Conducting gramicidin channel activity in phospholipid monolayers

Potential step amperometry (chronoamperometry) of the Tl(I)/Tl(Hg) electrochemical reduction process has been used to investigate the underlying mechanisms of gramicidin activity in phospholipid monolayers. The experiments were carried out at gramicidin-modified dioleoyl phosphatidylcholine (DOPC)-coated electrodes. Application of a potential step to the coated electrode system results in a current transient that can be divided into two regions. An initial exponential decay of current corresponds to the inactivation of monomer channel conductance and a longer time scale quasi-steady-state represents the diffusion of ions to a bimolecular surface reaction. Concentrations of monomer conducting channels are relatively low, and the results indicate that two or more forms of gramicidin are in equilibrium with each other in the layer. Aromatic/conjugated compounds incorporated into the monolayer increase the reduction current by decreasing the rate of channel inactivation and increasing the stability of the conducting channel. This effect is positively correlated with the degree of the compound's aromaticity. The anomalous influence of alkali metal ions on the reduction current is consistent with the model of gramicidin being speciated in the monolayer in more than one form. The results have implications on the lability of the peptide conformation in biological membranes and its dependence on lipid environment, solution composition, and applied potential.     

Effects of volatile anesthetic on channel structure of gramicidin A

Volatile anesthetic agent, 1-chloro-1,2,2-trifluorocyclobutane (F3), was found to alter gramicidin A channel function by enhancing Na(+) transport (. Biophys. J. 77:739-746). Whether this functional change is associated with structural alternation is evaluated by circular dichroism and nuclear magnetic resonance spectroscopy. The circular dichroism and nuclear magnetic resonance results indicate that at low millimolar concentrations, 1-chloro-1,2,2-trifluorocyclobutane causes minimal changes in gramicidin A channel structure in sodium dodecyl sulfate micelles. All hydrogen bonds between channel backbones are well maintained in the presence of 1-chloro-1,2,2-trifluorocyclobutane, and the channel structure is stable. The finding supports the notion that low affinity drugs such as volatile anesthetics and alcohols can cause significant changes in protein function without necessarily producing associated changes in protein structure. To understand the molecular mechanism of general anesthesia, it is important to recognize that in addition to structural changes, other protein properties, including dynamic characteristics of channel motions, may also be of functional significance.     

Inhibition of gramicidin channel activity by local anesthetics.

Ondrias et al. ((1986) Stud. Biophys. 115, 17-22) found that dibucaine, butacaine, and tetracaine reduce the conductance of membranes containing multiple (greater than 10(6)) gramicidin channels. Similar experiments with local anesthetics (LA's) added to the bath while gently stirring showed that the inhibition developed slowly over a time course of 5-10 min. We developed a many (10-20) channel membrane technique which demonstrated that when LA's were added to the bath and the membrane was repeatedly broken and reformed, the channel occurrence frequency declined promptly. In standard single-channel membrane experiments at lower gramicidin densities, the mean single channel conductance and lifetime distributions with LA's present in the bath did not differ from the controls. The predominant channel conductance amplitude was lower by 9.1% than those of controls, but channel amplitude distributions were also modified so that the net reduction in overall population channel conductance was only about 2.0%. Channel currents showed no evidence of flicker blocks. The lifetime histograms of control and LA-exposed channel populations were both satisfactorily fit by a single-exponential function with the same mean. Thus, inhibition is due primarily to a reduction in the frequency of occurrence of conducting channels, implying a reduced concentration of active monomers in the membrane.     

Redox-regulated ion channel activity of a cysteine-containing gramicidin A analogue

According to recent data, gramicidin A analogues having positively charged amino acid sequences at the C-termini exhibit two types of channel activity in lipid membranes: classical cation-selective channels and large unselective pores. The induction of unselective pores was shown here to strongly depend on the redox state of the membrane-bathing solution, if the gramicidin analogue contained a cysteine residue in the sequence GSGPKKKRKVC attached to the C-terminus. In particular, the addition of H₂O₂ led to an increase in the transmembrane current and the loss of cationic selectivity on planar bilayer lipid membranes and an increase in the carboxyfluorescein leakage of liposomes. The effect was observed at high concentration of the peptide while was absent at the single-channel level. It was concluded that oxidation led to possible formation of dimers of the peptide, which promoted the formation of large unselective pores.     

Redox-regulated ion channel activity of a cysteine-containing gramicidin A analogue

According to recent data, gramicidin A analogues having positively charged amino acid sequences at the C-termini exhibit two types of channel activity in lipid membranes: classical cation-selective channels and large unselective pores. The induction of unselective pores was shown here to strongly depend on the redox state of the membrane-bathing solution, if the gramicidin analogue contained a cysteine residue in the sequence GSGPKKKRKVC attached to the C-terminus. In particular, the addition of H2O2 led to an increase in the transmembrane current and the loss of cationic selectivity on planar bilayer lipid membranes and an increase in the carboxyfluorescein leakage of liposomes. The effect was observed at high concentration of the peptide while was absent at the single-channel level. It was concluded that oxidation led to possible formation of dimers of the peptide, which promoted the formation of large unselective pores.     

Effects of calcium on the gramicidin A single channel in phosphatidylserine membranes

In phosphatidylserine membranes the decrease in the conductance of the gramicidin A single channel caused by calcium is attributed to a reduction of surface potential and to a direct blocking of the pore (Apell et al. 1979).The aim of this paper is to make a, quantitative evaluation of these two effects. We recorded the conductance of gramicidin single channels in 100 mM KCl in the presence of different amounts of CaCl₂, MgCl₂ or TEACl.The ionic activities at the channel mouth were calculated using the Gouy-Chapman-Stern theory. Our experiments showed that even when the K⁺ activity at the channel mouth was estimated to be the same, the single channel conductance was lower if divalent cations were present. This effect is attributed to a blocking action of these ions.Abbreviations PS phosphatidylserine - TEA tetraethylammonium     

Comparison of gramicidin A and gramicidin M channel conductance dispersities

To explore the possible role of Trp side chains in gramicidin channel conductance dispersity, we studied the dispersity of gramicidin M (gM), a gramicidin variant in which all four tryptophan residues are replaced with phenylalanine residues, and its enantiomer, gramicidin M(-) (gM(-)), and compared them to that of gramicidin A (gA). The conductances of highly purified gM and gM(-) were studied in alkali metal solutions at a variety of concentrations and voltages, in seven different types of lipid, and in the presence of detergent. Like gA channels, the most common gM channel conductance forms a narrow band. However, unlike gA channels, where the remaining 5-30% of channel conductances are broadly distributed below (and slightly above) the main band, in gM there is a narrow secondary band with <50% of the main peak conductance. This secondary peak was prominent in NaCl and KCl, but significantly diminished in CsCl and RbCl. Under some conditions, minor components can be observed with conductances yet lower than the secondary peak. Interconversions between the primary conductance state and these yet lower conductance states were observed. The current-voltage relations for both primary and secondary gM channel types have about the same curvature. The mean lifetime of the secondary channel type is below one third that of the primary type. The variants represent state deviations in the peptide or adjacent lipid structure.     

Effect of avidin on channel kinetics of biotinylated gramicidin

Membrane protein functioning basically depends on the supramolecular structure of the proteins which can be modulated by specific interactions with external ligands. The effect of a water-soluble protein bearing specific binding sites on the kinetics of ionic channels formed by gramicidin A (gA) in planar bilayer lipid membranes (BLM) has been studied using three independent approaches: (1) sensitized photoinactivation, (2) single-channel, and (3) autocorrelation measurements of current fluctuations. As shown previously [Rokitskaya, T. I., et al. (1996) Biochim. Biophys. Acta 1275, 221], the time course of the flash-induced current decrease in most cases follows a single-exponential decay with an exponential factor (tau) that corresponds to the gA single-channel lifetime. Addition of avidin does not affect tau for gA channels, but causes a dramatic increase in tau for channels formed by gA5XB, a biotinylated analogue of gA. This effect is reversed by addition of an excess of biotin to the bathing solution. The average single-channel duration of gA5XB was about 3.6 s as revealed by single-channel recording of the BLM current. After prolonged incubation with avidin, a long-lasting open state of the gA5XB channel appeared which did not close for more than 10 min. The data on gA5XB photoinactivation kinetics and single-channel measurements were confirmed by analysis of the corresponding power spectra of the current fluctuations obtained in the control, in the presence of avidin, and after the addition of biotin. We infer that avidin produces a deceleration of gA5XB channel kinetics by motional restriction of gA5XB monomers and dimers upon the formation of avidin and gA5XB complexes, which would stabilize the channel state and thus increase the single-channel lifetime.     

Effects of green tea catechins on gramicidin channel function and inferred changes in bilayer properties

Green tea's health benefits have been attributed to its major polyphenols, the catechins: (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), and epicatechin (EC). Catechins (especially EGCG) modulate a wide range of biologically important molecules, including many membrane proteins. Yet, little is known about their mechanism(s) of action. We tested the catechins' bilayer-modifying potency using gramicidin A (gA) channels as molecular force probes. All the catechins alter gA channel function and modify bilayer properties, with a 500-fold range in potency (EGCG>ECG?EGC>EC). Additionally, the gallate group causes current block, as evident by brief downward current transitions (flickers).     

The channel properties of possible gramicidin dimers

Extending previous work (Sung & Jordan, 1987 a, Biophys. J. 51, 661-672; 1988, Biophys. J.54, 519-526), we describe channel properties of five possible gramicidin dimers by studying dimerization energies and axial electrical potentials. Unlike the head-to-head dimer (the predominant channel former), both tail-to-tail and head-to-tail dimers with the same beta-helical monomer structure as the head-to-head dimer only form four intermonomer hydrogen bonds and are much less stable. Were channels formed from these dimers to be observed, their electrical potential profiles suggest that they should be cation selective, probably conduct less than the head-to-head dimer, have a central cation binding site, bind cations preferentially if crystallizable, and in the case of the head-to-tail dimer, rectify. Like the antiparallel double stranded helical dimer (a possible minor conducting pathway) the parallel double stranded helical dimer has 28 interstrand hydrogen bonds, but its hydrogen bond network is quite distorted and it is much less stable. If it formed, its electrical potential profile suggests that it would be cation selective, bind anions preferentially if crystallizable, rectify, and at high enough voltages, might exhibit a conductance greater than that of the antiparallel form.     

Interfacial polar interactions affect gramicidin channel kinetics

Critical to biological processes such as secretion and transport, protein-lipid interactions within the membrane and at the membrane-water interface still raise many questions. Here we examine the role of lipid headgroups in these interactions by using gramicidin A (gA) channels in planar bilayers as a probe. We show that although headgroup demethylation from phosphatidylcholine (DOPC) to phosphatidylethanolamine decreases the lifetime of gA channels by an order of magnitude in accordance with the currently accepted hydrophobic mismatch mechanism, our findings with diether-DOPC suggest the importance of the headgroup-peptide interactions. According to our x-ray diffraction measurements, this lipid has the same hydrophobic thickness as DOPC but increases gA lifetime by a factor of 2. Thus we demonstrate that peptide-headgroup interactions may dominate over the effect of hydrophobic mismatch in regulating protein function.     

Effects of phenylalanine substitutions in gramicidin A on the kinetics of channel formation in vesicles and channel structure in SDS micelles

The common occurrence of Trp residues at the aqueous-lipid interface region of transmembrane channels is thought to be indicative of its importance for insertion and stabilization of the channel in membranes. To further investigate the effects of Trp-->Phe substitution on the structure and function of the gramicidin channel, four analogs of gramicidin A have been synthesized in which the tryptophan residues at positions 9, 11, 13, and 15 are sequentially replaced with phenylalanine. The three-dimensional structure of each viable analog has been determined using a combination of two-dimensional NMR techniques and distance geometry-simulated annealing structure calculations. These phenylalanine analogs adopt a homodimer motif, consisting of two beta6.3 helices joined by six hydrogen bonds at their NH2-termini. The replacement of the tryptophan residues does not have a significant effect on the backbone structure of the channels when compared to native gramicidin A, and only small effects are seen on side-chain conformations. Single-channel conductance measurements have shown that the conductance and lifetime of the channels are significantly affected by the replacement of the tryptophan residues (Wallace, 2000; Becker et al., 1991). The variation in conductance appears to be caused by the sequential removal of a tryptophan dipole, thereby removing the ion-dipole interaction at the channel entrance and at the ion binding site. Channel lifetime variations appear to be related to changing side chain-lipid interactions. This is supported by data relating to transport and incorporation kinetics.     

Formamidinium-induced dimer stabilization and flicker block behavior in homo- and heterodimer channels formed by gramicidin A and N-acetyl gramicidin A.

Compared to the N-formyl gramicidin A (GA), the N-acetyl gramicidin A (NAG) channel has unchanged conductance in 1 M NH4+ (gamma NN/gamma GG = 1, conductance ratio) but reduced conductance in 1 M K+ (gamma NN/gamma GG = 0.6) methylammonium (gamma NN/gamma GG = 0.3), and formamidinium (gamma NN/gamma GG = 0.1) solutions. Except with formamidinium, "flicker blocks" are evident even at low cutoff frequencies. For all cations studied, channel lifetimes of N-acetyl homodimers (NN) are approximately 50-fold shorter than those of the GA homodimer (GG). The novel properties of GA channels in formamidinium solution (supralinear current-voltage relations and dimer stabilization (Seoh and Busath, 1993)) also appear in NN channels. The average single channel lifetime in 1 M formamidinium solution at 100 mV is 6-7-fold longer than in K+ and methylammonium solutions and, like in the GA channel, significantly decreases with increasing membrane potential. Experiments with mixtures of the two peptides, GA and NAG, showed three main conductance peaks. Oriented hybrids were formed utilizing the principle that monomers remain in one leaflet of the bilayer (O'Connell et al., 1990). With GA at the polarized side and NAG at the grounded side, at positive potentials (in which case hybrids were designated GN) and at negative potentials (in which case hybrids were designated NG), channels had the same conductances and channel properties at all potentials studied. Flicker blocks were not evident in the hybrid channels, which suggests that both N-acetyl methyl groups at the junction of the dimer are required to cause flickers. Channel lifetimes in hybrids are only approximately threefold shorter than those of the GG channels, and channel conductances are similar to those of GG rather than NN channels. We suggest that acetyl-acetyl crowding at the dimeric junction in NN channels cause dimer destabilization, flickers, and increased selectivity in N-acetyl gramicidin channels.     

Effects of halothane on feeding motor activity in the snail Lymnaea stagnalis

Snails exposed to the general anaesthetic halothane show an increase in biting plus mouthing movements. Perfusion of the isolated CNS with halothane leads to a period of increased spiking activity, followed by suppression of activity in identified feeding motoneurones in the buccal ganglia. Synaptic inputs to motoneurones from interneurones of the buccal feeding pattern generator are differentially affected. Possible mechanisms underlying the generation of motoneuronal bursting in the presence of halothane are examined.     

The structure of the gramicidin A transmembrane channel

Gramicidin A is a linear polypeptide antibiotic that facilitates the diffusion of monovalent cations across lipid bilayer membranes by forming channels. It has been proposed that the conducting channel is a dimer which is in equilibrium with nonconducting monomers in the membrane. To directly test this model in several independent ways, we have prepared and purified a series of gramicidin C derivatives. All of these derivatives are fully active analogs of gramicidin A, and each derivative has a useful chromophore esterified to the phenolic hydroxyl of tyrosine #11. Simultaneous conductance and fluorescence measurements on planar lipid bi-layer membranes containing dansyl gramicidin C yielded four conclusions: (1) A plot of the logarithm of the membrane conductance versus the logarithm of the membrane fluorescence had a slope of 2.0 ± 0.3, over a concentration range for which nearly all the gramicidin was monomeric. Hence, the active channel is a dimer of the nonconducting species. (2) In a membrane in which nearly all of the gramicidin was dimeric, the number of channels was approximately equal to the number of dimers. Thus, most dimers are active channels and so it should be feasible to carry out spectroscopic studies of the conformation of the transmembrane channel. (3) The association constant for dimerization is more than 1,000-fold larger in a glycerolester membrane with 26 Å-hydrocarbon thickness than in a 47 Å-glycerolester membrane. The dimerization constant in a 48 Å-phosphatidyl choline membrane was 200 times larger than in a 47 Å-glycerolester membrane, showing that it depends on the type of lipid as well as on the thickness of the hydrocarbon core. (4) We were readily able to detect 10^?14 mole cm^?2 of dansyl gramicidin C in a bilayer membrane, which corresponds to 60 fluorescent molecules per square μm. The fluorescent techniques described here should be sufficiently sensitive for fluorescence studies of reconstituted gates and receptors in planar bilayer membranes. An alternative method of determining the number of molecules of gramicidin in the channel is to measure the fraction of hybrid channels present in a mixture of 2 chemically different gramicidins. The single-channel conductance of p-phenylazo-benzene-sulfonyl ester gramicidin C (PABS gramicidin C) was found to be 0.68 that of gramicidin A. In membranes containing a mixture of these 2 gramicidins, a hybrid channel was evident in addition to 2 pure channels. The hybrid channel conductance was 0.82 that of gramicidin A. Fluorescence energy transfer from dansyl gramicidin C to diethylamino-phenylazobenzene-sulfonyl ester gramicidin C (DPBS gramicidin C), provided an independent way to measure the fraction of hybrid channels on liposomes. For both techniques the fraction of hybrid channels was found to be 2ad where a² and d² were the fractions of the 2 kinds of pure channels. This result strongly supports a dimer channel and the hybrid data excludes the possibility of a tetramer channel. The study of hybrid species by conductance and fluorescence techniques should be generally useful in elucidating the subunit structure of oligomeric assemblies in membranes. The various models which have been proposed for the conformation of the gramicidin transmembrane channel are briefly discussed.     

Gravity dependency of the gramicidin A channel conductivity

Theoretical investigations involving the membrane-solution interface have revealed that the density of the solution varies appreciably within interfacial layers adjacent to charged membrane surfaces. The hypothesis that gravity interacts with this configuration and modifies transport rates across horizontal and vertical membranes differently was supported by initial experiments with gramicidin A channels in phosphatidylserine (PS) membranes in 0.1 M KCl. Channel conductivity was found to be about 1.6 times higher in horizontal membranes than in vertical membranes. Here we present the results of further experiments with gramicidin A channels (incorporated into charged PS- and uncharged phosphatidylcholine (PC) membranes in KCl- and CsCl-solutions) to demonstrate that the hypothesis is more generally applicable. Again, channel conductivity was found to be higher in horizontal PS membranes by a factor of between 1.20 and 1.75 in 0.1 M CsCl. No difference in channel conductivity was found for uncharged PC membranes in 0.1 M KCl and in 0.1 M CsCl. However, for PC membranes in 0.05 M KCl the channel conductivity was significantly higher in horizontal membranes by a factor of between 1.07 and 1.14. These results are consistent with the results of our model calculations of layer density and extension, which showed that the layer formation is enhanced by increasing membrane surface charge and decreasing electrolyte ion concentration. The mechanism of gravity interaction with membrane transport processes via interface reactions might be utilized by biological systems for orientational behaviour in the gravity field, which has been observed even for cellular systems. Received: 16 October 1995 / Accepted: 23 April 1996     

Influence of acylation on the channel characteristics of gramicidin A.

The influence of acylation on the conductance, average duration, and channel-forming potency of channels formed by gramicidin A analogues was investigated using single-channel and multichannel techniques. Lauroyl-, myristoyl-, palmitoyl-, stearoyl-, and oleoylgramicidin A were prepared by covalent coupling of that fatty acid to the C-terminal ethanolamine group. Acylation of gramicidin A does not affect the single-channel conductance or the minichannel frequency in diphytanoylphosphatidylcholine/n-decane black lipid membranes. However, the average duration of all acylgramicidin channels was increased approximately 5-fold as compared to unmodified gramicidin A, which has a duration of 0.9 s at 200-mV applied potential. Somewhat surprisingly the rate of channel formation of the acylgramicidins is decreased relative to gramicidin A: lauroyl- and stearoylgramicidin are approximately 200 times less effective in channel formation as compared to gramicidin A. We conclude that channels formed by the acylgramicidins and by gramicidin A are structurally and conformationally equivalent.     

Orientation of gramicidin A transmembrane channel. Infrared dichroism study of gramicidin in vesicles.

Polarized infrared spectroscopy has been used to investigate the orientation of gramicidin A incorporated in dimyristoylphosphatidylcholine liposomes. Dichroism measurements of the major lipid (C = O ester, PO2-, CH2) and peptide (amide A, I, II) bands were performed on liposomes (with or without gramicidin) oriented by air-drying. The mean orientation of the lipid groups and of the pi LD helix chain in the gramicidin has been determined. It can be inferred from infrared frequencies of gramicidin that the dominant conformation of the peptide in liposomes cannot be identified to the antiparallel double-helical dimer found in organic solution. No shift in lipid frequencies was observed upon incorporation of gramicidin in the liposomes. However, a slight reorganization of the lipid hydrocarbon chains which become oriented more closely to the normal to the bilayer is evidenced by a change in the dichroism of the CH2 vibrations. The infrared dichroism results of gramicidin imply a perpendicular orientation of the gramicidin transmembrane channel with the pi LD helix axis at less than 15 degrees with respect to the normal to the bilayer.