Dual-wavelength ratiometric fluorescence measurements of membrane potential

This work shows that the voltage across membranes in two very different preparations, lipid vesicles in suspension and individual HeLa cells under a microscope, is linearly related to the ratio of fluorescence excited from the two wings of the absorption spectrum of a voltage-sensitive dye. The dye di-4-ANEPPS [1-(3-sulfonatopropyl)-4-[beta-[2-(di-n-butylamino)-6-naphthyl] vin yl]pyridinium betaine] is well characterized from earlier investigations and responds via a rapid (less than millisecond) spectral shift to membrane potential changes. The resultant small change in fluorescence intensity monitored at a single wavelength is useful for measurements of temporally well-defined voltage transients such as action potentials. The dual-wavelength approach described in this work extends the usefulness of this fast potentiometric dye by filtering out complex or artifactual changes in fluorescence intensity and providing a voltage-dependent signal that is internally standardized. Thus, rapid measurements of membrane potential are made possible in nonexcitable cells.     

Non-linear relationship between fluorescence and membrane potential

The fluorescence intensity of the cynanine dye DiO-C₆-(3)_in 0.32% suspension of Ehrlich ascites tumor cells was determined under various potassium concentrations. In addition, the fluorescence levels of cell-free buffer solutions and those of supernatants were measured. For every potassium concentration the partition of the dye between cells and medium was calculated and its relation to the potassium gradient was given. A model was developed which assumes the fluorescence of a cell suspension to be the sum of the fluorescence signal of dye in the extracellular medium and that of cell-associated dye. A calibration curve of fluorescence vs. membrane potential was constructed. Neither the fluorescence of the cell suspension nor that of the supernatants was a linear function of the membrane potential. The limitations of membrane potential determination by fluorimetric methods are discussed.     

The effect of the lipid bilayer state on fluorescence intensity of fluorescein-PE in a saturated lipid bilayer

Fluorescein-PE is a fluorescence probe that is used as a membrane label or a sensor of surface associated processes. Fluorescein-PE fluorescence intensity depends not only on bulk pH, but also on the local electrostatic potential, which affects the local membrane interface proton concentration. The pH sensitivity and hydrophilic character of the fluorescein moiety was used to detect conformational changes at the lipid bilayer surface. When located in the dipalmitoylphosphatidylcholine (DPPC) bilayer, probe fluorescence depends on conformational changes that occur during phase transitions. Relative fluorescence intensity changes more at pretransition than at the main phase transition temperature, indicating that interface conformation affects the condition in the vicinity of the membrane. Local electrostatic potential depends on surface charge density, the local dielectric constant, salt concentration and water organisation. Initial increase in fluorescence intensity at temperatures preceding that of pretransition can be explained by the decreased value of the dielectric constant in the lipid polar headgroups region related in turn to decreased water organisation within the membrane interface. The abrupt decrease in fluorescence intensity at temperatures between 25 degrees C and 35 degrees C (DPPC pretransition) is likely to be caused by an increased value of the electrostatic potential, induced by an elevated value of the dielectric constant within the phosphate group region. Further increase in the fluorescence intensity at temperatures above that of the gel-liquid phase transition correlates with the calculated decreased surface electrostatic potential. Above the main phase transition temperature, fluorescence intensity increase at a salt concentration of 140 mM is larger than with 14 mM. This results from a sharp decline of the electrostatic potential induced by the phosphocholine dipole as a function of distance from the membrane surface.     

Liposomes as model for taste cells: receptor sites for bitter substances including N-C=S substances and mechanism of membrane potential changes

Various bitter substances were found to depolarize liposomes. The results obtained are as follows: (1) Changes in the membrane potential of azolectin liposomes in response to various bitter substances were monitored by measuring changes in the fluorescence intensity of 3,3'-dipropylthiocarbocyanine iodide [diS-C3(5)]. All the bitter substances examined increased the fluorescence intensity of the liposome-dye suspension, which indicates that the substances depolarize the liposomes. There existed a good correlation between the minimum concentrations of the bitter substances to depolarize the liposomes and the taste thresholds in humans. (2) The effects of changed lipid composition of liposomes on the responses to various bitter substances vary greatly among bitter substances, suggesting that the receptor sites for bitter substances are multiple. The responses to N-C=S substances and sucrose octaacetate especially greatly depended on the lipid composition; these compounds depolarized only liposomes having certain lipid composition, while no or hyperpolarizing responses to these compounds were observed in other liposomes examined. This suggested that the difference in "taster" and "nontaster" for these substances can be explained in terms of difference in the lipid composition of taste receptor membranes. (3) It was confirmed that the membrane potential of the planar lipid bilayer is changed in response to bitter substances. The membrane potential changes in the planar lipid bilayer as well as in liposomes in response to the bitter substances occurred under the condition that there is no ion gradient across the membranes. These results suggested that the membrane potential changes in response to bitter substances stem from the phase boundary potential changes induced by adsorption of the substances on the hydrophobic region of the membranes.     

The effects of membrane potential and lanthanides on the conformation of the Ca2+-transport ATPase in sarcoplasmic reticulum.

The effects of Ca2+, lanthanide ions (Gd3+, La3+ and Pr3+) and membrane potential on the fluorescence of tryptophan and covalently bound fluorescein were analysed in native and fluorescein isothiocyanate (FITC)-labelled sarcoplasmic reticulum vesicles. The binding of Ca2+ and lanthanides to the Ca2+-ATPase increases the fluorescence intensity of tryptophan and decreases the fluorescence intensity of FITC; the dependence of these effects on cation concentration is consistent with the involvement of the high-affinity Ca2+-binding sites of the Ca2+-ATPase in the cation-induced fluorescence changes. The fluorescence of FITC-labelled sarcoplasmic reticulum vesicles is also influenced by membrane potential changes induced by ion substitution. Inside positive potential increases, while inside negative potential decreases, the fluorescence of bound FITC. Smaller potential-dependent changes in tryptophan fluorescence were also observed. The effects of Ca2+, lanthanides and membrane potential on the fluorescence of tryptophan and FITC are discussed in terms of the two major conformations of the Ca2+-ATPase (E1 and E2), that are assumed to alternate during Ca2+ transport. The observations support the suggestion [Dux, Taylor, Ting-Beall & Martonosi (1985) J. Biol. Chem. 260, 11730-11743] that the vanadate-induced crystals of Ca2+-ATPase represent the E2, while the Ca2+ and lanthanide-induced crystals the E1, conformation of the enzyme.     

Cell suspensions from porcine olfactory mucosa. Changes in membrane potential and membrane fluidity in response to various odorants

A suspension of olfactory epithelial cells was prepared from porcine olfactory mucosa and the physiological functions of the suspension were examined. The membrane potential of the cell suspension, which was monitored by measuring the fluorescence changes of rhodamine 6G, was depolarized by an increase in the K+ concentration in the external medium. Various odorants depolarized the cell suspension in a dose-dependent fashion. The magnitude of depolarization by odorants was either unchanged or slightly increased by a reduction of the concentration of Na+, Ca2+, and Cl- in the external medium, which suggests that changes in the permeabilities of specific ions are not involved in depolarization by odorants. The application of various odorants to the cell suspension induced changes in the membrane fluidity at different sites of the membrane that were monitored with various fluorescent dyes [8-anilino-1-naphthalene sulfonate, n-(9-anthroyloxy) stearic acids, 12-(9-anthroyloxy) oleic acid, and (1,6-diphenyl-1,3,5-hexatriene)], which suggests that the odorants having different odors are adsorbed on different sites in the membrane. On the basis of these results, a possible mechanism of odor discrimination is discussed.     

Mechanism of ethonium effect on membrane preparations of brain Na+, K+-ATPase

The effect of ethonium, a local anesthetic, on the membrane preparations of brain N+, K+-ATPase was studied by fluorescent, radioisotopic, electron-microscopic and electrophysiological methods. Ethonium is established to affect the formation of an intermediate phosphorylated product and has no pronounced destructive effect on the membrane. It changes the fluorescence intensity of 2-toluidinonaphthalen-6-sulphonate (TNS), astrafloxin (AF) and fluorene probes in a suspension of the Na+, K+-ATPase preparations, which evidences for ethonium-induced changes in the structure of membrane fragments.     

The use of the potential-sensitive fluorescent probe bisoxonol in mast cells.

The regulation of the plasma membrane potential of rat peritoneal mast cells at the resting state and during activation was investigated using bisoxonol as a potential-sensitive fluorescent dye. Fluorescence microphotography showed that this negatively charged probe was not only present in the plasma membrane, but was also distributed in the cytoplasm. The intracellular localization of bisoxonol was confirmed by conducting experiments which showed that bisoxonol fluorescence was not enhanced in ATP-permeabilized mast cells. Rotenone (10(-7) M) and oligomycin (10(-6) M) did not change the fluorescence of bisoxonol showing, therefore, mitochondrial depolarization was not recorded with bisoxonol and suggesting that bisoxonol may represent a useful probe to study plasma membrane potential changes in the absence of exocytosis. We showed that, in non-stimulated mast cells, the blockade of the sodium pump enhanced the fluorescence of bisoxonol as did gramicidin a non selective ionophore used to fully depolarize the cells. High concentration of potassium (30 mM) as well as different ionic channel blockers did not significantly change the fluorescence intensity of bisoxonol, suggesting that ionic channel permeabilities were not involved in maintaining the resting plasma membrane potential of mast cells. Mast cells stimulated by compound 48/80 completely lost the fluorescence, shown by fluorescence microphotography, suggesting that exocytotic phenomena might induce a dye redistribution which is not only due to changes in the plasma membrane potential. In mast cells pretreated with pertussis toxin, which blocks mast cell-exocytosis, compound 48/80 induced a delayed (2 min) decrease of bisoxonol fluorescence which was shown to be dependent on the activity of the sodium pump. Considering that bisoxonol is a useful potential-sensitive probe in exocytosis-deprived mast cells, our results suggest that the sodium pump is mainly involved in the changes of plasma membrane potential of mast cells.     

Stimulation of triglyceride synthesis in mammalian liver and adipose tissue by two cytosolic compounds.

The enhancement of 1-anilino-8-naphthalenesulfonate fluorescence was observed followed by the binding of the probe to the $\text{E. coli}$ membrane. The fluorescence intensity of the probe is quenched upon energization of intact cells. The experiments with sonicated membrane particles, in which the orientation of the membrane is “inside-out”, showed an energy linked enhancement of the fluorescence intensity of the probe. The changes in the fluorescence intensity of fluorochrome-stained membranes can also be induced by generation of K⁺ ion diffusion potential.     

Electrostatic interaction between merocyanine 540 and liposomal and mitochondrial membranes

Summary The fluorescence of merocyanine 540 (MC) in liposomal and mitochondrial suspensions was measured under various conditions. Under a given condition, both the amount of dye bound to the membrane and the zeta potential were determined simultaneously. It was found that the fluorescence intensity was proportional to the amount of bound dye and correlated with the zeta potential of particles. The fluorescence intensity was represented quantitatively in terms of the Langmuir adsorption isotherm, when the electrostatic interaction acting between MC and membrane surface was properly taken into account. It was concluded that the changes in MC fluorescence in the liposomal and mitochondrial suspensions are mainly attributed to the changes in the surface potential of the membranes.     

Depolarization of synaptosomal membranes: a study of mechanism by which rhodamine 6G measures membrane potential

The fluorescence intensity of Rhodamine 6G in synaptosomal suspensions has been measured to monitor the membrane potential changes in pre-synaptic nerve terminals. The fluorescence response of the dye was seen to be a function of potential-dependent partitioning of dye molecules between the synaptosomes and the extracellular medium. Binding of dye molecules to the hydrophobic regions of membranes results in the quenching of fluorescence. Upon depolarization of the synaptosomal membrane, the dye molecules are released from the cells. The effect of changing extracellular ionic composition was also studied. The membrane potential increased linearly with log of [K]0. The resting membrane potential in buffer containing 5 mM K+ was calculated to be -60 mV. Raising the extracellular Ca2+ and Mg2+ from 1.2 mM to 10 mM did not change the membrane potential. Ca2+ ionophore A23187, in the presence of Ca2+ was found to depolarize the membranes.     

Optical response of the indicator chlortetracycline to membrane potential

Chlortetracycline is a fluorescent, Ca2+ indicator commonly used to monitor the internal Ca2+ concentration of membrane vesicles and organelles. We have found that the intensity of chlortetracycline fluorescence in the presence of Ca2(+)-loaded liposomes is dependent on the membrane potential of the vesicles as well as the intravesicular Ca2+ concentration. The fluorescence of chlortetracycline was lower when an inside-negative membrane potential was placed across the liposome membrane. Since chlortetracycline diffuses across the membrane in the zwitterionic form, the distribution of chlortetracycline across the membrane should not be strongly dependent on the membrane potential. However, because the proton permeability of phospholipid vesicles is relatively high, the intravesicular proton concentration is dependent on the membrane potential. The binding of Ca2+ to chlortetracycline is dependent on pH in the range of pH 6 to pH 8. Therefore, changes in the intravesicular pH as a result of a change in the membrane potential causes relatively large changes in the chlortetracycline fluorescence signal even when there isn't a change in the Ca2+ concentration.     

A comparative analysis of extrinsic fluorescence in nerve membranes and lipid bilayers

Changes in extrinsic fluorescence intensity, associated with step changes in membrane potential, have been studied in intracellularly or extracellularly stained squid axons, and in lipid bilayers, using six different aminonaphthalene dyes: 1,8-TNS; 2,6-TNS; 1,8-MANS; 2,6-MANS; 2,6-ANS and NPN. In all preparations the optical signals were found to be roughly proportional to the voltage applied. All signals had a very fast initial component, which was followed in some case by a slower change in the same direction. The slow component was observed only in intracellularly stained axons, and not for all chromophores studied. 1,8-TNS, 1,8-MANS and 2,6-MANS yielded the largest fluorescence signals in all preparations. The sign of these signals was independent of the type of membrane studied. However, the fluorescence changes of 2,6-MANS were opposite to those of 1,8-TNS and 1,8 MANS. Staining of both sides of the axolemma with 1,8-MANS or 2,6-MANS showed that these dyes yield larger signals when applied to the extracellular face. The changes in fluorescence light intensity of 2,6-TNS, 2,6-ANS and NPN were smaller and their sign depended on the membrane preparation studied. The comparison of the extrinsic fluorescence signals from the nerve membrane and the phosphatidylcholine bilayer suggests strong similarities between the basic structures of the two systems. The variety of observed signals cannot be easily interpreted in terms of changes in membrane structure. A possible alternative interpretation in terms of electrically induced displacements, rotations and changes in partition coefficient of bound chromophores, is discussed.Abbreviations 1,8-TNS 1-toluidinonaphthalene-8-sulfonate, and similarly, 2,6-TNS - 1,8-MANS 1-N-methylanilinonaphthalene-8-sulfonate, and similarly, 2,6-MANS - 1,8-ANS 1-anilinonaphthalene-8-sulfonate, and similarly, 2,6-ANS - NPN N-phenyl-1-naphthylamine     

Flow cytometric estimation of transmembrane potential of macrophages--a comparison with microelectrode measurements

Potential-dependent accumulation of the lipophilic cationic dye 3,3' dihexyloxacarbocyanine (DiOC6(3)) in macrophages has been investigated. Resulting fluorescence of cells was measured by flow cytometry. Alterations of membrane potential of macrophages were induced by ionophore treatment (valinomycin and gramicidin) in a dose-dependent (10(-5) M-10(-7) M) and time-dependent (0 min-45 min) manner. Resulting changes in relative fluorescence intensity were compared with changes of transmembrane potential measured by intracellular recordings obtained by applying glass microelectrodes. The comparative studies offer the possibility to calibrate the flow cytometric estimate of membrane potential of suspended cells. Equilibration of dye partition between cells and surrounding medium is strictly potential-dependent at dye concentrations between 5 X 10(-8) M and 10(-7) M and within an incubation interval from 10 min up to 30 min after addition of dye. Conclusions are drawn concerning the field of application of the optical method. Dynamics of electrical processes following ionophore treatment are discussed in terms of molecular mechanisms of altered ionic transport.     

Ion diffusion potentials across mycoplasma membranes determined by a novel method using a carbocyanine dye

The influence of transmembrane ion fluxes on mycoplasma membrane potentials was studied. Electric membrane potential was calibrated vs fluorescence intensity of a potential-sensitive carbocyanine dye according to delta psi = (RT/F) X log([aIN(1 - IN) - b]/Kint), where IN = I/I0, I0 = maximal fluorescence intensity (obtained for delta psi----infinity), and a and b are constants. Fluorescence intensity was calibrated vs membrane potential by inducing a K+ diffusion potential. The calibration procedure was based on the assumption that in the presence of valinomycin the membrane potential was determined entirely by K+ diffusion. Then the dependence of fluorescence intensity on the external K+ concentration, Kext, could be described by Ival = I0[1 + a/(Kext + b)]-1. For Mycoplasma mycoides subsp. capri and enterococci, the constants were determined from experimental data using nonlinear least-squares computer-assisted methods. The validity of our assumption was proved using the "null-point" method. Here the Ca2+ ionophore A23187 and varying external Ca2+ concentrations were used to change the membrane potential experimentally. K+ and Na+ diffusion potentials significantly contributed to mycoplasma membrane potential whereas Cl- had no influence. Under growth conditions the mycoplasma membrane potential was estimated to be delta psi = -68 mV.     

Liposomes as a model for olfactory cells: changes in membrane potential in response to various odorants

Various odorants were found to depolarize azolectin liposomes. The results obtained are as follows. (1) Changes in the membrane potential of azolectin liposomes in response to various odorants were monitored by measuring changes in the fluorescence intensity of 3,3'-dipropylthiocarbocyanine iodide [disS-C3(5)]. Ten odorants examined increased the fluorescence intensity of the liposome-dye suspensions in a dose-dependent manner, which indicates that odorants depolarize the liposomes. Concentrations of odorants that depolarized the liposomes greatly varied among the odorants. There existed a good correlation between the minimum concentrations of odorants to depolarize the liposomes and the thresholds of respective odorants in the frog or porcine olfactory responses. (2) Addition of sphingomyelin (SM) to azolectin led to a large enhancement of depolarizations by nonanol, citral, and n-amyl acetate. The results indicate that lipid composition of liposomes is one of the factors that control the sensitivity to odorants. (3) Odorants changed the membrane fluidity of the liposomes, which was monitored by changes in the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). The membrane fluidity was changed in concentration ranges of odorants similar to those where the membrane potential changes occurred, which suggests that changes in the membrane fluidity are related to generation of the membrane potential changes. (4) Changes in the membrane potential in response to odorants were electrically measured with the planar lipid bilayer made of an azolectin-SM (2:1 w/w) mixture. It was shown that odorants (nonanol, citral, and n-amyl acetate) depolarized the planar lipid bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

A study of the effect of adrenaline on the transmembrane potential of the plasma membrane of hepatocytes from rats of different age using fluorescent probes

The resting membrane potential of isolated hepatocytes from 2- and 20-month-old rats and its changes upon activation of cells by adrenaline have been studied by the method of quantitative microfluorimetry using diethyl derivatives of polymethine probes (H-510 and D-307). The potential was estimated by the Nernst equation adapted to lipophilic cationic probes. It was shown using both probes that the transmembrane potential of hepatocytes decreases with age. The microfluorimetry data were confirmed by the results of spectrofluorimetric measurements in a cell suspension. Changes in fluorescence occurring in adrenaline-activated single cells and suspensions were unidirectional, the effect of the hormone on the cells of old animals being less pronounced. The results indicate that the potential of the plasma membrane of individual hepatocytes can be adequately estimated by microfluorimetry, which can be used in metabolic and toxicologic investigations.     

Investigations of spectrin-lipid interactions using fluoresceinphosphatidylethanolamine as a membrane probe

The binding of human erythrocyte spectrin to large unilamellar vesicles (LUVET) formed by the extrusion technique has been studied using fluoresceinphosphatidylethanolamine (FPE) as a reporter of electrostatic membrane potential. Spectrin aliquots were added to a suspension of FPE-labelled LUVETs to elucidate both the type of charge involved and the dissociation constants for spectrin binding to various lipids. All binding experiments showed serial increases in FPE fluorescence intensity upon serial additions of spectrin, indicative of increasing positive charge at the membrane surface. This proves for the first time that although exhibiting an overall net negative charge, spectrin binds to lipid surfaces by presenting positive charges to the lipid surface. Binding curves were obtained from the change in fluorescence intensity upon each spectrin addition and analysed to determine dissociation constants. A K(d) of 0.14+/-0.12 microM was found for spectrin binding to FPE-labelled phosphatidylcholine/phosphatidylserine (PC/PS) LUVETs at 22 degrees C in high salt conditions. A similar K(d) of 0.17+/-0.11 microM was obtained for spectrin binding to neutral LUVETs composed of PC. However, binding was found to be much weaker for PC/PS LUVETs under low salt conditions with a K(d) of 1.22+/-0.48 microM.     

Dual-wavelength ratiometric fluorescence measurement of the membrane dipole potential.

The electrostatic potentials associated with cell membranes include the transmembrane potential (delta psi), the surface potential (psi s), and the dipole potential (psi D). psi D, which originates from oriented dipoles at the surface of the membrane, rises steeply just within the membrane to approximately 300 mV. Here we show that the potential-sensitive fluorescent dye 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6- naphthyl]vinyl]pyridinium betaine (di-8-ANEPPS) can be used to measure changes in the intramembrane dipole potential. Increasing the content of cholesterol and 6-ketocholestanol (KC), which are known to increase psi D in the bilayer, results in an increase in the ratio, R, of the dye fluorescence excited at 440 nm to that excited at 530 nm in a lipid vesicle suspension; increasing the content of phloretin, which lowers psi D, decreases R. Control experiments show that the ratio is insensitive to changes in the membrane's microviscosity. The lack of an isosbestic point in the fluorescence excitation and emission spectra of the dye at various concentrations of KC and phloretin argues against 1:1 chemical complexation between the dye and KC or phloretin. The macromolecular nonionic surfactant Pluronic F127 catalyzes the insertion of KC and phloretin into lipid vesicle and cell membranes, permitting convenient and controlled modulation of dipole potential. The sensitivity of R to psi D is 10-fold larger than to delta psi, whereas it is insensitive to changes in psi S. This can be understood in terms of the location of the dye chromophore with respect to the electric field profile associated with each of these potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of the electric diffusion potential on delayed fluorescence light curves of chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea

The potassium salt-induced transient increase of delayed fluorescence yield was studied in pea chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea.A simple kinetic model is proposed to account for the actinic light intensity dependence of the delayed fluorescence enhancement by the transmembrane diffusion potential induced by sudden salt addition. The electric field dependence of the rate constants for the recombination of primary separated charges with and without subsequent electronic excitation of reaction center chlorophyll was obtained.From the value of enhancement of delayed fluorescence by salt concentration gradients at saturating actinic light intensity, it is concluded that the distance, normal to thylakoid membrane surface, between the primary acceptor and the donor of Photosystem II is smaller than the membrane thickness.