Probing the lipid membrane dipole potential by atomic force microscopy

The electrostatic properties of biological membranes can be described by three parameters: the transmembrane potential, the membrane surface potential, and the membrane dipole potential. The first two are well characterized in terms of their magnitudes and biological effects. The dipole potential, however, is not well characterized. Various methods to measure the membrane dipole potential indirectly yield different values, and there is not even agreement on the source of the membrane dipole moment. This ambiguity impedes investigations into the biological effects of the membrane dipole moment, which should be substantial considering the large interfacial fields with which it is associated. Electrostatic analysis of phosphatidylcholine lipid membranes with the atomic force microscope reveals a repulsive force between the negatively charged probe tips and the zwitterionic lipids. This unexpected interaction has been analyzed quantitatively to reveal that the repulsion is due to a weak external field created by the internal membrane dipole potential. The analysis yields a dipole moment of 1.5 Debye per lipid with a dipole potential of +275 mV for supported phosphatidylcholine membranes. This new ability to quantitatively measure the membrane dipole moment in a noninvasive manner with nanometer scale spatial resolution will be useful in identifying the biological effects of the dipole potential.     

On the applicability of ising and other statistical models to phase transition in biomembranes

The current literature is critically examined as regards to the applicability of statistical mechanics to the dipole model in order to describe phase transition in biological membranes. The numerical calculations in this context are shown to be speculative and model dependent. Implications of the X-ray diffraction studies on membranes are discussed and it is deduced that only the perpendicular orientation of the dipoles in the polar layer is consistent with the X-ray diffraction data.The author wishes to thank Drs. L. David Roper and E. Keith Hege for many helpful comments.     

Effect of phloretin on chloride permeability: A structure-activity study

On the basis of data obtained with thin lipid membranes, phloretin's inhibition of chloride, urea and glucose transport in biological membranes has been suggested to be due to the effects of interfacial dipole fields on the translocator of these molecules (Andersen, O.S., Finkelstein, A., Katz,I. and Cass, A. (1976) J. Gen. Physiol. 67, 749–771).From the systematic analysis made in the present paper it effectively appears that the ability of phloretin analogs to inhibit chloride permeability in red-cell membrane depends on the capacity they have to alter the interfacial dipole potential: the magnitude of the potential change depending on the dipole moment of the molecule and its membrane concentration, it follows that the inhibitory capacity of a phloretin analog is a function of the dipole moment and the lipid solubility of the compound.     

Electrostatic field effects on membrane domain segregation and on lateral diffusion

Natural membranes are organized structures of neutral and charged molecules bearing dipole moments which generate local non-homogeneous electric fields. When subjected to such fields, the molecules experience net forces that can modify the lipid and protein organization, thus modulating cell activities and influencing (or even dominating) the biological functions. The energetics of electrostatic interactions in membranes is a long-range effect which can vary over distance within r⁻¹ to r⁻³. In the case of a dipole interacting with a plane of dipoles, e.g. a protein interacting with a lipid domain, the interaction is stronger than two punctual dipoles and depends on the size of the domain. In this article, we review several contributions on how electrostatic interactions in the membrane plane can modulate the phase behavior, surface topography and mechanical properties in monolayers and bilayers.     

Electrostatic modeling of ion pores. II. Effects attributable to the membrane dipole potential.

This paper presents calculations of the shielded dipole potential in the interior of a pore piercing a lipid membrane that is at a potential V0 with respect to the aqueous solution. Except in the case of long narrow pores, there is substantial shielding of the membrane dipole potential. The associated dipole field never extends a significant distance into the aqueous region. The fact that the single-channel conductance of gramicidin B is only twice as large in glyceryl monooleate membranes as in phosphatidyl choline (PC) membranes, even though PC is approximately 120 mV more positive with respect to water, is interpreted in terms of the potential energy profile calculated for a gramicidin-like channel. It is demonstrated that the membrane dipole potential can significantly affect channel conductance only if the pore is narrow and if the peak in the potential energy profile occurs in the pore interior.     

Conjugation of Cholesterol to HIV-1 Fusion Inhibitor C34 Increases Peptide-Membrane Interactions Potentiating Its Action

Recently, the covalent binding of a cholesterol moiety to a classical HIV-1 fusion inhibitor peptide, C34, was shown to potentiate its antiviral activity. Our purpose was to evaluate the interaction of cholesterol-conjugated and native C34 with membrane model systems and human blood cells to understand the effects of this derivatization. Lipid vesicles and monolayers with defined compositions were used as model membranes. C34-cholesterol partitions more to fluid phase membranes that mimic biological membranes. Importantly, there is a preference of the conjugate for liquid ordered membranes, rich in cholesterol and/or sphingomyelin, as observed both from partition and surface pressure studies. In human erythrocytes and peripheral blood mononuclear cells (PBMC), C34-cholesterol significantly decreases the membrane dipole potential. In PBMC, the conjugate was 14- and 115-fold more membranotropic than T-1249 and enfuvirtide, respectively. C34 or cholesterol alone did not show significant membrane activity. The enhanced interaction of C34-cholesterol with biological membranes correlates with its higher antiviral potency. Higher partitions for lipid-raft like compositions direct the drug to the receptor-rich domains where membrane fusion is likely to occur. This intermediary membrane binding step may facilitate the drug delivery to gp41 in its pre-fusion state.     

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.     

Lipid-protein interactions in rat renal subcellular membranes: a biophysical and biochemical study

The phase behavior of plasma membrane (PM), endoplasmic reticulum (ER), and nuclear membranes (NM) isolated from adult rat papillary cells was studied using the molecular probe Laurdan. The steady-state fluorescence data analysis was correlated with the lipid composition obtained by biochemical assays. The comparison between intact membranes and protein-free reconstituted vesicles using the whole lipid extract shows the essential role of proteins on the temperature response of natural membranes. The phospholipid (PL) and cholesterol (Cho) content was measured in the three membrane fractions, the PL/Cho molar ratio being between 1.5 and 1.9. However, Laurdan's parameters in NM show a fluid phase state pattern even at low temperature (5 degrees C), with a restricted dipole relaxation in comparison with that displayed in liquid crystalline phase state lipid model membranes. PM and ER are in a gel-like state at temperatures below 20 degrees C, showing increasing dipole relaxation with temperature. The curved fits obtained are characteristic of cholesterol-enriched membranes. The distinctive phase behavior of nuclear membranes vanishes when proteins are extracted. However, relaxation is still faster in this fraction, which correlates with the native lipid composition. NM has the lowest percentage of phosphatidylinositol and sphingomyelin-the latter being a highly saturated phospholipid- and the highest percentage of phosphatidylcholine and phosphatidylethanolamine (PE), nuclear PE being enriched in arachidonic acid. All these changes agree with the higher fluidity of NM compared with ER or PM in the conditions assayed.     

Dipole moment, enthalpy, and entropy changes of Hodgkin-Huxley type kinetic units.

Dipole moment, enthalpy, and entropy changes were calculated for hypothetical structural units which control the opening and closing of ionic channels in axon membranes. The changes of these thermodynamic functions were calculated both for activation (transition to intermediate complex) and for the structural transformation as a whole. The calculations are based on the experimentally determined Q10 values and the empirical formulae for the rate constants (alpha's and beta's) as functions of membrane potentials in Hodgkin-Huxley type models. From the calculated thermodynamic functions we suggest that the specific structural units of the axon membranes are probably of macromolecular (possible protein-like) dimensions with large dipole moments (hundreds of debyes). The calculated dipole moment changes of a single structural unit indicate that in many cases these dipole moments saturate at strong depolarizations or hyperpolarizations. The transitions in structural units show substantial activation enthalpies and entropies but the net enthalpy and entropy changes are practically negligible for the transition as a whole, i.e. the structural units presumably undergo displacements. While the calculated dipole moment changes associated with structural transformations in Loligo and Myxicola show similar potential dependencies, those for Rana usually show a different behavior. The relevance of the dipole moment changes to gating currents is discussed.     

Lipid regulation of cell membrane structure and function

Recent studies of structure-function relationships in biological membranes have revealed fundamental concepts concerning the regulation of cellular membrane function by membrane lipids. Considerable progress has been made in understanding the roles played by two membrane lipids: cholesterol and phosphatidyl-ethanolamine. Cholesterol has been shown to regulate ion pumps, which in some cases show an absolute dependence on cholesterol for activity. These studies suggest that an essential role that cholesterol plays in mammalian cell biology is to enable crucial membrane enzymes to provide function necessary for cell survival. Studies of phosphatidylethanolamine regulation of membrane protein activity and regulation of membrane morphology led to hypotheses concerning the roles for this particular lipid in biological membranes. New information on lipid-protein interactions and on the nature of the lipid head groups has permitted the development of mechanistic hypotheses for the regulation of membrane protein activity by phosphatidyl-ethanolamine. In addition, intermediates in the lamellar-nonlamellar phase transitions of membrane systems containing phosphatidylethanolamine, or other lipids with similar properties, have recently been implicated in facilitating membrane fusion. Finally, studies of transmembrane movement of lipids have provided new insight into the regulation of membrane lipid asymmetry and the biogenesis of cell membranes. These kinds of studies are harbingers of a new generation of progress in the field of cell membranes.     

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.     

Genistein derivatives decrease liposome membrane integrity--calcein release and molecular modeling study

The ability of newly synthesized genistein benzyl and glycosylated derivatives to permeabilize the liposome membrane was studied by calcein-leakage method. All studied derivatives appeared to be more effective than their parent compound--genistein. Comparing the experimental results with theoretical calculations we found that in the case of benzyl derivatives the dipole moment of added benzene ring (with its substitutions) might be important for the strength of flavonoids-lipid interactions. This conclusion may have some implications for QSAR studies in which mostly the dipole moments of entire molecules are considered.     

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.     

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.     

Differential effect of cholesterol and its biosynthetic precursors on membrane dipole potential

Dipole potential is the potential difference within the membrane bilayer, which originates due to the nonrandom arrangement of lipid dipoles and water molecules at the membrane interface. Cholesterol, a representative sterol in higher eukaryotic membranes, is known to increase membrane dipole potential. In this work, we explored the effects of immediate (7-DHC and desmosterol) and evolutionary (ergosterol) precursors of cholesterol on membrane dipole potential, monitored by the dual wavelength ratiometric approach utilizing the probe di-8-ANEPPS. Our results show that the effect of these precursors on membrane dipole potential is very different from that observed with cholesterol, although the structural differences among them are subtle. These results assume relevance, since accumulation of cholesterol precursors due to defective cholesterol biosynthesis has been reported to result in several inherited metabolic disorders such as the Smith-Lemli-Opitz syndrome. Interestingly, cholesterol (and its precursors) has a negligible effect on dipole potential in polyunsaturated membranes. We interpret these results in terms of noncanonical orientation of cholesterol in these membranes. Our results constitute the first report on the effect of biosynthetic and evolutionary precursors of cholesterol on dipole potential, and imply that a subtle change in sterol structure can significantly alter the dipolar field at the membrane interface.     

Gravity dependency of the gramicidin A channel conductivity

Theoretical investigations involving the membrane-solution interface have revealed that the density of the solution varies appreciably within interfacial layers adjacent to charged membrane surfaces. The hypothesis that gravity interacts with this configuration and modifies transport rates across horizontal and vertical membranes differently was supported by initial experiments with gramicidin A channels in phosphatidylserine (PS) membranes in 0.1 M KCl. Channel conductivity was found to be about 1.6 times higher in horizontal membranes than in vertical membranes. Here we present the results of further experiments with gramicidin A channels (incorporated into charged PS- and uncharged phosphatidylcholine (PC) membranes in KCl- and CsCl-solutions) to demonstrate that the hypothesis is more generally applicable. Again, channel conductivity was found to be higher in horizontal PS membranes by a factor of between 1.20 and 1.75 in 0.1 M CsCl. No difference in channel conductivity was found for uncharged PC membranes in 0.1 M KCl and in 0.1 M CsCl. However, for PC membranes in 0.05 M KCl the channel conductivity was significantly higher in horizontal membranes by a factor of between 1.07 and 1.14. These results are consistent with the results of our model calculations of layer density and extension, which showed that the layer formation is enhanced by increasing membrane surface charge and decreasing electrolyte ion concentration. The mechanism of gravity interaction with membrane transport processes via interface reactions might be utilized by biological systems for orientational behaviour in the gravity field, which has been observed even for cellular systems. Received: 16 October 1995 / Accepted: 23 April 1996     

Cholesterol sulfate inhibits the fusion of Sendai virus to biological and model membranes

Cholesterol sulfate is a component of several biological membranes. In erythrocytes, cholesterol sulfate inhibits hypotonic hemolysis, while in sperm, it can decrease fertilization efficiency. We have found cholesterol sulfate to be a potent inhibitor of Sendai virus fusion to both human erythrocyte and liposomal membranes. Cholesterol sulfate also raises the bilayer to hexagonal phase transition temperature of dielaidoyl phosphatidylethanolamine as demonstrated by differential scanning calorimetry and 31P nuclear magnetic resonance spectrometry. Although hexagonal phase structures are not readily found in biological membranes, there is a correlation between the effects of membrane additives on bilayer/non-bilayer equilibria and membrane stabilization. It is proposed that the ability of cholesterol sulfate to alter the physical properties of membranes contributes to its stabilization of biological membranes and the inhibition of membrane fusion.     

Does a lateral gradient of membrane potential on the plasma membrane of growing pollen tube of germinating pollen grain exist?

The data presented in the article by Breigina et al. (2009) "Changes in the membrane potential during pollen grain germination and pollen tube growth" (Tsitologiya. 51 (10): 815-823) and concerning the measurement of electric membrane potential (Delta Psi) on the plasma membrane of growing pollen tube of germinating pollen grain with the use of fluorescent potential-sensitive dye, di-4-ANEPPS, were critically analyzed in order to clarify whether a lateral gradient of Delta Psi on this membrane indeed exists. This analysis showed that the main conclusion of the authors of the above article on the existence of polar distribution of Delta Psi along the pollen tube plasma membrane is not in accordance with a number of known peculiarities of di-4-ANEPPS behavior in biological membranes and requires a significant revision. The findings in question reported by the authors, in my opinion, might be interpreted as evidence for the presence on the plasma membrane of growing pollen tube not only the membrane potential Delta Psi but also lateral gradient of so called intra-membrane dipole potential. Based on the comments made, another interpretation of the experimental results described by Breigina et al. has been offered. In addition, some drawbacks in the methodology used by the authors for measurement of Delta Psi with other fluorescent potential-sensitive dye, DiBAC3(3), are also shortly considered.