Formation of ionic channels in black lipid membranes by succinic derivatives of Gramicidin A

Summary Different succinyl derivatives of Gramicidin A were synthesized and their activity was investigated with different methods on lipid bilayer membranes. The succinyl derivatives of Gramicidin A can be classified as three different types, the O-succinyl derivative, the N-succinyl derivative and the N-O-succinyl derivative of Gramicidin A. An O-pyromellityl-N-succinyl gramicidin was synthesized which can be attributed to the latter class. It was found that O-succinyl gramicidin behaves like the unmodified Gramicidin A despite a charge effect on single-channel conductance, arising from the negative charge of the succinic residue, at the mouth of the channel. The activity of N-succinyl and N-O-succinyl gramicidin and of O-pyromellityl-N-succinyl-gramicidin depends strongly on the pH of the electrolyte solution. It is demonstrated that at low pH (5) the N-succinyl derivatives show high activity, whereas at high pH (7) the activity is sharply reduced or disappears totally. From these experiments it can be concluded that, for the formation of a dimeric gramicidin channel, the hydrogen of the formyl group can be replaced by a protonated carboxylic group of a succinic residue.Further results, obtained by measurement of the single-channel conductance and of the reaction rate constants for the channel formation, are discussed in terms of the structural basis of the single stranded model for the gramicidin channel. On this basis the double stranded helix can be, excluded and an interesting head-to-head single stranded (_L,D) helical channel is described which contains carboxyl groups at the head-to-head junction.     

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.     

The study of volt-ampere characteristics of ionic channels formed by gramicidin A

A method of measurement of the non-linearity coefficient of volt-ampere characteristics of the type i(U) approximately = U(1 + beta U2) has been developed for ionic channels formed by gramicidin A, using the third harmonic of the membrane current. The shape of the volt-ampere characteristics (VA) of ionic channels formed by gramicidin A did not depend on the antibiotic concentration in the membrane. The coefficient beta of non-linearity of VA of membranes modified by gramicidin A depended on electrolyte concentration "c" and it increased proportionally with the lg c from -17 V-2 at 0.03 mol/l KC1 to 8 V-2 at 3.4 mol/l KCl, and it was zero at co = 0.3 - 1 mol/l KCl. Egg lecithin and glycerol monooleate (GMO) membranes differ in their co values. The substitution of K+ for Li+ of the membrane solvent (n-heptane for n-hexadecane) did not influence the value of beta; the same applied for GMO membranes without any solvent. In a number of membranes, spontaneous change of the non-linearity coefficient with time observed after the membrane formation, as well as jumps of the non-linearity coefficient at a practically unchanged membrane conductivity. An analysis of some theoretical models of the ion transport through the channel has shown that, at voltages above 200 mV, these models provide rather small values of beta, or extremely high VA non-linearity.     

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.     

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.     

Correlation analysis of electrical noise in lipid bilayer membranes: kinetics of gramicidin A channels.

If a membrane contains ion-conducting channels which form and disappear in a random fashion, an electric current which is passed through the membrane under constant voltage shows statistical fluctuations. Information on the kinetics of channel formation and on the conductance of the single channel may be obtained by analyzing the electrical noise generated in a membrane containing a great number of channels. For this purpose the autocorrelation function of the current noise is measured at different concentrations of the channel-forming substance. As a test system for the application of this technique we have used lipid bilayer membranes doped with gramicidin A. From the correlation time of the current noise generated by the membrane, the rate constants of formation (k-R) and dissociation (k-D) of the channels could be determined. In addition, the mean square of the current fluctuations yielded the single-channel conductance lambda. The values of k-R, k-D, and lambda obtained from the noise analysis agreed closely with the values determined by relaxation measurments and single-channel experiments.     

Incorporation of ionic channels from yeast plasma membranes into black lipid membranes.

Recently, patch-clamping of yeast protoplasts has revealed the presence of plasma membrane K+ channels (Gustin, M. C., B. Martinac, Y. Saimi, M. R. Culberston, and C. Kung. 1986. Science (Wash. DC). 233:1195-1197). In this work we show that fusion of purified plasma membranes into planar bilayers allows the study of the yeast channels. The main cationic conductances detected were of 64 and 116 pS, however, larger and smaller conductances have been observed. The two main conductances were sensitive to the K+ channels blockers tetraethylammonium (TEA+) and Ba2+. Bionic experiments indicated that both conductances were K+ selective.     

1/f noise in membranes

The present situation of 1/f noise in the passage of ions across membranes is examined. A survey of biological and synthetic membranes is given at which a l/f frequency dependence has been observed in the spectrum of voltage or current fluctuations. Empirical relations and theories of 1/f noise in membranes are critically discussed. Supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 38 “Membranforschung”     

1/f noise in membranes.

The present situation of 1/f noise in the passage of ions across membranes is examined. A survey of biological and synthetic membranes is given at which a 1/f frequency dependence has been observed in the spectrum of voltage or current fluctuations. Empirical relations and theories of 1/f noise in membranes are critically discussed.     

Ion channels of various types induced in lipid membranes by gramicidin A derivatives carrying a cationic sequence at their C-termini

The channel-forming activity of gramicidin A derivatives carrying positively charged amino acid sequences at their C-termini was studied on planar bilayer lipid membranes and liposomes. We showed previously that, at low concentrations, these peptides form classical cation-selective pores typical of gramicidin A, whereas, at high concentrations, they form large nonselective pores. The ability of the peptides to form nonselective pores, which was determined by the efflux of carboxyfluorescein, an organic dye, from liposomes, decreased substantially as the length of the gramicidin fragment in the series of cationic analogues was truncated. CD spectra showed that large pores are formed by peptides having both beta6.3 single-stranded and beta5.6 double-stranded helical conformations of the gramicidin fragment, with the C-terminal cationic sequence being extended. The dimerization of the peptides by the oxidation of the terminal cysteine promoted the formation of nonselective pores. It was shown that nonselective pores are not formed in membranes of erythrocytes, which may indicate a dependence of the channel-forming ability on the membrane type. The results may be of interest for the directed synthesis of peptides with antibacterial activity.     

Ion channels formed by chemical analogs of gramicidin A.

Channel-forming peptides such as gramicidin A offer the opportunity to study the relationship between chemical structure and transport properties of an ion channel. This article summarizes a number of recent experiments with chemical analogs and derivatives of gramicidin A using artificial lipid bilayer membranes. The introduction of negative charges near the channel mouth leads to an increase in the cation transport rate. Hybrid channels consisting of a neutral and a negatively charged or of a positively and a negatively charged half-channel may be formed. The current-voltage characteristic of these hybrid channels exhibits a pronounced asymmetry.Experiments with charged derivatives of gramicidin A have been used in order to distinguish between different structural models of the dimeric channel; these studies strongly support Urry's model of a single-stranded, head-to-head associated helical dimer. In a further set of experiments gramicidin analogs with modified amino acid sequence were studied. It was found that a single substitution (tryptophan replaced by phenylalanine) leads to marked changes in the conductance of the channel. Analogs with a simplified amino acid sequence such as (L-Trp-D-Leu)7-L-Trp or L-Trp-Gly-(L-Trp-D-Leu)6-L-Trp are able to form cation permeable channels with similar properties as gramicidin A.     

Properties of gramicidin A channels in erythrocyte membranes

Studies for the cation permeability properties of the gramicidin A channel in erythrocyte membranes are presented. It is shown that gramicidin A interacts with the membrane in a cooperative manner, creating aggregates of the antibiotic molecules in the lipid lattice of the membrane. Cationic channels exist in these aggregates with the following order of selectivity: Rb+ greater than Cs+ greater K+ greater than Na+. The cation permeability of the channels depends on the media surrounding the membrane. This finding has been explained on the basis of Hodgkin-Keynes theory for single-file ion diffusion through extra-narrow pores.     

The ionic channels formed by cholera toxin in planar bilayer lipid membranes are entirely attributable to its B-subunit.

The interaction of cholera toxin with planar bilayer lipid membranes (BLM) at low pH results in the formation of ionic channels, the conductance of which can be directly measured in voltage-clamp experiments. It is found that the B-subunit of cholera toxin (CT-B) also is able to induce ionic channels in BLM whereas the A-subunit is not able to do it. The increase of pH inhibited the channel-forming activity of CT-B. The investigation of pH-dependences of both the conductance and the cation-anion selectivity of the CT-B channel allowed us to suggest that the water pore of this channel is confined to the B-subunit of cholera toxin. The effective diameter of the CT-B channels water pores was directly measured in BLM and is equal to 2.1 +/- 0.2 nm. The channels formed by whole toxin and its B-subunit exhibit voltage-dependent activity. We believe these channels are relevant to the mode of action of cholera toxin and especially to the endosomal pathway of the A-subunit into cells.     

A mechanism for 1/f noise in diffusing membrane channels.

The diffusion polarization effect is shown to produce 1/f (omega-1) noise in the conductance of membranes containing diffusing ion channels. The magnitude and frequency range of the effect are calculated.     

Radiation inactivation of ion channels formed by gramicidin A. Protection by lipid double bonds and by alpha-tocopherol.

The conductance induced by the channel-forming peptide gramicidin A in lipid membranes is reduced by many orders of magnitude on exposure of the membrane and its aqueous environment to ionizing radiation. This results from an interaction of free radicals of water radiolysis with the tryptophan residues of gramicidin A. The sensitivity of the ion channels towards irradiation is strongly reduced in the presence of either vitamin E or of highly unsaturated lipids. An increase of the D37 dose up to a factor of 50 was found. The phenomena are interpreted via a reduction of the effective concentration of free radicals (such as OH.) in the membrane by reaction with unsaturated fatty acid residues or with vitamin E.     

Photolysis of gramicidin A channels in lipid bilayers

We have tested the hypothesis that peptide tryptophan groups can control the ionic conductance of transmembrane channels. We report here that single gramicidin A channels change conductance state when the peptide tryptophans are flash photolyzed with ultraviolet light. The current flow through planar lipid bilayers containing multiple gramicidin A channels decreases irreversibly when exposed to ultraviolet light. The current-loss action spectrum peaks sharply at the 280 nm absorption maximum of the gramicidin A tryptophans. Gramicidin channel sensitivity to ultraviolet light is found to be about 20-fold higher than that of frog node sodium channels which is even more than expected based on the high tryptophan content of gramicidin. Channels which survive an ultraviolet light exposure exist in a wide variety of different low-conductance forms. The broad distribution of the single channel conductance of these partially photolyzed channels is attributable to the loss of different combinations of the dimer's normal complement of eight tryptophans per channel. Flash photolysis of single channels results in discrete conductance state changes. Partially photolyzed single channels manifest a further conductance cascade when exposed to a second flash of ultraviolet light. Analysis of the photolysis conductance turn-off process indicates that gramicidin A is a multistate electrochemical unit where the peptide tryptophan groups can modulate the flow of ions through the transmembrane channel.     

Ion channels of various types induced in lipid membranes by gramicidin a derivatives carrying a cationic sequence at their C-termini

The channel-forming activity of gramicidin A derivatives carrying positively charged amino acid sequences at their C-termini was studied on planar bilayer lipid membranes and liposomes. We showed previously (FEBS Lett., 2005, vol. 579, pp. 5247–5252) that, at low concentrations, these peptides form classical cation-selective pores typical of gramicidin A, whereas, at high concentrations, they form large nonselective pores. The ability of the peptides to form nonselective pores, which was determined by the efflux of carboxyfluorescein, an organic dye, from liposomes, decreased substantially as the length of the gramicidin fragment in the series of cationic analogues was truncated. CD spectra showed that large pores are formed by peptides having both β^6.3 single-stranded and β^5.6 double-stranded helical conformations of the gramicidin fragment, with the C-terminal cationic sequence being extended. The dimerization of the peptides by the oxidation of the terminal cysteine promoted the formation of nonselective pores. It was shown that nonselective pores are not formed in membranes of erythrocytes, which may indicate a dependence of the channel-forming ability on the membrane type. The results may be of interest for the directed synthesis of peptides with antibacterial activity.