Cations Mediate Interactions between the Nicotinic Acetylcholine Receptor and Anionic Lipids

Interactions between the nicotinic acetylcholine receptor (nAChR) and phosphatidic acid (PA) are bidirectional in that membranes containing PA are effective at stabilizing an agonist-responsive nAChR, whereas incorporation of the nAChR into the same membranes leads to a substantial increase in lipid lateral packing density. A previous study suggested that the ability of PA to adopt a dianionic ionization state is key. We monitored the ionization state of PA in both reconstituted and protein-free membranes. In model membranes composed of PA and 3:2 (mol/mol) phosphatidylcholine (PC)/PA, the monoanionic-to-dianionic transition of PA was detected with a pKa of 8.7 and 6.5, respectively. In the reconstituted 3:2 PC/PA membranes, however, PA was stabilized in a monoanionic state at pH values up to 10. Although dianionic PA does not play a role in nAChR function, we found that both the stabilization of monoanionic PA and the concentration of other cations at the bilayer surface can account for changes in bilayer physical properties that are observed upon incorporation of the nAChR into 3:2 PC/PA membranes. A nAChR-induced concentration of cations at the bilayer surface likely mediates interactions between the nAChR and the anionic lipids in its membrane environment.     

Assessing the lipid requirements of the Torpedo californica nicotinic acetylcholine receptor

The lipid requirements of the Torpedo californica nicotinic acetylcholine receptor (nAChR) were assessed by reconstituting purified receptors into lipid vesicles of defined composition and by using photolabeling with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) to determine functionality. Earlier studies demonstrated that nAChRs reconstituted into membranes containing phosphatidylcholine (PC), the anionic lipid phosphatidic acid (PA), and cholesterol (CH) are particularly effective at stabilizing the nAChR in the resting (closed) state that is capable of undergoing agonist-induced conformational transitions (i.e., functionality). The present studies demonstrate that (1) there is no obligatory requirement for PC, (2) increasing the CH content serves to increase the degree to which nAChRs are stabilized in the resting state, and this effect saturates at approximately 35 mol % (molar lipid percentage), and (3) the effect of increasing levels of PA saturates at approximately 12 mol % and in the absence of PA nAChRs are stabilized in the desensitized state (i.e., nonfunctional). Native Torpedo membranes contain approximately 35 mol % CH but less than 1 mol % PA, suggesting that other anionic lipids may substitute for PA. We report that (1) phosphatidylserine (PS) and phosphatidylinositol (PI), anionic lipids that are abundant in native Torpedo membranes, also stabilize the receptor in the resting state although with reduced efficacy (approximately 50-60%) compared to PA, and (2) for nAChRs reconstituted into PA/CH membranes at different lipid-protein molar ratios, receptor functionality decreases rapidly below approximately 65 lipids per receptor. Collectively, these results are consistent with a functional requirement of a single shell of lipids surrounding the nAChR and specific anionic lipid- and sterol (CH)-protein interactions.     

Behavioral differences between phosphatidic acid and phosphatidylcholine in the presence of the nicotinic acetylcholine receptor

It has been found experimentally that negatively charged phosphatidic acid (PA) lipids and cholesterol molecules stabilize the nicotinic acetylcholine receptor (nAChR) in a functional resting state that can participate in an agonist-induced conformational change. In this study, we compare phosphatidylcholine (PC) and PA lipid behavior in the presence of the nAChR to determine why PC lipids do not support a functional nAChR. For lipids that are located within 1.0 nm of the protein, both PC and PA lipids have very similar order-parameter and bilayer-thickness values, which indicate that the annular lipid properties are protein-dependent. The most significant difference between the PC and PA bilayers is the formation of a lipid domain around the protein, which is visible in the PA bilayer but not the PC bilayer. This suggests that the PA domain may help stabilize the nAChR resting state. The PA lipids in the microdomain have a decreased order compared to a homogeneous PA bilayer and the lipids near the protein attempt to increase the free space in their vicinity by residing in multiple lateral planes.     

Effects of cholesterol on the divalent cation-mediated interactions of vesicles containing amino and choline phospholipids

We have used assays of lipid probe mixing, contents mixing and contents leakage to monitor the divalent cation-mediated interactions between lipid vesicles containing phosphatidylserine (PS) as a minority component together with mixtures of phosphatidylethanolamine (PE), phosphatidylcholine (PC) or sphingomyelin, and cholesterol in varying proportions. The initial rates of calcium- and magnesium-induced lipid probe quenching between vesicles, which reflect primarily the rates of vesicle aggregation, are strongly reduced as progressively higher proportions of PC or sphingomyelin are incorporated into PE/PS vesicles. The initial rates of divalent cation-induced contents mixing and contents leakage for PE/PS vesicles are also strongly reduced when choline phospholipids are incorporated into the vesicles in even low molar proportions. Sphingomyelin has a more potent inhibitory effect on these processes than does PC at an equal level in the vesicle membranes. The inclusion of cholesterol in these vesicles, at levels up to 1:2 moles sterol/mole phospholipid, has little effect on the rates of calcium- or magnesium-induced vesicle aggregation. However, cholesterol significantly enhances the initial rates of vesicle contents mixing and contents leakage in the presence of divalent cations when the vesicles contain choline as well as amino phospholipids. This effect is substantial only when the level of cholesterol exceeds the level of choline phospholipids in the vesicles. These results may have significance for the fusion of certain cellular membranes in mammalian cells, whose cytoplasmic faces have lipid compositions very similar to those of the vesicles examined in this study.     

Effect of divalent cations on the assembly of neutral and charged phospholipid bilayers in patch-recording pipettes.

Monolayers of the negatively charged phospholipid phosphatidylserine (PS) and of the amphoteric phospholipid dioleoylphosphatidylethanolamine (DOPE) were used to assemble bilayers at the tip of patch-recording pipettes. PS bilayers, with seal resistances in the range of gigaohmns (gigaseals), could only be generated when millimolar concentration of divalent cations, Ca++, Mg++, or Ba++ were present in the pipette and bath solutions. In contrast, gigaseals of DOPE were independent of divalent ion concentration in the pH range where DOPE is predominantly neutral (pH 6.5) or positively charged (pH 1.5). At pH 10.0, when most DOPE molecules bear a net negative charge, gigaseals became divalent cation dependent, in a manner quantitatively similar to that of PS at neutral pH. The results indicate that divalent cations play an important role in stabilizing gigaseals of negatively charged lipid but are of no consequence in neutral or positively charged seals.     

Effect of phosphatidylserine on unitary conductance and Ba2+ block of the BK Ca2+-activated K+ channel: re-examination of the surface charge hypothesis

Incorporation of BK Ca2+-activated K+ channels into planar bilayers composed of negatively charged phospholipids such as phosphatidylserine (PS) or phosphatidylinositol (PI) results in a large enhancement of unitary conductance (gch) in comparison to BK channels in bilayers formed from the neutral zwitterionic lipid, phospatidylethanolamine (PE). Enhancement of gch by PS or PI is inversely dependent on KCl concentration, decreasing from 70% at 10 mM KCl to 8% at 1,000 mM KCl. This effect was explained previously by a surface charge hypothesis (Moczydlowski, E., O. Alvarez, C. Vergara, and R. Latorre. 1985. J. Membr. Biol. 83:273-282), which attributed the conductance enhancement to an increase in local K+ concentration near the entryways of the channel. To test this hypothesis, we measured the kinetics of block by external and internal Ba2+, a divalent cation that is expected to respond strongly to changes in surface electrostatics. We observed little or no effect of PS on discrete blocking kinetics by external and internal Ba2+ at 100 mM KCl and only a small enhancement of discrete and fast block by external Ba2+ in PS-containing membranes at 20 mM KCl. Model calculations of effective surface potential sensed by the K+ conduction and Ba2+-blocking reactions using the Gouy-Chapman-Stern theory of lipid surface charge do not lend support to a simple electrostatic mechanism that predicts valence-dependent increase of local cation concentration. The results imply that the conduction pore of the BK channel is electrostatically insulated from the lipid surface, presumably by a lateral distance of separation (>20 A) from the lipid head groups. The lack of effect of PS on apparent association and dissociation rates of Ba2+ suggest that lipid modulation of K+ conductance is preferentially coupled through conformational changes of the selectivity filter region that determine the high K+ flux rate of this channel relative to other cations. We discuss possible mechanisms for the effect of anionic lipids in the context of specific molecular interactions of phospholipids documented for the KcsA bacterial potassium channel and general membrane physical properties proposed to regulate membrane protein conformation via energetics of bilayer stress.     

Relevance of lipid polar headgroups on boron-mediated changes in membrane physical properties

Using liposomes composed of either brain phosphatidylcholine (PC), or binary mixtures of PC and phosphatidylserine (PS), galactolipids (GL), phosphatidylinositol (PI), cardiolipin (CL), phosphatidic acid (PA), or phosphatidylethanolamine (PE), we investigated the effects of graded amounts of boric acid (B, 0.5-1000 microM) on the following membrane physical properties: (a) surface potential, (b) lipid rearrangement through lateral phase separation, (c) fluidity, and (d) hydration. Incubation of the different populations of vesicles with B was associated with a small, but statistically significant, increase in membrane surface potential in PC, PC:PS, PC:GL, PC:PI, PC:PA, and PC:PE liposomes. B-induced lipid lateral rearrangement through lateral phase separation in PC, PC:PA, and PC:PE liposomes; but had no effects on PC:PS, PC:GL, and PC:PI liposomes. In PC liposomes B affected membrane fluidity at the water-lipid interface without affecting the hydrophobic core of the bilayer. In all the other binary liposomes studied, B increased membrane fluidity in both, the hydrophobic portion of the membrane and in the anionic domains. The above was associated with a decrease in the fluidity of the cationic domains. B (10-1000 microM) decreased membrane hydration regardless the composition of the liposomes. The obtained results demonstrate the ability of B to interact with membranes, and induce changes in membrane physical properties. Importantly, the extent of B-membrane interactions and the consequent effects were dependent on the nature of the lipid molecule; as such, B had greater affinity with lipids containing polyhydroxylated moieties such as GL and PI. These differential interactions may result in different B-induced modulations of membrane-associated processes in cells.     

Intramembrane Aromatic Interactions Influence the Lipid Sensitivities of Pentameric Ligand-gated Ion Channels

Although the Torpedo nicotinic acetylcholine receptor (nAChR) reconstituted into phosphatidylcholine (PC) membranes lacking cholesterol and anionic lipids adopts a conformation where agonist binding is uncoupled from channel gating, the underlying mechanism remains to be defined. Here, we examine the mechanism behind lipid-dependent uncoupling by comparing the propensities of two prokaryotic homologs, Gloebacter and Erwinia ligand-gated ion channel (GLIC and ELIC, respectively), to adopt a similar uncoupled conformation. Membrane-reconstituted GLIC and ELIC both exhibit folded structures in the minimal PC membranes that stabilize an uncoupled nAChR. GLIC, with a large number of aromatic interactions at the interface between the outermost transmembrane α-helix, M4, and the adjacent transmembrane α-helices, M1 and M3, retains the ability to flux cations in this uncoupling PC membrane environment. In contrast, ELIC, with a level of aromatic interactions intermediate between that of the nAChR and GLIC, does not undergo agonist-induced channel gating, although it does not exhibit the expected biophysical characteristics of the uncoupled state. Engineering new aromatic interactions at the M4-M1/M3 interface to promote effective M4 interactions with M1/M3, however, increases the stability of the transmembrane domain to restore channel function. Our data provide direct evidence that M4 interactions with M1/M3 are modulated during lipid sensing. Aromatic residues strengthen M4 interactions with M1/M3 to reduce the sensitivities of pentameric ligand-gated ion channels to their surrounding membrane environment.     

Adsorption of divalent cations to bilayer membranes containing phosphatidylserine

The Stern equation, a combination of the Langmuir adsorption isotherm, the Boltzmann relation, and the Grahame equation from the theory of the diffuse double layer, provides a simple theoretical framework for describing the adsorption of charged molecules to surfaces. The ability of this equation to describe the adsorption of divalent cations to membranes containing brain phosphatidylserine (PS) was tested in the following manner. Charge reversal measurements were first made to determine the intrinsic 1:1 association constants of the divalent cations with the anionic PS molecules: when the net charge of a PS vesicle is zero one-half of the available sites are occupied by divalent cations. The intrinsic association constant, therefore, is equal to the reciprocal of the divalent cation concentration at which the mobility of a PS vesicle reverses sign. The Stern equation with this association constant is capable of accurately describing both the zeta potential data obtained with PS vesicles at other concentrations of the divalent cations and the data obtained with with vesicles formed from mixtures of PS and zwitterionic phospholipids. Independent measurements of the number of ions adsorbed to sonicated PS vesicles were made with a calcium-sensitive electrode. The results agreed with the zeta potential results obtained with multilamellar vesicles. When membranes are formed at 20 degrees C in 0.1 M NaCl, the intrinsic 1:1 association constants of Ni, Co, Mn, Ba, Sr, Ca, and Mg with PS are 40, 28, 25, 20, 14, 12, and 8 M-1, respectively.     

Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of membrane lipid composition on the conformational equilibria of the nicotinic acetylcholine receptor

The effects of cholesterol (Chol) and an anionic lipid, dioleoylphosphatidic acid (DOPA) on the conformational equilibria of the nicotinic acetylcholine receptor (nAChR) have been investigated using Fourier transform infrared difference spectroscopy. The difference between spectra recorded in the presence and absence of agonist from the nAChR reconstituted into 3:1:1 egg phosphatidylcholine (EPC)/DOPA/Chol membranes exhibits positive and negative bands that serve as markers of the structural changes associated with the resting to desensitized conformational change. These markers are absent in similar difference spectra recorded from the nAChR reconstituted into EPC membranes lacking both Chol and DOPA, indicating that the nAChR cannot undergo conformational change in response to agonist binding. When low levels of either Chol or DOPA up to 25 mol % of the total lipid are included in the EPC membranes, the markers suggest the predominant stabilization of a conformation that is a structural intermediate between the resting and desensitized states. At higher levels of either Chol or DOPA, the nAChR is stabilized in a conformation that is capable of undergoing agonist-induced desensitization, although DOPA appears to be required for the nAChR to adopt a conformation fully equivalent to that found in native and 3:1:1 EPC/DOPA/Chol membranes. The ability of these two structurally diverse lipids, as well as others (Ryan, S. E., Demers, C. N., Chew, J. P., Baenziger, J. E. (1996) J. Biol. Chem. 271, 24590-24597), to modulate the functional state of the nAChR suggests that lipids act on the nAChR via an indirect effect on some physical property of the lipid bilayer. The data also suggest that anionic lipids are essential to stabilize a fully functional nAChR. We propose that membrane fluidity modulates the relative populations of nAChRs in the resting and desensitized states but that subtle structural changes in the presence of anionic lipids are essential for full activity.     

Divalent cation induced fusion and lipid lateral segregation in phosphatidylcholine-phosphatidic acid vesicles

The interactions of unilamellar vesicles containing phosphatidylcholine (PC) and phosphatidic acid (PA) in the presence of calcium and magnesium were examined by fluorometric assays of vesicle lipid mixing, contents mixing, and contents leakage and by spray-freezing freeze-fracture electron microscopy. These results were correlated with calorimetric and fluorometric measurements of divalent cation induced lateral segregation of lipids in these vesicles under comparable conditions. PA-PC vesicles in the presence of calcium show a rapid but limited intermixing of vesicle lipids and contents, the extent of which increases as the vesicle size decreases or the PA content increases. Calcium produces massive aggregation and efficient mixing of the contents of vesicles containing high proportions of dioleoyl-PA or egg PA, but vesicle coalescence in the latter case is followed rapidly by vesicle collapse and massive leakage of contents. The effects of magnesium are similar for vesicles of very high PA content. However, in the presence of magnesium, vesicles containing lower amounts of PA exhibit "hemifusion", a mode of interaction in which vesicles aggregate and mix approximately 50% of their lipids, apparently representing the lipids of the outer monolayer of each vesicle, without significant mixing of vesicle contents or collapse of the vesicles. Fluorometric measurements of lipid lateral segregation demonstrate that lateral redistribution of lipids in PA-PC vesicles begins at submillimolar concentrations of divalent cations and shows no abrupt change at the "threshold" divalent cation concentration, above which coalescence of vesicles is observed. By correlating calorimetric and fluorometric measurements of lipid lateral segregation and mixing of vesicle components, we can demonstrate that lipid segregation is at least strongly correlated with calcium-promoted coalescence of PA-PC vesicles and is essential to the magnesium-promoted interactions of vesicles of low PA contents.     

Electrostatic properties of membranes containing acidic lipids and adsorbed basic peptides: theory and experiment

The interaction of heptalysine with vesicles formed from mixtures of the acidic lipid phosphatidylserine (PS) and the zwitterionic lipid phosphatidylcholine (PC) was examined experimentally and theoretically. Three types of experiments showed that smeared charge theories (e.g., Gouy-Chapman-Stern) underestimate the membrane association when the peptide concentration is high. First, the zeta potential of PC/PS vesicles in 100 mM KCl solution increased more rapidly with heptalysine concentration (14.5 mV per decade) than predicted by a smeared charge theory (6.0 mV per decade). Second, changing the net surface charge density of vesicles by the same amount in two distinct ways produced dramatically different effects: the molar partition coefficient decreased 1000-fold when the mole percentage of PS was decreased from 17% to 4%, but decreased only 10-fold when the peptide concentration was increased to 1 microM. Third, high concentrations of basic peptides reversed the charge on PS and PC/PS vesicles. Calculations based on finite difference solutions to the Poisson-Boltzmann equation applied to atomic models of heptalysine and PC/PS membranes provide a molecular explanation for the observations: a peptide adsorbing to the membrane in the presence of other surface-adsorbed peptides senses a local potential more negative than the average potential. The biological implications of these "discreteness-of-charge" effects are discussed.     

Nicotinic acetylcholine receptor–lipid interactions: Mechanistic insight and biological function

Membrane lipids are potent modulators of the nicotinic acetylcholine receptor (nAChR) from Torpedo. Lipids influence nAChR function by both conformational selection and kinetic mechanisms, stabilizing varying proportions of activatable versus non-activatable conformations, as well as influencing the transitions between these conformational states. Of note, some membranes stabilize an electrically silent uncoupled conformation that binds agonist but does not undergo agonist-induced conformational transitions. The uncoupled nAChR, however, does transition to activatable conformations in relatively thick lipid bilayers, such as those found in lipid rafts. In this review, we discuss current understanding of lipid–nAChR interactions in the context of increasingly available high resolution structural and functional data. These data highlight different sites of lipid action, including the lipid-exposed M4 transmembrane α-helix. Current evidence suggests that lipids alter nAChR function by modulating interactions between M4 and the adjacent transmembrane α-helices, M1 and M3. These interactions have also been implicated in both the folding and trafficking of nAChRs to the cell surface. We review current mechanistic understanding of lipid–nAChR interactions, and highlight potential biological roles for lipid–nAChR interactions in modulating the synaptic response. This article is part of a Special Issue entitled: Lipid–protein interactions.     

The dependence of the conductance of the hemocyanin channel on applied potential and ionic concentration with mono- and divalent cations

Incorporation of Megatura crenulata hemocyanin into phosphatidylcholine black lipid membranes results in the formation of ion channels. Channel properties depend on many factors, three of which are examined in this work: type and concentration of electrolyte and applied voltage. Eight cations at different concentrations have been used. Instantaneous conductance of the channel is a saturating function of both applied voltage and ionic strength of the bathing solution with monovalent cations, but only of ionic strength with divalent cations. Steady-state voltage-conductance relations are nonlinear for both signs but show slight saturation with ionic strength. Relaxation towards the steady state can be fitted by two exponentials with different time constants. All experimental data are fitted postulating the existence of a mechanism of voltage gating of the channel, and of discrete negative charge near its mouth. Specific and nonspecific binding of cations is required.     

Effects of lipid composition on membrane permeabilization by sticholysin I and II, two cytolysins of the sea anemone Stichodactyla helianthus

Sticholysin I and II (St I and St II), two basic cytolysins purified from the Caribbean sea anemone Stichodactyla helianthus, efficiently permeabilize lipid vesicles by forming pores in their membranes. A general characteristic of these toxins is their preference for membranes containing sphingomyelin (SM). As a consequence, vesicles formed by equimolar mixtures of SM with phosphatidylcholine (PC) are very good targets for St I and II. To better characterize the lipid dependence of the cytolysin-membrane interaction, we have now evaluated the effect of including different lipids in the composition of the vesicles. We observed that at low doses of either St I or St II vesicles composed of SM and phosphatidic acid (PA) were permeabilized faster and to a higher extent than vesicles of PC and SM. As in the case of PC/SM mixtures, permeabilization was optimal when the molar ratio of PA/SM was ~1. The preference for membranes containing PA was confirmed by inhibition experiments in which the hemolytic activity of St I was diminished by pre-incubation with vesicles of different composition. The inclusion of even small proportions of PA into PC/SM LUVs led to a marked increase in calcein release caused by both St I and St II, reaching maximal effect at ~5 mol % of PA. Inclusion of other negatively charged lipids (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), or cardiolipin (CL)), all at 5 mol %, also elicited an increase in calcein release, the potency being in the order CL approximately PA > PG approximately PI approximately PS. However, some boosting effect was also obtained, including the zwitterionic lipid phosphatidylethanolamine (PE) or even, albeit to a lesser extent, the positively charged lipid stearylamine (SA). This indicated that the effect was not mediated by electrostatic interactions between the cytolysin and the negative surface of the vesicles. In fact, increasing the ionic strength of the medium had only a small inhibitory effect on the interaction, but this was actually larger with uncharged vesicles than with negatively charged vesicles. A study of the fluidity of the different vesicles, probed by the environment-sensitive fluorescent dye diphenylhexatriene (DPH), showed that toxin activity was also not correlated to the average membrane fluidity. It is suggested that the insertion of the toxin channel could imply the formation in the bilayer of a nonlamellar structure, a toroidal lipid pore. In this case, the presence of lipids favoring a nonlamellar phase, in particular PA and CL, strong inducers of negative curvature in the bilayer, could help in the formation of the pore. This possibility is confirmed by the fact that the formation of toxin pores strongly promotes the rate of transbilayer movement of lipid molecules, which indicates local disruption of the lamellar structure.     

Binding of a fluorescent dansylcadaverine-substance P analogue to negatively charged phospholipid membranes

We have investigated the binding of a new dansylcadaverine derivative of substance P (DNC-SP) with negatively charged small unilamellar vesicles composed of a mixture of phosphatidylcholine (PC) and either phosphatidylglycerol (PG) or phosphatidylserine (PS) using fluorescence spectroscopic techniques. The changes in fluorescence properties were used to obtain association isotherms at variable membrane negative charges and at different ionic strengths. The experimental association isotherms were analyzed using two binding approaches: (i) the Langmuir adsorption isotherm and the partition equilibrium model, that neglect the activity coefficients; and (ii) the partition equilibrium model combined with the Gouy-Chapman formalism that considers electrostatic effects. A consistent quantitative analysis of each DNC-SP binding curve at different lipid composition was achieved by means of the Gouy-Chapman approach using a peptide effective interfacial charge (v) value of (0.95 +/- 0.02), which is lower than the physical charge of the peptide. For PC/PG membranes, the partition equilibrium constant were 7.8 x 10(3) M(-1) (9/1, mol/mol) and 6.9 x 10(3) M(-1) (7/3, mol/mol), whereas for PC/PS membranes an average value of 6.8 x 10(3) M(-1) was estimated. These partition equilibrium constants were similar to those obtained for the interaction of DNC-SP with neutral PC membranes (4.9 x 10(3) M(-1)), as theoretically expected. We demonstrate that the v parameter is a determinant factor to obtain a unique value of the binding constant independently of the surface charge density of the vesicles. Also, the potential of fluorescent dansylated SP analogue in studies involving interactions with cell membranes is discussed.     

The effect of asymmetric surface potentials on the intramembrane electric field measured with voltage-sensitive dyes

Ratiometric imaging of styryl potentiometric dyes can be used to measure the potential gradient inside the membrane (intramembrane potential), which is the sum of contributions from transmembrane potential, dipole potential, and the difference in the surface potentials at both sides of the membrane. Here changes in intramembrane potential of the bilayer membranes in two different preparations, lipid vesicles and individual N1E-115 neuroblastoma cells, are calculated from the fluorescence ratios of di-4-ANEPPS and di-8-ANEPPS as a function of divalent cation concentration. In lipid vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) or from a mixture of the negatively charged lipid phosphatidylserine (PS) and PC, di-4-ANEPPS produces similar spectral changes in response to both divalent cation-induced changes in intramembrane potential and transmembrane potential. The changes in potential on addition of divalent cations measured by the fluorescence ratios of di-4-ANEPPS are consistent with a change in surface potential that can be modeled with the Gouy-Chapman-Stern theory. The derived intrinsic 1:1 association constants of Ba and Mg with PC are 1.0 and 0.4 M(-1); the intrinsic 1:1 association constants of Ba and Mg with PS are 1.9 and 1.8 M(-1). Ratiometric measurements of voltage sensitive dyes also allow monitoring of intramembrane potentials in living cells. In neuroblastoma cells, a tenfold increase of concentration of Ba, Mg, and Ca gives a decrease in intramembrane potential of 22 to 24 mV. The observed changes in potential could also be described by Gouy-Chapman theory. A surface charge density of 1 e(-)/115 A(2) provides the best fit and the intrinsic 1:1 association constants of Ba, Mg, and Ca with acidic group in the surface are 1.7, 6.1, and 25.3 M(-1).     

Role of membrane charge and semiquinone structure on naphthosemiquinone derivatives and 1,4-benzosemiquinone disproportionation and membrane-buffer distribution coefficients.

Semiquinone membrane/buffer partition coefficients have been determined for 1,2-naphthosemiquinone (ONQ.-), 1,4-naphthosemiquinone (NQ.-) and two of its hydroxylated derivatives, 5,8-dihydroxy-1,4-naphthosemiquinone (NZQ.-) and 5-hydroxy-1,4-naphthosemiquinone (JQ.-) as a function of membrane charge in multilamellar vesicles of phosphatidylcholine (PC) and equimolar mixtures of this lipid and phosphatidic acid (PC:PA) and cetyltrimethylammonium bromide (PC:CTAB) at physiological pH with the exception of values corresponding to PC:PA mixtures which were obtained at pH 9. These coefficients follow the order PC:PA < PC < PC:CTAB in agreement with the negative charge of the semiquinones. The disproportionation equilibria of the naphthosemiquinone derivatives are shifted to the semiquinone in the presence of neutral and positive membranes, being more pronounced in the latter. However, very low partition coefficients as well as small shifts in the semiquinone disproportionation equilibrium were observed for ONQ.- as compared to the other semiquinones. No partition of 1,4-benzosemiquinone (BQ.-) into the lipid phase was detected for either charged or neutral lipid membranes. The presence of lipid membranes decreases the BQ.- equilibrium concentration in the presence of all the types of membranes considered here.     

Dimethonium,a divalent cation that exerts only a screening effect on the electrostatic potential adjacent to negatively charged phospholipid bilayer membranes

Calcium and other alkaline earth cations change the electrostatic potential adjacent to negatively charged bilayer membranes both by accumulating in the aqueous diffuse double layer adjacent to the membrane and by adsorbing to the phospholipids. The effects of these cations on the electrostatic potential are described adequately by the Gouy-Chapman-Stern theory. We report the results of experiments with ethane-bis-trimethylammonium, a cation that has been termed "dimethonium" or "ethamethonium" in analogy with hexamethonium (hexane-1,6-bis-trimethylammonium) and decamethonium (decane-1,10-bis-trimethylammonium). We examined the effect of dimethonium on the zeta potential of multilamellar vesicles formed from the negative lipid phosphatidylserine (PS) and from 5:1 phosphatidylcholine/phosphatidylserine mixtures in solutions containing 0.1, 0.01 and 0.001 M sodium, cesium, or tetramethylammonium chloride. We also examined the effect of dimethonium on the conductance of planar PS bilayer membranes and the 31P NMR signal from sonicated PS vesicles formed in 0.1 M NaCl. We found no evidence that dimethonium adsorbs specifically to bilayer membranes. All the results, except for those obtained with vesicles of low charge density formed in a solution with a high salt concentration, are consistent with the predictions of the Gouy-Chapman theory. We conclude that dimethonium, which does not have the pharmacological effects of hexamethonium and decamethonium, is a useful divalent cation for physiologists interested in investigating electrostatic potentials adjacent to biological membranes.