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)     

Transmembrane proton-motive potential of Spiroplasma floricola

In Spiroplasma floricola, the transmembrane proton-motive potential delta p was studied. It is composed of a transmembrane electric potential difference, delta psi, and a transmembrane proton gradient, delta pH, according to delta p = delta psi - (Z.delta pH). Using a potential-sensitive carbocyanine dye and 5,5'-dimethyl[2-14C]oxazolidine-2,4-dione as probes, delta psi and delta pH were measured at different [H+] of the medium, and delta p was calculated to be remarkably constant at -123 mV +/- 16% over a wide range of external pH values. Inhibition experiments indicated that it is generated by a membrane-bound, electrogenic, proton-translocating ATPase.     

Existence of an adenosine 5'-triphosphate dependent proton translocase in bovine neurosecretory granule membrane

The addition of ATP to bovine neurohypophysial secretory granules suspended in isotonic sucrose medium induces a positive polarization, delta psi, of their interior without affecting their internal pH. In KCl-containing media, ATP failed to generate large delta psi but induced a pH gradient (delta pH; interior acidic). These observations are consistent with the existence in the neurosecretory granule membrane of an ATP-dependent inward electrogenic H+ translocase (H+ pump), capable in KCl-containing media of acidifying the granule matrix by H+-Cl- cotransport. The delta psi and delta pH generated by the H+ pump, defined as the ATP-induced changes sensitive to the H+ ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), were blocked by N,N'-dicyclohexylcarbodiimide, an inhibitor of all H+ pumps, and were insensitive to oligomycin, a mitochondrial ATPase inhibitor. In sucrose medium, measurements were complicated by a Donnan equilibrium reflecting the presence in the granule of peptide hormones and neurophysins which resulted in a CCCP-resistant resting delta pH. In KCl-containing media, the Donnan equilibrium was destroyed since the membrane is permeable to cations, but under these conditions a CCCP-resistant K+-diffusion potential was observed. The ATP-induced delta psi was also monitored by the extrinsic fluorescent probe bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol. The hypothesis of a granule H+ pump is further supported by the presence of an oligomycin-resistant ATPase in the preparation and the ultrastructural localization of such an activity on the granule membrane. The H+ pump has been found in both newly formed and aged neurosecretory granules. Its possible physiological function is discussed with reference to that of chromaffin granules, with which it has many similarities.     

Erythrocyte and ghost cytoplasmic resistivity and voltage-dependent apparent size.

Particle resistivity is explicitly included in the equations relating volume to voltage pulse, in electronic cell sizing or resistive pulse spectroscopy (RPS). It has long been known that in high electric fields cell resistivity decreases as the membrane undergoes dielectric breakdown. At sufficiently high electric field strengths, well past dielectric breakdown, the red cell membrane becomes electrically transparent, or nearly so, and apparent cell size becomes essentially a function of the cytoplasmic resistivity. Electronic cell sizing is traditionally carried out at low electric field strengths, and corrections made for the influence of cell shape by use of the Laplace equation. We find the Laplace solution to be still applicable at very high electric field strengths for purposes of calculating specific cytoplasmic resistivity from RPS measurements. Our value for discocytes, 220 omega X cm, is in good agreement with published results obtained by other researchers using other techniques. We have also applied these same procedures to determine the time course of voltage-dependent resistivity changes in ghosts and intact spherocytes, during the first 5 min after suspension in hypotonic medium. We believe these to be the first explicit calculations of particle specific resistivity from post-dielectric-breakdown apparent size, using traditional electronic sizing techniques.     

Imaging the permeability pore transition in single mitochondria.

In mitochondria the opening of a large proteinaceous pore, the "mitochondrial permeability transition pore" (MTP), is known to occur under conditions of oxidative stress and matrix calcium overload. MTP opening and the resulting cellular energy deprivation have been implicated in processes such as hypoxic cell damage, apoptosis, and neuronal excitotoxicity. Membrane potential (delta psi(m)) in single isolated heart mitochondria was measured by confocal microscopy with a voltage-sensitive fluorescent dye. Measurements in mitochondrial populations revealed a gradual loss of delta psi(m) due to the light-induced generation of free radicals. In contrast, the depolarization in individual mitochondria was fast, sometimes causing marked oscillations of delta psi(m). Rapid depolarizations were accompanied by an increased permeability of the inner mitochondrial membrane to matrix-entrapped calcein (approximately 620 Da), indicating the opening of a large membrane pore. The MTP inhibitor cyclosporin A significantly stabilized delta psi(m) in single mitochondria, thereby slowing the voltage decay in averaged recordings. We conclude that the spontaneous depolarizations were caused by repeated stochastic openings and closings of the transition pore. The data demonstrate a much more dynamic regulation of membrane permeability at the level of a single organelle than predicted from ensemble behavior of mitochondrial populations.     

Membrane potentials in reconstituted cytochrome c oxidase proteoliposomes determined by butyltriphenyl phosphonium cation distribution

Equilibration of the butyltriphenyl phosphonium (BTPP+) cation into cytochrome c oxidase reconstituted proteoliposomes was measured potentiometrically. The maximum membrane potential (delta psi) generated by oxidase activity was estimated to lie between -65 and -90 mV, vesicle interior negative, when internal BTPP+ binding is taken into account. Formation of delta psi was completely prevented by valinomycin and carbonyl-cyanide-p-trifluoromethoxyphenylhydrazone but only 10% inhibited by levels of N',N'-dicyclohexylcarbodiimide that abolish proton pumping by the oxidase. delta psi is thus maintained by at least one charge transfer process that does not involve proton movement. A nonlinear relationship was obtained between oxidase activity and steady-state delta psi. The value of delta psi estimated by BTPP+ distribution was lower than that calculated using the optical probes safranine and a carbocyanine dye. Possible reasons for this discrepancy are discussed.     

Dilation of the influenza hemagglutinin fusion pore revealed by the kinetics of individual cell-cell fusion events

We have monitored kinetics of fusion between cell pairs consisting of a single influenza hemaglutinin (HA)-expressing cell and a single erythrocyte (RBC) that had been labeled with both a fluorescent lipid (Dil) in the membrane and a fluorescent solute (calcein) in the aqueous space. Initial fusion pore opening between the RBC and HA-expressing cell produced a change in RBC membrane potential (delta psi) that was monitored by a decrease in Dil fluorescence. This event was followed by two distinct stages of fusion pore dilation: the flux of fluorescent lipid (phi L) and the flux of a large aqueous fluorescent dye (phi s). We have analyzed the kinetics of events that occur as a result of transitions between a fusion pore (FP) and a solute permissive fusion pore (FPs). Our data are consistent with a fusion pore comprising six HA trimers.     

Hydrogenosomes in the rumen protozoon Dasytricha ruminantium Schuberg.

The vacuo-lysosomes of Hevea brasiliensis (rubber tree) constitute a suitable model system for the study of active transport and energization at the level of the membrane of plant vacuoles. The pH gradient (delta pH) and the membrane potential (delta psi) of vacuo-lysosomes were determined by means of the weak base methylamine and the lipophilic cation tetraphenylphosphonium. The values obtained depended strongly on the experimental conditions such as medium pH or K+ concentration. Under experimental conditions, i.e., pH 7.5 outside and low K+, the delta pH amounts to about 0.9 unit, interior acid, and the delta psi to -120 mV, interior negative. The delta psi is presumably caused by the imposed K+ gradient, and the internal acidification might be a consequence of the passive proton inflow along the electric field. This explanation is sustained by the ineffectiveness of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in destroying the delta pH and delta psi, whereas higher K+ concentration decreased both. Under conditions existing in vivo, the membrane potential might be significantly lower. The presence of ATP increased the acidification of the intravesicular space by 0.5pH unit to a delta pH of up to 1.4 and shifts the membrane potential at least 60mV to a more positive value. The change of the protonmotive potential did not occur with ADP; the pH-dependence of the change was identical with the pH-dependence of a vacuo-lysosomal membrane-bound ATPase, and the effect of ATPase was prevented by the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The change of protonmotive potential difference, brought about by the ATPase, was at least 90 mV. This is evidence that a vacuo-lysosomal ATPase in plants can function as an electrogenic proton pump that transfers protons into the vacuo-lysosomal space.     

Glutamate uptake into synaptic vesicles of bovine cerebral cortex and electrochemical potential difference of proton across the membrane.

Measurements have been made of the ATP-dependent membrane potential (delta psi) and pH gradient (delta pH) across the membranes of the synaptic vesicles purified from bovine cerebral cortex, using the voltage-sensitive dye bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxanol and the delta pH-sensitive fluorescent dye 9-aminoacridine respectively. A pre-existing small delta pH (inside acidic) was detected in the synaptic vesicles, but no additional significant contribution by MgATP to delta pH was observed. In contrast, delta psi (inside positive) increased substantially upon addition of MgATP. This ATP-dependent delta psi was reduced by thiocyanate anion (SCN-), a delta psi dissipator, or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a protonmotive-force dissipator. Correspondingly, a substantially larger glutamate uptake occurred in the presence of MgATP, which was inhibited by SCN- and FCCP. A nonhydrolysable analogue of ATP, adenosine 5'-[beta gamma-methylene]triphosphate, did not substitute for ATP in either delta psi generation or glutamate uptake. The results support the hypothesis that a H+-pumping ATPase generates a protonmotive force in the synaptic vesicles at the expense of ATP hydrolysis, and the protonmotive force thus formed provides a driving force for the vesicular glutamate uptake. The delta psi generation by ATP hydrolysis was not affected by orthovanadate, ouabain or oligomycin, but was inhibited by N-ethylmaleimide, quercetin, trimethyltin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid. These results indicate that the H+-pumping ATPase in the synaptic vesicle is similar to that in the chromaffin granule, platelet granule and lysosome.     

Membrane potential estimation by flow cytometry

Membrane potential (delta psi) is generated and maintained by concentration gradients of ions such as sodium, potassium, chloride, and hydrogen. Changes in cytoplasmic delta psi in the course of surface-receptor-mediated processes related to the development, function, and pathology of many cell types often play a role in transmembrane signaling. Cytoplasmic delta psi is also reduced to zero when the membrane is ruptured by chemical or physical agents. Mitochondrial delta psi is reduced when energy metabolism is disrupted, notably in apoptosis. In bacteria, which lack mitochondria, delta psi reflects both the state of energy metabolism and the physical integrity of the cytoplasmic membrane. Flow cytometry can be used to estimate membrane potential in eukaryotic cells, mitochondria in situ, isolated mitochondria, and bacteria. Older methods, using lipophilic cationic dyes such as the cyanines and rhodamine 123 or lipophilic anionic dyes such as the oxonols can detect relatively large changes in delta psi and identify heterogeneity of response in subpopulations comprising substantial fractions of a cell population. Newer ratiometric techniques allow precise measurement of delta psi to within 10 mV or less. Among other factors, action of efflux pumps, changes in membrane structure, and changes in protein or lipid concentration in the medium in which cells are suspended can produce changes in cellular fluorescence which may be misinterpreted as changes in delta psi. Techniques for estimation and measurement of Delta Psi therefore typically require careful control of cell and reagent concentrations and incubation times and selection of appropriate controls if they are to provide accurate information.     

Dependence of Electroporation Detection Threshold on Cell Radius: An Explanation to Observations Non Compatible with Schwan’s Equation Model

It is widely accepted that electroporation occurs when the cell transmembrane voltage induced by an external applied electric field reaches a threshold. Under this assumption, in order to trigger electroporation in a spherical cell, Schwan’s equation leads to an inversely proportional relationship between the cell radius and the minimum magnitude of the applied electric field. And, indeed, several publications report experimental evidences of an inverse relationship between the cell size and the field required to achieve electroporation. However, this dependence is not always observed or is not as steep as predicted by Schwan’s equation. The present numerical study attempts to explain these observations that do not fit Schwan’s equation on the basis of the interplay between cell membrane conductivity, permeability, and transmembrane voltage. For that, a single cell in suspension was modeled and the electric field necessary to achieve electroporation with a single pulse was determined according to two effectiveness criteria: a specific permeabilization level, understood as the relative area occupied by the pores during the pulse, and a final intracellular concentration of a molecule due to uptake by diffusion after the pulse, during membrane resealing. The results indicate that plausible model parameters can lead to divergent dependencies of the electric field threshold on the cell radius. These divergent dependencies were obtained through both criteria and using two different permeabilization models. This suggests that the interplay between cell membrane conductivity, permeability, and transmembrane voltage might be the cause of results which are noncompatible with the Schwan’s equation model.     

Active electrogenic transport H+ in plasma membrane vesicles of cow parsnip phloem cells

Generation of electric (delta psi) and chemical (delta pH) components of electrochemical proton gradient delta muH+, in plasma membrane vesicles of Heracleum sosnovskyi phloem cells was investigated. ATP-dependent generation of delta psi at pH 6.0 in the presence of Mg2+ and K+ was established with the help of fluorescent probes AU+ and ANS-. Protonophore CCCP and proton ATPase inhibitor DCCD suppressed generation, whereas oligomycin, the inhibitor of mitochondrial ATPases did not affect it. Measurings of delta psi value indicated its oscillations within the limits from 10 to 60 mV. ATP-dependent generation of delta pH was established by means of fluorescent probe 9-AA. The effect was eliminated by CCCP and stimulated by K+, that may testify to the transformation of a part of delta psi into delta pH at antiport H+/K+. Existence of H+-ATPase in the plasma membranes of higher plant cells insuring generation of delta muH+ is supposed.     

Membrane conductance of an electroporated cell analyzed by submicrosecond imaging of transmembrane potential.

Transmembrane potential was induced in a sea urchin egg by applying a microsecond electric pulse across the cell. The potential was imaged at a submicrosecond time resolution by staining the cell membrane with the voltage-sensitive fluorescent dye RH292. Under moderate electric fields, the spatial distribution of the induced potential as well as its time dependence were in accord with the theoretical prediction in which the cell membrane was regarded as an insulator. At higher field intensities, however, the potential apparently did not fully develop and tended to saturate above a certain level. The saturation is ascribed to the introduction of a large electrical conductance, in the form of aqueous openings, in the membrane by the action of the induced potential (electroporation). Comparison of the experimental and theoretical potential profiles indicates that the two regions of the membrane that opposed the electrodes acquired a high membrane conductance of the order of 1 S/cm2 within 2 microseconds from the onset of the external field. The conductance was similar in the two regions, although permeability in the two regions of the membrane long after the pulse treatment appeared quite different.     

Electrical properties of an excitable epithelium

The exumbrellar epithelium of the hydromedusa, Euphysa japonica, is composed of a single layer of broad (70 micrometers), thin (1--2 micrometers) cells which are joined by gap junctions and simple appositions. Although the epithelium lacks nerves, it is excitable; electrically stimulating the epithelium initiates a propagated action potential. The average resting potential of the epithelial cells is -46 mV. The action potential, recorded with an intracellular electrode, is an all-or-nothing, positive, overshooting spike. The epithelial cells are electrically coupled. The passive electrical properties of the epithelium were determined from the decrement in membrane hyperpolarization with distance from an intracellular, positive current source. The two-dimensional space constant of the epithelium is 1.3 mm, the internal longitudinal resistivity of the cytoplasm and intercellular junctions is 196 omega cm, and the resistivity of both apical and basal cell membranes is greater than 23 k omega cm2. Although the membrane resistivity is high, the transverse resistivity of the epithelium is quite low (7.5 omega cm2), indicating that the epithelium is leaky with a large, transverse, paracellular shunt.     

Preprotein translocation creates a halide anion permeability in the Escherichia coli plasma membrane.

The electrochemical potential drives the translocation of the precursor form of outer membrane protein A (proOmpA) and other proteins across the plasma membrane of Escherichia coli. We have measured the electrical potential, delta psi, across inverted membrane vesicles during proOmpA translocation. delta psi, generated by the electron transport chain, is substantially dissipated by proOmpA translocation. delta psi dissipation requires SecA, ATP, and proOmpA. proOmpA which, due to the covalent addition of a folded protein to a cysteinyl side chain, is arrested during its translocation, can nevertheless cause the loss of delta psi. Thus the movement of charged amino acyl residues is not dissipating the potential. This translocation-specific reduction in delta psi is only seen in the presence of halide anions, although halide anions are not needed for proOmpA translocation per se. We therefore propose that translocation intermediates directly increase the membrane permeability to halide anions.     

Time courses of cell electroporation as revealed by submicrosecond imaging of transmembrane potential.

Changes in the membrane conductance of sea urchin eggs, during the course of electroporation, were investigated over the time range of 0.5 microsecond to 1 ms by imaging the transmembrane potential at a submicrosecond resolution with the voltage-sensitive fluorescent dye RH292. When a rectangular electric pulse of moderate intensity was applied across an egg, a position-dependent potential developed synchronously with the pulse, as theory predicts for a cell with an insulating membrane. From the rise and fall times, the membrane capacitance of unfertilized eggs was estimated to be 0.95 microF/cm2 and the intracellular conductance 220 omega.cm. Under an electric pulse of much higher intensity, the rise of the induced potential stopped at a certain level and then slowly decreased on the microsecond time scale. This saturation and subsequent reversal of the potential development was ascribed to the introduction of finite membrane conductance, or permeabilization of the membrane, by the action of the intense pulse (electroporation). Detailed analysis indicated the following: already at 0.5 microsecond in the rectangular electric pulse, the two sides of the egg facing the positive and negative electrodes were porated and gave a high membrane conductance in the order of 1 S/cm2; the conductance on the positive side appeared higher. Thereafter, the conductance increased steadily, reaching the order of 10 S/cm2 by 1 ms. This increase was faster on the negative-electrode side; by 1 ms the conductance on the negative side was more than twice that on the positive side. The recovery of the porated membrane after the pulse treatment was assessed from the membrane conductance estimated in a second electric pulse of a small amplitude. At least two recovery processes were distinguished, one with a time constant of 7 microseconds and the other 0.5 ms, at the end of which the membrane conductance was already < 0.1 S/cm2.     

Determination of intracellular conductivity from electrical breakdown measurements

The intracellular resistivity (conductivity) of cells can be easily calculated with high accuracy from electrical membrane breakdown measurements. The method is based on the determination of the size distribution of a cell suspension as a function of the electrical field strength in the orifice of a particle volume analyser (Coulter counter). The underestimation of the size distribution observed beyond the critical external field strength leading to membrane breakdown represents a direct access to the intracellular resistivity as shown by the theoretical analysis of the data. The potential and the accuracy of the method is demonstrated for red blood cells and for ghost cells prepared by electrical haemolysis. The average value of 180 omega X cm for the intracellular resistivity of intact red blood cells is consistent with the literature.     

Transmembrane electric potential as a regulator of functional activity of biomembranes

The role of the transmembrane electric potential difference in producing structural tension of biological membranes is analysed. A suggestion is made that delta psi can optimize conditions of protein-protein interactions by ordering the membrane lipid. For energy-coupling membranes this makes it possible to explain the dependence of ATP synthesis on both electron transport and delta psi in terms of local bioenergetic coupling. The energized biomembrane is considered as a self-supporting structure, delta psi being a regulator of efficiency of molecular machines performing different membrane functions, including energization as well.     

Factors and processes involved in membrane potential build-up in yeast: diS-C3(3) assay.

No methods are currently available for fully reliable monitoring of membrane potential changes in suspensions of walled cells such as yeast. Our method using the Nernstian cyanine probe diS-C3(3) monitors even relatively fast changes in membrane potential delta psi by recording the shifts of probe fluorescence maximum lambda max consequent on delta psi-dependent probe uptake into, or exit from, the cells. Both increased [K+]out and decreased pHout, but not external NaCl or choline chloride depolarise the membrane. The major ion species contributing to the diS-C3(3)-reported membrane potential in S. cerevisiae are thus K+ and H+, whereas Na+ and Cl- do not perceptibly contribute to measured delta psi. The strongly pHout-dependent depolarisation caused by the protonophores CCCP and FCCP, lack of effect of the respiratory chain inhibitors rotenone and HQNO on the delta psi, as well as results obtained with a respiration-deficient rho- mutant show that the major component of the diS-C3(3)-reported membrane potential is the delta psi formed on the plasma membrane while mitochondrial potential forms a minor part of the delta psi. Its role may be reflected in the slight depolarisation caused by the F1F0-ATPase inhibitor azide in both rho- mutant and wildtype cells. Blocking the plasma membrane H(+)-ATPase with the DMM-11 inhibitor showed that the enzyme participates in delta psi build-up both in the absence and in the presence of added glucose. Pore-forming agents such as nystatin cause a fast probe entry into the cells signifying membrane damage and extensive binding of the probe to cell constituents reflecting obviously disruption of ionic balance in permeabilised cells. In damaged cells the probe therefore no longer reports on membrane potential but on loss of membrane integrity. The delta psi-independent probe entry signalling membrane damage can be distinguished from the potential-dependent diS-C3(3) uptake into intact cells by being insensitive to the depolarising action of CCCP.     

Voltage-sensitive cyanine dye fluorescence signals in lymphocytes: plasma membrane and mitochondrial components

The origin of the cyanine dye fluorescence signal in murine and human peripheral blood leukocytes was investigated using the oxa- and indo-carbocyanines di-O-C5(3) and di-I-C5(3). Fluorescence signals from individual cells suspended with nanomolar concentrations of the dyes were measured in a flow cytometer modified to permit simultaneous four-parameter analysis (including two-color fluorescence or fluorescence polarization measurements). The contributions of mitochondrial membrane potential (psi m) and plasma membrane potential (psi pm) to the total voltage-sensitive fluorescence signal were found to depend on the equilibrium extracellular dye concentration, manipulated in these experiments by varying the ratio of dye to cell density. Hence, conditions could be chosen that amplified either the psi m or the psi pm component. Selective depolarization of lymphocytes or polymorphonuclear leukocytes (PMN) in mixed cell suspensions demonstrated that defining the partition of dye between cells and medium is requisite to assessing the heterogeneity of cell responses by cyanine dye fluorescence. At extracellular dye concentrations exceeding 5 nM in equilibrated cell suspensions, both mitochondrial and plasma membrane dye toxicity were observed. In murine splenic lymphocytes, plasma membrane toxicity (dye-induced depolarization) was selective for the B lymphocytes. Certain problems in calibration of psi pm with valinomycin at low dye concentrations and perturbations of psi pm by mitochondrial inhibitors are presented. These findings address the current controversy concerning psi m and psi pm measurement in intact cells by cyanine dye fluorescence. The finding of selective toxicity at low cyanine dye concentrations suggest that purported differences in resting psi m among cells or changes in psi pm with cell activation may reflect variable susceptibility to dye toxicity rather than intrinsic cell properties.