increase dramatically with denervation. The membrane protein patterns on SDS gels were less obvious than the changes observed in the functional components. The major effects appeared after only one week and seemed to be restricted to high molecular weight membrane proteins, especially in the 100,000 to 250,000 daltons range. This effect was more prominent in slow-twitch membranes with an apparent semiquantitative decrease in stain at 240,000 daltons. In gels of membranes from fast-twitch muscles a decreased stain in the range of 100,000 to 110,000 daltons occurred, and this became more obvious with longer periods of denervation. The results suggest that considerable influence on the macromolecular components of active cation transport in skeletal muscle is exerted by the motor nerve. No appreciable difference was found in this effect when the two major types of skeletal muscle, fast-twitch and slow-twitch, were compared, suggesting that motor nerve regulation of this membrane property is qualitatively the same.

Lack of effect of fusicoccin on passive transport in living plant tissues and in artificial phospholipid membranes

The finding that fusicoccin (FC) markedly stimulates in higher plant tissues the energy-dependent transport of protons, monovalent cations, amino acids and sugars, suggested the possibility that it might specifically influence some general physicochemical characteristic of the cell membrane. The effect of FC on passive solute transport in vivo and on the electric conductance of artificial phospholipid membranes (BLM) was thus investigated. FC did not influence passive transport (uptake) of urea and thiourea and water permeability (influx and efflux) in pea internode, maize coleoptile and maize root segments. Also the electric conductance of BLM (soybean lecithin), and valinomycin-mediated K + transport across the BLM were not influenced by FC. These results are in agreement with the hypothesis that the effects of FC on solute transport depend on its interaction with some specific protein component, rather than on its capacity to induce specific changes of the lipid component of the cell membrane.

NaK-ATPase in rat pancreatic islets

Shifts in the distribution of the monovalent cations Na+ and K+ between the extra- and intracellular space seem to be important for the secretory response of the beta-cell. An attempt was therefore made to study the enzyme responsible for monovalent cation transport, the (NaK)-activated ATPase. In the presence of NaN3 as inhibitor of the mitochondrial Mg-ATPase, a NaK-ATPase with a specific activity of 72 mU X mg protein-1 could be demonstrated in crude membrane preparations of rat pancreatic islets. The enzyme, which was inactive in the absence of Mg++, needed both Na+ and K+ for activation and was inhibited by ouabain and PCMB. The main part of the NaK-ATPase was localized in the microsomal fraction. Glucose, sulphonylureas, somatostatin and diazoxide were without effect on NaK-ATPase.

Macrocyclic ligands composed of tetrahydrofuran for selective transport of monovalent cations through liquid membranes

Macrocyclic ligands composed of tetrahydrofuran for selective transport of monovalent cations through liquid membranes

Rubidium transport and ouabain binding in normal and virally transformed mouse fibroblasts

Possible importance of the monovalent cation transport in oncogenic cell transformation was evaluated by determining changes in the rate of cation transport and the number of translocating macromolecules in normal and virally transformed mouse fibroblasts. Ouabain-sensitive 86Rb + uptake was measured in 3T3, SV 40-3T3 and MSV-3T3 cells to estimate the rate of cation transport. The initial rates of 86Rb + uptake in the oncogenic virus transformed cells were several-fold greater than the normal 3T3 cells. This increased rate of accumulation of cation was due to the increase in the V max and not the apparent K m for Rb + or K +. The changes in the number of (Na + + K +)-ATPase molecules was estimated by measuring the binding of [ 3H]ouabain to different cell populations. There was a decrease in the binding of [ 3H]ouabain in the virus-transformed cells but, the apparent affinity of different cell lines for ouabain did not significantly change. These results suggest some association between the alterations of (Na + + K +)-ATPase activity and oncogenic cell transformation, but an increase in the rate of monovalent cation transport does not appear to precede oncogenic cell transformation.

Ionophore A23187: the effect of H+ concentration on complex formation with divalent and monovalent cations and the demonstration of K+ transport in mitochondria mediated by A23187.

The two-phase extraction technique has been used to study the equilibrium between A23187, metal cations, and H+. Under these conditions the ionophore forms charge neutral isostoichiometric complexes with divalent cations in which both carboxylate groups of the 2:1 A23187:M2+ complexes are deprotonated. In ethanol, however, the methyl ester of A23187 also binds divalent cations indicating that protonated complexes between A23187 and cations should also exist. With monovalent cations, A23187 forms two charge-neutral complexes of stoichiometries and relative stabilities: A2HM greater than AM. Examination of energy utilization K+ and H+ movements, and light scattering capacity of mitochondria in the presence of divalent cation chelators, A23187, and valinomycin demonstrates that A23187 can act as a nigericin type K+ ionophore under appropriate conditions. Formation constants for the A2HM complexes with monovalent cations indicate that with appropriate conditions transport of Li+ and Na+ mediated by A23187 would also be expected. The binding constant data and associated free energies of complex formation are compared as a function of ionic radius and of cation charge. The data indicate that lack of conformational mobility in A23187 is responsible for the high cation size selectivity of this compound. To explain the transport selectivity of A23187 for divalent cations, it is proposed that this ionophore forms a family of five complexes, isostoichiometric between cations of different valence but of which only charge-neutral species are permeant to membranes. The charge of a given complex is in turn determined by that of the cation. The concept is consistent with the divalent cation transport specificity of A23187, explains the observed monovalent cation transport, and is useful in rationalizing the differences in charge selectivity between A23187 and X-537A.

Post recognition ion dependent events in mitogen induced lymphocyte proliferation and in cytotoxic effector cell responses.

This review discusses the increased permeability and enhanced uptake transport of monovalent and divalent cations following mitogen-induced lymphocyte proliferation. The observed Ca2+ uptake is discussed in terms of gated Ca2+ channels. The importance of divalent cations, particularly Ca2+, is discussed in terms of triggering of cytotoxic effector cell responses for three model systems (antibody dependent cytotoxicity, mitogen-induced cytotoxicity and cell mediated cytotoxicity); (85).

Effects of (Na+ + K+)-ATPase-specific antibodies of enzymatic activity and monovalent cation transport.

Antibodies have been obtained that specifically interact with the transport enzyme (Na+ + K+)-activated ATPase. The antigen used was purified (Na+ + Ka+)-ATPase from canine renal medulla. Purified gamma globulin from immunized animals, but not from control animals or preimmune serum, inhibited (Na+ + Ka+)-ATPase from canine renal medullar with reduction of activity to 33 +/- 4 (SD)% in concentration-dependent manner. Maximum inhibition occurred in less than 5 minutes at 37 degrees C. The Mg++ -dependent, nonouabain inhibited component of activity (Mg++ -ATPase) was unaffected. Fab fragments obtained by papain cleavage of the gamma globulin fraction had similar inhibitory activity and specificity. These antibodies also produced varying degrees of concentration-related inhibition of canine myocardial, calf brain, and human red cell ghost (Na++ + Ka+)-ATPase, but not Mg++-ATPase activity.