Effect of the dipole potential of a bilayer lipid membrane on gramicidin channel dissociation kinetics.

A technique of measuring of the light-induced transients of the gramicidin-mediated electric current across a membrane in the presence of a photosensitizer has been applied for the study of the effect of agents modifying the dipole potential of a bilayer lipid membrane (phloretin, 6-ketocholestanol, and RH421) on the processes of the gramicidin channel dissociation and formation. It is shown that phloretin, known to lower the dipole potential, decelerates the flash-induced decrease in the current, whereas 6-ketocholestanol and RH421, known to raise the dipole potential, accelerate the current decrease. It is revealed that the addition of phloretin leads to a decrease in the dissociation rate constant, whereas addition of either 6-ketocholestanol or RH421 causes an increase in this constant. Single-channel data show that phloretin brings about an increase in the lifetime of the gramicidin channels, whereas RH421 produces a more complicated effect. It is conclude that the dipole potential affects the process of channel dissociation, presumably via the influence on the movement of the dipoles of gramicidin molecules through the layer of the dipole potential drop near the membrane-water interface.     

Solid-state 15N NMR of oriented lipid bilayer bound gramicidin A'

Highly oriented samples of lipid and gramicidin A' (8:1 molar ratio) have been prepared with the samples extensively hydrated (approximately 70% water v/w). These preparations have been shown to be completely in a bilayer phase with a transition temperature of 28 degrees C, and evidence is presented indicating that the gramicidin is in the channel conformation. An estimate of the disorder in the alignment of the bilayers parallel with the glass plates used to align the bilayers can be made from the asymmetry of the nuclear magnetic resonances (NMR). Such an analysis indicates a maximal range of disorder of +/- 3 degrees. Uniformly 15N-labeled gramicidin has been biosynthesized by Bacillus brevis grown in a media containing 15N-labeled Escherichia coli cells as the only nitrogen source. When prepared with labeled gramicidin, the oriented samples result in high-resolution 15N NMR spectra showing 12 resonances for the 20 nitrogen sites of the polypeptide. The frequency of the three major multiple resonance peaks has been interpreted to yield the approximate orientation of the N-H bonds in the peptide linkages with respect to the magnetic field. These bond orientations are only partially consistent with the extant structural models of gramicidin.     

Cholesterol-dependent gramicidin A channel inactivation in red blood cell membranes and lipid bilayer membranes

The exchange diffusions of tracer cations (22Na+, 86Rb+) are studied on gramicidin-A-treated red blood cell (RBC) membranes. A time-dependent decrease in cation permeability has been observed and has been considered to be the result of a channel inactivation process. The channel inactivation appears at 20 and 30 degrees C but not at a temperature as low as 6 degrees C. The gramicidin A channel inactivation can be monitored by a conductivity decay of molecular lipid membranes (BLM) prepared either from cholesterol or from a mixture of cholesterol and phospholipids but not of pure phosphatidylethanolamine. The role of cholesterol in the channel inactivation is discussed.     

Interaction of gramicidin S analogs with lipid bilayer membrane.

One of the side chains of Orn residues in gramicidin S (GS) was connected with alanine (AGS), sarcosine (SGS), or histidine (HGS) residue, aiming at developing membrane-active artificial enzymes by virtue of the membrane-associating property of GS. The conformation of the GS analogs was similar to that of GS. However, the affinity of GS and its analogs for dipalmitoylphosphatidylcholine (DPPC) vesicles decreased in the order of GS greater than SGS greater than HGS congruent to AGS. The addition of GS analogs at 10 microM to DPPC vesicles decreased the membrane fluidity, indicating that GS analogs did not disrupt the vesicular structure of DPPC vesicles. On the other hand, GS analogs enhanced carboxyfluorescein-leakage from DPPC vesicles. It was therefore considered that the GS analogs induced the phase-separation of the lipid bilayer membrane. Hydrolytic reactions of HGS in the presence of DPPC vesicles were studied using N-methylindoxyl alkanoate as substrate. HGS reacted only with N-methylindoxyl hexanoate below the phase-transition temperature of the membrane. The substrate specificity of HGS was ascribed to the condensation of HGS in the neighbourhood of the substrate in the lipid bilayer membrane due to the phase-separation below the phase-transition temperature of the membrane.     

Water permeability of gramicidin A-treated lipid bilayer membranes

In membranes containing aqueous pores (channels), the osmotic water permeability coefficient, P f, is greater than the diffusive water permeability coefficient, P d. In fact, the magnitude of P f/P d is commonly used to determine pore radius. Although, for membranes studied to date, P f/P d monotonically declines with decreasing pore radius, there is controversy over the value it theoretically assumes when that radius is so small that water molecules cannot overtake one another within the channel (single-file transport). In one view it should equal 1, and in another view it should equal N, the number of water molecules in the pore. Gramicidin A forms, in lipid bilayer membranes, narrow aqueous channels through which single-file transport may occur. For these channels we find that P f/P d approximately 5. In contrast, for the wider nystatin and amphotericin B pores, P f/P d approximately 3. These findings offer experimental support for the view that P f/P d = N for single-file transport, and we therefore conclude that there are approximately five water molecules in a gramicidin A channel. A similar conclusion was reached independently from streaming potential data. Using single-channel conductance data, we calculate the water permeability of an individual gramicidin A channel. In the Appendix we report that there is a wide range of channel sizes and lifetimes in cholesterol-containing membranes.     

The action of a carbonsuboxide dimerized gramicidin A on lipid bilayer membranes.

Gramicidin A was dimerized with carbonsuboxide as bifunctional reagent. The effect of the resulting malonyl-bis-desformylgramicidin on lipid bilayer membranes was investigated and compared with the effect of the monomer gramicidin. It was found that the single channel conductance and the ion selectivity are very similar to the behaviour of the monomer molecule, whereas the channel forming kinetics and the life time of the single channel of the malonyl-bis-desformylgramicidin differ strongly from the behaviour of the monomer gramicidin. The electrical relaxations are very small and possibly associated with some structural changes of the membrane after a voltage jump. The single channel lifetime of the malonyl-bis-desformylgramicidin is measured in minutes, whereas for the same lipid system the single channel lifetime in the case of the monomer gramicidin is restricted to 1-2 s. It is concluded that the malonyl-bis-desformylgramicidin-molecule itself (as a single molecule) forms an ionic channel without further association.     

Orientation of the valine-1 side chain of the gramicidin transmembrane channel and implications for channel functioning. A 2H NMR study.

The orientation of the valine-1 side chain of gramicidin was determined by solid-state 2H NMR using valine-1-deuterated (d8) gramicidin. The peptide was incorporated into DMPC bilayers that were oriented between glass plates. When the plates were oriented with their normal perpendicular to the magnetic field, four quadrupolar splittings were observed of 106, 68, 9.7, and 2.0 kHz. These resonances were assigned to C alpha D, C beta D, and the deuterons of each of the C gamma D3 methyl groups, respectively. The average orientation of the various C-D bonds was calculated with respect to the helix axis. The angle obtained for the C alpha-D resonance was consistent with a single-stranded beta 6.3-helical model for the backbone but not with double-helical models. The angles of the side chain were then fitted to a model for the right-handed beta 6.3-helix. Rotation of the valine-1 side chain yielded a set of torsion angles that matched the angles as determined from the 2H NMR measurements. The corresponding orientation of the valine-1 side chain (chi 1 = -5 degrees) was found to be quite unusual, but it explains well the importance of a branched side chain at position 1 for channel formation and stability. A van der Waals interaction between valine-1 of one monomer and alanine-5 of the other helps to stabilize the gramicidin dimer.     

13C solid-state NMR of gramicidin A in a lipid membrane.

The natural-abundance 13C NMR spectrum of gramicidin A in a lipid membrane was acquired under magic-angle spinning conditions. With fast sample spinning (15 kHz) at approximately 65 degrees C the peaks from several of the aliphatic, beta-, alpha-, aromatic, and carbonyl carbons in the peptide could be resolved. The resolution in the 13C spectrum was superior that observed with 1H NMR under similar conditions. The 13C linewidths were in the range 30-100 Hz, except for the alpha- and beta-carbons, the widths of which were approximately 350 Hz. The beta-sheet-like local structure of gramicidin A was observed as an upfield shift of the gramicidin alpha and carbonyl resonances. Under slow sample spinning (500 Hz), the intensity of the spinning sidebands from 13C in the backbone carbonyls was used to determine the residual chemical shift tensor. As expected, the elements of the residual chemical shift tensor were consistent with the single-stranded, right-handed beta6.3 helix structure proposed for gramicidin A in lipid membranes.     

Molecular dynamics simulations and 2H NMR study of the GalCer/DPPG lipid bilayer

Molecular dynamics simulations were performed on a two-component lipid bilayer system in the liquid crystalline phase at constant pressure and constant temperature. The lipid bilayers were composed of a mixture of neutral galactosylceramide (GalCer) and charged dipalmitoylphosphatidylglycerol (DPPG) lipid molecules. Two lipid bilayer systems were prepared with GalCer:DPPG ratio 9:1 (10%-DPPG system) and 3:1 (25%-DPPG system). The 10%-DPPG system represents a collapsed state lipid bilayer, with a narrow water space between the bilayers, and the 25%-DPPG system represents an expanded state with a fluid space of approximately 10 nm. The number of lipid molecules used in each simulation was 1024, and the length of the production run simulation was 10 ns. The simulations were validated by comparing the results with experimental data for several important aspects of the bilayer structure and dynamics. Deuterium order parameters obtained from (2)H NMR experiments for DPPG chains are in a very good agreement with those obtained from molecular dynamics simulations. The surface area per GalCer lipid molecule was estimated to be 0.608 +/- 0.011 nm(2). From the simulated electron density profiles, the bilayer thickness defined as the distance between the phosphorus peaks across the bilayer was calculated to be 4.21 nm. Both simulation systems revealed a tendency for cooperative bilayer undulations, as expected in the liquid crystalline phase. The interaction of water with the GalCer and DPPG oxygen atoms results in a strong water ordering in a spherical hydration shell and the formation of hydrogen bonds (H-bonds). Each GalCer lipid molecule makes 8.6 +/- 0.1 H-bonds with the surrounding water, whereas each DPPG lipid molecule makes 8.3 +/- 0.1 H-bonds. The number of water molecules per GalCer or DPPG in the hydration shell was estimated to be 10-11 from an analysis of the radial distribution functions. The formation of the intermolecular hydrogen bonds was observed between hydroxyl groups from the opposing GalCer sugar headgroups, giving an energy of adhesion in the range between -1.0 and -3.4 erg/cm(2). We suggest that this value is the contribution of the hydrogen-bond component to the net adhesion energy between GalCer bilayers in the liquid crystalline phase.     

Effect of bilayer lipid membrane thickness, composition, and tension on gramicidin channel parameters

Dependence of channel parameters formed by gramicidin A (conductivity and mean life time) on thickness, composition and tension of planar bilayer lipid membranes (BLM) was studied. BLM were obtained from solutions of alpha-monoglycerides of fatty acids in n-alkanes. It has been shown that channel conductivity depends on the length of lipid radical hydrocarbon and is insensitive to the isomerization of lipid and to the change of solvent. There was no direct relationship between the life time, thickness and composition of BLM. Logarithm tau for all the systems studied is proportional to BLM tension, which points to a significant role of surface phenomena in the formation by grammicidine A of a conducting pore in the lipid bilayer.     

The effect of changes in gramicidin conformation on bilayer lipid properties.

The effects of two different gramicidin conformations on lipid phase behaviour and dynamics are compared. Samples of chain-perdeuterated dimyristoylphosphatidylcholine containing gramicidin were first prepared with gramicidin in a state having a circular dichroism spectrum generally identified as corresponding to the non-channel conformation. The effects, on bilayer lipid properties, of gramicidin in this conformation were then determined using deuterium nuclear magnetic resonance measurements of acyl chain orientational order and transverse relaxation times as a function of temperature. These samples were then incubated at 65 degrees C to convert the gramicidin to a state with a circular dichroism spectrum of the type generally identified with the channel conformation. The nuclear magnetic resonance measurements were then repeated. In the gel phase, it was found that transverse relaxation time and chain orientational order of the lipid were insensitive to gramicidin conformation. In the liquid crystalline phase, gramicidin in the channel conformation was found to have a slightly larger effect on transverse relaxation and orientational order than gramicidin in the non-channel conformation. The perturbation of the phase behavior by gramicidin was found to be relatively insensitive to gramicidin conformation.     

Analysis of simulated NMR order parameters for lipid bilayer structure determination

The conventional formula for relating CD2 average order parameters to average methylenic travel is flawed when compared to molecular dynamics simulations of dipalmitoylphosphatidylcholine. Inspired by the simulated probability distribution functions, a new formula is derived that satisfactorily relates these quantities. This formula is used to obtain the average chain length , and the result agrees with the direct simulation result for . The simulation also yields a hydrocarbon thickness 2. The result = is consistent with a model of chain packing with both early chain termination and partial interdigitation of chains from opposing monolayers. The actual simulated area per lipid is easily obtained from the order parameters. However, when this method is applied to NMR order parameter data from dimyristoylphosphatidylcholine, the resulting is 10% larger than the currently accepted value.     

Channel formation kinetics of gramicidin A in lipid bilayer membranes

Summary Previous studies have given evidence that the active form of gramicidin A in lipid bilayer membranes is a dimer which acts as an ion channel; it has been further shown that the mean lifetime of the channel strongly depends on the membrane thickness. As the thickness slightly decreases when a voltage is applied to the membrane, the equilibrium between conducting dimers and nonconducting monomers may be displaced by a voltage jump. From the relaxation of the electrical current after the voltage jump, information about the kinetics of channel formation is obtained. For a dioleoyllecithin/n-decane membrane the rate constant of association is found to be 2×10¹⁴ cm² mole^–1 sec^–1, which is by three orders of magnitude below the limiting value of a diffusion-controlled reaction in a two-dimensional system. The dissociation rate constant is equal to 2 sec^–1, a value which is consistent with the channel lifetime as obtained from electrical fluctuation measurements.     

Brief closures of gramicidin A channels in lipid bilayer membranes

Brief closures, so called flickers, gramicidin A channels were observed for glycerol monooleate/n-decane membranes for cesium chloride and hydrochloric acid solutions. The flickers, similar in nature to the flickers observed for physiological channels, were of the order of 1 ms and the interval between flickers was of the order of 50 ms. The flicker-duration and interval between flickers both decrease with voltage. The field dependence of the flickers is consistent with the hypothesis that the membrane forms a dimple when accomodating a dimer in the membrane and that the monomers, on breaking up, are associated over displacements of the order of 2 nm. For similar measurements for glycerol monoleate/hexadecane membranes only rare occurrences of flickers were observed. It is suggested that the flicker phenomenon is governed by the physical and chemical properties of the membrane and the influence of membrane thickness and interfacial free energy is emphasized.     

Voltage-induced thickness changes of lipid bilayer membranes and the effect of an electrin field on gramicidin A channel formation.

The thickness changes of black lipid membranes of different composition after a voltage jump were investigated. In a second series of electrical relaxation experiments the kinetics of channel formation by gramicidin A were measured. The time course of the membrane current was compared with the time course of the thickness change of the membranes. We found that the time course of the current as a consequence of channel formation by gramicidin A did not correlate with the thickness change of the lipid membranes. A possible direct influence of the electric field is discussed.     

Two phases of gramicidin photoinactivation in bilayer lipid membranes in the presence of a photosensitizer

The kinetics of the light-induced decrease in the gramicidin-mediated current across a bilayer lipid membrane in the presence of a photosensitizer has been shown to include a slow phase with a characteristic time of the order of 1 s and a fast phase. Based on the dependence of the slow phase relative amplitude and characteristic time on the gramicidin-mediated stationary conductance we concluded that the slow phase reflected the establishment of an equilibrium between gramicidin monomers and dimers in the membrane after the distortion of this equilibrium resulting from modification of a portion of gramicidin molecules by reactive oxygen species generated upon excitation of the photosensitizer. The dependence of the fast phase contribution to the overall kinetics on the stationary conductance allowed us to conclude that the fast phase is associated with transition of gramicidin dimers into a nonconducting state. The characteristic time of the fast phase measured with nanosecond laser excited pulses is 1.5 ms. The slow phase of the decrease in the gramicidin-mediated current was considerably decelerated in the presence of Rose Bengal. The results obtained indicate that adsorption of Rose Bengal on the bilayer interface leads to a reduction of the dipole potential drop at the membrane-solution boundary, similarly to the action of phloretin.     

The action of a carbonsuboxide dimerized gramicidin A on lipid bilayer membranes

Gramicidin A was dimerized with carbonsuboxide as bifunctional reagent. The effect of the resulting malonyl-bis-desformylgramicidin on lipid bilayer membranes was investigated and compared with the effect of the monomer gramicidin. It was found that the single channel conductance and the ion selectivity are very similar to the behaviour of the monomer molecule, whereas the channel forming kinetics and the life time of the single channel of the malonyl-bis-desformylgramicidin differ strongly from the behaviour of the monomer gramicidin.The electrical relaxations are very small and possibly associated with some structural changes of the membrane after a voltage jump. The single channel lifetime of the malonyl-bis-desformylgramicidin is measured in minutes, whereas for the same lipid system the single channel lifetime in the case of the monomer gramicidin is restricted to 1–2 s. It is concluded that the malonyl-bis-desformylgramicidin-molecule itself (as a single molecule) forms an ionic channel without further association.     

Modulation of a gated ion channel admittance in lipid bilayer membranes.

A future class of amperometric biosensors may utilize gated ion channels such as acetylcholine and glutamate receptors as chemical detection components. In this study, bilayer lipid membranes containing voltage-dependent anion channels (VDAC) were used to model an ion-channel-based biosensor which could continuously monitor AC amperometric changes resulting from induced changes in channel conductance. The in-phase and quadrature components of the induced alternating membrane current were monitored as a function of the applied DC offset voltage which was superimposed on the sinusoidal test voltage. The accuracy and sensitivity of the AC-measured VDAC response was dependent on the magnitude of the AC test voltage relative to the DC offset necessary for channel closure. The VDAC channel appears to be a suitable model protein for AC impedance-based biosensor fabrication.