gramicidin-a and 1-2-diphytanoylphosphatidylcholine

In contrast to expectations that unsaturated fatty acids contribute to oxidative stress by providing a source of lipid peroxides, we demonstrated the protective effect of double bonds in lipids on oxidative damage to membrane proteins. Photodynamic inactivation of gramicidin channels was decreased in unsaturated lipid compared to saturated lipid bilayers. By estimating photosensitizer (boronated chlorine e6 amide) binding to the membrane with the current relaxation technique, the decrease in gramicidin photoinactivation was attributed to singlet oxygen scavenging by double bonds in lipids rather than to the reduction in photosensitizer binding. Gramicidin protection by unsaturated lipids was also observed upon induction of oxidative stress with tert-butyl hydroperoxide.

Topics: Adsorption; Fatty Acids, Unsaturated; Gramicidin; Lipid Bilayers; Membrane Potentials; Oxidants; Oxidation-Reduction; Oxidative Stress; Phosphatidylcholines; Singlet Oxygen; tert-Butylhydroperoxide

Linear rate-equilibrium (RE) relations, also known as linear free energy relations, are widely observed in chemical reactions, including protein folding, enzymatic catalysis, and channel gating. Despite the widespread occurrence of linear RE relations, the principles underlying the linear relation between changes in activation and equilibrium energy in macromolecular reactions remain enigmatic. When examining amphiphile regulation of gramicidin channel gating in lipid bilayers, we noted that the gating process could be described by a linear RE relation with a simple geometric interpretation. This description is possible because the gating process provides a well-understood reaction, in which structural changes in a bilayer-embedded model protein can be studied at the single-molecule level. It is thus possible to obtain quantitative information about the energetics of the reaction transition state and its position on a spatial coordinate. It turns out that the linear RE relation for the gramicidin monomer-dimer reaction can be understood, and the quantitative relation between changes in activation energy and equilibrium energy can be interpreted, by considering the effects of amphiphiles on the changes in bilayer elastic energy associated with channel gating. We are not aware that a similar simple mechanistic explanation of a linear RE relation has been provided for a chemical reaction in a macromolecule. RE relations generally should be useful for examining how amphiphile-induced changes in bilayer properties modulate membrane protein folding and function, and for distinguishing between direct (e.g., due to binding) and indirect (bilayer-mediated) effects.

Topics: Algorithms; Capsaicin; Chromans; Energy Transfer; Genistein; Gramicidin; Hydrophobic and Hydrophilic Interactions; Ion Channel Gating; Ion Channels; Kinetics; Lipid Bilayers; Membrane Lipids; Models, Chemical; Octoxynol; Phosphatidylcholines; Protein Folding; Rosiglitazone; Thiazolidinediones; Troglitazone

Many drugs are amphiphiles that, in addition to binding to a particular target protein, adsorb to cell membrane lipid bilayers and alter intrinsic bilayer physical properties (e.g., bilayer thickness, monolayer curvature, and elastic moduli). Such changes can modulate membrane protein function by altering the energetic cost (DeltaG(bilayer)) of bilayer deformations associated with protein conformational changes that involve the protein-bilayer interface. But amphiphiles have complex effects on the physical properties of lipid bilayers, meaning that the net change in DeltaG(bilayer) cannot be predicted from measurements of isolated changes in such properties. Thus, the bilayer contribution to the promiscuous regulation of membrane proteins by drugs and other amphiphiles remains unknown. To overcome this problem, we use gramicidin A (gA) channels as molecular force probes to measure the net effect of amphiphiles, at concentrations often used in biological research, on the bilayer elastic response to a change in the hydrophobic length of an embedded protein. The effects of structurally diverse amphiphiles can be described by changes in a phenomenological bilayer spring constant (H(B)) that summarizes the bilayer elastic properties, as sensed by a bilayer-spanning protein. Amphiphile-induced changes in H(B), measured using gA channels of a particular length, quantitatively predict changes in lifetime for channels of a different length--as well as changes in the inactivation of voltage-dependent sodium channels in living cells. The use of gA channels as molecular force probes provides a tool for quantitative, predictive studies of bilayer-mediated regulation of membrane protein function by amphiphiles.

Topics: Algorithms; Capsaicin; Cell Line; Cell Membrane; Genistein; Gramicidin; Humans; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Ion Channel Gating; Ion Channels; Isoflavones; Kinetics; Lipid Bilayers; Membrane Potentials; Membrane Proteins; Octoxynol; Phloretin; Phosphatidylcholines; Protein Conformation

Gramicidin D was incorporated in a biomimetic membrane consisting of a lipid bilayer tethered to a mercury electrode via a hydrophilic spacer, and its behavior was investigated in aqueous 0.1 M KCl by potential-step chronocoulometry and electrochemical impedance spectroscopy. The impedance spectra, recorded from 0.1 to 1 x 10(5) Hz over a potential range of 0.7 V, were fitted to a series of RC meshes, which were related to the different substructural elements of the biomimetic membrane. These impedance spectra were compared with those obtained by incorporating valinomycin, under otherwise identical conditions. The potential dependence of the stationary currents reported on bilayer lipid membranes by Bamberg and Läuger (Bamberg, E.; Läuger, P. J. Membrane Biol. 1973, 11, 177-194) as well as those extracted from potential-step chronocoulometric measurements was interpreted by relating the increase in gramicidin dimerization to a progressive increase in single-file K+ flux along the dimeric channels. An analogous approach was adopted in explaining the difference between the impedance spectra obtained with gramicidin D and those obtained with valinomycin. It is concluded that gramicidin has a low tendency to form dimers in the absence of ionic flux.

Topics: Algorithms; Biomimetics; Electrochemistry; Gramicidin; Lipid Bilayers; Metals; Phosphatidylcholines; Potassium Chloride; Spectrum Analysis; Valinomycin

Tethered membranes have been proven during recent years to be a powerful and flexible biomimetic platform. We reported in a previous article on the design of a new architecture based on the self-assembly of a thiolipid on ultrasmooth gold substrates, which shows extremely good electrical sealing properties as well as functionality of a bilayer membrane. Here, we describe the synthesis of lipids for a more modular design and the adaptation of the linker part to silane chemistry. We were able to form a functional tethered bilayer lipid membrane with good electrical sealing properties covering a silicon oxide surface. We demonstrate the functional incorporation of the ion carrier valinomycin and of the ion channel gramicidin.

Topics: Biophysics; Electric Impedance; Electrochemistry; Gold; Gramicidin; Ion Channels; Ionophores; Ions; Lipid Bilayers; Lipids; Microscopy, Atomic Force; Models, Chemical; Phosphatidylcholines; Phytol; Protein Array Analysis; Silanes; Silicon; Silicon Dioxide; Spectrophotometry; Substrate Specificity; Temperature; Time Factors; Valinomycin

Ordered porous alumina substrates with pore diameters of 55 and 280 nm, respectively, were produced and utilized as a support to prepare membranes suspending the pores of the material. Highly ordered porous alumina was prepared by an anodization process followed by dissolution of the remaining aluminum and alumina at the backside of the pores. The dissolution process of Al(2)O(3) at the backside of the pores was monitored by electrical impedance spectroscopy ensuring the desired sieve-like structure of the porous alumina. One side of the porous material with an area of 7 mm(2) was coated with a thin gold layer followed by chemisorption of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol. The hydrophobic monolayer on top of the upper surface was a prerequisite for the formation of suspending membranes, termed nano-black lipid membranes (nano-BLMs). The formation process, and long-term and mechanical stability of the nano-BLMs were followed by electrical impedance spectroscopy indicating the formation of lipid bilayers with typical specific membrane capacitances of (0.65 +/- 0.2) micro F/cm(2) and membrane resistances of up to 1.6 x 10(8) Omega cm(2). These high membrane resistances allowed for single-channel recordings. Gramicidin as well as alamethicin was successfully inserted into the nano-BLMs exhibiting characteristic conductance states.

Topics: Adsorption; Alamethicin; Aluminum Oxide; Biocompatible Materials; Electric Impedance; Electrochemistry; Gramicidin; Lipid Bilayers; Membrane Potentials; Membranes, Artificial; Nanotechnology; Permeability; Phosphatidic Acids; Phosphatidylcholines; Porosity

Clustering of membrane proteins, in particular of ion channels, plays an important role in their functioning. To further elucidate the mechanism of such ion channel activity regulation, we performed experiments with a model system comprising the negatively-charged gramicidin analog, O-pyromellitylgramicidin (OPg) that forms ion channels in bilayer lipid membrane (BLM), and polycations. The effect of polylysines on the kinetics of OPg channels in BLM was studied by the method of sensitized photoinactivation. As found in our previous work, the interaction of polylysine with OPg led to the deceleration of the OPg photoinactivation kinetics, i.e., to the increase in the characteristic time of OPg photoinactivation. It was shown here that in a certain range of polylysine concentrations the photoinactivation kinetics displayed systematic deviations from a monoexponential curve and was well described by a sum of two exponentials. The deviations from the monoexponential approximation were more pronounced with polylysines having a lower degree of polymerization. These deviations increased also upon the elevation of the ionic strength of the bathing solution and the addition of calcium ions. A theoretical model is presented that relates the OPg photoinactivation kinetics at different concentration ratios of OPg and polylysine to the distribution of OPg molecules among OPg-polylysine clusters of different stoichiometry. This model is shown to explain qualitatively the experimental results, although the quantitative description of the whole body of evidence requires further development, assuming that the interaction of polylysine with OPg causes segregation of membrane domains enriched in OPg channels. The single-channel data, which revealed the insensitivity of the single-channel lifetime of OPg to the addition of polylysine, are in good agreement with the theoretical model.

Topics: Cations; Gramicidin; Ion Channels; Kinetics; Light; Lipid Bilayers; Membrane Microdomains; Models, Chemical; Phosphatidylcholines; Photochemistry; Polylysine; Sensitivity and Specificity; Static Electricity

The interactions between an aliphatic or phenyl side chain and an indole ring in a phospholipid environment were investigated by synthesizing and characterizing gramicidins in which Trp(9) was ring-labeled and D-Leu(10) was replaced by D-Val, D-Ala, or D-Phe. All three analogues form conducting channels, with conductances that are lower than that of gramicidin A (gA) channels. The channel lifetimes vary by less than 50% from that of gA channels. Circular dichroism spectra and size-exclusion chromatography show that the conformation of each analogue in dimyristoylphosphatidylcholine (DMPC) vesicles is similar to the right-handed beta(6.3)-helical conformation that is observed for gA. (2)H NMR spectra of oriented samples in DMPC show large changes for the Trp(9) ring when residue 10 is modified, suggesting a steric interaction between D-Leu(10) and Trp(9), in agreement with previous acylation studies (R. E. Koeppe II et al. (1995) Biochemistry 34, 9299-9307). The outer quadrupolar splitting for Trp(9) is unchanged with D-Phe(10), at approximately 153 kHz, but increases by approximately 25 kHz with D-Val(10) and decreases by approximately 10 kHz with D-Ala(10). With D-Ala(10) or D-Val(10), the outer resonance splits into two in a temperature-dependent manner. The NMR spectra indicate that the side chain torsion angles chi1 and chi2 for Trp(9) change when residue 10 is substituted. The changes in chi1 are small, in all cases less than 10 degrees, as is Deltachi2 when D-Ala(10) is introduced, but with D-Val(10) and D-Phe(10) Deltachi2 is at least 25 degrees. We conclude that D-Leu(10) helps to stabilize an optimal orientation of Trp(9) in gA channels in lipid bilayers and that changes in Trp orientation alter channel conductance and lifetime without affecting the basic channel fold.

Topics: Amino Acid Sequence; Amino Acid Substitution; Chromatography, Gel; Circular Dichroism; Deuterium; Gramicidin; Ion Channels; Leucine; Lipid Bilayers; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylcholines; Protein Conformation; Tryptophan

Photodynamic inactivation of gramicidin channels in bilayer lipid membranes induced by single flashes of the visible light in the presence of phthalocyanine has been studied. The kinetic curves of the flash-induced decrease in the gramicidin-mediated electric current are used for determination of the rate constants of formation and termination of gramicidin channels in terms of the channel dimer model. It is revealed that the kinetics of the sensitized photoinactivation of gramicidin in the membrane is altered by agents which modify the dipole potential drop at the membrane-water interface. Addition of phloretin, which is known to decrease the dipole potential drop, slows down the kinetics, whereas the addition of RH421 or 6-ketocholestanol, which increase the dipole potential drop, accelerates the kinetics. It is shown that the photoinactivation kinetics is also slowed down upon the addition of the thyroid hormone L-thyronine, which reduces the dipole potential drop similar to phloretin, as it was found earlier (M. V. Tsybulskaya, Yu. N. Antonenko, A. E. Tropsha, and L. S. Yaguzhinsky, Biofizika 29:801-805 (1984) (in Russian)). It is demonstrated that the changes in the dissociation rate constant of gramicidin dimers under the action of different dipole modifiers correlate with the changes in the dipole potential drop. It is concluded that the process of the gramicidin channel termination corresponding to the dimer dissociation is sensitive to the dipole potential drop. This conclusion is supported by the data on the effect of dipole modifiers on the lifetime of single gramicidin channels.

Topics: Gramicidin; Indoles; Isoindoles; Ketocholesterols; Kinetics; Lipid Bilayers; Phloretin; Phosphatidylcholines; Pyridinium Compounds; Radiation-Sensitizing Agents; Styrenes; Thyronines

We manipulate lipid bilayer surface charge and gauge its influence on gramicidin A channel conductance by two strategies: titration of the lipid charge through bulk solution pH and dilution of a charged lipid by neutral. Using diphytanoyl phosphatidylserine (PS) bilayers with CsCl aqueous solutions, we show that the effects of lipid charge titration on channel conductance are masked 1) by conductance saturation with Cs+ ions in the neutral pH range and 2) by increased proton concentration when the bathing solution pH is less than 3. A smeared charge model permits us to separate different contributions to the channel conductance and to introduce a new method for "bilayer pKa" determination. We use the Gouy-Chapman expression for the charged surface potential to obtain equilibria of protons and cations with lipid charges. To calculate cation concentration at the channel mouth, we compare different models for the ion distribution, exact and linearized forms of the planar Poisson-Boltzmann equation, as well as the construction of a "Gibbs dividing surface" between salt bath and charged membrane. All approximations yield the intrinsic pKain of PS lipid in 0.1 M CsCl to be in the range 2.5-3.0. By diluting PS surface charge at a fixed pH with admixed neutral diphytanoyl phosphatidylcholine (PC), we obtain a conductance decrease in magnitude greater than expected from the electrostatic model. This observation is in accord with the different conductance saturation values for PS and PC lipids reported earlier (, Biochim. Biophys. Acta. 552:369-378) and verified in the present work for solvent-free membranes. In addition to electrostatic effects of surface charge, gramicidin A channel conductance is also influenced by lipid-dependent structural factors.

Topics: Gramicidin; Ion Channels; Lipid Bilayers; Membrane Potentials; Models, Biological; Models, Molecular; Models, Theoretical; Molecular Conformation; Phosphatidylcholines; Protein Conformation; Surface Properties

The question, whether the conducting channels formed by the linear gramicidins are dimers (as is generally believed) or tetramers (as has been recently proposed [Stark G., M. Strässle, and Z. Takacz. 1986. J. Membr. Biol. 89:23-37; Strässle, M., G. Stark, M. Wilhelm, P. Daumas, F. Heitz, and R. Lazaro. 1989. Biochim. Biophys. Acta. 980:305-314]) has been addressed in single-channel experiments. The experimental approach was based on the ability of electrophysiological (single-channel) experiments to resolve the number of hybrid channel types that could form between gramicidin A or C and O-pyromellityl-gramicidin A or C (in which a pyromellitic acid residue has been esterified to the ethanolamine-OH group [Apell, H.-J., E. Bamberg, H. Alpes, and P. Läuger. 1977. J. Membr. Biol. 31:171-188]). The presence of the bulky, negatively charged pyromellityl group at the channel entrances endows the hybrid channels with characteristically different features and thus facilitates the resolution of the different hybrid channel types. Only two hybrid channel types were detected, indicating that the conducting channels are membrane-spanning dimers. There was likewise no evidence for lateral association between conducting channels and nonconducting monomers. These results can be reconciled with those of Stark et al. (op. cit.) if gramicidin channel formation involves a (slow) folding into beta 6.3-helical monomers followed by the dimerization step.

Topics: Gramicidin; Ion Channels; Kinetics; Lipid Bilayers; Macromolecular Substances; Mathematics; Models, Biological; Models, Structural; Phosphatidylcholines

To determine whether amino acid side-chain substitutions in linear gramicidins after the structure of membrane-spanning channels formed by the modified peptides, we have developed a quantitative measure of structural equivalence of the peptide backbone among gramicidin channels based on functional (single-channel) measurements. The experiments exploit the fact that gramicidin channels are symmetrical dimers, and that channels formed by different gramicidin analogues can be distinguished on the basis of their single-channel current amplitudes or durations. It is thereby possible to determine whether hybrid channels can form between chemically dissimilar peptides, i.e. whether the peptides can adapt to each other. Further, since the relative rates of channel formation as well as the relative concentrations of pure and hybrid channel types can be measured in the same membrane, these experiments provide a quantitative measure of the energetic cost of hybrid channel formation relative to the formation of the pure channels. For a wide variety of different side-chains, we find that substitutions as extreme as glycine to phenylalanine at position 1, at the join between the two monomers in a membrane-spanning dimer, incur no energetic cost for channel formation, which implies that channels formed by each of the modified peptides are structurally equivalent. In addition, the average durations of the hybrid channels (except those having tyrosine or hexafluorovaline at position 1) are intermediate to the average durations of the respective pure channel types, thus providing further evidence for structural equivalence among channels formed by sequence-substituted gramicidins.

Topics: Amino Acid Sequence; Gramicidin; Ion Channels; Kinetics; Lipid Bilayers; Mathematics; Models, Biological; Molecular Sequence Data; Phosphatidylcholines; Protein Conformation; Structure-Activity Relationship; Thermodynamics

The structure of membrane-associated gramicidins can depend on the solvent in which they were dissolved prior to membrane incorporation (LoGrasso, P. V., F. Moll, and T. A. Cross 1988. Biophys. J. 54:259-267; Killian, J. A., K. U. Prasad, D. Hains, and D. W. Urry. 1988. Biochemistry. 27:4848-4855). The peptide's solvent history might thus affect the functional characteristics of gramicidin channels (op. cit.). We tested this proposal by examining the properties (conductance, conductance dispersity, and average duration) of channels formed by [Val1]gramicidin A that had been dissolved in eight different solvents. The peptide was incorporated into lipid bilayers either by addition to the aqueous phase (and subsequent adsorption to the membrane) or by cosolubilization with the membrane-forming phospholipid. When the peptide was cosolubilized with the phospholipid, the channel properties did not vary with the solvent used. When the peptide was dissolved in chloroform, benzene, or trifluoroethanol and added through the aqueous phase, the channel properties differed from those found when gramidicin was dissolved in methanol, ethanol, dioxane, dimethylsulfoxide, or ethylacetate. The changes observed with the former three solvents were reproduced by adding them to the aqueous phase, and are therefore due to the ability of these solvents to partition into the membrane and alter the channels' behavior.

Topics: Gramicidin; Ion Channels; Membrane Lipids; Models, Biological; Phosphatidylcholines; Phospholipids; Solvents

A study of the temperature dependence of gramicidin A conductance of K+ in diphytanoyllecithin/n-decane membranes shows the plot of In (single channel conductance) as a function of reciprocal temperature to be nonlinear for the most probable set of conductance states. These results are considered in terms of a series of barriers, of the dynamics of channel conformation, vis-a-vis the peptide libration mechanism, and of the effect of lipid viscosity on side chain motions again as affecting the energetics of peptide libration.

Topics: Gramicidin; Ion Channels; Liposomes; Mathematics; Models, Biological; Peptides; Phosphatidylcholines; Potassium; Thermodynamics

The patch-clamp technique of Mueller (1975, Ann. N.Y. Acad. Sci., 274:247-264) and Neher and Sakmann (1976, Nature (Lond.), 260:799-802) was modified to be suitable for single-channel measurements in lipid bilayers at potentials up to 500 mV. This method was used to study gramicidin A single-channel current-voltage characteristics. It was found that the sublinear current-voltage behavior normally observed at low permeant ion concentrations and rather low potentials (V less than or equal to 200 mV) continues to be seen all the way up to 500 mV. This phenomenon is characteristic of the low permeant ion situation in which the channel is far from saturation, and implies that the overall rate constant for association between ion and channel is very weakly, if at all, voltage dependent. The magnitude of the single channel currents at 500 mV is consistent with the notion that the aqueous convergence conductance is a significant factor in determining the permeability characteristics of the gramicidin A channel.

Topics: Chemical Phenomena; Chemistry; Electric Stimulation; Evoked Potentials; Glycerides; Gramicidin; Ion Channels; Lipid Bilayers; Mathematics; Models, Molecular; Phosphatidylcholines; Time Factors