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.     

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.     

Hydrophobic ion hydration and the magnitude of the dipole potential

The magnitude of the dipole potential of lipid membranes is often estimated from the difference in conductance between the hydrophobic ions, tetraphenylborate, and tetraphenylarsonium or tetraphenylphosphonium. The calculation is based on the tetraphenylarsonium-tetraphenylborate hypothesis that the magnitude of the hydration energies of the anions and cations are equal (i.e., charge independent), so that their different rates of transport across the membrane are solely due to differential interactions with the membrane phase. Here we investigate the validity of this assumption by quantum mechanical calculations of the hydration energies. Tetraphenylborate (Delta G(hydr) = -168 kJ mol(-1)) was found to have a significantly stronger interaction with water than either tetraphenylarsonium (Delta G(hydr) = -145 kJ mol(-1)) or tetraphenylphosphonium (Delta G(hydr) = -157 kJ mol(-1)). Taking these differences into account, literature conductance data were recalculated to yield values of the dipole potential 57 to 119 mV more positive in the membrane interior than previous estimates. This may partly account for the discrepancy of at least 100 mV generally observed between dipole potential values calculated from lipid monolayers and those determined on bilayers.     

The Effects of Aluminum on the Influx of Calcium,Potassium, Ammonium,Nitrate, and Phosphate in an Aluminum-Sensitive Cultivar of Barley (Hordeum vulgare L.)

The mechanism by which aluminum interferes with ion influx is not known. In this study, the effects of aluminum on the influx of the cations calcium, potassium, and ammonium and the anions nitrate and phosphate were measured in an aluminum-sensitive cultivar of barley (Hordeum vulgare L.). Aluminum (100 [mu]M) was found to inhibit the influx of the cations calcium (69%), ammonium (40%), and potassium (13%) and enhancing the influx of the anions nitrate (44%) and phosphate (17%). Aluminum interfered with the binding of the cations in the cell wall by the same order of magnitude as their respective influxes, whereas phosphate binding was strongly enhanced. The results are consistent with a mechanism whereby aluminum binds to the plasma membrane phospholipids, forming a positively charged layer that influences ion movement to the binding sites of the transport proteins. A positive charge layer would retard the movement of cations and increase the movement of anions to the plasma membrane in proportion to the charges carried by these ions.     

A critical assessment of the use of lipophilic cations as membrane potential probes

A critical review has been made of the literature on the use of lipophilic cations, such as triphenylmethyl phosphonium (TPMP⁺) as membrane potential probes in prokaryotes, uekaryote organelles in vitro, and eukaryote cells. An ideal lipophilic cation should be capable of penetrating through a biological membrane and obey the Nernst equation between a membrane bound phase and its environment. Many different forms of the Nernst equation are presented, useful in the calculation equilibrium potentials of lipophilic cations across membranes. Lipophilic cations appear to behave as valid membrane potential probes in prokaryotes and eukaryote organelles in vitro and even in vivo although some technical difficulties may be involved. On the other hand in valid forms of the Nernst equation have often been used to calculate the equilibrium potential of lipophilic cations across the plasma membranes of eukaryotic cells. In particular, the problem of intracellular compartmentation of lipophilic cations has often not been appreciated. Lipophilic cations do not appear to behave as reliable plasma membrane potential probes in eukaryotic cells. Some other avenues are discussed which might be useful in the determination of the plasma membrane potentials of small eukaryotic cells, e.g. the use of lipophilic anions as membrane potential probes.     

Effects of internal and external ionic environment on excitability of squid giant axon. A macromolecular approach

The effects of ten cations and fifteen anions on the excitability of the squid giant axon were studied. The method of intracellular perfusion used in these investigations is described in detail. Empirical criteria were established for evaluating the relative favorability of any salt solution for maintaining the normal excitability of the membrane of the axon. It was found that both cations and anions could be ordered in sequences of relative favorability, which are directly related to the classic lyotropic sequences found for protein macromolecules and in colloid chemistry in general. The effects of concentration, salt mixtures, non-electrolyte carriers, enzymes, metabolic inhibitors, pH, and external media were also studied. The results are interpreted in terms of current concepts of the interactions between water structure, charged macromolecules, and their ionic environments. A macromolecular approach is given to the physicochemical nature of the "two stable states" of the excitable membrane, to describe the time-dependent potential changes observed.     

Effect of phloretin on the permeability of thin lipid membranes

Phloretin dramatically increases cation conductances and decreases anion conductances of membranes treated with ion carriers (nonactin, valinomycin, carbonyl-cyanide-m-chlorophenylhydrazone [CCCP], and Hg(C6F5)2) or lipophilic ions (tetraphenylarsonium [tphAs+] and tetraphenylborate [TPhB-]). For example, on phosphatidylethanolamine membranes, 10(-4) M phloretin increases K+ -nonactin and TPhAs+ conductances and decreases CCCP- and TPhB- conductances 10(3)-fold; on lecithin: cholesterol membranes, it increases K+-nonactin conductance 10(5)-fold and decreases CCCP- conductance 10(3)-fold. Similar effects are obtained with p- and m-nitrophenol at 10(-2) M. These effects are produced by the un-ionized form of phloretin and the nitrophenols. We believe that phloretin, which possesses a large dipole moment, adsorbs and orients at the membrane surface to introduce a dipole potential of opposite polarity to the preexisting positive one, thus increasing the partition coefficient of cations into the membrane interior and decreasing the partition coefficient of anions. (Phloretin may also increase the fluidity of cholesterol-containing membranes; this is manifested by its two- to three-fold increase in nonelectrolyte permeability and its asymmetrical effect on cation and anion conductances in cholesterol-containing membranes.) It is possible that pholoretin's inhibition of chloride, urea, and glucose transport in biological membranes results from the effects of these intense intrafacial dipole fields on the translocator(s) of these molecules.     

Influence of cations on the blue to purple transition of bacteriorhodopsin. Comparison of Ca2+ and Hg2+ binding and their effect on the surface potential

We have investigated the effect of Ca2+ and Hg2+ binding on various properties of the blue membrane prepared by deionization of the Halobacterium halobium purple membrane. Binding of radioactive 45Ca2+ and 203Hg2+ was monitored by a filtration technique. Five high and medium affinity sites for Ca2+ and seven low affinity sites for Hg2+ were found per bacteriorhodopsin. Competitive binding was observed only for three Ca2+ and three Hg2+. Visible absorption studies indicated that Ca2+ binding could restore the purple color of bacteriorhodopsin while Hg2+ was inefficient. Hg2- could partially reverse to blue the Ca2+-regenerated purple membrane in parallel with the displacement of three Ca2+. Effects of cation binding on the surface potential of the membrane were measured by Electron Spin Resonance spectroscopy using a cationic spin-labeled amphiphile. Cations such as La3+, Ca2+, Mg2+, or Na+ strongly increased (i.e. rendered less negative) the surface potential. An univocal correlation was found between the cation-induced variation of surface potential and the extent of regeneration of the purple color. Hg2+ induced a smaller increase in surface potential than that corresponding to the effective divalent cations. This lower effect appears to be due to binding to sites not related to those of other cations.     

Surface density of calcium ions and calcium spikes in the barnacle muscle fiber membrane

The effects of various divalent cations in the external solution upon the Ca spike of the barnacle muscle fiber membrane were studied using intracellular recording and polarizing techniques. Analysis of the maximum rate of rise of the spike potential indicates that different species of divalent cations bind the same membrane sites competitively with different dissociation constants. The overshoot of the spike potential is determined by the density of Ca (Sr) ions in the membrane sites while the threshold membrane potential for spike initiation depends on the total density of divalent cations. The order of binding among different divalent and trivalent cations is the following: La+++, UO2++ > Zn++, Co++, Fe++ > Mn++ > Ni++ > Ca++ > Mg++, Sr++.     

Relative role of anions and cations in the stabilization of halophilic malate dehydrogenase.

Halophilic malate dehydrogenase unfolds at low salt, and increasing the salt concentration stabilizes, first, the folded form and then, in some cases, destabilizes it. From inactivation and fluorescence measurements performed on the protein after its incubation in the presence of various salts in a large range of concentrations, the apparent effects of anions and cations were found to superimpose. A large range of ions was examined, including conditions that are in general not of physiological relevance, to explore the physical chemistry driving adaptation to extreme environments. The order of efficiency of cations and anions to maintain the folded form is, for the low-salt transition, Ca(2+) approximately Mg(2+) > Li(+) approximately NH(4)(+) approximately Na(+) > K(+) > Rb(+) > Cs(+), and SO(4)(2)(-) approximately OAc(-) approximately F(-) > Cl(-), and for the high-salt transition, NH(4)(+) approximately Na(+) approximately K(+) approximately Cs(+) > Li(+) > Mg(2+) > Ca(2+), and SO(4)(2)(-) approximately OAc(-) approximately F(-) > Cl(-) > Br(-) > I(-). If a cation or anion is very stabilizing, the effect of the salt ion of opposite charge is limited. Anions of high charge density are always the most efficient to stabilize the folded form, in accordance with the order found in the Hofmeister series, while cations of high charge density are the most efficient only at the lower salt concentrations and tend to denature the protein at higher salt concentrations. The stabilizing efficiency of cations and anions can be related in a minor way to their effect on the surface tension of the solution, but the interaction of ions with sites only present in the folded protein has also to be taken into account. Unfolding at high salt concentrations corresponds to interactions of anions of low charge density and cations of high charge density with the peptide bond, as found for nonhalophilic proteins.     

Conversion of Escherichia coli cell-produced metabolic energy into electric form.

The formation of membrane potential in energized E. coli cells has been investigated by means of ionic penetrants. The fluxes of anions and cations in opposite directions have been observed: anions moved out and cations moved into the cells. The energy-linked uptake of cations was stoichiometrically coupled with the outflow of H+ ions from the cells. The value of a membrane potential in the energized cells calculated from a distribution of permanent cations was in the range of -140 mV (inside minus). The uptake of penetrating cations by deenergized cells has been observed following the non-enzymatic generation of a membrane potential. The influx of synthetic and natural (lactose) penetrants collapsed the non-enzymatic membrane potential. The effect of lactose was sensitive to N-ethyl maleimide. These results are in favour of the conception that in the energized E. coli cells an energy-linked H+-pump generates a membrane potential which is a driving force for the transport of synthetic and some natural penetrants.     

Transport of hydrophobic ions in erythrocyte membrane: I. Zero membrane potential properties

Summary The permeability and partition coefficients of tetraphenylarsonium (TPA) and several other organic cations were studied in the human erythrocyte using an ion-selective electrode. The permeability constant for the different cations could be explained quite well by differences in oil/water partition coefficients. No evidence for facilitated transport could be found. Binding of the organic ions occurred to both the cell membrane and to intracellular contents. Partitioning to the membrane remained relatively constant despite variation from ion intracellular binding with blood samples from different donors. TPA flux is stimulated by substoichiometric amounts of tetraphenylboron and other organic anions, suggesting an ion-pairing mechanism.     

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.     

Ion Channels in the Xylem Parenchyma of Barley Roots (A Procedure to Isolate Protoplasts from This Tissue and a Patch-Clamp Exploration of Salt Passageways into Xylem Vessels

To identify mechanisms for the simultaneous release of anions and cations into the xylem sap in roots, we investigated voltage-dependent ion conductances in the plasmalemma of xylem parenchyma cells. We applied the patch-clamp technique to protoplasts isolated from the xylem parenchyma by differential enzymic digestion of steles of barley roots (Hordeum vulgare L. cv Apex). In the whole-cell configuration, three types of cation-selective rectifiers could be identified: (a) one activated at membrane potentials above about -50 mV; (b) a second type of outward current appeared at membrane potentials above +20 to +40 mV; (c) below a membrane potential of approximately -110 mV, an inward rectifier could be distinguished. In addition, an anion-specific conductance manifested itself in single-channel activity in a voltage range extending from about -100 to +30 mV, with remarkably slow gating. In excised patches, K+ channels activated at hyperpolarization as well as at depolarization. We suggest that salt is released from the xylem parenchyma into the xylem apoplast by simultaneous flow of cations and anions through channels, following electrochemical gradients set up by the ion uptake processes in the cortex and, possibly, the release and reabsorption of ions on their way to the xylem.     

CONTRIBUTION TO THE THEORY OF PERMEABILITY OF MEMBRANES FOR ELECTROLYTES

From experiments on such membranes as apple skin, parchment paper membrane, and a membrane of completely dry collodion, results have been obtained which could be interpreted by the assumption that these membranes are less permeable for anions than for cations. In parchment paper there is only a relative diminution of the mobility of the anions, in the apple skin and in the dry collodion membrane there is practically no permeability for anions at all. The theory is developed which explains how the decrease or complete lack of mobility of anions influences the electromotive effects of the membrane and the diffusibility of electrolytes across a membrane. The results of the theory are compared with the experimental results. In membranes impermeable for anions the permeability for cations gives the same order of cations as for the mobilities in a free aqueous solution. But the differences of the mobilities are enormously magnified, e.g. the mobilities of H^• and Li^•, which are in the proportion of about 1:10 in aqueous solution, are in proportion of about 1:900 in the collodion membrane. The general cause for the retardation of ionic mobility within the membrane may be supposed to be the increased friction of the water envelope dragged along by the ion in the capillary canals of the membrane. The difference of the effect on the cations and on the anions may be attributed to the electric charge of the walls of the canals.     

“Russian doll” complexes of [n]cycloparaphenylenes: a theoretical study

The formation of "Russian doll" complexes consisting of [n]cycloparaphenylenes was predicted using quantum chemistry tools. The electronic structures of multiple inclusion complexes containing up to four macrocycles were explored at the M06-2X/6-31G* level of theory. The binding energy between the macrocycles increases from the center to the periphery of the complex and can be >60?kcal?mol(-1) for macrocycles containing 14 and 19 repeating units. It has been demonstrated that additional electrostatic interactions originating from the asymmetric electron density distribution observed when comparing the concave and convex macrocycle sides are responsible for the high binding energies in these Russian doll complexes. Oxidation or reduction of the Russian doll complexes creates polarons that are delocalized across the complexes. In the case of polaron cations, most of the polarons are localized at the macrocycle with the smallest ionization potential; for polaron anions, the negative charge is localized across the outer rings of the complex. Because anion polarons are more delocalized than cation polarons, the relaxation energies of the polaron anions were found to be smaller than those of the polaron cations.     

Permeation of both cations and anions through a single class of ATP-activated ion channels in developing chick skeletal muscle.

Micromolar concentrations of extracellular adenosine 5'-triphosphate (ATP) elicit a rapid excitatory response in developing chick skeletal muscle. Excitation is the result of a simultaneous increase in membrane permeability to sodium, potassium, and chloride ions. In the present study we quantify the selectivity of the ATP response, and provide evidence that a single class of ATP-activated ion channels conducts both cations and anions. Experiments were performed on myoballs using the whole-cell patch-clamp technique. We estimated permeability ratios by measuring the shift in reversal potential when one ion was substituted for another. We found that monovalent cations, divalent cations, and monovalent anions all permeate the membrane during the ATP response, and that there was only moderate selectivity between many of these ions. Calcium was the most permeant ion tested. To determine if ATP activates a single class of channels that conducts both cations and anions, or if ATP activates separate classes of cation and anion channels, we analyzed the fluctuations about the mean current induced by ATP. Ionic conditions were arranged so that the reversal potential for cations was +50 mV and the reversal potential for anions was -50 mV. Under these conditions, if ATP activates a single class of channels, ATP should not evoke an increase in noise at the reversal potential of the ATP current. However, if ATP activates separate classes of cation and anion channels, ATP should evoke a significant increase in noise at the reversal potential of the ATP current. At both +40 and -50 mV ATP elicited a clear increase in noise, but at the reversal potential of the ATP current (-5 mV), no increase in noise above background was seen. These results indicate that there is only a single class of excitatory ATP-activated channels, which do not select by charge. Based on analysis of the noise spectrum, the conductance of individual channels is estimated to be 0.2-0.4 pS.     

Proton-linked transport systems as sensors of changes in the membrane surface potential

The kinetic properties of proton linked transport systems and their relation to the membrane surface potential were studied in yeast cells. (1) The negative surface potential of cells rich in anionic phospholipids was found to be 2-times higher than that of control cells; in agreement with their 2-fold increase in the anionic/zwitterionic phospholipid ratio (A/Z). (2) At low external concentration of substrates (high-affinity systems), higher uptake activities were observed for the anions, glutamate, aspartate and phosphate; the zwitterion glycine and the cations lysine and arginine, in both phosphatidylserine and phosphatidylinositol rich cells when compared to control cells. (3) On the other hand, at high external concentration of substrates (low-affinity systems), lower uptake activities were observed for glutamate, aspartate, phosphate and glycine in the cells rich in anionic phospholipids. (4) A decrease in Km without significant alteration in Vmax was found in the high-affinity transport systems that can be explained by the increase in proton concentration at the interface caused by the enhancement in negative surface charge of the cells rich in anionic phospholipids. (5) The mechanisms of the high-affinity proton linked transport systems are compatible with a model which is necessarily ordered, protons before anions. The low-affinity transport systems, on the other hand, follow a random order of binding. The transport systems studied behave as sensors of the changes in surface potential. The reduction of the surface potential reversed the transport alterations with the following sequence: monovalent cations less than divalent cations less than cationic local anesthetics.     

The interaction of various lanthanide ions and some anions with phosphatidylcholine vesicle membranes. A 31P NMR study of the surface potential effects

The interaction of various lanthanide ions with vesicles of phosphatidylcholine from egg yolk has been followed by 31P NMR at 30 degrees C. From known magnetic properties of these ions, separation of the paramagnetic shift into a pure contact and a pseudo-contact part was carried out. Binding curves for the contact contribution (F curves) were obtained from vesicles in solutions of sodium salts with monovalent anions over a wide concentration range. These curves should be insensitive to any conformational effects due to ion binding. Indication of a conformational change in the lipid head group at low ion binding was obtained by studying the ratio between the contact and the pseudo-contact contributions. Besides the adsorption of lanthanide ions, specific anion binding to the surface was introduced to account for the enhanced chemical shifts (Cl- < Br- < NO3-). The results were analyzed in terms of the theory for the diffuse double layer (Gouy-Chapman-Grahame) with equilibrium conditions for the adsorbing cations and anions. Simulations of the titration curves furnished parameters for the ion-lipid interactions. The synergism between the cations and anions follows from the potential effects. Comparison of results with lanthanide ions and Ca2+ indicates that the anion adsorption probably depends on the nature of the adsorbed cation. Lanthanide ion binding to L-glycerophosphorylcholine is not influenced by sodium salts. The binding constant for this complex is weaker than with phosphatidylcholine. The chemical shifts for the lanthanide ion complexes with these two phosphorus compounds seem to be about the same.     

Membrane transport of electrolyte ions and time-dependent membrane potential

Equations are derived for the transport of a symmetrical electrolyte, consisting of cations and anions of equal valency, through a neutral membrane that separates two solutions of finite volume under quasi-steady-state conditions. The time-dependent membrane potential produced by the flow of ions is taken into account. Deviation of the time course of the solute concentrations from that of neutral solutes is found to be determined by the permeability ratio of cations and anions (when this ratio equals unity, the derived membrane transport equations reduce to those for neutral substances). Simple approximate expressions for the solute concentrations and of the membrane potential as functions of time are proposed, which are in excellent agreement with the exact numerical results.