Intramembrane molecular dipoles affect the membrane insertion and folding of a model amphiphilic peptide.

The relationship between the dipole potential and the interaction of the mitochondrial amphipathic signal sequence known as p25 with model membranes has been studied using 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octyl-amino)-6-naphthyl]viny l] pyridinium betaine (di-8-ANEPPS) as a fluorescent probe. The dipole potential of phosphatidylcholine membranes was modified by incorporating into the bilayer the sterols phloretin and 6-ketocholestanol (KC), which decrease and increase the dipole potential, respectively. The results derived from the application of a dual-wavelength ratiometric fluorescence method for following the variation of the membrane dipole potential have shown that when p25 inserts into the lipidic bilayer, a decrease in the dipole potential takes place. The magnitude of this decrease depends on the initial value of the dipole potential, i.e., before interaction with the peptide. Thus, when KC was incorporated into the bilayer, the decrease caused by the membrane insertion of p25 was larger than that caused in PC membranes. Alternatively, in the presence of phloretin, the decrease in the potential caused by the peptide insertion was smaller. Complementary studies involving attenuated total reflectance-Fourier transform infrared spectroscopy of the peptide membrane interactions have shown that modification of the dipole potential affects the conformation of the peptide during the course of its interaction with the membrane. The presence of KC induces a higher amount of helicoidal structure. The presence of phloretin, however, does not appear to affect the secondary structure of the peptide. The differences observed in the dipole potential decreases caused by the presence of the peptide with the PC membranes and phloretin-PC membranes, therefore, must involve differences in the tertiary and, perhaps, quaternary conformations of p25.     

Internal electrostatic potentials in bilayers: measuring and controlling dipole potentials in lipid vesicles.

The binding and translocation rates of hydrophobic cation and anion spin labels were measured in unilamellar vesicle systems formed from phosphatidylcholine. As a result of the membrane dipole potential, the binding and translocation rates for oppositely charged hydrophobic ions are dramatically different. These differences were analyzed using a simple electrostatic model and are consistent with the presence of a dipole potential of approximately 280 mV in phosphatidylcholine. Phloretin, a molecule that reduces the magnitude of the dipole potential, increases the translocation rate of hydrophobic cations, while decreasing the rate for anions. In addition, phloretin decreases the free energy of binding of the cation, while increasing the free energy of binding for the anion. The incorporation of 6-ketocholestanol also produces differential changes in the binding and translocation rates of hydrophobic ions, but in an opposite direction to those produced by phloretin. This is consistent with the view that 6-ketocholestanol increases the magnitude of the membrane dipole potential. A quantitative analysis of the binding and translocation rate changes produced by ketocholestanol and phloretin is well accounted for by a point dipole model that includes a dipole layer due to phloretin or 6-ketocholestanol in the membrane-solution interface. This approach allows dipole potentials to be estimated in membrane vesicle systems and permits predictable, quantitative changes in the magnitude of the internal electrostatic field in membranes. Using phloretin and 6-ketocholestanol, the dipole potential can be altered by over 200 mV in phosphatidylcholine vesicles.     

Effect of gramicidin A on the dipole potential of phospholipid membranes.

The effect of channel-forming peptide gramicidin A on the dipole potential of phospholipid monolayers and bilayers has been studied. Surface pressure and surface potential isotherms of monolayers have been measured with a Langmuir trough equipped with a Wilhelmy balance and a surface potential meter (Kelvin probe). Gramicidin has been shown to shift pressure-area isotherms of phospholipids and to reduce their monolayer surface potentials. Both effects increase with the increase in gramicidin concentration and depend on the kind of phosphatidylcholine used. Application of the dual-wavelength ratiometric fluorescence method using the potential-sensitive dye RH421 has revealed that the addition of gramicidin A to dipalmitoylphosphatidylcholine liposomes leads to a decrease in the fluorescence ratio of RH421. This is similar to the effect of phloretin, which is known to decrease the dipole potential. The comparison of the concentration dependences of the fluorescence ratio for gramicidin and phloretin shows that gramicidin is as potent as phloretin in modifying the membrane dipole potential.     

Delta pH, H+ diffusion potentials, and Mg2+ ATPase in neurosecretory vesicles isolated from bovine neurohypophyses

The proton gradient (delta pH) and electrical potential (delta psi) across the neurosecretory vesicles were measured using the optical probes 9-aminoacridine and Oxanol VI, respectively. The addition of neurosecretory vesicles to 9-aminoacridine resulted in a rapid quenching of the dye fluorescence which was reversed when the delta pH was collapsed with ammonium chloride or K+ in the presence of nigericin. From fluorescence quenching data and the intravesicular volume, delta pH across the membrane was calculated. Mg2+ ATP caused a marked carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive change in the membrane potential measured using Oxanol VI (plus 100 mV inside positive), presumably due to H+ translocation across the neurosecretory vesicle membrane. Imposition of this membrane potential was responsible for the lysis of vesicles in the presence of permeant anions. The effectiveness of these anions to support lysis reflected the relative permeability of the anion which followed the order acetate greater than I- greater than Cl greater than F- greater than SO4- = isethionate = methyl sulfate. These data showed that the neurosecretory vesicles possess a membrane H+-translocating system and prompted the study of Mg2+-dependent ATPase activities in the vesicle fractions. In intact vesicles a Mg2+ ATPase appeared to be coupled to electrogenic proton translocation, since the enzyme activity was enhanced by uncoupling the electrical potential, using proton ionophores. Inhibition of this enzyme with dicyclohexylcarbodiimide also inhibited the carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive delta psi across the vesicle membrane caused by H+ translocation. A second Mg2+ ATPase was also found on the vesicle membranes which is sensitive to vanadate. Complete inhibition of this enzyme with vanadate had little effect on the proton ionophore-uncoupled ATPase activity or on the Mg2+ ATP-induced membrane potential change.     

Spectrophotometric measurements of transmembrane potential and pH gradients in chromaffin granules

The electrical potential (delta psi) and proton gradient (alpha pH) across the membranes of isolated bovine chromaffin granules and ghosts were simultaneously and quantitatively measured by using the membrane- permeable dyes 3,3'dipropyl-2,2'thiadicarbocyanine (diS-C3-(5)) to measure delta psi and 9-aminoacridine or atebrin to measure delta pH. Increases or decreases in the delta psi across the granular membrane could be monitored by fluorescence or transmittance changes of diS-C3- (5). Calibration of the delta psi was achieved by utilization of the endogenous K+ and H+ gradients, and valinomycin or carbonyl cyanide-p- trifluoromethoxyphenylhydrazone (FCCP), respectively, with the optical response of diS-C3-(5) varying linearly with the Nernst potential for H+ and K+ over the range -60 to +90 mV. The addition of chromaffin granules to a medium including 9-aminoacridine or atebrin resulted in a rapid quenching of the dye fluorescence, which could be reversed by agents known to cause collapse of pH gradients. From the magnitude of the quenching and the intragranular water space, it was possible to calculate the magnitude of the alpha pH across the chromaffin granule membrane. The time-course of the potential-dependent transmittance response of diS-C3-(5) and the delta pH-dependent fluorescence of the acridine dyes were studied simultaneously and quantitatively by using intact and ghost granules under a wide variety of experimental conditions. These results suggest that membrane-permeable dyes provide an accurate method for the kinetic measurement of delta pH and delta psi in an amine containing subcellular organelle.     

Cyanine and safranine dyes as membrane potential probes in cytochrome c oxidase reconstituted proteoliposomes

Safranine and the cyanine dye, 3',3'-dipropylthiadicarbocyanine (diSC3-5), were examined as membrane potential probes in cytochrome c oxidase vesicles. The spectra of the vesicle-associated dyes resemble those of the same dyes in organic solvents, indicating that safranine and diSC3-5 probably dissolve in a hydrophobic region of the proteoliposomal membrane. This binding of safranine to proteoliposomes occurs with a dye-membrane dissociation constant in the micromolar range. The binding of safranine and of diSC3-5 to liposomes or proteoliposomes is accompanied by fluorescence enhancement. This enhanced fluorescence is quenched by respiration or by the establishment of a K+ diffusion potential by valinomycin (negative interior). An optimal dye/lipid ratio was required to secure maximum fluorescence quenching of the dyes, whether that quenching was active or passive. Calibrations of both the safranine and the diSC3-5 responses with K+ diffusion potentials were also affected by the dye/lipid ratio. At lower dye/lipid ratios, the calibration curve was linear at higher potentials but deviated from linearity at lower potentials. The converse was true at higher dye/lipid ratios. The non-linearity of the calibration curve at higher potential was attributed to a 'saturation' effect; it may also involve increased permeability of proteoliposomal membrane to protons. Destacking of dye at the lower dye/lipid ratio was probably responsible for the non-linearity of the calibration curves at lower potentials. When all these effects are taken into account, the steady-state value of delta psi generated during maximal proteoliposomal respiration was calculated to be between 140 and 160 mV (interior negative) when measured with either safranine or diSC3-5. We conclude that quantitative estimates of delta psi values can be made using these probes in cytochrome c oxidase reconstituted proteoliposomes provided that appropriate precautions are taken.     

Simultaneous determination of membrane potential and pH gradient by photodiode array spectroscopy

Membrane potential (delta psi) and pH difference (delta pH) were simultaneously determined in liposomes using a photodiode array spectrophotometer. By the use of a cyanine dye (DiS-C3(5)) and 9-aminoacridine for delta psi and delta pH probes, respectively, both changes of delta psi and delta pH could be successfully determined by photodiode array spectrometry. Each dye did not disturb the fluorescence spectrum of the other probe when its concentration was lower than 5 microM. The K+-diffusion potential-driven, FCCP(protonophore)-mediated H+-influx process in the K+-loaded liposomes was analyzed by this method. Results indicate that the kinetic behavior of H+ influx changes at a FCCP concentration of approx. 30 nM. The rate of delta pH formation increased quantitatively with increasing concentrations of FCCP up to 30 nM, but was markedly enhanced at higher concentrations, although the maximal delta pH attained was about 3 pH units in any case when a K+-diffusion potential of -180 mV was applied.     

Comparison of excitation and emission ratiometric fluorescence methods for quantifying the membrane dipole potential

We are interested in developing fluorescence methods for quantifying lateral variations in the dipole potential across cell surfaces. Previous work in this laboratory showed that the ratio of fluorescence intensities of the voltage-sensitive dye di-8-ANEPPS using excitation wavelengths at 420 and 520 nm correlates well with measurements of the dipole potential. In the present work we evaluate the use of di-8-ANEPPS and an emission ratiometric method for measuring dipole potentials, as Bullen and Saggau (Biophys. J. 65 (1999) 2272-2287) have done to follow changes in the membrane potential in the presence of an externally applied field. Emission ratiometric methods have distinct advantages over excitation methods when applied to fluorescence microscopy because only a single wavelength is needed for excitation. We found that unlike the excitation ratio, the emission ratio does not correlate with the dipole potential of vesicles made from different lipids. A difference in the behaviour of the emission ratio in saturated compared to unsaturated lipid vesicles was noted. Furthermore, the emission ratio did not respond in the same way as the excitation ratio when cholesterol, 6-ketocholestanol, 7-ketocholesterol, and phloretin were added to dimyristoylphosphatidylcholine (DMPC) vesicles. We attribute the lack of correlation between the emission ratio and the dipole potential to simultaneous changes in membrane fluidity caused by changes in membrane composition, which do not occur when the electric field is externally applied as in the work of Bullen and Saggau. Di-8-ANEPPS can, thus, only be used via an excitation ratiometric method to quantify the dipole potential.     

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.     

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.     

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.     

An electrical and structural characterization of H+/OH- currents in phospholipid vesicles

Paramagnetic amphiphiles have been utilized to measure and characterize electrogenic H+/OH- ion transport in a series of model membrane systems. Membrane conductivity to H+/OH- ions varies with the method of vesicle preparation and with the level of saturation of the membrane phospholipid. Small sonicated vesicles have the lowest conductivities by approximately an order of magnitude compared to reverse-phase or ether-injection vesicle systems. This conductivity is particularly sensitive to the presence of polyunsaturated lipids in the vesicle membrane. The current-delta pH dependence of the H+/OH- conductivity shows a nonideal behavior and renders the phenomenological membrane permeability dependent upon the experimental value of delta pH that is chosen. These factors can account for much, if not all, of the variability in the published values for the H+/OH- permeability of model membranes. A procedure has been developed to establish and estimate changes in the dipole potential of vesicle bilayers. Using this method, we demonstrate that H+/OH- currents are insensitive to alterations in the membrane dipole field, a result that suggests that these currents are not rate limited by diffusion over simple electrostatic barriers in the membrane interior. In addition, conduction in D2O has been examined, and we find that there is little difference in the magnitudes of D+/OD- currents compared to H+/OH- currents in vesicle systems.     

Comparison of excitation and emission ratiometric fluorescence methods for quantifying the membrane dipole potential

We are interested in developing fluorescence methods for quantifying lateral variations in the dipole potential across cell surfaces. Previous work in this laboratory showed that the ratio of fluorescence intensities of the voltage-sensitive dye di-8-ANEPPS using excitation wavelengths at 420 and 520 nm correlates well with measurements of the dipole potential. In the present work we evaluate the use of di-8-ANEPPS and an emission ratiometric method for measuring dipole potentials, as Bullen and Saggau (Biophys. J. 65 (1999) 2272-2287) have done to follow changes in the membrane potential in the presence of an externally applied field. Emission ratiometric methods have distinct advantages over excitation methods when applied to fluorescence microscopy because only a single wavelength is needed for excitation. We found that unlike the excitation ratio, the emission ratio does not correlate with the dipole potential of vesicles made from different lipids. A difference in the behaviour of the emission ratio in saturated compared to unsaturated lipid vesicles was noted. Furthermore, the emission ratio did not respond in the same way as the excitation ratio when cholesterol, 6-ketocholestanol, 7-ketocholesterol, and phloretin were added to dimyristoylphosphatidylcholine (DMPC) vesicles. We attribute the lack of correlation between the emission ratio and the dipole potential to simultaneous changes in membrane fluidity caused by changes in membrane composition, which do not occur when the electric field is externally applied as in the work of Bullen and Saggau. Di-8-ANEPPS can, thus, only be used via an excitation ratiometric method to quantify the dipole potential.     

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.     

Characterization of a H+-ATPase in rat brain synaptic vesicles. Coupling to L-glutamate transport

Synaptic vesicles contain a H+-ATPase that generates a proton electrochemical gradient (delta mu H+) required for the uptake of neurotransmitters into the organelles. In this study, the synaptic vesicle H+-ATPase was examined for structural and functional similarities with other identified ATPases that generate a delta mu H+ across membranes. The synaptic vesicle H+-ATPase displayed immunological similarity with the 115-, 72-, and 39-kDa subunits of a vacuolar-type H+-ATPase purified from chromaffin granules. Functionally, the ATP-dependent H+ pumping across synaptic vesicles and ATP hydrolysis were sensitive to the sulfhydryl-modifying reagents, N-ethylmaleimide and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, at concentrations known to affect vacuolar-type H+-ATPases. In addition, as with vacuolar-type H+-ATPases, the presence of NO3-, SO4(2-), or F- inhibited the generation of a delta mu H+, but addition of vanadate or oligomycin had no effect. The delta mu H+ is a function of the pH gradient (delta pH) and membrane potential (delta psi sv) across the synaptic vesicle. Acidification (delta pH) of the synaptic vesicle interior was enhanced in the presence of permeant anions, such as Cl-, or the K+ ionophore, valinomycin. In the absence of permeant anions, the H+-ATPase generated a delta psi sv that effected the transport of L-glutamate into the synaptic vesicles. Dissipation of delta psi sv by incubation with increased external Cl- or nigericin resulted in the abolition of glutamate uptake, despite the continued maintenance of a delta mu H+ across the synaptic vesicle as a substantial delta pH. The results suggest that the synaptic vesicle H+-ATPase is of a vacuolar type and energizes the uptake of anionic glutamate by virtue of the delta psi sv component of the delta mu H+ it generates.     

Use of oxonol V as a probe of membrane potential in proteoliposomes containing cytochrome oxidase in the submitochondrial orientation

Absorbance changes in the anionic dye bis[3-phenyl-5-oxoisoxazol-4-yl]pentamethineoxonol (oxonol V) can be used to monitor the membrane potential of liposomes and cytochrome c containing cytochrome oxidase proteoliposomes (c-loaded COV). Diffusion potentials (positive inside the vesicles) cause an increase in the dye extinction, with a maximum at 640 nm. A similar increase is seen upon energization of internally facing cytochrome oxidase molecules in c-loaded COV. Both "passive" and "active" responses are only seen when the dye is fully bound to the vesicle membrane. Calibration curves using potassium or n-butyltriphenylphosphonium ion (BTPP+) diffusion potentials are linear up to 100 mV and pass through the origin. Diffusion potentials (positive inside) also cause an increase and red shift in the oxonol V fluorescence emission spectrum. However, potentials of the same sign induced by cytochrome oxidase turnover induce a large fluorescence quenching in c-loaded COV. A similar anomaly has been observed with submitochondrial particles [Smith, J. C., Russ, P., Cooperman, B. S., & Chance, B. (1976) Biochemistry 15, 5094-5105]. A model is proposed consistent with these responses. It is suggested that the dye molecules move further into the membrane phase upon energization, causing the absorbance increase. In the presence of active enzyme, anionic dye molecules are attracted to a positive dipole on each enzyme molecule, causing self-quenching of the fluorescence.     

The lysosomal proton pump is electrogenic

Lysosomes were purified approximately 40-fold from rat kidney cortex by differential and Percoll density gradient centrifugation. In a sucrose medium, the lysosomes quenched the fluorescence of the potential sensitive dye diS-C3-(5) (3,3'-dipropylthiocarbo-cyanine iodide) in a time-dependent manner, indicating that the dye accumulates within the lysosomal interior. After treatment of the lysosomes with valinomycin, the dye fluorescence displayed a logarithmic dependence upon the external K+ concentration; thus, the fluorescence signal provides a semiquantitative measure of the lysosomal membrane potential (delta psi). In the absence of valinomycin, lysosomal quenching of diS-C3-(5) fluorescence was partially reversed by agents which collapse the lysosomal pH gradient (ammonium sulfate, chloroquine, and K nigericin), suggesting that the proton gradient across the lysosomal membrane contributes to delta psi. A rapid increase in diS-C3-(5) fluorescence, indicative of an increase in delta psi, was observed upon the addition of Mg-ATP to the lysosomes. The ATP-dependent fluorescence change was inhibited by protonophores, K valinomycin, permeable anions, and N-ethylmaleimide, but was unaffected by ammonium sulfate, K nigericin, or sodium vanadate. Oligomycin had no effect at concentrations below 2 micrograms/ml; at higher concentrations, oligomycin partially inhibited the fluorescence response to Mg-ATP, but it also inhibited the fluorescence response to K valinomycin, suggesting that it had modified the permeability of the lysosomal membrane. Dicylohexylcarbodiimide behaved similarly to oligomycin. Mg-ATP also altered the lysosomal distribution of 86Rb+ (in the presence of valinomycin) and S[14C]CN-, consistent with an increase in the potential of the lysosomal interior of 40-50 mV. The results demonstrate that the lysosomal proton pump is electrogenic.     

Phloretin-induced changes of lipophilic ion transport across the plasma membrane of mammalian cells.

The adsorption of the hydrophobic anion [W(CO)(5)CN](-) to human lymphoid Jurkat cells gave rise to an additional anti-field peak in the rotational spectra of single cells, indicating that the cell membrane displayed a strong dielectric dispersion in the kilohertz to megahertz frequency range. The surface concentration of the adsorbed anion and its translocation rate constant between the two membrane boundaries could be evaluated from the rotation spectra of cells by applying the previously proposed mobile charge model. Similar single-cell electrorotation experiments were performed to examine the effect of phloretin, a dipolar molecule known to influence the dipole potential of membranes, on the transport of [W(CO)(5)CN](-) across the plasma membrane of mammalian cells. The adsorption of [W(CO)(5)CN](-) was significantly reduced by phloretin, which is in reasonable agreement with the known phloretin-induced effects on artificial and biological membranes. The IC(50) for the effect of phloretin on the transport parameters of the lipophilic ion was approximately 10 microM. The results of this study are consistent with the assumption that the binding of phloretin reduces the intrinsic dipole potential of the plasma membrane. The experimental approach developed here allows the quantification of intrinsic dipole potential changes within the plasma membrane of living cells.     

The adsorption of phloretin to lipid monolayers and bilayers cannot be explained by langmuir adsorption isotherms alone.

Phloretin and its analogs adsorb to the surfaces of lipid monolayers and bilayers and decrease the dipole potential. This reduces the conductance for anions and increases that for cations on artificial and biological membranes. The relationship between the change in the dipole potential and the aqueous concentration of phloretin has been explained previously by a Langmuir adsorption isotherm and a weak and therefore negligible contribution of the dipole-dipole interactions in the lipid surface. We demonstrate here that the Langmuir adsorption isotherm alone is not able to properly describe the effects of dipole molecule binding to lipid surfaces--we found significant deviations between experimental data and the fit with the Langmuir adsorption isotherm. We present here an alternative theoretical treatment that takes into account the strong interaction between membrane (monolayer) dipole field and the dipole moment of the adsorbed molecule. This treatment provides a much better fit of the experimental results derived from the measurements of surface potentials of lipid monolayers in the presence of phloretin. Similarly, the theory provides a much better fit of the phloretin-induced changes in the dipole potential of lipid bilayers, as assessed by the transport kinetics of the lipophilic ion dipicrylamine.     

The rate of ATP-synthesis as a function of delta pH and delta psi catalyzed by the active, reduced H(+)-ATPase from chloroplasts.

The H(+)-ATPase from chloroplasts was brought into the active, reduced state. Then, an electrochemical potential difference of protons across the thylakoid membranes was generated by an acid-base transition, delta pH, combined with a K+/valinomycin diffusion potential, delta psi. The initial rate of ATP synthesis was measured with a rapid-mixing quenched-flow apparatus in the time-range between 20-150 ms. The rate of ATP synthesis depends in a sigmoidal way on delta pH. Increasing diffusion potentials shifts the delta pH-dependencies to lower delta pH values. Analysis of the data indicate that the rate of ATP synthesis depends on the electrochemical potential difference of protons irrespective of the relative contribution of delta pH and delta psi.