Characterization of micellar-packaged gramicidin A channels.

The effects of heating, on an aqueous gramicidin A lysolecithin system, were examined by carbon-13 nuclear magnetic resonance (¹³C-NMR), circular dichroism (CD), and sodium-23 nuclear magnetic resonance (²³Na-NMR), and the results are collectively interpreted to indicate micellar-packaging of gramicidin channels and cation occupancy in the channel. ¹³C-NMR of the gramicidin-lysolecithin system demonstrates a decrease in mobility of the micellar lipid on heating which is indicative of incorporation of gramicidin into the hydrophobic core of the micelle. A unique and reproducible CD spectrum is obtained for the heat incorporated state. Sodium-23 spin-lattice relaxation times (T₁) demonstrated sodium interaction to be dependent on heat incorporation. The T₁ identified interaction is blocked by silver ion which is known to block sodium transport through the channel in lipid bilayer studies. The temperature dependence of the sodium-23 line width defines an exchange process with an activation energy of 6.8 kcal/mole which is essentially the same as the activation energy reported for transport through the channel in lecithin bilayer studies, and the sodium exchange process is blocked by thallium ion which is also known to block sodium transport through the channel.     

Formation of non-beta 6.3-helical gramicidin channels between sequence-substituted gramicidin analogues.

Using the linear gramicidins as an example, we have previously shown how the statistical properties of heterodimeric (hybrid) channels (formed between the parent [Val1]gramicidin A (gA) and a sequence-altered analogue) can be used to assess whether the analogue forms channels that are structurally equivalent to the parent channels (Durkin, J. T., R. E. Koeppe II, and O. S. Andersen. 1990. J. Mol. Biol. 211:221-234). Generally, the gramicidins are tolerant of amino acid sequence alterations. We report here an exception. The optically reversed analogue, gramicidin M- (gM-) (Heitz, F., G. Spach, and Y. Trudelle. 1982. Biophys. J. 40:87-89), forms channels that are the mirror-image of [Val1]gA channels; gM- should thus form no hybrid channels with analogues having the same helix sense as [Val1]gA. Surprisingly, however, gM- forms hybrid channels with the shortened analogues des-Val1-[Ala2]gA and des-Val1-gC, but these channels differ fundamentally from the parent channels: (a) the appearance rate of these heterodimers is only approximately 1/10 of that predicted from the random assortment of monomers into conducting dimers, indicating the existence of an energy barrier to their formation (e.g., monomer refolding into a new channel-forming conformation); and (b), once formed, the hybrid channels are stabilized approximately 1,000-fold relative to the parent channels. The increased stability suggests a structure that is joined by many hydrogen bonds, such as one of the double-stranded helical dimers shown to be adopted by gramicidins in organic solvents (Veatch, W. R., E. T. Fossel, and E. R. Blout. 1974. Biochemistry. 13:5249-5256).     

Electrostatic interactions in gramicidin channels

A model based on the solution of the electrostatic potential for a geometry of three dielectric regions associated with a gramicidin A channel (GA) is presented. The model includes a cylindrical dielectric layer to represent the peptide backbone and dipole rings to account for dipolar side chains. Image potential and dipolar contributions for different orientations and positions along the channel are analyzed. The conductance of GA and two analogues obtained by substituting the amino acid at position 1 are studied. The numerical simulation reproduces experimental results (Barrett et al. 1986, Biophys J 49, 673–686) and supports the idea that electrostatic dipole-ion interactions are of primary importance in gramicidin channel function. Correspondence to: G. Martinez     

Spring constants for channel-induced lipid bilayer deformations. Estimates using gramicidin channels.

Hydrophobic interactions between a bilayer and its embedded membrane proteins couple protein conformational changes to changes in the packing of the surrounding lipids. The energetic cost of a protein conformational change therefore includes a contribution from the associated bilayer deformation energy (DeltaGdef0), which provides a mechanism for how membrane protein function depends on the bilayer material properties. Theoretical studies based on an elastic liquid-crystal model of the bilayer deformation show that DeltaGdef0 should be quantifiable by a phenomenological linear spring model, in which the bilayer mechanical characteristics are lumped into a single spring constant. The spring constant scales with the protein radius, meaning that one can use suitable reporter proteins for in situ measurements of the spring constant and thereby evaluate quantitatively the DeltaGdef0 associated with protein conformational changes. Gramicidin channels can be used as such reporter proteins because the channels form by the transmembrane assembly of two nonconducting monomers. The monomerleft arrow over right arrow dimer reaction thus constitutes a well characterized conformational transition, and it should be possible to determine the phenomenological spring constant describing the channel-induced bilayer deformation by examining how DeltaGdef0 varies as a function of a mismatch between the hydrophobic channel length and the unperturbed bilayer thickness. We show this is possible by analyzing experimental studies on the relation between bilayer thickness and gramicidin channel duration. The spring constant in nominally hydrocarbon-free bilayers agrees well with estimates based on a continuum analysis of inclusion-induced bilayer deformations using independently measured material constants.     

Voltage-dependent formation of gramicidin channels in lipid bilayers.

The formation kinetics of gramicidin A channels in lipid bilayer membranes has been characterized as a function of voltage for different solution conditions and membrane composition. The frequency of channel events was measured during the application of voltage ramps and counted in given intervals, a procedure that eliminated the effects of drift in gramicidin concentration. The formation rate was found to increase strongly with voltages up to approximately 50 mV and then to level off slightly. The shape of the voltage dependence was independent of lipid solvent and ramp speed but differed for different ions and different solution concentrations. This suggested an ion occupancy effect on the formation rate that was further supported by the fact that the minimum of the formation rate was shifted toward the equilibrium potential in asymmetric solution concentrations. The effects are explained in terms of a model that contains two contributions to the voltage dependence, a voltage-dependent ion binding to the monomers and a polarization of monomers by the applied electric field and by the occupied ions. The theory is found to give a good fit to experimental data.     

Tandem gramicidin channels cross-linked by streptavidin

The interaction of biotin-binding proteins with biotinylated gramicidin (gA5XB) was studied by monitoring single-channel activity and sensitized photoinactivation kinetics. It was discovered that the addition of streptavidin or avidin to the bathing solutions of a bilayer lipid membrane (BLM) with incorporated gA5XB induced the opening of a channel characterized by approximately doubled single-channel conductance and extremely long open-state duration. We believe that the deceleration of the photoinactivation kinetics observed here with streptavidin and previously (Rokitskaya, T.I., Y.N. Antonenko, E.A. Kotova, A. Anastasiadis, and F. Separovic. 2000. Biochemistry. 39:13053-13058) with avidin reflects the formation of long-lived channels of this type. Both opening and closing of the double-conductance channels occurred via a transient sub-state of the conductance coinciding with that of the usual single-channel transition. The appearance of the double-conductance channels after the addition of streptavidin was preceded by bursts of fast fluctuations of the current with the open state duration of the individual events of 60 ms. The streptavidin-induced double-conductance channels appeared to be inherent only to the gramicidin analogue with a biotin group linked to the COOH terminus through a long linker arm. Including biotinylated phosphatidylethanolamine into the BLM prevented the formation of the double-conductance channels even with the excess streptavidin. In view of the results obtained here, it is suggested that the double-conductance channel represents a tandem of two neighboring gA5XB channels with their COOH termini being cross-linked by the bound streptavidin at both sides of the BLM. The finding that streptavidin induces the formation of the tandem gramicidin channel comprising two channels functioning in concert is considered to be relevant to the physiologically important phenomenon of ligand-induced receptor oligomerization.     

Small iminium ions block gramicidin channels in lipid bilayers.

Guanidinium and acetamidinium, when added to the bathing solution in concentrations of approximately 0.1M, cause brief blocks in the single channel potassium currents from channels formed in planar lipid bilayers by gramicidin A. Single channel lifetimes are not affected indicating that the channel structure is not modified by the blockers. Guanidinium block durations and interblock times are approximately exponential in distribution. Block frequencies increase with guanidinium concentration whereas block durations are unaffected. Increases in membrane potential cause an increase in block frequency as expected for a positively charged blocker but a decrease in block duration suggesting that the block is relieved when the blocker passes through the channel. At low pH, urea, formamide, and acetamide cause similar blocks suggesting that the protonated species of these molecules also block. Arginine and several amines do not block. This indicates that only iminium ions which are small enough to enter the channel can cause blocks in gramicidin channels.     

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.     

The influence of phospholipid polar groups on gramicidin channels.

The influence of well-defined changes in the polar part of phospholipid molecules on the properties of black lipid membranes was studied using a series of phospholipids with identical hydrocarbon chains, but systematically changed polar groups. The hydrocarbon tails of the lipids under study were composed of 1,2-dipentadecylmethylidene glycerol. The polar parts differed in the degree of N-methylation and comprised phosphocholine, -N,N-dimethylethanolamine, -N-methylethanolamine and ethanolamine. Stable black lipid membranes could be formed with the solvents octane, decane, dodecane, tetradecane and hexadecane. The properties of gramicidin-induced single ionic channels changed systematically in membranes from the phosphatidylcholine to the phosphatidylethanolamine analogue, as indicated by an increase in the amplitude lambda of the unit conductance step and a decrease in the average channel life-time or duration tau. The series of tau-values was opposite to that expected from hydrocarbon thickness (specific capacitance). It is suggested that the surface tension gamma is a relevant parameter for the prediction of tau-values.     

Streaming potentials in gramicidin channels measured with ion-selective microelectrodes.

Streaming potentials have been measured for gramicidin channels with a new method employing ion-selective microelectrodes. It is shown that ideally ion-selective electrodes placed at the membrane surface record the true streaming potential. Using this method for ion concentrations below 100 mM, approximately seven water molecules are transported whenever a sodium, potassium, or cesium ion, passes through the channel. This new method confirms earlier measurements (Rosenberg, P.A., and A. Finkelstein. 1978. Interaction of ions and water in gramicidin A channels. J. Gen. Physiol. 72:327-340) in which the streaming potentials were calculated as the difference between electrical potentials measured in the presence of gramicidin and in the presence of the ion carriers valinomycin and nonactin.     

On the helix sense of gramicidin A single channels.

In order to resolve whether gramicidin A channels are formed by right- or left-handed beta-helices, we synthesized an optically reversed (or mirror image) analogue of gramicidin A, called gramicidin A-, to test whether it forms channels that have the same handedness as channels formed by gramicidin M- (F. Heitz et al., Biophys. J. 40:87-89, 1982). In gramicidin M- the four tryptophan residues have been replaced with phenylalanine, and the circular dichroism (CD) spectrum therefore reflects almost exclusively contributions from the polypeptide backbone. The CD spectrum of gramicidin M- in dimyristoylphosphatidylcholine vesicles is consistent with a left-handed helical backbone folding motif (F. Heitz et al., Biophys. Chem. 24:149-160, 1986), and the CD spectra of gramicidins A and A- are essentially mirror images of each other. Based on hybrid channel experiments, gramicidin A- and M- channels are structurally equivalent, while gramicidin A and A- channels are nonequivalent, being of opposite helix sense. Gramicidin A- channels are therefore left-handed, and natural gramicidin A channels in phospholipid bilayers are right-handed beta 6.3-helical dimers.     

Induction of conductance heterogeneity in gramicidin channels

In previous work from our laboratory, 5-10% of the channels formed by [Val1]gramicidin A have conductances that fall outside the narrow range that conventionally has defined the standard gramicidin channel [e.g., see Russell et al. (1986) Biophys. J. 49, 673]. Reports from other laboratories, however, show that up to 50% of [Val1]gramicidin channels have conductances that fall outside the range for standard channels [e.g., see Prasad et al. (1986) Biochemistry 25, 456]. This laboratory-to-laboratory variation in the distribution of gramicidin single-channel conductances suggests that the conductance variants are induced by some environmental factor(s) [Busath et al. (1987) Biophys. J. 51, 79]. In order to test whether extrinsic agents can induce such conductance heterogeneity, we examined the effects of nonionic or zwitterionic detergents upon gramicidin channel behavior. In phospholipid bilayers, detergent addition induces many changes in gramicidin channel behavior: all detergents tested increase the channel appearance rate and average duration; most detergents decrease the conductance of the standard channel; and all but one of the detergents increase the conductance heterogeneity. These results show that the conductance heterogeneity can result from environmental perturbations, thus providing a possible explanation for the laboratory-to-laboratory variation in the heterogeneity of gramicidin channels. In addition, the differential detergent effects suggest possible mechanisms by which detergents can induce the conformational perturbations that result in gramicidin single-channel conductance variations.     

An alternative to Urry's model for the gramicidin transmembrane channels

It is suggested that the conducting transmembrane channels formed by the linear gramicidins A, B and C may be head-to-head (formyl end-to-formyl end) dimers of double-stranded parallel β-helices. This possibility is not in conflict with any of the various experimental findings bearing on the molecular organization of the channels and represents a plausible alternative to Urry's model of a head-to-head dimer of single-stranded β^6.3-helices.     

Asymmetric gramicidin channels: heterodimeric channels with a single F6Val1 residue.

Substitution of Val1 by 4,4,4,4',4',4'-F6Val in [Val1]gramicidin A ([Val1]gA) produces channels in which the effects of amino acid replacements on dimer stability and ion permeation are nonadditive. If only one Val1 (in a symmetric [Val1]gA channel) is substituted by F6Val, the resulting heterodimeric channels are destabilized relative to both homodimeric parent channels and the single-channel conductance of the heterodimeric channels is reduced relative to the parent channels (Russell, E. W. B., L. B. Weiss, F. I. Navetta, R. E. Koeppe II, and O. S. Andersen. 1986. Single-channel studies on linear gramicidins with altered amino acid side chains. Effects of altering the polarity of the side chain at position #1 in gramicidin A. Biophys. J. 49:673; Durkin, J. T., R. E. Koeppe II, and O. S. Andersen. 1990. Energetics of gramicidin hybrid channel formation as a test for structural equivalence. Side-chain substitutions in the native sequence. J. Mol. Biol. 211:221-234). To understand the basis for this destabilization, we have examined further the characteristics of [F6Val1]/[Xxx1]gA heterodimers, where Xxx = Gly, Val, and Ala. These heterodimeric channels show rapid current transitions between (at least) two current levels and display asymmetric i-V characteristics. The orientation of the heterodimers relative to the applied potential was determined by asymmetric addition of the gramicidin analogs, one to each side of a preformed bilayer. The current transitions are most clearly illustrated for [F6Val1]/[Gly1]gA heterodimers, which possess two finite and well defined current levels. Based on the existence of these two conductance states and the analysis of duration and interval distributions, we conclude that the transitions between the two current levels correspond to conformational transitions in "stable" heterodimers. In the case of [F6Val1]/[Val1]gA and [F6Val1]/[Ala1]gA heterodimers, the low-conductance state is indistinguishable from zero. The two (or more) conductance states presumably correspond to different orientations of the dipolar F6Val1 side chain. The distribution between the high- and the low-conductance states varies as a function of potential in [F6Val1]/[Gly1]gA channels. These characteristics cause the [F6Val1]/nonpolar (Val, Ala, Gly)gA hybrid channels to serve as a "simple" model for understanding gating transitions in membrane-spanning channels.     

Uniformly oriented gramicidin channels embedded in thick monodomain lecithin multilayers.

Phosphatidylcholine multilayers, containing 20% water by total sample weight and gramicidin/lipid molar ratios up to 1:40 were aligned by low temperature annealing (less than 60 degrees C) and mechanical stressing. We were able to obtain large (greater than 80 micron thick X 40 mm2 area) monodomain defect-free multilayers containing approximately 10(17) uniformly oriented gramicidin channels. The alignment of lipid multilayers was monitored by conoscopy and polarized microscopy. The smectic defects, which appeared during the alignment process, were identified and dissolved. The incorporation of gramicidin into the multilayers in the form of transmembrane channels was indicated by its circular dichroic (CD) spectrum. A well-defined CD spectrum of uniformly oriented gramicidin channels was obtained. The oriented samples will allow spectroscopic studies of the ion channel in its conducting state and diffraction studies of the channel-channel organization in the membrane.     

Solvent drag across gramicidin channels demonstrated by microelectrodes

The competition of ion and water fluxes across gramicidin channels was assessed from the concentration distributions of both pore-impermeable and -permeable cations that were simultaneously measured by double-barreled microelectrodes in the immediate vicinity of a planar bilayer. Because water movement across the membrane led to accumulation of solutes on one side of the membrane and depletion on the other, the permeable cation was not only pushed by water across the channel (true solvent drag); it also flowed along its concentration gradient (pseudo-solvent drag). For the demonstration of true solvent drag, a difference between the bulk concentrations on the hypertonic and the hypotonic sides of the membrane was established. It was adjusted to get equal cation concentrations at both membrane/water interfaces. From the sodium and potassium fluxes measured along with membrane conductivity under these conditions, approximately five water molecules were found to be transported simultaneously with one ion through the channel. In diphytanoyl phosphatidylcholine membranes, a single-channel hydraulic permeability coefficient of 1.6 x 10(-14) cm(3) s(-1) was obtained.     

A molecular dynamics study of gating in dioxolane-linked gramicidin A channels.

The gating transition of the RR and SS dioxolane ring-linked gramicidin A channels were studied with molecular dynamics simulations using a detailed atomic model. It was found that the probable reaction path, describing the transition of the ring from the exterior to the interior of the channel where it blocked the permeation pathway, involved several steps including the isomerization of the transpeptide plane dihedral angle of Val1. Reaction coordinates along this pathway were defined, and the transition rates between the stable conformers were calculated. It was found, in good accord with experimental observations, that the calculated blocking rate for the RR-linked channel was 280/s with a mean blocking time of 0.04 ms, whereas such blocking did not occur in the case of the SS-linked channel. An important observation is that the resulting lifetime for the blocked state of the RR-linked channel was in good accord with the experimental observations only when the calculations were performed in the presence of a potassium ion inside the channel.     

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.     

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.