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PubMed1970

Contributions of the sodium pump and ionic gradients to the membrane potential of a molluscan neurone.

1. The membrane potential of the gastro-oesophageal giant neurone of the marine mollusc, Anisodoris nobilis, was examined during changes of temperature and of the ionic medium.2. The response of the membrane potential to rapid changes in the external K concentration was prompt, stable, and reversible up to 200 mM-K, and was independent of the external Cl concentration.3. Warming the cell produced a prompt hyperpolarization that was approximately 10 times greater than predicted by the Nernst or constant field equations. Electrogenic activity of the Na-K exchange pump was shown to be responsible for this effect.4. At temperatures below 5 degrees C, the relationship between the membrane potential and the external K concentration could be predicted by a constant field equation.5. At temperatures above 5 degrees C, the membrane potential could not be predicted by the constant field equation except after inhibition of the electrogenic Na pump with ouabain or the reduction of internal Na.6. Inhibition of the electrogenic Na pump by low external K concentrations was dependent upon the external Na concentration.7. It is concluded that the membrane potential is the sum of ionic and metabolic components, and that the behaviour of the ionic component can be predicted by a constant field type equation.

Action Potentials↗
PubMed1994

A conformational change in the lactose permease of Escherichia coli is induced by ligand binding or membrane potential.

Lactose transport in membrane vesicles containing lactose permease with a single Cys residue in place of Val 315 is inactivated by N-ethylmaleimide in a manner that is stimulated by substrate or by a H+ electrochemical gradient (delta microH+; Sahin-Tóth M, Kaback HR, 1993, Protein Sci 2:1024-1033). The findings are confirmed and extended in this communication. Purified, reconstituted Val 315-->Cys permease reacts with N-ethylmaleimide or hydrophobic fluorescent maleimides but not with a membrane impermeant thiol reagent, and beta-galactosides specifically stimulate the rate of labeling. Furthermore, the reactivity of purified Val 315-->Cys permease is enhanced by imposition of a membrane potential (delta psi, interior negative). The results indicate that either ligand binding or delta psi induces a conformational change in the permease that brings the N-terminus of helix X into an environment that is more accessible from the lipid phase.

Cysteine↗
PubMed1992

Sodium-dependent effects of melatonin on membrane potential of neonatal rat pituitary cells.

Melatonin inhibits GnRH-stimulated release of LH from neonatal rat pituitary cells, probably by inhibiting GnRH-induced elevation of intracellular Ca2+. This effect of melatonin seems to involve inhibition of Ca2+ influx through voltage-sensitive channels. Accordingly, it is possible that melatonin could act by hyperpolarizing pituitary cells, which would close these channels. This issue was addressed here by determining if melatonin influences membrane potential. Membrane potential and intracellular Ca2+ were studied in neonatal rat pituitary cells in suspension, using bis-oxonol and Fluo-3 as fluorescent indicators, respectively. It was found that treatment with melatonin alone causes membrane hyperpolarization and that it has a repolarizing effect after GnRH-induced membrane depolarization. This effect on membrane potential appears to be mediated by high affinity melatonin receptors and a pertussis toxin-sensitive Na(+)-dependent mechanism; it is not dependent upon Ca2+, Cl-, or bicarbonate. This may be the molecular basis of action of melatonin in other tissues with high affinity melatonin receptors.

Aging↗
PubMed1992

The plasma membrane electrical gradient (membrane potential) in Leishmania donovani promastigotes and amastigotes.

The equilibrium distribution of tetraphenylphosphonium bromide was used to measure the membrane potential in Leishmania donovani amastigotes and promastigotes and to investigate mechanisms underlying the maintenance of membrane potential. At pH 7.0, membrane potential ranges between -90 and -113 mV. Increasing the external concentrations of hydrogen or potassium ions decreased membrane potential as did treatments with carbonylcyanide chlorophenylhydrazone or valinomycin. These observations are consistent with a membrane potential set by hydrogen and potassium ion diffusion gradients. Anaerobiosis lowered membrane potential, suggesting the involvement of ATPase(s) in maintaining membrane potential. Membrane potential was insensitive to treatment with ouabain, demonstrating the absence of a Na+/K(+)-ATPase. Treatment with dicyclohexylcarbodiimide caused a temporary hyperpolarization of the membrane suggesting the participation of a proton ATPase in the maintenance of membrane potential. Determination of the membrane potential makes it possible to quantitate the total proton motive force which is the force for active transport across the parasite membrane.

Adenosine Triphosphatases↗
PubMed1975

Effects of Na+ and K+ on the resting membrane potential of the rabbit sinoatrial node cell.

To estimate the "resting potential" of the sinoatrial node cell, membrane potentials in quiescent states under various conditions were compared with the holding potential at which no net membrane current flowed during the voltage clamp. The membrane potential of the rabbit sinoatrial node cell was recorded using a conventional microelectrode method. The single sucrose gap method and the double microelectrode method were employed for the voltage clamp. The membrane potential immediately before the resumption of the spontaneous activity in normal Tyrode solution after a quiescence in high K+ concentration, in low Na+ concentration or in low temperature was approximately - 37 m V and was similar to that of the temporarily quiescent sinoatrial node cells after dissection. The holding potential at which no net membrane current flowed was - 38.4 m V, which coincided with the membrane potential during quiescence. The resting potential in quiescence increased by approximately 17 m V for a tenfold decrease in the extracellular Na+ concentration, and 22 m V for a tenfold increase in the K+ concentration. The relative membrane conductance decreased transiently on removing Na+ from the bathing medium. These findings suggest that a potential equivalent to the resting potential existss in the S-A node cell approximately 20 m V positive to the maximum diastolic potential, and this low value might be due to the high Na+ conductance of the cell membrane.

Animals↗
PubMed1967

A method to relate steady-state ionic currents, conductances, and membrane potential in ion exchange membranes with unknown thermodynamic properties.

A method is presented by which the steady-state properties of an homogeneous, permselective membrane at uniform temperature can be predicted without knowledge of its thermodynamic properties other than assuming that they are functions only of local mole fractions in the membrane. By making this assumption, it is shown how the ionic conductances can be calculated at any point in the membrane from two sets of measurements, (a) R(symm), the steady-state resistance of the membrane measured between identical solutions and (b) V(0), the potential difference between nonidentical solutions for zero current. These two parameters are measured at different external solution compositions (e.g. a varying sodium-potassium ratio ranging from zero to infinity). From these measurements it is shown how the flux equations may be integrated without a knowledge of mobilities, activity coefficients, and other interior membrane parameters. The application of the method to fixed site membranes with variable mobilities is described and the theory for this particular case has also been verified experimentally in glass membranes.1 A possible application to biological membranes is discussed and a comparison is made between the present treatment and previous treatments used to calculate the steady-state properties of cell membranes, notably the theory of Teorell, Meyer, and Sievers and the constant field theory.

Cell Membrane↗
PubMed1978

Measurements of membrane potentials using the dye safranine.

A metachromatic shift in the spectrum of the cationic dye safranine occurs upon induction of electrical potentials across the mitochondrial membrane by adding respiratory substrate, ATP or a cation conductor valinomycin (when a potassium gradient exists across the membrane) to a mitochondrial suspension. The extent of spectral change correlates linearily to the membrane potential. During the spectral change safranine is taken up by the mitochondria and most of the dye can be recovered in the pellet after centrifugation. By measuring the spectral changes a fairly good estimate of membrane potentials in mitochondria and bacteria is obtained. This method is compared to other optical methods of studying membrane potentials and is possible applications in different systems in the future is discussed.

Adenosine Triphosphate↗
PubMed1980

Sodium dependence of membrane potential oscillation induced by veratrine.

The dependence of the membrane potential oscillation on extracellular sodium concentration was studied in the frog sartorius muscle. (1) When Na concentration of the Ringer solution was decreased to zero, the membrane potential oscillation cesed and the membrane tended to repolarize. This inhibitory effect was reversible. (2) Tetrodotoxin, 10(-7) g/ml, inhibited the development of membrane potential oscillation or stopped within 2--4 min the membrane potential oscillation already developed, the membrane then became partly repolarized maximum to -72mV. The inhibitory effect of tetrodotoxin was also reversible. (3) When the Na concentration of the Ringer solution was reduced to 60 mM the latency time of the membrane potential oscillation did not change. By further decreasing the Na concentration the latency time increased in a hyperbolic function. In case of 10 mM Na the latency period was 7 times longer than the control. (4) The depolarizing effect of veratrine was linearly related to the logarithm of Na concentration in a range from 10 to 120 mM Na. (5). By decreasing the [Na]c from 120 to 10 mM the frequency of oscillation decreased and the amplitude increased linearly as a function of the logarithm of Na concentration.

Animals↗
PubMed1978

Evidence that catecholamine transport into chromaffin vesicles is coupled to vesicle membrane potential.

The effects of ATP, Mg(2+), and various agents on pH gradient, membrane potential, and catecholamine transport across membranes of intact bovine chromaffin vesicles were investigated. Methylamine and thiocyanate (SCN(-)) distributions across the vesicle membrane were used to estimate the H(+) concentration gradient and membrane potential, respectively. The H(+) concentration ratio (intravesiculanmedium) equals 16 when the medium pH is 6.9 and is unaltered by ATP and Mg(2+). In the absence of ATP and Mg(2+), the steady-state intravesicular S(14)CN(-) concentration is lower than the medium concentration. ATP and Mg(2+) cause an increased influx and a decreased efflux of SCN(-) that results in SCN(-) being concentrated in the vesicles 6- to 8-fold over the medium. The findings are consistent with an ATP,Mg(2+)-induced potential of approximately 50 mV (intravesicular side positive). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a H(+) translocater, and N-ethylmaleimide (NEM), a sulfhydryl reagent, decrease the SCN(-) ratio and, thus, the membrane potential in the presence of ATP and Mg(2+). They have no effect on the H(+) concentration gradient. The rate of catecholamine uptake into vesicles is increased 4- to 6-fold by ATP and Mg(2+). The ATP,Mg(2+)-stimulated uptake is inhibited by FCCP and NEM over the same concentration ranges that reduce the SCN(-) distribution (membrane potential). FCCP increases and NEM decreases vesicular membrane ATPase activity. Thus, catecholamine uptake is correlated to an inside-positive membrane potential, and not to ATPase activity. If catecholamine uptake is coupled to membrane potential, then a charged species must be involved in the transport mechanism. Reserpine and rotenone inhibit catecholamine influx but have no effect on the H(+) electrochemical gradient; they probably act at a step before coupling to the membrane potential (or the H(+) electrochemical gradient). Atractyloside, an inhibitor of nucleotide transport, has no effects on catecholamine transport or the H(+) electrochemical gradient.

Adenosine Triphosphatases↗
PubMed1981

Membrane potential dependence of potential oscillation induced by cevadine in striated muscle.

The dependence of the membrane potential oscillation induced by cevadine on the actual transmembrane potential was studied in the frog sartorius muscle. 1. If the membrane potential oscillation is recorded for hours, its amplitude is seen to decrease slowly and smoothly and the membrane potential measured during the resting period among the waves of oscillation also decreases simultaneously. This means that the depolarization increases. 2. The increase of depolarization results not only in a decrease in the amplitude of the oscillation but the oscillation ceases between -40 and -55 mV as well. 3. The phenomenon reappears if the membrane is partially repolarized on a cevadine treated muscle fibre on which the membrane potential oscillation has already ceased as a consequence of the relatively marked depolarization. 4. Changing the membrane potential either to a value more positive than -40 mV, or to one more negative than -90 mV the developed oscillation activity may reversibly be suspended. 5. According to the above results, the amplitude of the membrane potential oscillation depends on the actual membrane potential. If other factors which may influence the oscillation parameters are unchanged, the relationship between the oscillation amplitude and the membrane potential can be characterized by a linear equation. In these cases there is a close correlation between the measured values and the calculated ones.

Animals↗
PubMed1974

Mitochondrial membrane potential: evidence from studies with a fluorescent probe.

The fluorescence of the probe 3,3'-dihexyl-2,2'-oxacarbocyanine (CC(6)) has been found to indicate potential across cell membranes. Results obtained in the present study using CC(6) and Drosophila mitochondria are in agreement with membrane potentials previously measured by Tupper and Tedeschi using microelectrodes. The results of both studies with Drosophila suggest that the potential across the mitochondrial membrane does not play a significant role in oxidative phosphorylation.

Acetates↗
PubMed1980

Energy transduction in intact synaptosomes. Influence of plasma-membrane depolarization on the respiration and membrane potential of internal mitochondria determined in situ.

A method is described, based on the differential accumulation of Rb+ and methyltriphenylphosphonium, for the simultaneous estimation of the membrane potentials across the plasma membrane of isolated nerve endings (synaptosomes), and across the inner membrane of mitochondria within the synaptosomal cytoplasm. These determinations, together with measurements of respiratory rates, and ATP and phosphocreatine concentrations, are used to define the bioenergetic behaviour of isolated synaptosomes under a variety of conditions. Under control conditions, in the presence of glucose, the plasma and mitochondrial membrane potentials are respectively 45 and 148mV. Addition of a proton translocator induces a 5-fold increase in respiration, and abolishes the mitochondrial membrane potential. The addition of rotenone to inhibit respiration does not affect the plasma membrane potential, and only lowers the mitochondrial membrane potential to 128mV. Evidence is presented that ATP synthesis by anaerobic glycolysis is sufficient under these conditions to maintain ATP-dependent processes, including the reversal of the mitochondrial ATP synthetase. Addition of oligomycin under non-respiring conditions leads to a complete collapse of the mitochondrial potential. Even under control conditions the plasma membrane (Na+ + K+)-dependent ATPase is responsible for a significant proportion of the synaptosomal ATP turnover. Veratridine greatly increases respiration, and depolarizes the plasma membrane, but only slightly lowers the mitochondrial membrane potential. High K+ and ouabain also lower the plasma membrane potential without decreasing the mitochondrial membrane potential. In non-respiring synaptosomes, anaerobic glycolysis is incapable of maintaining cytosolic ATP during the increased turnover induced by veratridine, and the mitochondrial membrane potential collapses. It is concluded that the internal mitochondria must be considered in any study of synaptosomal transport.

Adenosine Triphosphate↗
PubMed1994

Intracellular microelectrode membrane potential measurements in tobacco cell-suspension protoplasts and barley aleurone protoplasts: interpretation and artifacts.

Intracellular microelectrode measurements in plant cell protoplasts have been widely used to study hormone signal transduction processes. However, the interpretation and reliability of such measurements are largely dependent on a detailed evaluation of the measurement conditions, as investigated in the present paper. Upon microelectrode penetration of tobacco cell suspension protoplasts and of barley aleurone protoplasts a fast negative going impalement-induced potential transient of less than a few ms duration could be observed. After reaching a steady-state potential at the ms time scale the measured potential hyperpolarized again and, in most cases, subsequently depolarized to a new steady-state value. Analysis of the electrical equivalent circuit of the measurement configuration showed that the occurrence of the impalement-induced potential transient indicates that these measurements suffer from a microelectrode-induced shunt resistance which loads the measurement. In addition, it is shown that the peak-value of the potential transient is the most reliable indicator of the true membrane potential and of true membrane potential changes of the protoplast, since this value is rather membrane resistance independent. For correct interpretation of steady-state measurements of membrane potential and stimulus-induced membrane potential changes data on membrane and shunt resistance are essential. As an example of the measurement of membrane potential changes the effects of 1-NAA on measured potential values in tobacco protoplasts and the effect of extracellular pH changes on barley aleurone protoplasts are analyzed with regard to the above described conclusions.

Hordeum↗
PubMed1997

Estimation of membrane potential deltapsi in reconstituted plasma membrane vesicles using a numerical model of oxonol VI distribution.

A model of membrane potential-dependent distribution of oxonol VI to estimate the electrical potential difference deltapsi across Schizosaccharomyces pombe plasma membrane vesicles (PMV) has been developed. deltapsi was generated by the H+-ATPase reconstituted in the PMV. The model treatment was necessary since the usual calibration of the dye fluorescence changes by diffusion potentials (K+ + valinomycin) failed. The model allows for fitting of fluorescence changes at different vesicle and dye concentrations, yielding deltapsi in ATP-energized PMV of 80 mV. The described model treatment to estimate deltapsi may be applicable for other reconstituted membrane systems.

Cell Membrane↗
PubMed1988

Platelet membrane potential as a modulator of aggregating mechanisms.

The membrane potential of platelets suspended in physiological medium and membrane potential changes induced by high potassium concentrations, ouabain and cooling have been measured using a cyanine fluorescent dye (3,3'-dipropylthiodicarbocyanine) [corrected]. The membrane potential of platelets suspended in physiological medium was -63.8 mV. High potassium concentrations, ouabain and cooling induced depolarization of platelet membrane. Depolarization using the above procedures enhanced platelet aggregation induced by ADP, adrenaline and collagen. These results suggest that the membrane potential could modulate platelet activity.

Adult↗
PubMed1991

Dependence of Na+ pump current on external monovalent cations and membrane potential in rabbit cardiac Purkinje cells.

1. The effect of membrane potential and various extracellular monovalent cations on the Na+ pump current (Ip) was studied on isolated, single Purkinje cells of the rabbit heart by means of whole-cell recording. 2. Ip was identified as current activated by external K+ or its congeners NH4+ and Tl+. The current was blocked by dihydroouabain (1-5 x 10(-4) M) over the whole range of membrane potentials tested. 3. In Na(+)-containing solution half-maximum Ip activation (K0.5) occurred at 0.4 mM-Tl+, 1.9 mM-K+ and 5.7 mM-NH4+ (holding potential, -20 mV). 4. The pump current (Ip)-voltage (V) relationship of the cells in Na(+)-containing media with K+ or its congeners at the tested concentrations greater than K0.5 displayed a steep positive slope at negative membrane potentials between -120 and -20 mV. Little voltage dependence of Ip was observed at more positive potentials up to +40 mV. At even more positive potentials Ip measured at 2 and 5.4 mM-K+ decreased. 5. Lowering the concentration of K+ or its congeners below the K0.5 value in Na(+)-containing solution induced a region of negative slope of the Ip-V curve at membrane potentials positive to -20 mV. 6. The shape of the Ip-V relationship remained unchanged when the K+ concentration (5.4 mM) of the Na(+)-containing medium was replaced by NH4+ or Tl+ concentrations of similar potency to activate Ip (20 mM-NH4+ or 2 mM-Tl+). 7. In Na(+)-free, choline-containing solution half-maximum Ip activation occurred at 0.13 mM-K+ (holding potential, -20 mV). 8. At negative membrane potentials the positive slope of the Ip-V curve was flatter in Na(+)-free than in Na(+)-containing media. A reduced voltage dependence of Ip persisted, regardless of whether choline ions or Li+ were used as a Na+ substitute. 9. Lowering the K+ concentration of the Na(+)-free, choline-containing solution to 0.05 mM evoked an extended region of negative slope in the Ip-V relationship at membrane potentials between -40 and +60 mV. 10. It is concluded that the apparent affinity of the Na(+)-K+ pump towards K+ in cardiac Purkinje cells depends on both the membrane potential and the extracellular Na+ concentration. 11. The region of negative slope of the Ip-V curve observed in cells which were superfused with media containing low concentrations of K+ or its congeners strongly suggests the existence of at least two voltage-sensitive steps in the cardiac Na(+)-K+ pump cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1980

Membrane potential measurements during rat liver regeneration.

Membrane potential was measured in perfused rat liver and was shown to increase from -33 +/- 1.0 mV in livers from normal rats to -50 +/- 1.1 mV in livers from rats 12 hr after partial hepatectomy. The hyperpolarization of the membrane in regenerating liver was no longer evident after perfusion with 1 mM ouabain for 5 min. Ouabain had a small (4 mV) depolarizing effect on membrane potential in normal liver. The potential measured in normal and regenerating liver decreased as a function of the external potassium concentration above 5 mM; however, the potential was more electronegative in regenerating liver compared to normal liver at all values of external potassium concentration, and the differences in potential between the two kinds of cells did not decrease at higher concentrations of external potassium. Thus, a plot of membrane potential vs external potassium concentration resulted in approximately parallel curves for the two different cell types. We conclude that hyperpolarization of the liver cell membrane is an early event during rat liver regeneration and results from an electrogenic Na-K pump.

Animals↗
PubMed2002

Development of a high throughput screening assay for mitochondrial membrane potential in living cells.

The mitochondrion plays a pivotal role in energy metabolism in eukaryotic cells. The electrochemical potential across the mitochondrial inner membrane is regulated to cope with cellular energy needs and thus reflects the bioenergetic state of the cell. Traditional assays for mitochondrial membrane potential are not amenable to high-throughput drug screening. In this paper, I describe a high-throughput assay that measures the mitochondrial membrane potential of living cells in 96- or 384-well plates. Cells were first treated with test compounds and then with a fluorescent potentiometric probe, the cationic-lipophilic dye tetramethylrhodamine methyl ester (TMRM). The cells were then washed to remove free compounds and probe. The amount of TMRM retained in the mitochondria, which is proportional to the mitochondrial membrane potential, was measured on an LJL Analyst fluorescence reader. Under optimal conditions, the assay measured only the mitochondrial membrane potential. The chemical uncouplers carbonylcyanide m-chlorophenyl hydrazone and dinitrophenol decreased fluorescence intensity, with IC(50) values (concentration at 50% inhibition) similar to those reported in the literature. A Z' factor of greater than 0.5 suggests that this cell-based assay can be adapted for high-throughput screening of chemical libraries. This assay may be used in screens for drugs to treat metabolic disorders such as obesity and diabetes, as well as cancer and neurodegenerative diseases.

Animals↗