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.     

Conformation of gramicidin in relation to its ability to form bilayers with lysophosphatidylcholine

The ability of gramicidin to induce bilayer formation in lysophosphatidylcholine (LPC) systems was investigated as a function of the conformation of the peptide. The conformation was varied by using different solvents to cosolubilize gramicidin and lipid. Using circular dichroism (CD), it was found that when codissolved in trifluoroethanol (TFE), after drying and subsequent hydration, gramicidin is mainly present in the single-stranded beta 6.3-helical configuration, whereas when using chloroform/methanol or ethanol as the solvent, it is proposed that the dominant conformation of gramicidin in the membrane is that of the double-stranded antiparallel dimer. Employing 31P NMR, the stoichiometry for bilayer formation was found to be 6 to 7 lipid molecules per gramicidin monomer, when samples were prepared from TFE, whereas a stoichiometry of 4 was found when chloroform/methanol or ethanol was the solvent. Upon heating the latter samples, a conversion was observed in the CD pattern toward that indicative of the beta 6.3-helical configuration. This change was accompanied by an increase in the extent of bilayer formation. Next, it was investigated whether the conformation of gramicidin and its ability to induce bilayer formation were dependent on the lipid acyl chain length. CD measurements of samples prepared from TFE indicated that gramicidin, independent of acyl chain length, was present in the beta 6.3-helical configuration but the intensity of the ellipticities at 218 nm increased with the length of the acyl chain. The extent of bilayer formation in these samples was found to be largely chain length independent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Permeability of acetic acid across gel and liquid-crystalline lipid bilayers conforms to free-surface-area theory.

Solubility-diffusion theory, which treats the lipid bilayer membrane as a bulk lipid solvent into which permeants must partition and diffuse across, fails to account for the effects of lipid bilayer chain order on the permeability coefficient of any given permeant. This study addresses the scaling factor that must be applied to predictions from solubility-diffusion theory to correct for chain ordering. The effects of bilayer chemical composition, temperature, and phase structure on the permeability coefficient (Pm) of acetic acid were investigated in large unilamellar vesicles by a combined method of NMR line broadening and dynamic light scattering. Permeability values were obtained in distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dilauroylphosphatidylcholine bilayers, and their mixtures with cholesterol, at various temperatures both above and below the gel-->liquid-crystalline phase transition temperatures (Tm). A new scaling factor, the permeability decrement f, is introduced to account for the decrease in permeability coefficient from that predicted by solubility-diffusion theory owing to chain ordering in lipid bilayers. Values of f were obtained by division of the observed Pm by the permeability coefficient predicted from a bulk solubility-diffusion model. In liquid-crystalline phases, a strong correlation (r = 0.94) between f and the normalized surface density sigma was obtained: in f = 5.3 - 10.6 sigma. Activation energies (Ea) for the permeability of acetic acid decreased with decreasing phospholipid chain length and correlated with the sensitivity of chain ordering to temperature, [symbol: see text] sigma/[symbol: see text](1/T), as chain length was varied. Pm values decreased abruptly at temperatures below the main phase transition temperatures in pure dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine bilayers (30-60-fold) and below the pretransition in dipalmitoylphosphatidylcholine bilayers (8-fold), and the linear relationship between in f and sigma established for liquid-crystalline bilayers was no longer followed. However, in both gel and liquid-crystalline phases in f was found to exhibit an inverse correlation with free surface area (in f = -0.31 - 29.1/af, where af is the average free area (in square angstroms) per lipid molecule). Thus, the lipid bilayer permeability of acetic acid can be predicted from the relevant chain-packing properties in the bilayer (free surface area), regardless of whether chain ordering is varied by changes in temperature, lipid chain length, cholesterol concentration, or bilayer phase structure, provided that temperature effects on permeant dehydration and diffusion and the chain-length effects on bilayer barrier thickness are properly taken into account.     

Mechanosensitivity of gramicidin A channels in semispherical bilayer membranes at constant tension

Mechanoelectrical transduction in gramicidin A channels was studied in macroscopic planar lipid bilayer membranes bulged at constant tension. We found a supralinear increase in the single channel activity, which was proportional to the square of membrane radius but could not be accounted for by the increase in membrane surface area or by recruitment of new channels. When being extrapolated to biological membranes, these observations may suggest that the activity of permeability of ion channels can be influenced simply by changing the shape of the membrane, with or without stretching.     

Fluorescence lifetimes of carbocyanine lipid analogues in phospholipid bilayers

The fluorescence lifetimes for the 1,1'-dialkyl-3,3,3',3'-tetramethylindocarbocyanine (CNdiI) dyes (N = 12, 18, and 22) in a variety of lipid bilayer membranes were measured. Effects of bilayer physical state, probe chain length, probe concentration, charge, lipid head group, and cholesterol concentration were examined. Even in single-phase membranes these probes did not exhibit single-exponential decays. Rather, the data were fit by biexponential decays with lifetimes of approximately 0.3-0.4 and approximately 0.9-1.3 ns with no significant improvement in chi 2 convergence with the addition of a third component. Average lifetimes were dependent upon lipid phase and to a lesser degree surface charge and the phospholipid head group. In dipalmitoyl-phosphatidylcholine (DPPC)-cholesterol membranes, the C18diI lifetime was sensitive to membrane reorganizations at both 20 and approximately 33 mol % cholesterol. In egg phosphatidylcholine (EPC) bilayers, the C18diI lifetime was essentially independent of its concentration below 1:10(3).     

Lipid bilayer pressure profiles and mechanosensitive channel gating

The function of membrane proteins often depends on the proteins' interaction with their lipid environment, spectacularly so in the case of mechanosensitive channels, which are gated through tension mediated by the surrounding lipids. Lipid bilayer tension is distributed quite inhomogeneously, but neither the scale at which relevant variation takes place nor the effect of varying lipid composition or tension has yet been investigated in atomic detail. We calculated lateral pressure profile distributions in lipid bilayers of various composition from all-atom molecular dynamics simulations totaling 110.5 ns in length. Reproducible pressure profile features at the 1 A length scale were determined. Lipids with phosphatidylcholine headgroups were found to shift the lateral pressure out of the hydrophobic core and into the headgroup region by an amount that is independent of area per lipid. POPE bilayers simulated at areas smaller than optimal exerted dramatically higher lateral pressure in a narrow region at the start of the aliphatic chain. Stretching of POPC bilayers increased tension predominantly in the same region. A simple geometric analysis for the gating of the mechanosensitive channel MscL suggests that pressure profiles affect its gating through the second moment of the profile in a tension-independent manner.     

Identification of the hydrophobic thickness of a membrane protein using fluorescence spectroscopy: studies with the mechanosensitive channel MscL

The hydrophobic thickness of a membrane protein is an important parameter, defining how the protein sits within the hydrocarbon core of the lipid bilayer that surrounds it in a membrane. Here we show that Trp scanning mutagenesis combined with fluorescence spectroscopy can be used to define the hydrophobic thickness of a membrane protein. The mechanosensitive channel of large conductance (MscL) contains two transmembrane alpha-helices, of which the second (TM2) is lipid-exposed. The region of TM2 that spans the hydrocarbon core of the bilayer when MscL is reconstituted into bilayers of dioleoylphosphatidylcholine runs from Leu-69 to Leu-92, giving a hydrophobic thickness of ca. 25 A. The results obtained using Trp scanning mutagenesis were confirmed using Cys residues labeled with the N-methyl-amino-7-nitroben-2-oxa-1,3-diazole [NBD] group; both fluorescence emission maxima and fluorescence lifetimes for the NBD group are sensitive to solvent dielectric constant over the range (2-40) thought to span the lipid headgroup region of a lipid bilayer. Changing phospholipid fatty acyl chain lengths from C14 and C24 results in no significant change for the fluorescence of the interfacial residues, suggesting very efficient hydrophobic matching between the protein and the surrounding lipid bilayer.     

Simulation study of a gramicidin/lipid bilayer system in excess water and lipid. I. Structure of the molecular complex

This paper reports on a simulation of a gramicidin channel inserted into a fluid phase DMPC bilayer with 100 lipid molecules. Two lipid molecules per leaflet were removed to insert the gramicidin, so the resulting preparation had 96 lipid molecules and 3209 water molecules. Constant surface tension boundary conditions were employed. Like previous simulations with a lower lipid/gramicidin ratio (Woolf, T. B., and B. Roux. 1996. Proteins: Struct., Funct., Genet. 24:92-114), it is found that tryptophan-water hydrogen bonds are more common than tryptophan-phospholipid hydrogen bonds. However, one of the tryptophan NH groups entered into an unusually long-lived hydrogen bonding pattern with two glycerol oxygens of one of the phospholipid molecules. Comparisons were made between the behavior of the lipids adjacent to the channel with those farther away. It was found that hydrocarbon chains of lipids adjacent to the channel had higher-order parameters than those farther away. The thickness of the lipid bilayer immediately adjacent to the channel was greater than it was farther away. In general, the lipids adjacent to the membrane had similar orientations to those seen by Woolf and Roux, while those farther away had similar orientations to those pertaining before the insertion of the gramicidin. A corollary to this observation is that the thickness of the hydrocarbon region adjacent to the gramicidin was much thicker than what other studies have identified as the "hydrophobic length" of the gramicidin channel.     

Mechanosensitivity of gramicidin A channels in bulged bilayer membranes at constant tension

Mechanoelectrical transduction in gramicidin A channels was studied in macroscopic planar lipid bilayer membranes bulged at constant tension. We found a supralinear increase in the single channel activity that was proportional to the square of membrane radius, but could not be accounted for by the increase in membrane surface area, or by recruitment of new channels. Extrapolated to biological membranes, these observations may suggest that the permeability of ion channels can be influenced simply by changing shape of the membrane, with or without stretching. Published in Russian in Biofizika, 2006, Vol. 51, No. 6, pp. 1014–1018. The text was submitted by the authors in English.     

The influence of n-alkanols and cholesterol on the duration and conductance of gramicidin single channels in monoolein bilayers

The mean lifetime of gramicidin A channels in bilayers formed from monoolein and squalane was sharply reduced by the absorption of a range of n-alkanols and cholesterol. Results are shown for n-hexanol, n-octanol, n-decanol, n-dodecanol, n-tetradecanol, n-hexadecanol, n-octadecanol and cholesterol. The longer chain n-alkanols were apparently more effective than the shorter members and cholesterol was the most effective of the substances examined. The single channel conductance was also affected, though to a much lesser extent than the mean channel lifetime, the n-alkanols producing increases and cholesterol a decrease. It is suggested that membrane fluidity changes are not likely to be primarily responsible for the reductions in channel lifetimes but that the bilayer tension, which is known to be increased by n-octanol, could be significant.     

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.     

Synthetic analogues of alamethicin: effect of C-terminal residue substitutions and chain length on the ion channel lifetimes.

In a previous study, a synthetic analogue of the peptaibol alamethicin, in the sequence of which all alpha-aminoisobutyric acid (Aib) were substituted by leucine residues and the C-terminal residue modified, was shown to display the same single-channel behaviour as alamethicin in planar lipid bilayer, except that the sublevel lifetimes were much reduced. New analogues differing in their C-terminal residue (Phe-NH2, Pheol, Trp-NH2) have now been tested for their single channel properties in neutral lipid bilayers. The conductance amplitudes and open channel lifetimes do not differ significantly from the previous analogue. Thus, the nature of the last residue, which may be located near the membrane interface, does not seem to play an important role in the destabilisation of the conducting aggregate observed after the Aib substitution by Leu. Since the deletion of one residue (Glu18) in the 14-20 moiety induces a slight decrease of the increment between the conductance levels, but has no effect upon the channel lifetimes, this residue and the length of this segment do not interfer much with the channel lifetime of peptaibols. In conclusion the factors influencing the aggregate stability may be sought in the helix-helix interactions.     

Thermodynamics of mechanosensitivity

Mechanosensitivity of ion channels is conventionally interpreted as being driven by a change of their in-plane cross-sectional area A(msc). This, however, does not include any factors relating to membrane stiffness, thickness, spontaneous curvature or changes in channel shape, length or stiffness. Because the open probability of a channel is sensitive to all these factors, we constructed a general thermodynamic formalism. These equations provide the basis for the analysis of the behaviour of mechanosensitive channels in lipids of different geometric and chemical properties such as the hydrophobic mismatch at the boundary between the protein and lipid or the effects of changes in the bilayer intrinsic curvature caused by the adsorption of amphipaths. This model predicts that the midpoint gamma(1/2) and the slope(1/2) of the gating curve are generally not independent. Using this relationship, we have predicted the line tension at the channel/lipid border of MscL as approximately 10 pN, and found it to be much less than the line tension of aqueous pores in pure lipid membranes. The MscL channel appears quite well matched to its lipid environment. Using gramicidin as a model system, we have explained its observed conversion from stretch-activated to stretch-inactivated gating as a function of bilayer thickness and composition.     

Shaped Apertures in Photoresist Films Enhance the Lifetime and Mechanical Stability of Suspended Lipid Bilayers

Planar lipid bilayers suspended in apertures provide a controlled environment for ion channel studies. However, short lifetimes and poor mechanical stability of suspended bilayers limit the experimental throughput of bilayer electrophysiology experiments. Although bilayers are more stable in smaller apertures, ion channel incorporation through vesicle fusion with the suspended bilayer becomes increasingly difficult. In an alternative bilayer stabilization approach, we have developed shaped apertures in SU8 photoresist that have tapered sidewalls and a minimum diameter between 60 and 100 μm. Bilayers formed at the thin tip of these shaped apertures, either with the painting or the folding method, display drastically increased lifetimes, typically >20 h, and mechanical stability, being able to withstand extensive perturbation of the buffer solution. Single-channel electrical recordings of the peptide alamethicin and of the proteoliposome-delivered potassium channel KcsA demonstrate channel conductance with low noise, made possible by the small capacitance of the 50 μm thick SU8 septum, which is only thinned around the aperture, and unimpeded proteoliposome fusion, enabled by the large aperture diameter. We anticipate that these shaped apertures with micrometer edge thickness can substantially enhance the throughput of channel characterization by bilayer lipid membrane electrophysiology, especially in combination with automated parallel bilayer platforms.     

A quantitative explanation of the effects of some alcohols on gramicidin single-channel lifetime

The effects of n-decanol, n-hexadecanol, n-octyl(oxyethylene)3 alcohol and cholesterol on gramicidin single-channel lifetime in planar lipid bilayers have been determined. The bilayers used were formed from a solution of monoolein in squalene. Measurements have also been made of the above compounds' effects on membrane thickness (as measured by electrical capacity and optical reflectance technique) and surface tension (as derived from bulk interfacial tension and bilayer-lens contact angle measurements). The reduction in single-channel lifetime caused by the n-alkanols may be accounted for quantitatively in terms of the effects of these compounds on bilayer thickness and surface tension. The n-octyl(oxyethylene)3 alcohol caused an increase in single-channel lifetime which is also consistent with the thickness/tension theory. The reduction in channel lifetime caused by cholesterol, however, was much larger than would be predicted from its effects on bilayer thickness and surface tension.     

Influence of membrane thickness and ion concentration on the properties of the gramicidin a channel. Autocorrelation, spectral power density, relaxation and single-channel studies.

The properties of the gramicidin A channel in membranes made from a series of monoglycerides have been studied. In agreement with previous studies, the dissociation rate constant kD of the dimeric channel was found to increase strongly with increasing chain length of the monoglyceride, corresponding to a decrease of the mean life-time of the channel. The value of kD, however, was not strictly correlated with the membrane thickness, as seen from a comparison of membranes with different solvent content. Furthermore, the life-time of the channel increased with the concentration of the permeable ion. This effect was tentatively explained by an electrostatic stabilization of the channel. The single-channel conductance lambda was found to decrease with increasing membrane thickness d, if d was varied by increasing the chain length of the lipid. On the other hand, if d was changed by varying the solvent content of the membranes formed from one and the same lipid, lambda remained constant. These observations were explained by the assumption of local inhomogeneities in the membrane thickness. A striking difference between the lambda values obtained from autocorrelation analysis in the presence of many presence of many channels (lambda a) and those obtained from single-channel experiments (lambda sc) occurred with membranes from longer chain-length monoglycerides. This difference disappeared at low ion concentrations. Electrostatic interactions between channels in local clusters were proposed for an interpretation of these findings.     

The influence of dioxan on the bilayer and monolayer phase transition of phospholipids

The influence of 1.4.-dioxan on the bilayer phase transition of various phospholipids was studied by differential scanning calorimetry and turbidity measurements. The addition of 1.4.-dioxan to lipid bilayers decreases the transition temperature T_m increases the transition enthalpy of the transition. The cooperativity of the transition is unaffected. The phospholipid monolayer transition from the liquid-condensed to the liquid-expanded phase was measured by recording area versus temperature curves at constant surface pressure (isobars). The monolayer transition temperature at constant surface pressure is increased when 1.4.-dioxan is added to the subphase. The change in molecular area becomes larger. A comparison of monolayer isobars on water and water/dioxan as subphase at constant surface tension rather than surface pressure leads to a decrease of the transition temperature on water/dioxan as subphase. This decrease as well as the larger change in molecular area at the monolayer transition can be correlated to the decrease in T_m and the increase in the transition enthalpy of the corresponding bilayer system. 1.4.-Dioxan seems to accumulate at the lipid head group/water interface, thus lowering the tension of the bilayer membrane. This cyclic ether can be used for altering the characteristics of bilayer membranes without disturbing the lipid chain organization.     

Interactions of phospholipids with the potassium channel KcsA

The potassium channel KcsA from Streptomyces lividans has been reconstituted into bilayers of phosphatidylcholines and fluorescence spectroscopy has been used to characterize the response of KcsA to changes in bilayer thickness. The Trp residues in KcsA form two bands, one on each side of the membrane. Trp fluorescence emission spectra and the proportion of the Trp fluorescence intensity quenchable by I(-) hardly vary in the lipid chain length range C10 to C24, suggesting efficient hydrophobic matching between KcsA and the lipid bilayer over this range. Measurements of fluorescence quenching for KcsA reconstituted into mixtures of brominated and nonbrominated phospholipids have been analyzed to give binding constants of lipids for KcsA, relative to that for dioleoylphosphatidylcholine (di(C18:1)PC). Relative lipid binding constants increase by only a factor of three with increasing chain length from C10 to C22 with a decrease from C22 to C24. Strongest binding to di(C22:1)PC corresponds to a state in which the side chains of the lipid-exposed Trp residues are likely to be located within the hydrocarbon core of the lipid bilayer. It is suggested that matching of KcsA to thinner bilayers than di(C24:1)PC is achieved by tilting of the transmembrane alpha-helices in KcsA. Measurements of fluorescence quenching of KcsA in bilayers of brominated phospholipids as a function of phospholipid chain length suggest that in the chain length range C14 to C18 the Trp residues move further away from the center of the lipid bilayer with increasing chain length, which can be partly explained by a decrease in helix tilt angle with increasing bilayer thickness. In the chain length range C18 to C24 it is suggested that the Trp residues become more buried within the hydrocarbon core of the bilayer.     

Incorporation of the V-ATPase inhibitors concanamycin and indole pentadiene in lipid membranes. Spin-label EPR studies

The incorporation of concanamycin A, a potent inhibitor of vacuolar ATPases, into membranes of dimyristoyl phosphatidylcholine has been studied by using EPR of spin-labelled lipid chains. At an inhibitor/lipid ratio of 1:1 mol/mol, concanamycin A broadens the chain-melting transition of the phospholipid bilayer membrane, and effects the lipid chain motion in the fluid phase. The outer hyperfine splitting of a spin label at the C-5 position and the line widths of a spin label at the C-14 position of the lipid chain are increased by concanamycin A. Considerably larger membrane perturbations are caused by equimolar admixture of a designed synthetic 5-(5,6-dichloro-2-indolyl)-2,4-pentadienoyl V-ATPase inhibitor. These results indicate that concanamycin A intercalates readily between the lipid chains in biological membranes, with minimal perturbation of the bilayer structure. Essentially identical results are obtained with concanamycin A added to preformed membranes as a concentrated solution in DMSO, or mixed with lipid in organic solvent prior to membrane formation. Therefore, the common mode of addition in V-ATPase inhibition assays ensures incorporation of concanamycin into the lipid bilayer milieu, which provides an efficient channel of access to the transmembrane domains of the V-ATPase.     

Deformation free energy of bilayer membrane and its effect on gramicidin channel lifetime.

The deformation free energy of a lipid bilayer is presented based on the principle of a continuum theory. For small deformations, the free energy consists of a layer-compression term, a splay-distortion term, and a surface-tension term, equivalent to the elastic free energy of a two-layer smectic liquid crystal with surface tension. Minimization of the free energy leads to a differential equation that, with boundary conditions, determines the elastic deformation of a bilayer membrane. When a dimeric gramicidin channel is formed in a membrane of thickness greater than the length of the channel, the membrane deformation reduces the stability of the channel. Previously this effect was studied by comparing the variation of channel lifetime with the surface tension of bilayers (Elliott, J. R., D. Needham, J. P. Dilger, and D. A. Hayden, 1983, Biochim. Biophys. Acta, 735:95-103). The tension was assumed to pull a dimer for a distance z before the channel loses ion conductivity. To account for the data, z was found to be 18 A. With the deformation free energy, the data can be accounted for with z less than or approximately to 1 A, which is consistent with the breaking of hydrogen bonds in a dimer dissociation. Increasing the strength of lipid-protein interactions is not the only consequence of the complete free energy compared with the previous discussions. It also changes the shape of membrane deformation around an embedded channel from convex to concave, and increases the range of deformation from less than 10 A to greater than 20 A. Clearly these will be important factors in the general considerations of lipid-protein interactions and membrane-mediated interactions between proteins. In addition, thermal fluctuations of a membrane are calculated; in particular, we calculate the relations between the intrinsic thickness and the experimentally measured values. The experimental parameters of monoolein-squalene membranes are used for quantitative analyses.