Leakage-channel conductance of single (Na+ + K+)-ATPase molecules incorporated into planar bilayers by fusion of liposomes

By fusing liposomes which contain in the mean only one pump unit (one intramembranous particle) to planar bilayers, and provoking the ouabain-blockable leakage conductance by the presence of n-decane, the predominant unit leakage conductance associated with one pump unit was estimated to be 40–50 pS, indicating the channel nature of the leakage pathway.     

Effects of the plant alkaloid sanguinarine on cation transport by human red blood cells and lipid bilayer membranes

Summary The plant alkaloid, sanguinarine, inhibits the ouabain-sensitive K–Na pump and increases the downhill, ouabain-insensitive movements of K and Na in human red cells. These two effects have different temporal and concentration dependencies and are mediated by two different chemical forms of sanguinarine. The oxidized, charged form (5×10^–5 m) promptly inhibits the pump but does not affect leakage of K and Na. The reduced, uncharged form of sanguinarine causes lysis of red cells but does not inhibited the pump. Sanguinarine also increases the conductance of bilayers formed from sheep red cell lipids. The effect is produced by the uncharged but not by the charged form of sanguinarine. Bilayer conductance increases as the fourth power of sanguinarine concentration when the compound is present on both sides of the membrane and as the second power of concentration when present on only one side. Conductance also increasee-fold for each 34 mV increase in the potential difference imposed across the membrane. The results suggest that the uncharged forms of sanguinarine produce voltage-dependent channels in bilayers.     

The Effect of Cu2+ on Ion Transport Systems of the Plant Cell Plasmalemma

Changes in plasmalemma permeability caused by excessive Cu2+ levels were examined in cells of a freshwater alga (Nitella flexilis) using a conventional microelectrode voltage-clamp technique. A rapid Cu2+-induced increase of plasmalemma conductance starting from 5 [mu]M Cu2+ was shown. Cu2+-induced plasmalemma conductance (ClGm) was nonselective and potential-independent, resembling the conductance of nonselective ionic leakage of the plasmalemma. The K+ channel conductance was shown to be unaltered by Cu2+, and a decrease in plasmalemma Cl- channel conductance at Cu2+ concentrations above 5 [mu]M was found. The depression of Cl- channels and ClGm were time-, dosage-, and Ca2+-dependent processes, revealing a great similarity in all parameters, with Ca2+ causing the preventive effect by shifting the effective Cu2+ concentrations to higher levels. This phenomenon may be explained by the same Cu2+-modified target on the plasmalemma both for ClGm and Cl- channel depression. In addition, a reversible, inhibitory effect of Cu2+ (>10 [mu]M) on the light-stimulated H+-ATPase electrogenic pump in the plasmalemma was demonstrated. This effect was Ca2+- independent, which made it possible to distinguish it from ClGm. Therefore, the Cu2+-induced dramatic alterations in plant cell plasmalemma permeability are caused mainly by nonselective conductance increases and electrogenic pump inhibition.     

Studies of insulin action on the amphibian oocyte plasma membrane using NMR, electrophysiological and ion flux techniques

Insulin (0.1-10 microM) reinitiates the meiotic divisions in Rana oocytes and produces a 14-20 mV negative-going hyperpolarization of the plasma membrane as well as a 0.25 unit increase in intracellular pH during the first 90 min. During hyperpolarization, the Na+ conductance of the membrane decreases by 40-50% with a concomitant increase in 22Na+ uptake from the medium. The increased uptake of Na+ during a period of decreasing Na+ conductance is apparently due to an increase in fluid phase turnover associated with insulin-mediated endocytosis. Both membrane hyperpolarization and increase in pHi are Na+-dependent and are blocked by the serine proteinase inhibitor, phenylmethylsulfonyl fluoride. The membrane potential of the prophase oocyte has a significant electrogenic component with potential but not conductance sensitive to glycosides and substitution of Li+ for Na+. Insulin hyperpolarizes Li+ or glycoside-treated oocytes whereas glycosides do not affect insulin-hyperpolarized oocytes. [3H]Ouabain binding by the plasma membrane of the untreated oocyte shows at least two K+-sensitive components (Kd = 42 and 2000 nM) linked to inhibition of the Na+ pump. Insulin-treated oocytes show a single class of intermediate-affinity ouabain sites (Kd = 490 nM) which appear to result from insulin-induced internalization of membrane-bound ouabain. [125I]Insulin binding to the plasma membrane shows a class of high-affinity sites (Kd = 87 nM) with 40-50 pump sites per insulin-binding site. Our results suggest that insulin-induced mediator peptides stimulate Na+-H+ exchange resulting in an increase in intracellular pH and Na+ uptake concomitant with an increase in receptor-mediated endocytosis and a decrease in Na+ conductance and associated membrane hyperpolarization. The net result appears to be a down-regulation of the Na+ pump which together with a decrease in Na+ conductance may divert high-energy phosphate compounds from cation regulation to anabolic processes of meiosis.     

Inhibitors of proton pumping: effect on passive proton transport

Reported inhibitors of the Characean plasmalemma proton pump were tested for their ability to inhibit the passive H⁺ conductance which develops in Chara corallina Klein ex Willd. at high pH. Diethylstilbestrol inhibits the proton pump and the passive H⁺ conductance with about the same time course, at concentrations that have no effect on cytoplasmic streaming. N-Ethylmaleimide, a sulfhydryl reagent which is small and relatively nonpolar, also inhibits both pumping and passive conductance of H⁺. However, it also inhibits cytoplasmic streaming with about the same time course, and therefore could not be considered a specific ATPase inhibitor. p-Chloromercuribenzene sulfonate (PCMBS), a sulfhydryl reagent which is large and charged and hence less able to penetrate the membrane, does not inhibit pumping or conductance at low concentration. At high concentration, PCMBS sometimes inhibits pumping without affecting H⁺ conductance, but since streaming is also inhibited, the effect on the pump cannot be said to be specific. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, a water soluble carbodiimide, weakly inhibits both pump and conductance, apparently specifically.     

Activation of respiration and loss of thermodynamic control in hyperthyroidism. Is it due to increased slipping in mitochondrial proton pumps?

T3 administration increases the extent of non-linearity in the flow-force relationship between pump proton conductance and protonmotive force. The effect is present also at the ATPase proton pump. These effects are not accompanied by changes in passive proton conductance. Incubation of mitochondria at 45 degrees C also causes an increased non-linearity, accompanied by a partial increase of proton conductance. It appears that the increase of respiratory activity following T3 administration is due to loss of thermodynamic control within or at the proton pumps, an effect which might be attributed to increased slipping.     

Single-channel currents produced by the serotonin transporter and analysis of a mutation affecting ion permeation.

Single-channel activities were observed in outside-out patches excised from oocytes expressing a mammalian 5-hydroxytryptamine (5-HT) transporter. Channel conductance was larger for a mutant in which asparagine177 of the third putative transmembrane domain was replaced by glycine, suggesting that this residue lies within or near the permeation pathway. The N177G mutant enables quantitative single-channel measurements; it displays two conducting states. One state, with conductance of approximately 6 pS, is induced by 5-HT and is permeable to Na+. The other state (conductance of approximately 13 pS) is associated with substrate-independent leakage current and is permeable to both Na+ and Li+. Cl- is not a major current carrier. Channel lifetimes under all conditions measured are approximately 2.5 ms. The single-channel phenomena account for previously observed macroscopic electrophysiological phenomena, including 5-HT-induced transport-associated currents and substrate-independent leakage currents. The channel openings occur several orders of magnitude less frequently than would be expected if one such opening occurred for each transport cycle and therefore do not represent an obligatory step in transport. Nevertheless, single-channel events produced by neurotransmitter transporters indicate the functional and structural similarities between transporters and ion channels and provide a new tool, at single-molecule resolution, for detailed structure-function studies of transporters.     

Simulation of the light-induced oscillations of the membrane potential in Potamogeton leaf cells

Summary An attempt has been made to simulate the light-induced oscillations of the membrane potential of Potamogeton lucens leaf cells in relation to the apoplastic pH changes. Previously it was demonstrated that the membrane potential of these cells can be described in terms of proton movements only. It is hypothesized that the membrane potential is determined by an electrogenic H⁺-ATPase with a variable H⁺/ATP stoichiometry. The stoichiometry shifts from a value of two in the dark to a value of one in the light. Moreover, this H⁺ pump shows the characteristics of either a pump or a passive H⁺ conductance: the mode of operation of the H⁺ translocator is considered to be regulated by the external pH. The pump conductance is assumed to be dominant at low or neutral pH, while the passive H⁺ conductance becomes more significant at alkaline pH. The pH dependence of the transport characteristic is expressed by protonation reactions in the plasma membrane. The proposed model can account for most features of the light-induced oscillations but not for the absolute level of the membrane potential.This research was supported by the Foundation of Biophysics, part of the Dutch Organization for Scientific Research (NWO) ECOTRANS publication No. 34.     

A microscopic model for the current-voltage behaviour of the Na,K-pump

The current voltage characteristic of the Na, K pump is described on the basis of a modified Post-Albers cycle. The voltage dependence of the rate constants is derived from the elementary chargetranslocations associated with the single reaction steps. Charge displacements result from movements of the sodium- or potassium-loaded binding sites, as well as from motions of polar groups in the pump molecule. If part of the transmembrane voltage drops between the alkali-ion binding sites and the aqueous solution, the binding constants become voltage-dependent. Depending on the values of the microscopic parameters, the current-voltage characteristic may assume a variety of different shapes. Saturating behaviour results when one or more voltage-independent reaction steps become rate limiting. Non-monotonic current-voltage curves exhibiting regions of negative pump conductance are predicted when, at least in one of the transitions, charge is moved against the direction of overall charge-translocation. The theoretical predictions are compared with recent experimental studies of voltage-dependent pump currents.     

Mechanism of loss of thermodynamic control in mitochondria due to hyperthyroidism and temperature.

Incubation of normal mitochondria at 45 degrees C results in increases of respiration and of total apparent proton conductance (TAPC, respiration/proton motive force) and in an upward shift of the flow-force relationships. Similar effects are observed during operation of the redox proton pumps at different sites of the respiratory chain. These effects are accompanied by an almost equivalent increase of the passive proton conductance (PPC, proton leakage/proton motive force). In mitochondria from 3,3,5-triiodo-L-thyronine (T3)-treated rats there are also increases of respiration and of TAPC and an upward shift of flow-force relationships, more pronounced at the level of the cytochrome oxidase proton pump. However, at variance from the incubation at 45 degrees C, in mitochondria from T3-treated rats there is only a slight increase of PPC. Addition of bovine serum albumin to normal mitochondria incubated at 45 degrees C results in a marked depression of TAPC in the nonlinear range of the flow-force relationships. An equivalent effect is not observed in mitochondria from T3-treated rats. The experimental results have been compared with computer simulations obtained on the basis of a chemiosmotic model of energy transduction. The increase of TAPC following incubation at high temperature is apparently due to changes of the proton conductance mainly at the level of PPC, while the increase of TAPC following T3 administration is rather due to changes presumably at the level of the redox or ATPase proton pumps.     

The effect of variations in sodium conductances on pacemaking in a dopaminergic retinal neuron model

Dopaminergic neurons in the retina show spontaneous tetrodotoxin-sensitive pacemaking, which has been explained by a reduced Hodgkin-Huxley-type computer model. The present study used this model to investigate the effect of variations in transient and persistent sodium conductance values on pacemaking, under variable leakage conductance levels. This study indicated that transient sodium conductance plays an indispensable role in pacemaking, which occurs under conditions in which only a persistent sodium conductance is considerably reduced, thus contributing to a detailed understanding of the relationship between sodium conductance and pacemaking.     

Flux ratios and pump stoichiometries at sites II and III in liver mitochondria. Effect of slips and leaks.

Addition of bovine serum albumin to state 4 mitochondria results in a depression of the proton leak and of the resting respiration of 70 and 25%, respectively. The conductance membrane potential diagram, both in the ohmic and in the non-ohmic region, shows that in the presence of bovine serum albumin the level of ohmic conductance is lowered while that of non-ohmic conductance is increased toward higher delta psi values. The same effect is observed during operation of the different proton pumps. Addition of chloroform affects the conductance membrane potential diagram in the following manner: there is no effect in the ohmic region with all pumps, while there is an effect in the non-ohmic region either at site III or at sites II plus III but not at site II. This suggests a possible effect of chloroform at the level of the cytochrome oxidase proton pump. During titration with oligomycin of the ATPase proton pump the conductance potential diagram shows a region of non-ohmicity only in the presence but not in the absence of an ATP-regenerating system. Protonophoric uncouplers such as carbonyl cyanide p(trifluoromethoxy)phenylhydrazone and intrinsic uncouplers such as chloroform have different effects on the relationship between rates of charge translocation and of oxygen consumption, and thus on the pump stoichiometries, in that the slope of the diagram is modified by the latter but not by the former. The differential effects of protonophores and of intrinsic uncouplers on the stoichiometries have been analyzed by computer simulations and represent an additional criterion to distinguish between extrinsic and intrinsic mechanisms of uncoupling.     

Protein AQ_1862 from the hyperthermophilic bacterium Aquifex aeolicus is a porin and contains two conductance pathways of different selectivity

The "hypothetical protein" AQ_1862 was isolated from the membrane fraction of Aquifex aeolicus and identified as the major porin. In experiments with one conducting unit (molecule) a conductance of 1.4 nS was observed in 0.1 M KCl at pH 7.5. This stable (basic) conductance was superimposed by conductance fluctuations of approximately 0.25 nS. Because both events were always observed simultaneously, it is suggested that they are caused by the same molecular entity. Nonetheless they show very different properties. The basic conductance is anion selective at neutral pH with a conductance sequence Cl- approximately Br- approximately NO3->F->gluconate approximately acetate approximately propionate and does not saturate up to 0.5 M KCl. At alkaline pH and in the presence of large anions, it becomes unselective and the conductance saturates at low concentrations (Km approximately 20 mM). In contrast the fluctuating component is mainly cation selective with a conductance sequence K+ approximately Rb+>NH4+>Na+ approximately Li+ approximately Cs+. It saturates at low salt concentrations (Km approximately 15 mM) and is not affected by pH. In view of the diverging properties of both conductance components, it seems appropriate to assume that AQ_1862 has two different conducting pathways rather than one with two different open states.     

Current-voltage characteristics of planar lipid membranes with attached Halobacterium cell-envelope vesicles

We have studied the photoactivity of a system consisting of large, planar, essentially solvent free bilayers bearing adsorbed cell-envelope vesicles prepared from Halobacterium halobium (strain L 33). The system was made conductive by addition of a proton carrier (SF-6847). We observed photocurrents which were linearly dependent upon transmembrane voltage. Current-voltage curves were found to be well described by an equivalent circuit with the following significant parameters: planar bilayer conductance, planar bilayer-vesicle contact area conductance, cell-envelope vesicle conductance, and chloride pump equivalent voltage-generator potential. These parameters are uniquely obtained as a result of a few independent current measurements. The stationary photovoltage was dependent upon chloride concentration, and from this dependence an active transport (pump) affinity of the system for chloride was calculated to be about 50 mM.     

The effect of shoot architecture on hydraulic conductance in beech (Fagus sylvatica L.)

 Shoot hydraulic conductance was measured in beech (Fagus sylvatica L.) that had previously been exposed to high levels of nitrogen input. Whole-shoot hydraulic conductance, conductance per unit pressure gradient and leaf specific conductance were negatively correlated with the number of bud scars per unit length, a morphological parameter of tree decline. We propose a negative feedback mechanism by which stress induced alterations in shoot morphology can cause a lasting reduction of tree vigour. Received: 2 July 1997 / Accepted: 25 June 1998     

Analyzing Thermal Stability of Cell Membrane of Salmonella Using Time-Multiplexed Impedance Sensing

Heat treatment is one of the most widely used methods for inactivation of bacteria in food products. Heat-induced loss of bacterial viability has been variously attributed to protein denaturation, oxidative stress, or membrane leakage; indeed, it is likely to involve a combination of these processes. We examine the effect of mild heat stress (50–55°C for ≤12 min) on cell permeability by directly measuring the electrical conductance of samples of Salmonella enterica serovar Typhimurium to answer a fundamental biophysical question, namely, how bacteria die under mild heat stress. Our results show that when exposed to heat shock, the cell membrane is damaged and cells die mainly due to the leakage of small cytoplasmic species to the surrounding media without lysis (confirmed by fluorescent imaging). We measured the conductance change, ΔY, of wild-type versus genetically modified heat-resistant (HR) cells in response to pulse and ramp heating profiles with different thermal time constants. In addition, we developed a phenomenological model to correlate the membrane damage, cytoplasmic leakage, and cell viability. This model traces the differential viability and ΔY of wild-type and HR cells to the difference in the effective activation energies needed to permeabilize the cells, implying that HR cells are characterized by stronger lateral interactions between molecules, such as lipids, in their cell envelope.     

A kinetic analysis of the electrogenic pump ofChara corallina: I. Inhibition of the pump by DCCD

Summary The current-voltage curve of theChara membrane was obtained by applying a slow ramp depo- and hyperpolarization by use of voltage clamp. With the progress of poisoning by DCCD (dicyclohexylcarbodiimide) theI–V curve moved by about 50 mV (depolarization) along the voltage axis, reducing its slope, and finally converged to thei _d -V curve of the passive diffusion channel. Changes ofi _p -V curve of the electrogenic pump channel could be obtained by subtracting the latter from the former.The sigmoidali _p -V curve could be simulated satisfactorily by adopting a simple reaction kinetic model. Kinetic parameters of the successive changes of state of the H⁺ ATPase could be evaluated. Changes of these kinetic parameters during inhibition gave useful information about the molecular mechanism of the electrogenic pump.Depolarization of the membrane potential, decrease of membrane conductance, and decrease of pump current during inhibition of the pump with DCCD are caused mainly by the decrease of conductance of the pump channel. The decrease of this pump conductance is caused principally by a marked decrease of the rate constant for releasing H⁺ to the outside.     

Regulation of the H+ pump activity in the plasma membrane of internally perfused Chara corallina

The role of cytoplasm for the maintenance of the H+ pump activity in Chara corallina internodal cells was examined by the intracellular perfusion technique. Cytoplasm-rich and -poor states were obtained by changing the perfusion time, short-term (less than 2 min) and long-term (more than 5 min), respectively. A large portion of cytoplasm was left by short-term perfusion but most of the cytoplasm was removed by long-term perfusion. The activities of the H+ pump of these two different conditions were examined by measuring current-voltage relation (I-V curve) and conductance-voltage relation (G-V curve) under voltage clamp conditions. The H+ pump conductance decreased to 37%, 9% and zero by short-term, long-term and hexokinase perfusion, respectively, whereas the passive channel conductance decreased to 71%, 39% and 73% by short-term, long-term and hexokinase perfusion, respectively. On the other hand, the electromotive-force of the H+ pump (approximately -260 mV) and the passive channel (approximately -130 mV) were not affected by either short- or long-term perfusion. It is indicated that the cytoplasm plays an essential role to regulate the activity of both the H+ pump and the passive channel together with ATP.     

N-Ethylmaleimide Blocks the H+ Pump in the Plasma Membrane of Chara corallina Internodal Cells

The sulfhydryl (SH) modifying reagent N-ethylmaleimide (NEM)was applied to the internodal cells of Chara corallina to studythe role of SH residues in the activity of the plasma membraneH⁺ pump. NEM (1 µM) caused a marked depolarizing shiftof the resting potential by 6410mV (n=7) together with depressionof the conductance peak at around —200 mV, indicatinga marked depression of the H⁺ pump activity. This effect ofNEM was partly reversible, the membrane repolarized and theconductance peak was restored after extracellular washing. TheH⁺ pump inhibitor, dicyclohexylcarbodiimide (DCCD), caused noadditive membrane depolarization and/or depression of the H⁺pump conductance, in the presence of NEM. This suggests thatNEM blocks the H⁺ pump and that SH residues play a pivotal rolein maintaining the H⁺ pump activity in Chara corallina. (Received April 10, 1993; Accepted July 29, 1993)     

A kinetic analysis of the electrogenic pump ofChara corallina: III. Pump activity during action potential

Summary The current-voltage curve (I–V curve) of theChara membrane was obtained by applying a slow ramp hyper- and depolarization by use of voltage clamp. By inhibiting the electrogenic pump with 50m DCCD (dicyclohexylcarbodiimide), theI–V curve approached a steadyI–V curve within two hours, which gave thei _d -V curve of the passive diffusion channel. Thei _p -V curve of the electrogenic pump channel was obtained by subtracting the latter from the former. The sigmoidali _p -V curve could be simulated satisfactorily with a simple reaction kinetic model which assumes a stoichiometric ratio of 2. The emf of the pump (E _p ) is given as the voltage at which the pump current changes its sign. The conductance of the pump (g _p ) can be calculated as the chord conductance from thei _p -V curve, which is highly voltage dependent having a peak at a definite voltage. The changes of emf and conductance during excitation were determined by use of the current clamp (I=0). Since theE _p andg _p (V) are known, the changes, during excitation, of emf (E _d ) and conductance (g _d ) of the passive diffusion channel can be calculated. The marked increase of the membrane conductance and the large depolarization during the action potential are caused by the marked increase of the conductance of the passive diffusion channel and the large depolarization of its emf. The conductance of the electrogenic pump decreases to about half at the peak of action potential, while the pump current increases almost to a saturated level.