Alamethicin channels incorporated into frog node of ranvier: calcium-induced inactivation and membrane surface charges

Alamethicin, a peptide antibiotic, partitions into artificial lipid bilayer membranes and into frog myelinated nerve membranes, inducing a voltage-dependent conductance. Discrete changes in conductance representing single-channel events with multiple open states can be detected in either frog node or lipid bilayer membranes. In 120 mM salt solution, the average conductance of a single channel is approximately 600 pS. The channel lifetimes are roughly two times longer in the node membrane than in a phosphatidylethanolamine bilayer at the same membrane potential. With 2 or 20 mM external Ca and internal CsCl, the alamethicin-induced conductance of frog nodal membrane inactivates. Inactivation is abolished by internal EGTA, suggesting that internal accumulation of calcium ions is responsible for the inactivation, through binding of Ca to negative internal surface charges. As a probe for both external and internal surface charges, alamethicin indicates a surface potential difference of approximately -20 to -30 mV, with the inner surface more negative. This surface charge asymmetry is opposite to the surface potential distribution near sodium channels.     

Study of lipid dependence of glucose-6-phosphatase from rat liver and hepatoma using lipid exchange proteins]

In order to study the lipid dependence of glucose-6-phosphatase the lipid composition of microsomes from rat liver and hepatoma was modified using lipid exchange proteins. It was shown that the enzyme activity depends on the presence of phosphatidyl ethanolamine and phosphatidyl serine, but is unaffected by the enrichment of the microsomes with phosphatidyl choline. On the basis of the data obtained it was assumed that the aminophospholipids are required for the functioning of the protein carrier; however, they do not affect the activity of the catalytic component of the glucose-6-phosphatase system on the inner surface of the microsomal membrane.     

Surface potential and reaction of the membrane-bound electron transfer components. II. Integrity of the chloroplast membrane and reaction of P-700

Electrostatic characteristics of the membrane in the vicinity of P-700 were estimated by analyzing the salt and detergent effects on its reaction rate with ionic reagents using the Gouy-Chapman diffuse double layer theory in various preparations of chloroplasts. Upon disruption of thylakoid membranes by sonic treatment or by treatment with digitonin, the reaction rate markedly increased, while the estimated surface charge density became smaller. It was concluded that the membrane surface which determines the reaction rate between P-700 and the ionic reagents changed as the disruption of thylakoid structure. The outer thylakoid surface had more negative charges than the inner one. Changes in the electrical potential profile across the thylakoid membrane during the illumination were also discussed from these results.     

Surface potential and reaction of the membrane-bound electron transfer components. II. Integrity of the chloroplast membrane and reaction of P-700

Electrostatic characteristics of the membrane surface in the vicinity of P-700 were estimated by analyzing the salt and detergent effects on its reaction rate with ionic reagents using the Gouy-Chapman diffuse double layer theory in various preparations of chloroplasts.

Upon disruption of thylakoid membranes by sonic treatment or by treatment with digitonin, the reaction rate markedly increased, while the estimated surface charge density became smaller.

It was concluded that the membrane surface which determines the reaction rate between P-700 and the ionic reagents changed as the disruption of thylakoid structure. The outer thylakoid surface had more negative charges than the inner one.

Changes in the electrical potential profile across the thylakoid membrane during the illumination were also discussed from these results.     

Aging of human erythrocytes: the role of membrane perturbations induced by in vitro ATP-depletion.

The in vitro metabolic depletion of the erythrocytes has been often utilized to mimic the oxidative stress responsible of their in vivo senescence process. The present study is focused to detect the extent to which the modifications of the whole cell and/or of its membrane, consequent to in vitro ATP-depletion, could trigger a mechanism similar to that verified during in vivo aging. From our data it appears that the shape changes and the increased osmotic fragility could be related to physico-chemical alterations that take place at membrane level. Main alterations concern the significant decrease of negative charges of membrane surface, as evidenced by fluorescence intensity enhancement of membrane-associated ANS, and of the increase of fluidity in the lipid/protein membrane region, suggesting a close analogy with the progressive in vivo senescence of the RBC. Furthermore, the ATP-depletion erythrocyte can efficiently remove exogenous hydrogen peroxide differently from old RBC that showed, as well reported, a severe decrease of enzymic protection against oxidative insult.     

Calcium-induced inactivation of alamethicin in asymmetric lipid bilayers

This paper discusses a calcium-dependent inactivation of alamethicin- induced conductance in asymmetric lipid bilayers. The bilayers used were formed with one leaflet of phosphatidyl ethanolamine (PE) and one of phosphatidyl serine (PS). Calcium, initially confined to the neutral lipid (PE) side, can pass through the open alamethicin channel to the negative lipid (PS) side, where it can bind to the negative lipid and reduce the surface potential. Under appropriate circumstances, the voltage-dependent alamethicin conductance is thereby inactivated. We have formulated a model for this process based on the diffusion of calcium in the aqueous phases and we show that the model describes the kinetic properties of the alamethicin conductance under various circumstances. EGTA on the PS side of the membrane reduces the effects of calcium dramatically as predicted by the model.     

Sidedness of inhibition of energy transduction in oxidative phosphorylation in rat liver mitochondria by ethidium bromide.

Ethidium bromide, a new type of inhibitor of energy transduction in oxidative phosphorylation, inhibited ATP synthesis in intact mitochondria but not in submitochondrial particles, the latter being inside-out relative to the membranes of intact mitochondria. Ethidium bromide incorporated inside the submitochondrial particles inhibited ATP synthesis in the particles. The decrease of the membrane potential by valinomycin (plus KCl) inhibited only slightly the energy-dependent binding of ethidium bromide to the mitochondria. The present results show clearly that ethidium bromide inhibited energy transduction in oxidative phosphorylation by acting on the outer side (C-side) of the inner mitochondrial membrane, perhaps by neutralizing negative charges created on the surface of the C-side, and that it had no inhibitory activity on the inner side (M-side) of the membrane. Th present results show also that the energy-dependent binding of ethidium is not due to electrophoretic transport down the membrane potential; ethidium may bind to negative charges on the surface of the C-side. The present study suggest that an anisotropic distribution of electric charge in the inner mitochondrial membrane is an intermediary high energy state of oxidatvie phosphorylation.     

Tunable membrane binding of the intrinsically disordered dehydrin Lti30, a cold-induced plant stress protein

Dehydrins are intrinsically disordered plant proteins whose expression is upregulated under conditions of desiccation and cold stress. Their molecular function in ensuring plant survival is not yet known, but several studies suggest their involvement in membrane stabilization. The dehydrins are characterized by a broad repertoire of conserved and repetitive sequences, out of which the archetypical K-segment has been implicated in membrane binding. To elucidate the molecular mechanism of these K-segments, we examined the interaction between lipid membranes and a dehydrin with a basic functional sequence composition: Lti30, comprising only K-segments. Our results show that Lti30 interacts electrostatically with vesicles of both zwitterionic (phosphatidyl choline) and negatively charged phospholipids (phosphatidyl glycerol, phosphatidyl serine, and phosphatidic acid) with a stronger binding to membranes with high negative surface potential. The membrane interaction lowers the temperature of the main lipid phase transition, consistent with Lti30's proposed role in cold tolerance. Moreover, the membrane binding promotes the assembly of lipid vesicles into large and easily distinguishable aggregates. Using these aggregates as binding markers, we identify three factors that regulate the lipid interaction of Lti30 in vitro: (1) a pH dependent His on/off switch, (2) phosphorylation by protein kinase C, and (3) reversal of membrane binding by proteolytic digest.     

Increased adherence of oxidant-treated human and bovine erythrocytes to cultured endothelial cells

Bovine erythrocytes, which normally lack phosphatidyl choline in their membranes, when treated with either H2O2 or diamide (1-3 mM), showed a partial appearance of phosphatidyl ethanolamine (PE 40%) and phosphatidyl serine (PS, 30-33%) in the external leaflet of the bilayer and a concomitant increased (four- to five-fold) propensity to adhere to cultured bovine aortic endothelial cells. Similar treatment of normal human erythrocytes caused an alteration in the organization of the phospholipid bilayer and also resulted in their increased adherence to endothelial cells derived either from human umbilical vein or bovine aorta. Treatment of RBCs with H2O2 at low concentration (0.5 mM) resulted in cross-linking of spectrin without significant changes in the orientation of aminophospholipids but the RBCs exhibited 15-20% increase in adherence to endothelial cells. Pretreatment of either human or bovine erythrocytes with antioxidants such as vitamin E (2 mM) prevented both oxidant-induced reorganization of phospholipids in the bilayer and enhancement of adherence to endothelial cells. Introduction of either phosphatidyl serine or phosphatidyl ethanolamine but not phosphatidyl choline into erythrocyte membranes increased their adherence to endothelial cells threefold. Oxidant-treated RBCs exhibited enhanced binding and fluorescence of Merocyanine 540 dye (MC-540), which is sensitive to the packing of lipids in the lipid bilayer. On flow cytometric analysis, 78% of H2O2 (0.5 mM)-treated erythrocytes compared to 30% of untreated RBCs exhibited MC-540 binding and fluorescence, indicating differences in the lipid packing in the outer leaflet of the bilayer. Oxidant-treated erythrocytes adhere preferentially to endothelial cells rather than to bovine aortic smooth muscle cells and skin fibroblasts. It is suggested that the alterations in the erythrocyte membrane surface due to spectrin cross-linking and the organization of the phospholipids concomitant with less ordered packing in the external leaflet of the bilayer, either induced by oxidative manipulation in normal RBC or in pathological erythrocytes, play a role in erythrocyte-endothelial cell interaction.     

Effect of lipid composition changes on carbocyanine dye fluorescent response

Egg yolk phosphatidyl choline liposomes containing variable amounts of phosphatidyl ethanolamine, phosphatidyl inositol or phosphatidyl serine demonstrated important variations in the fluorescence of 3.3' dipropylthiodicarbocyanine. When the membrane contained no cholesterol, fluorescence was not correlated with membrane fluidity as measured by diphenyl hexatriene polarization. Increasing cholesterol concentration in valinomycin containing liposome membranes decreased the potassium induced apparent membrane potential and prevented sorption of dye to the membrane. Discontinuity in the apparent potential occurred at 30 mol% cholesterol but could not be correlated with changes in microviscosity. These results indicate that great care should be taken when correlating rapid variations of fluorescence to changes in membrane potential. We propose that changes in phospholipid metabolism could well explain fluorescent changes when monitoring the fluorescence of cyanine dye molecules sorbed to biological membranes.     

Membrane potential and Donnan potential

The Nernst-Planck-Poisson equations for the potential profile across a membrane are exactly solved without recourse to the assumption of constant field within the membrane. It is assumed that the membrane core of thickness dc is covered by a surface layer of thickness ds in which the membrane-fixed charges are distributed at a uniform density N. The membrane boundary potentials as well as the diffusion potentials contribute to the membrane potential. It is shown that for ds greater or similar 1/k, k being the Debye-Hückel parameter, the potential in the membrane surface layer except in the region very near the membrane/solution boundary is effectively equal to the Donnan potential and that its contribution to the membrane potential becomes dominant as N increases. For low N, on the other hand, the membrane potential arises mostly from the diffusion potential.     

Mechanism of hemolysis and erythrocyte transformation caused by lipogrammistin-A, a lipophilic and acylated cyclic polyamine from the skin secretion of soapfishes (Grammistidae).

The mechanism of hemolysis and erythrocyte transformation caused by lipogrammistin-A (LGA), a lipophilic and acylated cyclic polyamine from the skin secretion of soapfishes (Grammistidae), was investigated. The dependency of hemolysis on the erythrocyte concentration indicated that the amount of membrane-bound LGA required for 50% hemolysis is about 13% of the total phospholipids in erythrocytes on a molar basis. A synthetic analogue which lacked a long alkyl chain exhibited much less activity, suggesting that the alkyl chain is important for membrane-binding. In addition, microscopic observations showed that LGA elicited the invagination of erythrocytes at sublytic concentrations, which makes LGA one of the most potent agents with this transforming activity known to date. Its protonated secondary amino group is responsible for the unequal distribution of LGA in the inner leaflet of the lipid bilayer, which leads to invagination, since acetylation at the amino group markedly reduced the invagination activity. Furthermore, the size of LGA-induced lesions on erythrocyte membrane was estimated to be 7-29 A based on osmotic protection experiments, where the external addition of isotonic molecules in this size range gradually increased the effective dose of LGA. Based on these lines of evidence, the mode of LGA action on erythrocytes is deduced to be as follows. First, LGA molecules bind to erythrocyte membrane by lipophilicity. Second, the molecules accumulate in the inner leaflet of the lipid bilayer by interaction of their cationic ammonium groups with acidic residues of membrane lipid in the inner surface. This uneven distribution of LGA distorts the bilayer structure, and results in a change in cell shape and consequent small lesions. Third, small solutes permeate through the lesions, which induces an osmotic change across the membrane, which leads to colloid-osmotic rupture. This mode of action of LGA on erythrocytes accompanied by cell invagination is the first reported example for natural defense substances.     

Voltage-dependent lipid flip-flop induced by alamethicin.

Alamethicin appears to allow voltage-dependent lipid exchange ("flip-flop") between leaflets of a planar bilayer. In membranes with one leaflet of phosphatidyl serine and one of phosphatidyl ethanolamine, the shape of the nonactin current-voltage curve accurately reports the difference in surface potential between the two sides of the membrane. The surface potential is itself a good measure of membrane asymmetry. Alamethicin added to the bathing solutions of an asymmetric membrane does not per se reduce the membrane asymmetry, but turning on the alamethicin conductance by application of a voltage pulse does. Immediately after application of a voltage pulse, large enough to turn on the alamethicin conductance, the asymmetry of the nonactin-K+ current voltage curve decreases, in some cases, nearly to zero. During the pulse, the alamethicin conductance activates if a decrease in surface potential favors turn-on of the alamethicin conductance or inactivates if a decrease in surface potential favors turn-off of the alamethicin conductance. After the pulse, the nonactin-K+ asymmetry returns to its original value if the alamethicin conductance is not turned on. The time-course of this return allows an estimate of the diffusion constant of lipid in the planar bilayer. The value obtained is 5.1 x 10(-8) cm2/s.     

Babesia bovis--effects of phospholipid translocation and adenosine tri-phosphate consumption

The adenosine tri-phosphate concentration of Babesia bovis-infected erythrocytes was significantly (P less than 0.001) less than that of pre-infection erythrocytes. In addition, phosphatidyl serine was detected on the plasmatic surface of the infected erythrocyte. These two related findings could play important roles in the microvascular stasis characteristic of acute B. bovis infection.     

A metabolic osmotic model of human erythrocytes

A metabolic osmotic model of red blood cells is presented which takes into account the main reaction steps of glycolysis and the passive and active fluxes of ions across the cell membrane. Cellular energy metabolism and osmotic behaviour are linked by the ATP consumption for the active transport of cations as well as by the osmotic action of the glycolytic intermediate 2,3-diphosphoglycerate (2,3-DPG). The model is based on a system of differential equations describing the metabolic reactions and transport processes. Further, two algebraic conditions for the osmotic equilibrium and the electroneutrality of the cell are considered. Using realistic system parameters the model allows the calculation of a great number of dependent variables, among them the cell volume, the concentrations of metabolites and ions and the transmembrane potential. Only stationary states are considered.The parameter dependence of important model variables is characterized by control coefficients. The main results are: (a) The volume of erythrocytes is mainly determined by the permeabilities of the leak fluxes of cations, the content of hemoglobin and the activity of the hexokinase-phosphofructokinase system of glycolysis; (b) Changes of volume affect the glycolytic rate mainly by changing the concentration of ATP which is a regulator of glycolysis; (c) A change in the membrane area may affect the other cell properties only if it is connected with variations of the number of active and leak sites of the membrane.     

Respiration induces variable porosity to polyols in the mitochondrial inner membrane

The osmotic basis of low and high amplitude swelling in mitochondria was investigated in detail using sucrose and mannitol as external osmolytes. Osmotic behaviour of mitochondria in various respiratory states was consistent with significant changes in the porosity of the inner membrane corresponding to the rate of respiration. The stoichiometry of oxidative phosphorylation was confirmed to be dependent on the physical state (i.e., osmotic stretch) of the inner membrane regardless of the external polyol used. High amplitude swelling in polyol media was shown to arise from a sequential disruption of the outer and inner mitochondrial membranes, due to a dynamic instability induced by a combination of respiration, unscreened (fixed) surface charge density and the consequent variable porosity of the inner membrane. These novel experimental findings based on the physical theory of osmosis emphasize the need to define the fine structural changes of the inner membrane associated with oxidative phosphorylation to arrive at a comprehensive mechanism.     

RelA1 gene control of Escherichia coli lipid structure and cell performance during glucose limited fed-batch conditions

At increasing glucose limitation, typical for fed-batch cultivation performance, cultivation of Escherichia coli (relA1) results in development of a lipid structure that radically differs from the wild type and is characterised by accumulation of neutral phospholipids and saturated fatty acids. The mutant can, furthermore, not change the level of cardiolipin, which is generally the hallmark of changes to severe glucose limitation. The result suggests an increased negative control in the mutant with respect to the flux to phosphatidyl glycerol and cardolipin as well as to unsaturated fatty acids. Opposite to the wild type, the cardiolipin-depleted membrane is more fragile with respect to sonication and osmotic chock, at severe limitation, and results in extensive foaming during the process. Protein leakage and cell lysis is, however, lower in the mutant most likely due to the increased amounts of saturated fatty acids, which might be a possible strategy to overcome the reduced amounts of membrane-strengthening cardiolipin. The membrane potential of the outer surface is negative, however less negative for the mutant. This was supported by aqueous two-phase extraction experiments which, furthermore indicated a difference in outer surface hydrofobicity. These findings suggest that the relA1 gene has a defined, but ppGpp-independent, role in cells with a slowly decreasing metabolism of glucose to control the membrane morphology.     

Differential effects of osmotic pressure on mitochondrial respiratory chain and indices of oxidative phosphorylation

Oxidative phosphorylation was critically evaluated in terms of activities which are sensitive and insensitive to variations in external osmotic pressure in mitochondria. Integrity of mitochondria was determined in terms of a variety of parameters, including the latency of the occluded enzymes, by careful titrations as a function of external osmotic pressure as well as detergent concentrations. The evidence indicated that the rate-limiting step in respiratory states 2 and 4 would be osmotically insensitive, as opposed to the osmotically sensitive respiration of states 1 and 3 and uncoupler-stimulated respiration with glutamate + malate and succinate. Cytochrome oxidase activity in mitochondria as well as in purified reconstituted systems exhibited osmotic insensitivity but marked sensitivity to ionic strength, offering an interesting model to study the osmotically insensitive respiration. Cytochrome oxidase activity led to permeation of mannitol across the mitochondrial inner membrane. Stimulation of cytochrome oxidase activity by uncouplers did not require an intact membrane.     

Spectrin, red cell shape and deformability. II. The antagonistic action of spectrin and sialic acid residues in determining membrane curvature in genetic spectrin deficiency in mice

In a companion paper, the shapes of spectrin deficient mouse erythrocytes were described; in contrast to previous assumptions, spherules with tethered microvesicles rather than true "spherocytes" were found. Thence, spectrin deficient mouse erythrocytes are endowed with an excess of surface area for the given volume but the membrane is assuming a highly positive curvature. Observations during and after the action of enzymes cleaving the red cell surface charge (Neuraminidase, Trypsin, Chymotrypsin) showed that the previously positive membrane curvature, as well as the tendency of the membrane to flow into fingerlike protrusions was completely abolished. The erythrocytes of the spectrin deficient, desialylated mouse erythrocytes assumed a variety of shapes, often discocytic or even stomatocytic, i.e. their membrane presented with negative curvature. However, while these desialylated membranes could be easily deformed (elongated) by shear flow they did not recoil elastically into any definitive configuration after removal of the deforming forces. It is concluded from these observations that spectrin (acting on the inner interface between membrane and cytoplasm) and sialic acid residues (acting on the outer interface between membrane and plasma) exert antagonizing effects on membrane curvature and membrane bending elasticity. Sialic acid residues, strongly charged and situated on the outer side of the cell, produce positive membrane curvature; this observation can most readily be explained by assuming that this mechanical effect is caused by repulsive coulombic forces expanding the outer half of the bilayer. To explain the effect of the spectrin-complex in counteracting positive or in producing negative membrane curvature, a similar expansive coulombic force acting between the highly charged residues has been postulated. Thence, a model for explaining the overall elastic behaviour of the normal mammalian red cell is developed which is based on the assumption of elastic interactions of proteinacous membrane components coupled to the lipid bilayer of the membrane.     

Asymmetric distribution of phospholipids in membranes from Mycobacterium phlei

The distribution of phospholipids in the membranes of Mycobacterium phlei has been studied by the use of phospholipase C and trinitrobenzenesulfonic acid. In inverted membrane vesicles, whose external surface apparently corresponds topologically to the cytoplasmic surface of the membrane in intact cells, 80% of the phosphatidyl ethanolamine, 24% of diphosphatidyl glycerol, and 13% of phosphatidyl inositol are accessible to cleavage by phospholipase C. These results are in agreement with the finding that 70–75% of phosphatidyl ethanolamine in the membrane is accessible to chemical modification by trinitrobenzenesulfonic acid or dimethylsuberimidate at 4 °C. It can be inferred that in the inverted membrane the majority of phosphatidyl ethanolamine is present on the outer half of the lipid bilayer while inner half constitutes primarily other phospholipids namely phosphatidyl inositol and diphosphatidyl glycerol. Phospholipase C treatment of ETP membranes selectively impairs the active transport of Ca²⁺ without affecting the generation of a proton gradient, respiration, and coupled phosphorylation.