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.     

Temperature-jump and voltage-jump experiments at planar lipid membranes support an aggregational (micellar) model of the gramicidin A ion channel

Summary The kinetics of formation and dissociation of channels formed by gramicidin A and two analogues in planar lipid membranes was studied using a laser temperature-jump technique developed earlier [Brock, W., Stark, G., Jordan, P.C. (1981),Biophys. Chem. 13:329–348]. The time course of the electric current was found to agree with a single exponential term plus a linear drift. In case of gramicidin A the relaxation time was identical to that reported for V-jump experiments [Bamberg, E., Läuger, P. (1973),J. Membrane Biol. 11:177–194], which were interpreted on the basis of a dimerization reaction. The same results were obtained for gramicidin A and for chemically dimerized malonyl-bis-desformylgramicidin. It is therefore suggested that the dimerization represents a parallel association of two dimers to a tetramer. There is evidence that the tetramer, contrary to the presently favored dimer hypothesis, is the smallest conductance unit of an active gramicidin channel. An additional V-jump-induced relaxation process of considerably larger time constant is interpreted as a further aggregation of gramicidin dimers.Abbreviations GA gramicidin A - OPG O-pyromellitylgramicidin A - MBDG malonyl-bis-desformylgramicidin     

Gramicidin tryptophans mediate formamidinium-induced channel stabilization.

Compared with alkali metal cations, formamidinium ions stabilize the gramicidin A channel molecule in monoolein bilayers (Seoh and Busath, 1993a). A similar effect is observed with N-acetyl gramicidin channel molecules in spite of the modified forces at the dimeric junction (Seoh and Busath, 1993b). Here we use electrophysiological measurements with tryptophan-to-phenylalanine-substituted gramicidin analogs to show that the formamidinium-induced channel molecule stabilization is eliminated when the four gramicidin tryptophans are replaced with phenylalanines in gramicidin M-. This suggests that the stabilization is mediated by the tryptophan side chains. Tryptophan residues 9, 13, and 15 must cooperate to produce the effect because replacement of any one of the three with phenylalanine significantly reduces stabilization; replacement of Trp-11 with phenylalanine causes negligible decrease in stabilization. In addition, formamidinium-related current-voltage supralinearity and open-channel noise are absent with gramicidin M-. When the lipid bilayer was formed with monoolein ether rather than monoolein ester, the channel lifetimes were reduced markedly and, at low voltage and relative to those in KCl solution, were decreased by a factor of 2, whereas the open-channel noise was unaffected and the current-voltage relation was only modestly affected. These results suggest that formamidinium modifies the state of the tryptophan side chains, which, in turn, affects channel lifetime, current-voltage supralinearity, and open-channel noise through interactions with water or lipid headgroup atoms including the lipid ester carbonyl.     

Preparation and properties of O-dansyltyrosine gramicidin C.

Gramicidins A, B, and C are a family of poly-peptide antibiotics which facilitate the passive diffusion of alkali cations and protons through lipid bilayer membranes. It is clear that gramicidin forms a multimeric transmembrane channel and it has been suggested that the channel is an io-conducting dimer in equilibrium on the membrane with non-conducting monomer. We describe the preparation and purification of a derivative of gramicidin C in which the phenolic hydroxyl of the tyrosine at position 11 has been esterified to 8-dimethylaminonaphthalene-1-sulfonate (dansyl). This derivative fluoresces strongly in the visible with an emission maximun in dioxane of 530 nm, an emission lifetime of 16 ns, and a quantum yield of 0.8. Veatch et al. ((1975),J. Mol. Biol. 99, 75) have shown this 0-dansyltyrosine gamicidin C to be a fully active analogue of gramicidin A in artificial lipid bilayer membranes. We here utilize this derivative to further characterize the state of aggregation and rotational mobility of the four interconvertible conformational species formed by gramicidin in nonpolar organic solvents (Veatch et al. (1974), Biochemsitry 13, 5249; Veatch and Blout (1974), Biochemistry 13, 5257). Fluorescence energy transfer from the tryptophans of gramicidin A to the 0-dansyltyrosine of this derivatives supports the conclusion that all of these gramicidin isolated species are aggregates. Decay of fluorescence polarization anisotropy measurements yield a rotational correlation time of 1 ns for the 0-dansyltyrosine chromophore in ethanol in good agreement with the more detailed information previously obtained by 13C-nuclear magnetic resonance for the monomer in dimethyl sulfoxide (Fossel et al. (1974), Biochemistry 13, 5264). However, it is likely that the chromophore has much more rotational mobility than the rest of the gramicidin molecule in the aggregated comformational states.     

Effect of dipole modifiers on the kinetics of sensitized photoinactivation of gramicidin channels in bilayer lipid membranes

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

Single channel properties of D-Leu2-gramicidin A. Side chain modulation of channel lifetime

The channel forming properties of synthetic gramicidin A and DLeu2-gramicidin A were compared in black lipid membranes. The most probable single channel conductance was identical for both derivatives but in each case a distribution of smaller channel sizes was observed. However, the lifetime of the channel formed by DLeu2-gramicidin A was considerably shorter than for gramicidin A. The DLeu2 substitution is considered to interfere with the head to head hydrogen bonding which forms the conducting dimer, thus destabilizing the dimeric structure of the channel and reducing the lifetime. This represents the first demonstration of side-chain modulation of channel lifetime.     

A single-channel sensor based on gramicidin controlled by molecular recognition at bilayer lipid membranes containing receptor

A novel ion-channel sensor based on a membrane bound receptor and a single gramicidin channel is described, in which the binding of an analyte to the membrane bound receptor modulates the single-channel activity of gramicidin. The sensor is composed of a planar bilayer lipid membrane (BLM) containing biotin-labeled phosphatidylethanolamine as receptor for avidin and gramicidin as signal transducer. When the receptor catches an analyte (avidin or ferritin-labeled avidin (FA)) at the membrane surface, the bilayer structure is locally distorted and the gramicidin monomer/dimer kinetics is modulated in a manner that the fraction of channel opening with a short lifetime ( < or = 100 ms) to the total opening events increases. The fraction was found to increase with the concentration of avidin from 1.0 x 10(-9) to 1.0 x 10(-6) M and of FA from 1.0 x 10(-9) to 1.0 x 10(-8) M. With dinitrophenyl-labeled PE embedded as receptor in the BLM for monoclonal anti-dinitrophenyl antibody (anti-DNP), the fraction of channel openings ( < or = 100 ms) increased with the concentration of anti-DNP from 2.0 x 10(-9) to 2.0 x 10(-7) g/ml. Bovine serum albumin (BSA) and anti-BSA antibody caused no changes in the channel opening. The possible mechanism of analyte-induced modulation of single-channel activity of gramicidin is also discussed.     

Calcium-dependent association of annexins with lipid bilayers modifies gramicidin A channel parameters

In order to examine whether calcium-dependent binding of annexin to acidic phospholipids could change the lipid bilayer environment sufficiently to perturb channel-mediated transmembrane ion-transport, gramicidin A channel activity in planar lipid bilayers was investigated in the presence of calcium and annexins II, III or V. The experiments were performed with membranes consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in 300 mM KCl solution buffered to pH 7.4 and with either 0.1 or 1 mM calcium added to the solution. Annexin (1 microM) was subsequently applied to the cis side of the membrane. All three annexins (II, III and V) when tested at 1 mM calcium decreased the gramicidin single-channel conductance. Annexins II and III increased the mean lifetime of the channels whereas annexin V seemed to have no influence on the mean lifetime. Since the lifetime of gramicidin A channels is a function of the rate constant for dissociation of the gramicidin dimer, which is dependent on the physical properties of the lipid phase, binding of annexins II and III seems to stabilize the gramicidin channel owing to a change of the bilayer structure.     

Single channel properties of d-Leu2-gramicidin A side chain modulation of channel lifetime

The channel forming properties of synthetic gramicidin A and dLeu²-gramicidin A were compared in black lipid membranes. The most probable single channel conductance was identical for both derivatives but in each case a distribution of smaller channel sizes was observed. However, the lifetime of the channel formed by dLeu²-gramicidin A was considerably shorter than for gramicidin A. The dLeu² substitution is considered to interfere with the head to head hydrogen bonding which forms the conducting dimer, thus destabilizing the dimeric structure of the channel and reducing the lifetime. This represents the first demonstration of side-chain modulation of channel lifetime.     

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.     

Monovalent and Multivalent Binding of Streptavidin to Biotinylated Gramicidin Affects the Kinetic Properties of the Ion Channel

Biotin-avidin (or streptavidin) high affinity binding has been widely applied as a universal tool for basic research as well as diagnostic and therapeutic purposes. Here we studied the interaction of streptavidin with ionic channels formed by biotinylated gramicidin in planar bilayer lipid membranes (BLM) using the method of sensitized photoinactivation. As shown previously, the addition of streptavidin leads to a profound increase in the lifetime (tau) of gA5XB, a biotinylated analog of gramicidin A with a linker arm of five aminocaproyl groups (Rokitskaya et al. (2000) Biochemistry, 39, 13053-13058). The present study has revealed that the increase in tau is related to multivalent interaction of streptavidin with biotinylated gramicidin, i.e., to formation of a complex of streptavidin with several gramicidin channels, whereas binding of streptavidin to a single channel does not change the value of tau. A rather long linker arm attaching biotin to the C-terminus of gramicidin appeared to be required for the multivalent interaction of streptavidin with gramicidin channels, as the increase in tau was not observed with channels formed by gA2XB, the biotinylated gramicidin analog with a linker arm comprising only two aminocaproyl groups. However, the formation of a stoichiometric (1 : 1) complex of streptavidin with gA2XB apparently occurred. The multivalent interaction of streptavidin with gA5XB disappeared if biotinylated lipids were included into the diphytanoylphosphatidylcholine membrane. It is suggested that the slowing of gramicidin channel kinetics provoked by streptavidin binding is due to membrane-mediated elastic interactions between two neighboring channels.     

Ionic channels in Langmuir-Blodgett films imaged by a scanning tunneling microscope.

The molecular structure of channels formed by gramicidin A in a lipid membrane was imaged by a scanning tunneling microscope operating in air. The mono- and bimolecular films of lipid with gramicidin A were deposited onto a highly oriented pyrolitic graphite substrate by the Langmuir-Blodgett technique. It has been shown that under high concentration gramicidin A molecules can form in lipid films a quasi-regular, densely packed structure. Single gramicidin A molecules were imaged for the first time as well. The cavity of 0.4 +/- 0.05 nm in halfwidth was found on the scanning tunneling microscopy image of the gramicidin A molecule. The results of direct observation obtained by means of scanning tunneling microscope are in good agreement with the known molecular model of gramicidin A. It was shown that gramicidin A molecules can exist in a lipid monolayer as individual molecules or combined into clusters. The results demonstrate that scanning tunneling microscope can be used for high spatial resolution study of ionic channel structure.     

Low conductance gramicidin A channels are head-to-head dimers of beta 6.3-helices.

Weakly conductive, atypical channels were observed to form from highly purified Val1-gramicidin A in planar lipid bilayer membranes. The structure of these low-conductance channels (minis) was investigated by a detailed study of their channel forming characteristics. The possibility that minis originate from primary structural analogs or degradation products of gramicidin was considered and ruled out. In particular, spontaneous conductance changes in single channels demonstrated that minis can derive directly and reversibly from "standard" channels having the most common conductance level. The fraction of channels which are minis does not vary with changes in membrane gramicidin concentration, indicating that mini and standard channels have the same molecularity, that is, both are dimers. The mean lifetime of mini channels is only slightly shorter than that of standard channels, indicating that the six hydrogen bonds that stabilize the head-to-head dimer are minimally affected in minis. The fraction of channels which are minis is unaffected by the ionic strength, ionic composition, or pH of the bathing solution; it is also unaffected by the lipid composition of the bilayer. These findings are consistent with the hypothesis that minis arise from minor changes in the conformation of the Val1-gramicidin A molecule near the channel entrance or exit.     

Hydrophobic coupling of lipid bilayer energetics to channel function

The hydrophobic coupling between membrane-spanning proteins and the lipid bilayer core causes the bilayer thickness to vary locally as proteins and other "defects" are embedded in the bilayer. These bilayer deformations incur an energetic cost that, in principle, could couple membrane proteins to each other, causing them to associate in the plane of the membrane and thereby coupling them functionally. We demonstrate the existence of such bilayer-mediated coupling at the single-molecule level using single-barreled as well as double-barreled gramicidin channels in which two gramicidin subunits are covalently linked by a water-soluble, flexible linker. When a covalently attached pair of gramicidin subunits associates with a second attached pair to form a double-barreled channel, the lifetime of both channels in the assembly increases from hundreds of milliseconds to a hundred seconds--and the conductance of each channel in the side-by-side pair is almost 10% higher than the conductance of the corresponding single-barreled channels. The double-barreled channels are stabilized some 100,000-fold relative to their single-barreled counterparts. This stabilization arises from: first, the local increase in monomer concentration around a single-barreled channel formed by two covalently linked gramicidins, which increases the rate of double-barreled channel formation; and second, from the increased lifetime of the double-barreled channels. The latter result suggests that the two barrels of the construct associate laterally. The underlying cause for this lateral association most likely is the bilayer deformation energy associated with channel formation. More generally, the results suggest that the mechanical properties of the host bilayer may cause the kinetics of membrane protein conformational transitions to depend on the conformational states of the neighboring proteins.     

Mixed monolayers of linear gramicidins and phospholipid. Surface pressure and surface potential studies.

The behavior of two gramicidins incorporated into lipid monolayers is analyzed on the basis of the force and surface potential area curves. It is shown that the position of the gramicidins (helical axis parallel or perpendicular to the interface) depends on the monolayer pressure and that these molecules are not miscible with dioleoylphosphatidylcholine. Surface potential measurements suggest the existence of a relationship between the single channel characteristics and the surface potential and indicate that the tryptophans are essential for lowering the lipid surface potential in agreement with the single channel behaviour of both gramicidin A and gramicidin M.     

Synthesis and characterization of 1-(13) C-D X Leu12, 14 gramicidin A

The 13C-D-Leu12, 14 gramicidin A was synthesized by the solid phase method incorporating 13C-D-leucine in positions 12 and 14 with about 25 and 50% enrichment, respectively. The pentadecapeptide was removed from the resin by ethanolamine treatment, with the N-protecting group (Boc) still on. After removal of the protecting group, the peptide was formylated and purified by preparative t.l.c. to obtain 13C-D-Leu12, 14 gramicidin A in a very pure state in an overall yield of about 12.5%. The peptide was then thoroughly characterized by HPLC which gave one single peak with the same retention time as that of Val1-gramicidin A of the natural gramicidin mixture. The CD spectra of the synthetic and the HPLC purified natural Val1-GA were obtained and found to be identical, indicating the optical purity of the sample. The synthetic GA was characterized by 13C n.m.r. spectrum and compared with that of natural GA. Single channel conductance parameters of the synthetic GA were determined and found to be indistinguishable from those of natural Val1-GA in lipid bilayer membranes and the mean channel lifetime was found to be as reported earlier by others.     

Bilayers containing calcium ionophore A23187 form channels.

For the first time, based on bilayer membrane conductance experiments, it has been shown that A23187, a carboxylic calcium ionophore, incorporated in lipid bilayers gives single channel currents similar to the well known gramicidin channel. The current characteristics indicate the possibility that the transmembrane ion transport by this important calcium ionophore is initially by a carrier mechanism but with time is by a channel or pore mechanism due to the aggregation of the molecule in a lipid matrix.     

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.     

Gramicidin-induced hexagonal HII phase formation in negatively charged phospholipids and the effect of N- and C-terminal modification of gramicidin on its interaction with zwitterionic phospholipids

The effect of gramicidin on macroscopic structure of the negatively charged membrane phospholipids cardiolipin, dioleoylphosphatidylglycerol and dioleoylphosphatidylserine in aqueous dispersions was investigated and compared with the effect of gramicidin on dioleoylphosphatidylcholine. It was shown by small-angle X-ray diffraction, 31P nuclear magnetic resonance and freeze-fracture electron microscopy that in all these lipid systems gramicidin is able to induce the formation of a hexagonal HII phase. 31P-NMR measurements indicated that the extent of HII phase formation in the various lipids ranged from about 40% to 60% upon gramicidin incorporation in a molar ratio of peptide to lipid of 1 : 10. Next, the following charged analogues of gramicidin were prepared: desformylgramicidin, N-succinylgramicidin and O-succinylgramicidin. The synthesis was verified with 13C-NMR and the effect of these analogues on lipid structure was investigated. It was shown that, as with gramicidin itself, the analogues induce HII phase formation in dioleoylphosphatidylcholine, lower and broaden the bilayer-to-HII phase transition in dielaidoylphosphatidylethanolamine and form lamellar structures upon codispersion with palmitoyllysophosphatidylcholine. Differential scanning calorimetry measurements indicated that, again like gramicidin, in phosphatidylethanolamine the energy content of the gel-to-liquid-crystalline phase transition is not affected by incorporation of the analogues, whereas in phosphatidylcholine a reduction of the transition enthalpy is found. These observations were explained in terms of a similar tendency to self-associate for gramicidin and its charged analogues. The results are discussed in the light of the various factors which have been suggested to be of importance for the modulation of lipid structure by gramicidin.     

Single channel and monolayer studies of acylated gramicidin A: influence of the length of the alkyl group

Three different gramicidin A analogues bearing acyl chains of various length on the ethanolamine moiety have been studied by investigating their single channel behaviour and their monolayer properties. It is shown that the single channel conductance does not depend on the substitution of the ethanolamine OH group and that the channel lifetime is roughly proportional to the length of the alkyl chain. The monolayer study indicates that acylation of gramicidin A produces compounds which have medium-dependent conformations. These acylated compounds are miscible with lipids, while GA is not, and the surface potential is not modified by the esterification of the alcohol group. Offprint requests to: F. Heitz