Probes of membrane electrostatics: synthesis and voltage-dependent partitioning of negative hydrophobic ion spin labels in lipid vesicles.

Two spin-labeled derivatives of the hydrophobic anion trinitrophenol have been synthesized and characterized in lipid vesicles. In the presence of lipid vesicles, the electron paramagnetic resonance (EPR) spectra of these probes are a composite of both membrane-bound and aqueous populations; as a result, the membrane-aqueous partitioning can be determined from their electron paramagnetic resonance spectra. The effect of transmembrane potentials on the membrane-aqueous partitioning of these spin-labeled hydrophobic ions was examined in phosphatidylcholine vesicles formed by extrusion. Inside positive membrane potentials promote an increase in the binding of these probes that is quantitatively accounted for by a simple thermodynamic model used previously to describe the partitioning of paramagnetic phosphonium ions. The transmembrane migration rates of these ions are dependent on the dipole potential, indicating that these ions transit the membrane in a charged form. The partitioning of the probe is also sensitive to the membrane surface potential, and this dependence is accurately accounted for using the Gouy-Chapman Stern formalism. As a result of the membrane dipole potential, these probes exhibit a stronger binding and a more rapid transmembrane migration rate compared with positive hydrophobic ion spin labels and provide a new set of negatively charged hydrophobic ion probes to investigate membrane electrostatics.     

Effect of headgroup on the dipole potential of phospholipid vesicles

The dipole potentials, ψ _d, of phospholipid vesicles composed of pure dimyristoylphosphatidylcholine (DMPC) or vesicles in which 50 mol% of the DMPC was substituted by dimyristoylphosphatidylserine (DMPS), dimyristoylphosphatidylglycerol (DMPG), dimyristoylethanolamine (DMPE), dimyristoylphosphatidic acid (DMPA) or monomyristoylphosphatidylcholine (MMPC) were measured via a fluorescent ratiometric method utilizing the probe di-8-ANEPPS. The PS and PG headgroups were found to cause only minor changes in ψ _d. PE caused an increase in ψ _d of 51 mV. This could be explained by a decrease in the dielectric constant of the glycerol backbone region as well as a movement of the P⁻–N⁺ dipole of the less bulky PE headgroup to a position more parallel to the membrane surface than in PC. The negatively charged PA headgroup increases ψ _d by 215 mV relative to PC alone. This indicates that the positive pole of the dipole predominantly responsible for the dipole potential is located at a position closer to the interior of the membrane than the phosphate group. The increase in the charge of the negative pole of the dipole by the phosphate group of PA increases the electrical potential drop across the lipid headgroup region. The incorporation of the single chain lipid MMPC into the membrane causes a decrease in ψ _d of 142 mV. This can be explained by a decrease in packing density within the membrane of carbonyl dipoles from the sn-2 chain of DMPC. The results presented should contribute to a better understanding of the electrical effect of lipid headgroups on the functioning of membrane proteins.     

The application of a potential-sensitive cyanine dye to rat small intestinal brush border membrane vesicles

The sensitivity of the fluorescent dye, 3,3′-diethylthiadicarbocyanine (DiS-C₂(5)), was too low for the detection of membrane potential changes in rat small intestinal membrane vesicles. Only after adding LaCl₃ or after fractionation of the intestinal membranes by free-flow electrophoresis could the dye be used to monitor electrogenic Na⁺-dependent transport systems. It is concluded that the response of this potential-sensitive dye is influenced by the negative surface charge density of the vesicles.     

Light-induced proton permeability changes in retinal rod photoreceptor disk membranes.

We have used the membrane-permeant charged fluorescent dye, 3,3'-dipropylthiadicarbocyanine iodide (diS-C3[5]), to monitor electrical potentials across the membranes of isolated bovine disks. Calibration curves obtained from experiments where a potential was created across the disk membrane by a potassium concentration gradient and valinomycin showed an approximately linear relation between dye fluorescence and calculated membrane potential from 0 to -120 mV. Light exposure in the presence of the permeant buffer, imidazole, caused a rapid decay of the membrane potential to a new stable level. Addition of CCCP, a proton ionophore, in the dark produced the same effect as illumination. When the permeant buffer, imidazole, was replaced by the impermeant buffer, Hepes, neither light nor CCCP discharged the gradient. We interpret the changes in membrane potential measured upon illumination to be the result of a light-induced increase in the permeability of the disk membrane to protons. A permeant buffer is required to prevent the build-up of a pH gradient which would inhibit the sustained proton flow needed for an observable change in membrane potential.     

A selective cholesterol-dependent induction of H+/OH- currents in phospholipid vesicles by amphotericin B

The effect of amphotericin B on the proton/hydroxide permeability of small unilamellar vesicles has been investigated by using potential-dependent paramagnetic probes. Amphotericin B at 1-10 molecules/vesicle causes a modest 4-8-fold increase in the background H+/OH- permeability of egg phosphatidylcholine (egg PC) vesicles. However, in the presence of cholesterol, amphotericin B promotes a dramatic increase in the H+/OH- permeability of more than 2 orders of magnitude. Surprisingly, this is not observed in vesicle membranes containing ergosterol. In membranes composed of 5-15 mol% ergosterol, amphotericin B is even less effective at promoting H+/OH- currents than in pure egg PC vesicles. The K+ current promoted by amphotericin B in vesicles formed from egg PC and from egg PC plus cholesterol or ergosterol was measured. No significant sterol dependence was found for the K+ current. These results strongly suggest that different mechanisms, or amphotericin B/sterol complexes, are responsible for the induction of H+/OH- and K+ currents. These results have important implications for understanding the therapeutic and toxic effects of amphotericin B.     

Evidence supporting a model of voltage-dependent uptake of auxin into Cucurbita vesicles

The accumulation of [¹⁴C]indole-3-acetic acid (IAA), of [³H]tetra-phenyl phosphonium ion as a membrane potential probe, and of [¹⁴C]butyric acid as probe for pH gradients was studied with membrane vesicles from etiolated hypocotyls of Cucurbita pepo. Ion gradients (K⁺, H⁺) were applied in the presence and absence of specific ionophores e.g. valinomycin or carbonylcyanide m-chlorophenylhydrazone. In all cases tested, the accumulation of [¹⁴C]IAA equals neither potential probe nor pH-probe accumulation, but represents. an intermediate between the two. Auxin molecules seem to be taken up as positively charged ions and a pH gradient is required for accumulation. The uptake mechanism thus appears to be a specific, carrier-mediated cotransport of the anion of IAA and no less than two protons. The initial rates of auxin uptake by the saturable influx carrier, of permeation through the membrane, and of efflux by the phytotropin-affected efflux carrier were analysed.Abbreviations BA butyric acid - CCCP carbonylcyanid-3-chlorophenylhydrazone - CPD 2-carboxylphenyl-3-phenylpropan-1,3-dion - IAA indole-3-acetic acid - IAA anion of IAA - IAAH undissociated form of IAA - 2-NAA 2-naphthaleneacetic acid - NPA 1-N-naphthylphthalamic acid - TPP⁺ tetra-phenyl phosphonium ion     

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.     

Potential dependent riigidity changes in lipid membrane vesicles

Steady-state fluorescence depolarization measurements of the hydrophobic probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), embedded into lipid membrane vesicles, reveal an increase in the membrane rigidity in the presence of transmembrane potential gradients. The effect depends on the lipid head-group structure and its charge. Additionally, the curvature of the vesicle membrane leads to an asymmetry in the observed phenomena. In consistence with several theories and experiments, it is concluded that besides a possible electrostriction of the membrane the electric field causes alterations in the orientation of the surface dipoles of the lipid molecules. The biological significance of these observations is briefly discussed.     

Transport properties of membrane vesicles fromAcholeplasma laidlawii

Membrane vesicles obtained from Acholeplasma laidlawii accumulate glucose as well as maltose and fructose against their concentration gradient in the absence of exogenous energy sources. Glucose uptake by membrane vesicles is inhibited by anaerobiosis and by electron transfer inhibitors, such as rotenone and amytal, but not by 2-heptyl-4-hydroxyquinoline N-oxide, antimycin A, cyanide and azide. Rotenone, cyanide and amytal also produce a rapid efflux of glucose from the membrane vesicles. Arsenate, oligomycin and N,N'-dicyclohexylcarbodimide do not inhibit glucose transport. Transport of glucose is markedly inhibited by proton conductors such as CCCP and pentachlorophenol. It is concluded that glucose transport can be driven by a high-energy state of the membrane or by the membrane potential.     

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 Transport in Isolated Vesicles from Sugarbeet Taproot : II. Evidence for a Sucrose/H-Antiport

The process of sucrose transport was investigated in sealed putative tonoplast vesicles isolated from sugarbeet (Beta vulgaris L.) taproot. If the vesicles were allowed to develop a steady state pH gradient by the associated transport ATPase and 10 millimolar sucrose was added, a transient flux of protons out of the vesicles was observed. The presence of an ATPase produced pH gradient allowed [¹⁴C]sucrose transport into the vesicles to occur at a rate 10-fold higher than the rate observed in the absence of an imposed pH gradient. Labeled sucrose accumulated into the sealed vesicles could be released back to the external medium if the pH gradient was dissipated with carbonylcyanide-m-chlorophenyl hydrazone (CCCP). When the kinetics of ATP dependent [¹⁴C]sucrose uptake were examined, the kinetic profile followed the simple Michaelis-Menten relationship and a Michaelis constant of 12.1 millimolar was found. When a transient, inwardly directed sucrose gradient was imposed on the vesicles in the absence of charge compensating ions, a transient interior negative membrane potential was observed. This membrane potential could be prevented by the addition of CCCP prior to sucrose or dissipated by the addition of CCCP after sucrose was added. These results suggest that an electrogenic H⁺/sucrose antiport may be operating on the vesicle membrane.     

Ratiometric fluorescence measurements of membrane potential generated by yeast plasma membrane H-ATPase reconstituted into vesicles

Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Δψ) generated by the Schizosaccharomyces pombe plasma membrane H⁺-ATPase reconstituted into vesicles. Oxonol VI was used for quantitative measurements of the Δψ because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V. However, oxonol V has a superior sensitivity to Δψ at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution. Oxonol VI was found to be a good emission-ratiometric probe. We have shown that the reconstituted H⁺-ATPase generates Δψ of about 160 mV on the vesicle membrane. The generated Δψ was stable at least over tens of minutes. An influence of the H⁺ membrane permeability on the Δψ buildup was demonstrated by manipulating the H⁺ permeability with the protonophore CCCP. Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H⁺-ATPase-mediated Δψ buildup.     

Energy coupling of L-glutamate transport and vacuolar H(+)-ATPase in brain synaptic vesicles

Energy coupling of L-glutamate transport in brain synaptic vesicles has been studied. ATP-dependent acidification of the bovine brain synaptic vesicles was shown to require CI-, to be accelerated by valinomycin and to be abolished by ammonium sulfate, nigericin or CCCP plus valinomycin, and K+. On the other hand, ATP-driven formation of a membrane potential (positive inside) was found to be stimulated by ammonium sulfate, not to be affected by nigericin and to be abolished by CCCP plus valinomycin and K+. Like formation of a membrane potential, ATP-dependent L-[3H]glutamate uptake into vesicles was stimulated by ammonium sulfate, not affected by nigericin and abolished by CCCP plus valinomycin and K+. The L-[3H]glutamate uptake differed in specificity from the transport system in synaptic plasma membranes. Both ATP-dependent H+ pump activity and L-glutamate uptake were inhibited by bafilomycin and cold treatment (common properties of vacuolar H(+)-ATPase). ATP-dependent acidification in the presence of L-glutamate was also observed, suggesting that L-glutamate uptake lowered the membrane potential to drive further entry of H+. These results were consistent with the notion that the vacuolar H(+)-ATPase of synpatic vesicles formed a membrane potential to drive L-glutamate uptake. ATPase activity of the vesicles was not affected by the addition of Cl-, glutamate or nigericin, indicating that an electrochemical H+ gradient had no effect on the ATPase activity.     

Role of pH gradient and membrane potential in dipeptide transport in intestinal and renal brush-border membrane vesicles from the rabbit. Studies with L-carnosine and glycyl-L-proline

We examined the role of pH gradient and membrane potential in dipeptide transport in purified intestinal and renal brush-border membrane vesicles which were predominantly oriented right-side out. With an intravesicular pH of 7.5, changes in extravesicular pH significantly affected the transport of glycyl-L-proline and L-carnosine, and optimal dipeptide transport occurred at an extravesicular pH of 5.5-6.0 in both intestine and kidney. When the extravesicular pH was 5.5, glycyl-L-proline transport was accelerated 2-fold by the presence of an inward proton gradient. A valinomycin-induced K+ diffusion potential (interior-negative) stimulated glycyl-L-proline transport, and the stimulation was observed in the presence and absence of Na+. A carbonyl cyanide p-trifluoromethoxyphenylhydrazone-induced H+ diffusion potential (interior-positive) reduced dipeptide transport. It is suggested that glycyl-L-proline and proton(s) are cotransported in intestinal and renal brush-border membrane vesicles, and that the process results in a net transfer of positive charge.     

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.     

Generation of a transmembrane electric potential during respiration by Azotobacter vinelandii membrand vesicles.

Membrane vesicles isolated from Azotobacter vinelandii strain O by lysis of spheroplasts in potassium of sodium phosphate buffer develop a transmembrane electric potential during respiration. The magnitude of this potential was determined by three independent methods: (i) fluorescence of 3,3'-dipropylthiodicarbocyanine and 3,3'-dihexyloxacarbocyanine; (ii) uptake of 86Rb+ in the presence of valinomycin; and (iii) uptake of [3H]triphenylmethyl phosphonium. In method (i), the relative fluorescence of these cyanine dyes in the presence of intact cells or derived vesicles is quenched during oxication of electron donors. A linear relationship between this quenching and a potassium diffusion potential was employed to calibrate the probe response. In method (ii), the steady-state concentration ratio of rubidium across the vesicle membrane during oxidation of L-malate was converted to potential by the Nernst equation. In method (iii), the steady-state concentration ratio of this lipophilic cation was likewise converted to a potential. With the exception of 3,3'-dihexyloxacarbocyanine fluorescence, these methods gave good agreement for the potential developed during L-malate oxidation by membrane vesicles. A value of 75 to 80 mV (inside negative) was obtained for vesicles prepared in potassium phosphate, and 104 mV (inside negative) was obtained for vesicles prepared in sodium phosphate. Electrogenic expulsion of hydrogen ion was observed during L-malate oxidation, and the amount of proton exodus was greater in potassium rather than the sodium-containing vesicles. This indicates the presence of a sodium-proton antiport mechanism. In addition, D-glucose uptake was observed during development of a potassium diffusion potential that was artificially imposed across the vesicle membrane. These observations suggest the presence of a glucose-proton symport mechanism in accordance with the principles of Mitchell.     

A test of discreteness-of-charge effects in phospholipid vesicles: measurements using paramagnetic amphiphiles

A new series of negatively charged, paramagnetic alkylsulfonate probes was synthesized and can be used to measure both the internal and the external surface potentials of model membrane systems. We tested for discreteness-of-charge effects in lipid membranes by comparing the surface potentials, estimated by use of these negatively charged amphiphiles, with that of a series of positively charged alkylammonium nitroxides in charged membranes. From the partitioning of these probes, the membrane surface potential was estimated in phosphatidylcholine membranes containing either phosphatidylserine or didodecyldimethylammonium bromide. The surface potentials, estimated with either positive or negative probes, were identical, within experimental error, in either positive or negative membranes, and they were well accounted for by a simple Gouy-Chapman-Stern theory. This symmetry, with respect to the sign of the charge, indicates that discreteness-of-charge effects are not significant in determining the potential-sensitive phase partitioning of these probes in model membranes. Thus, despite the fact that charge on membranes is discrete, models that assume a uniform density of charge in the plane of the membrane adequately account for the potentials measured by these amphiphilic probes.     

Use of fluorescent probes that form intramolecular excimers to monitor structural changes in model and biological membranes.

1,3-dipyrenylpropane (PC3P) and bis(4-biphenylmethyl)ether, two molecules that form intramolecular excimers, were embedded in phospholipid vesicles and biological membranes to monitor dynamic properties of membrane lipids. Excimer formation was evaluated from determinations of excimer to monomer emission intensity ratios (ID/IM). ID/IM values of PC3P and bis(4-biphenylmethyl)ether were reduced when cholesterol was added to egg lecithin vesicles. PC3P was sensitive to the temperature-induced crystalline to liquid-crystalline phase transition in dimyristoyl phosphatidylcholine vesicles. For studies of cellular membranes, membranes, PC3P was used exclusively, because of the fluorescence of tryptophan residues of membrane proteins interferes with the responses bis(4-biphenylmethyl)ether. Microviscosities of membrane interiors were calculated from standard curves of IM/ID plotted against solvent viscosity. Microviscosity values of egg lecithin vesicles and biological membranes, especially those obtained with PC3P, were more than an order of magnitude lower than values obtained by other techniques. We concluded that the intramolecular process leading to the formation of the excimer is influenced differently in isotropic solvents than in anisotropic environments, such as lipid bilayers. Although distinguishable ID/IM ratios can be obtained for different biological membranes (mitochondrial, microsomal, and plasma membranes were studied), this parameter may be phenomenological and not simply related to membrane microviscosity. As such, fluorescent probes that form intramolecular excimers are of value in making qualitative comparisons of different membranes and in studying the relative effects of physical changes and chemical agents on membrane structure. These probes may also be valuable for studying structural anisotropy of biological membranes.