Effects of lipophilic dications on planar bilayer phospholipid membrane and mitochondria

In this paper, we studied effects of phosphonium dications P2C5 and P2C10 on bilayer planar phospholipid membrane (BLM) and rat liver mitochondria. In line with our previous observations [M.F. Ross, T. Da Ros, F.H. Blaikie, T.A. Prime, C.M. Porteous, I.I. Severina, V.P. Skulachev, H.G. Kjaergaard, R.A. Smith, M.P. Murphy, Accumulation of lipophilic dications by mitochondria and cells, Biochem. J. 400 (2006) 199-208], we showed both P2C5 and P2C10 are cationic penetrants for BLM. They generated transmembrane diffusion potential (Delta Psi), the compartment with a lower dication concentration positive. However, the Delta Psi values measured proved to be lower that the Nernstian. This fact could be explained by rather low BLM conductance for the cations at their small concentrations and by induction of some BLM damage at their large concentrations. The damage in question consisted in appearance of non-Ohmic current/voltage relationships which increased in time. Such a non-Ohmicity was especially strong at Delta Psi >100 mV. Addition of penetrating lipophilic anion TPB, which increases the BLM conductance for lipophilic cations, yielded the Nernstian Delta Psi, i.e. 30 mV per ten-fold dication gradient. In the State 4 mitochondria, dications stimulated respiration and lowered Delta Psi. Moreover, they inhibited the State 3 respiration with succinate or glutamate and malate (but not with TMPD and ascorbate) in an uncoupler-sensitive fashion. Effect on the in State 4 mitochondria, similarly to that on BLM, was accounted for by a time-dependent membrane damage. On the other hand, the State 3 effect was most probably due to inhibition of the respiratory chain Complex I and/or Complex III. The damaging and inhibitory activities of lipophilic dications should be taken into account when one considers a possibility to use them as a vehicle to target antioxidants or other compounds to mitochondria.     

Effects of lipophilic dications on planar bilayer phospholipid membrane and mitochondria

In this paper, we studied effects of phosphonium dications P2C5 and P2C10 on bilayer planar phospholipid membrane (BLM) and rat liver mitochondria. In line with our previous observations [M.F. Ross, T. Da Ros, F.H. Blaikie, T.A. Prime, C.M. Porteous, I.I. Severina, V.P. Skulachev, H.G. Kjaergaard, R.A. Smith, M.P. Murphy, Accumulation of lipophilic dications by mitochondria and cells, Biochem. J. 400 (2006) 199-208], we showed both P2C5 and P2C10 are cationic penetrants for BLM. They generated transmembrane diffusion potential (ΔΨ), the compartment with a lower dication concentration positive. However, the ΔΨ values measured proved to be lower that the Nernstian. This fact could be explained by rather low BLM conductance for the cations at their small concentrations and by induction of some BLM damage at their large concentrations. The damage in question consisted in appearance of non-Ohmic current/voltage relationships which increased in time. Such a non-Ohmicity was especially strong at ΔΨ > 100 mV. Addition of penetrating lipophilic anion TPB, which increases the BLM conductance for lipophilic cations, yielded the Nernstian ΔΨ, i.e. 30 mV per ten-fold dication gradient. In the State 4 mitochondria, dications stimulated respiration and lowered ΔΨ. Moreover, they inhibited the State 3 respiration with succinate or glutamate and malate (but not with TMPD and ascorbate) in an uncoupler-sensitive fashion. Effect on the in State 4 mitochondria, similarly to that on BLM, was accounted for by a time-dependent membrane damage. On the other hand, the State 3 effect was most probably due to inhibition of the respiratory chain Complex I and/or Complex III. The damaging and inhibitory activities of lipophilic dications should be taken into account when one considers a possibility to use them as a vehicle to target antioxidants or other compounds to mitochondria.     

Effect of different drugs on a cytochrome c--phospholipid bilayer membrane.

Liposomes were prepared from dipalmitoyl phosphatidylcholine and dicetylphosphate and their interaction with the extrinsic membrane protein cytochrome c examined in terms of changes in 22Na permeability, electrophoretic mobility, protein binding, and motion of an incorporated spin label. The amount of cytochrome c bound displays no significant temperature dependence over the temperature range studied (from 30 to 55 degrees C) whereas cytochrome c causes an increase in 22Na efflux only above the phospholipid phase transition temperature. Interaction of the protein with the lipid vesicles causes no significant disturbance in the bilayer interior as monitored by the motion of the incorporated spin probe. The drugs 2,4-dinitrophenol and ethacrynic acid, both of which increase the magnitude of the vesicle negative charge, enhance both cytochrome c binding and its effect on 22Na permeability. In contrast, the local anesthetic dibucaine, which induces a positive surface charge on these liposomes, reduces both protein binding and the protein-induced increase in 22Na efflux. Finally, the chemicals butylated hydroxytoluene, 2-tert-butylphenol, and tert-butylbenzene, all of which cause early 'melting' of the phospholipid fatty acyl chains, block the capacity of cytochrome c to enhance 22Na permeability while having no effect on its binding to the vesicles.     

Ion-channels reconstituted into lipid bilayer from human sperm plasma membrane

Ion environment and ionic fluxes through membrane are thought to be important in the spermatozoa's maturation, capacitation, and the initiating process of gamete interaction. In this work, the membrane proteins isolated from human sperm plasma membrane were reconstituted into planar lipid bilayers via fusion, and the ion channels activities were observed under voltage clamp mode. In cis 200 // trans 100 mM KCl solution, a TEA-sensitive cation-selective channel with a unit conductance of 40 pS was recorded. In a gradient of 200//100 mM NaCl solutions, a Na⁺-selective channel with a unit conductance of 26 pS was recorded. In both cases, reversal potential was about −18 mV, which is close to the predicated value of a perfect Nernst K⁺ or Na⁺ electrode. In 50//10 mM CaCl₂ solution, a cation channel activity with a unit conductance of 40 pS and reversal potential of about −20 mV was usually observed. In 200//100 mM NMDG(N-methyl-D-glucamine)-Cl solution, where the cation ions were substituted with NMDG, a 30-pS anion-selective channel activity was also detected. The variety in the types of ion channels observed in human spermatozoa plasma membrane suggests that ion channels may play a range of different roles in sperm physiology and gamete interaction. Mol. Reprod. Dev. 50:354–360, 1998. © 1998 Wiley-Liss, Inc.     

A model of diffusion of anti-cataract drugs into the lens tissue

For modeling anticataract drugs diffusion into the lens surface tension of drugs, their solubility in nonpolar solvent and drug adsorption at air-water and water-lipid monolayer surface were measured. It has been shown that diffusion of many drugs into the lens is limited by their low solubility in a monolayer phase of the membrane. Catachrom concentration in the lens should be the highest, because it can concentrate at interfaces.     

Uptake of safranine and other lipophilic cations into model membrane systems in response to a membrane potential

Lipophilic cations such as safranine and methyltriphenylphosphonium (MTPP⁺) are commonly employed to obtain measures of the membrane potential (Δψ) exhibited by energized biological membrane systems. These probes reflect the presence of Δψ (inside negative) by accumulating in the interior of the membrane bound system to achieve transmembrane distributions dictated by the Nernst equation. In this work, we characterize the ability of model membrane large unilamellar vesicle systems to accumulate safranine and other biologically active lipophilic cations in response to a K⁺ diffusion potential (interior negative) across the large unilamellar vesicle membrane. We show that safranine, MTPP⁺, chlorpromazine and vinblastine can be rapidly accumulated to achieve interior lipophilic cation concentrations which may be more than two orders of magnitude higher than exterior concentrations. In the case of safranine, for example, incubation of 2 mM safranine with large unilamellar vesicle systems exhibiting a Δψ of −100 mV or more can lead to interior safranine concentrations in excess of 120 mM. This accumulation appears to proceed as an antiport K⁺-safranine exchange process, and the optical ‘safranine response’ observed can be attributed to precipitation of the dye inside the vesicle as the interior concentrations of safranine exceeds its solubility (96 mM). These observations are discussed in terms of the utility of probes such as safranine and MTPP⁺ for determinations of Δψ as well as their implications for the equilibrium transbilayer distributions of biologically active lipophilic cations in vivo.     

Insertion of short amino-functionalized single-walled carbon nanotubes into phospholipid bilayer occurs by passive diffusion

Carbon nanotubes have been proposed to be efficient nanovectors able to deliver genetic or therapeutic cargo into living cells. However, a direct evidence of the molecular mechanism of their translocation across cell membranes is still needed. Here, we report on an extensive computational study of short (5 nm length) pristine and functionalized single-walled carbon nanotubes uptake by phospholipid bilayer models using all-atom molecular dynamics simulations. Our data support the hypothesis of a direct translocation of the nanotubes through the phospholipid membrane. We find that insertion of neat nanotubes within the bilayer is a "nanoneedle" like process, which can often be divided in three consecutive steps: landing and floating, penetration of the lipid headgroup area and finally sliding into the membrane core. The presence of functional groups at moderate concentrations does not modify the overall scheme of diffusion mechanism, provided that their deprotonated state favors translocation through the lipid bilayer.     

Interactions of liposomes carrying lipophilic prodrugs in the bilayer with blood plasma proteins

Interactions of 100-nm liposomes prepared from egg yolk phosphatidylcholine and baker’s yeast phosphatidylinositol carrying diglyceride ester conjugates of melphalan (Mlph-liposomes) and methotrexate (MTX-liposomes) in the bilayer with blood plasma proteins were studied with Western blotting. Earlier hemocompatibility tests have demonstrated that the liposomes did not affect main blood cells, but MTX-liposomes, and not Mlph-liposomes, induced complement (C) activation in vitro. Here, we show that introduction of polyethylene glycol conjugate (instead of phosphatidylinositol as a stabilizing lipid) or a targeting carbohydrate conjugate has little effect on interaction of Mlph-liposomes with major C components and apolipoproteins, as well as the total protein binding ability of the liposomes. Liposomes loaded with Mlph prodrug did not trigger fragmentation of C3 protein, the central component of the complement, while MTX-liposomes did so, which agrees with our previous findings. Analysis of MTX-liposome binding with C3 protein and its fragments, regulatory C proteins, and immunoglobulins allowed for the conclusion that MTX-liposomes activate complement via the alternative activation pathway. As shown earlier, the decrease in the prodrug concentration in the bilayer to the level corresponding to MTX low-dose treatment regimen allows avoiding C activation.     

Alignment of cholesterol in the membrane bilayer

Freeze-fracture replicas of filipin-treated samples of guinea pig colon mucosa reveal areas in the membrane of the goblet cell granules labeled by filipin-cholesterol complexes (FCC) intermingled with regions patterned by "lines." The FCC and "lines" are arranged in an approximately rhombic pattern. Other membranes of the same cell or of other cells display either FCC only, aligned and occasionally ordered in "rhombs," "lines" only, with a similar pattern, or randomly distributed FCC. Optical diffraction was used to analyze and compare replicas of membranes with ordered FCC and "lines", as well as randomly distributed FCC. The results demonstrate that all these structures are reciprocally related through a common distribution pattern in the membrane. This observation supports the assumption that cholesterol has a preferential ordered distribution within the membrane bilayer.     

Effects of drugs on pigeon erythrocyte membrane and asymmetric control of adenylate cyclase by the lipid bilayer

In pigeon erythrocyte membrane, the β-adrenergic receptor and the enzyme adenylate cyclase can be uncoupled in two different ways depending on the type of drug used.Cationic drugs: chlorpromazine, methochlorpromazine, tetracaine, $\text{n-}\text{octylamine}$ and a neutral alcohol, octanol, abolished alprenolol receptor binding ability and in the same range of concentration of the drug, sensitized adenylate cyclase to fluoride or Gpp(NH)p stimulation. Anionic drugs: di- and trinitrophenols, indomethacin and octanoic acid did not affect the total number of β-adrenergic receptor sites and, with the exception of trinitrophenol, did not change the association constant for alprenolol but they abolished the stimulation of adenylate cyclase by isoproterenol, fluoride or Gpp(NH)p. These modifications of the adenylate cyclase system occurred in a range of drug concentration where cell shape and protection against hemolysis were also affected.As chemical composition varies widely from one drug to another, it is suggested that these effects are largely nonspecific and mediated by the lipid bilayer. They are probably related to a preferential sidedness of action of the drugs in the lipid bilayer, displaying the role of an asymmetric control of the adenylate cyclase system in the membrane by the two halves of this bilayer.     

Amphiphile-induced tubular budding of the bilayer membrane

Amphiphile-induced tubular budding of the erythrocyte membrane was studied using transmission electron microscopy. No chiral patterns of the intramembraneous particles were found, either on the cylindrical buds, or on the tubular nanoexovesicles. In agreement with these observations, the tubular budding may be explained by in-plane ordering of anisotropic membrane inclusions in the buds where the difference between the principal membrane curvatures is very large. In contrast to previously reported theories, no direct external mechanical force is needed to explain tubular budding of the bilayer membrane.     

Partition of chlorpromazine into lipid bilayer membranes: the effect of membrane structure and composition

Partition coefficients, kp, of chlorpromazine between the aqueous phase and lipid bilayer vesicles were determined as function of drug concentration, lipid chain length, cholesterol content and temperature encompassing the range of the lipid phase transition. Radioactivity and absorption measurements were performed to determine the kp values. Up to a concentration of 3 . 10(-5) M, the partition coefficient is independent of chlorpromazine concentration, whereas it decreases drastically at higher chlorpromazine concentrations, at which membrane lysis is observed. Membrane structure is not disturbed at less than 3 . 10(-5) M chlorpromazine, as was concluded from electron paramagnetic resonance studies measuring TEMPO partitioning and order degree. However, the lipid phase-transition temperature decreases and is broadened at higher chlorpromazine concentrations. From fluorescence measurements, we conclude the formation of chlorpromazine micelles at concentrations higher than 5 . 10(-5) M in chlorpromazine in the absence of lipids and the formation of mixed micelles in the presence of lipids. The effect of lipid chain length on kp values was investigated. The partition coefficient decreases from 8100 in dilauroyl- to 3400 in dipalmitoylphosphatidylcholine vesicles, both at 50 degrees C, that is, above their corresponding phase-transition temperature tt. At t less than tt the kp values are strongly reduced, by at least a factor of 10, depending on lipid chain length and membrane composition. It is possible to establish a lipid phase-transition curve from the temperature-dependent measurements of the kp values. Cholesterol within the lipid membrane strongly decreases kp. At 20 mol% cholesterol in dipalmitoylphosphatidylcholine membranes, the partition coefficient is reduced from 3400 to 2300. This value is well comparable to the kp value obtained in erythrocyte ghosts. In contradiction to earlier experiments by Conrad and Singer (Biochemistry 20 (1981) 808-818), this value in a biological membrane could be obtained by the hygroscopic desorption as well as the centrifugation method. From our experiments we are justified in further considering artificial bilayer membranes as models for biological membranes.     

Incorporation of membrane proteins into large single bilayer vesicles. Application to rhodopsin

A general procedure to incorporate membrane proteins in a native state into large single bilayer vesicles is described. The results obtained with rhodopsin from vertebrate and invertebrate retinas are presented. The technique involves: (a) the direct transfer of rhodopsin-lipid complexes from native membranes into ether or pentane, and (b) the sonication of the complex in apolar solvent with aqueous buffer followed by solvent evaporation under reduced pressure. The spectral properties of rhodopsin in the large vesicles are similar to those of rhodopsin in photoreceptors; furthermore, bleached bovine rhodopsin is chemically regenerable with 9-cis retinal. These results establish the presence of photochemically functional rhodopsin in the large vesicles. Freeze-fracture replicas of the vesicles reveal that both internal and external leaflets contain numerous particles approximately 80 A in diameter, indicating that rhodopsin is symmetrically distributed within the bilayer. More than 75% of the membrane area is incorporated into vesicles larger than 0.5 micron and approximately 40% into vesicles larger than 1 micron.     

Catalytic properties and conformation of hydrophobized alpha-chymotrypsin incorporated into a bilayer lipid membrane

Surfactant proteins A and D are collectins which are considered to play an important role in the innate immunity of lungs. Our aim was to investigate whether surfactant protein A or D is expressed in the porcine Eustachian tube originating from the upper airways. Both surfactant proteins A and D were present in the epithelial cells of the Eustachian tube, as shown by strong immunostaining. Using RT-PCR and Northern hybridization, these collectins were detected in the Eustachian tube. The present study is the first report demonstrating surfactant protein gene expression in the Eustachian tube. Surfactant proteins A and D may be important in the antibody-independent protection of the middle ear.     

Insertion kinetics of a denatured alpha helical membrane protein into phospholipid bilayer vesicles

Membrane protein folding has suffered from a lack of detailed kinetic studies, particularly with regard to the insertion of denatured protein into lipid bilayers. We present a detailed in vitro kinetic study of the association of a denatured, transmembrane alpha helical protein with lipid vesicles. The mechanism of folding of Escherichia coli diacylglycerol kinase from a partially denatured state in urea has been investigated. The protein associates with lipid vesicles to give a protein, vesicle complex with an apparent association constant of 2 x 10(6) M(-1) s(-1). This association rate approaches the diffusion limit of the protein, vesicle reaction. The association of the protein with lipid vesicles is followed by a slower process occurring at observed rate of 0.031 s(-1), involving insertion into the bilayer and generation of a functional oligomer of diacylglycerol kinase. Protein aggregation competes with vesicle insertion. The urea-denatured protein monomers begin to aggregate as soon as the urea is diluted. This aggregation is faster than the association of the protein with vesicles so that most protein aggregates before it inserts into a vesicle. Increasing the vesicle concentration favours insertion of protein monomers, but at high vesicle concentrations monomers are primarily in separate vesicles and do not associate to form functional oligomers. Irreversible aggregation limits the yield of functional protein, while the data also suggest that lipid vesicles can reverse another aggregation reaction, leading to the recovery of correctly folded protein.