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 gramicidin on the potassium conductance of an isolated frog muscle fiber]

The antibiotic gramicidin A (1.10(-6) M) increases the K+ conductance of normal and detubulated frog skeletal muscle fibres in isotonic K2SO4 solution to a steady-state level, which is reached in 6--9 min, and corresponds to 8058 +/- 1669 and 5767 +/- 902 Om-1. 10(-6)/cm2, resp. There is no correlation between the initial K+ conductance and the value of the steady state of gramicidin A-induced conductance (r = 0.24). According to the dimer hypothesis, the dissociation rate constant of the garmicidin channels was found to be 0.006 +/- 0.0001 sec-1. This result supports the suggestion of a higher stability of gramicidin channels in muscle compared to the bimolecular lipid membranes.     

Relaxation spectra of gramicidin dimerization in a lipid bilayer membrane

The kinetics of formation and dissociation of gramicidin dimers in a lipid bilayer membrane have been studied by pressure-jump and electric field-jump methods. The traditional AC-coupled pressure-jump apparatus has been modified so that a known DC-voltage drop is maintained across a Teflon cell divided by a septum with a hole for membrane formation. From the response of the amplified output voltage after the pressure release, information about the kinetics of channel (dimer) formation is obtained. In addition, using the same apparatus, electric field-jump measurements were performed on the gramicidin/membrane system. In asolectin/7-dehydrocholesterol (5:1) membranes at 25 +/- 0.1 degrees C, the best fit to the pressure-jump data gives a dimer dissociation rate constant of 0.5 +/- 0.3 s-1. The standard volume change for dimerization determined from the amplitude of the pressure-jump experiments is -66 +/- 35 cm3/mol. Rate data determined by the electric field-jump method are consistent with the pressure-jump values; results obtained with either technique are compatible with other determinations of the kinetics of dimerization on gramicidin/membrane systems.     

Location of ion-binding sites in the gramicidin channel by X-ray diffraction.

We report the first X-ray diffraction on gramicidin in its membrane-active form by using uniformly aligned multilayer samples of membranes containing gramicidin and ions (T1+, K+, Ba2+, Mg2+ or without ions). From the difference electron density profiles, we found a pair of symmetrically located ion-binding sites for T1- at 9.6 (+/- 0.3) A and for Ba2+ at 13.0 (+/- 0.2) A from the midpoint of the gramicidin channel. The location of Ba(2+)-binding sites is near the ends of the channel, consistent with the experimental observation that divalent cations do not permeate but block the channel. The location of T1(+)-binding sites is somewhat of a surprise. It was generally thought that monovalent cations bind to the first turn of the helix from the mouth of the channel. (It is now generally accepted that the gramicidin channel is a cylindrical pore formed by two monomers, each a single-stranded beta 6.3 helix and hydrogen-bonded head-to-head at their N termini.) But our experiment shows that the T1(+)-binding site is either near the bottom of or below the first helix turn.     

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.     

Dynamic properties of gramicidin A in phospholipid membranes

The flexibility of the tryptophan side chains of gramicidin A and the rotational diffusion of the peptide in methanolic solution and in three membrane systems were studied with deuterium nuclear magnetic resonance (NMR). Gramicidin A was selectively deuterated at the aromatic ring systems of its four tryptophan side chains. In methanolic solution, the tryptophan residues remained immobile and served as a probe for the overall rotation of the peptide. The experimentally determined rotational correlation time of tau c = 0.6 X 10(-9) s was consistent with the formation of gramicidin A dimers. For gramicidin A incorporated into bilayer membranes, quite different results were obtained depending on the chemical and physical nature of the lipids employed. When mixed with 1-palmitoyl-sn-glycero-3-phosphocholine (LPPC) at a stoichiometric lipid:peptide ratio of 4:1, gramicidin A induced the formation of stable bilayer membranes in which the lipids were highly fluid. In contrast, the gramicidin A molecules of this membrane remained completely static over a large temperature interval, suggesting strong protein-protein interactions. The peptide molecules appeared to form a rigid two-dimensional lattice in which the interstitial spaces were filled with fluidlike lipids. When gramicidin A was incorporated into bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) above the lipid phase transition, the deuterium NMR spectra were motionally narrowed, indicating large-amplitude rotational fluctuations. From the measurement of the quadrupole echo relaxation time, a rotational correlation time of 2 X 10(-7) s was estimated, leading to a membrane viscosity of 1-2 P if the rotational unit was assumed to be a gramicidin A dimer. (ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The divalent cation-binding sites of gramicidin A transmembrane ion-channel.

The conductance of the gramicidin A single channels in glycerolmonooleate membranes is strongly reduced in the presence of Mn2+ cations. The nmr experiments were performed for N-terminal to N-terminal gramicidin A dimer formed by two right-handed single-stranded helixes incorporated into the sodium dodecyl sulfate micelles in the presence of Mn2+ ions. Dependence of the nonselective spin-lattice relaxation rates of the gramicidin A protons on Mn2+ concentration was analyzed to determine coordinates of the divalent cation binding sites. It is inferred that Mn2+ ions are bound at the channel mouths at distances of 6.4, 8.6, and 8.8 A (+/- 2 A) from the oxygen atoms of exposed carbonyl groups of D-Leu 12, 14, and 10, respectively. The bounded Mn2+ retains its hydrate shell, the size of which (approximately 6 A) exceeds the inner pore diameter (approximately 4 A). That makes the gramicidin A channel impermeable for divalent cations.     

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     

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.     

Effect of temperature on the inward rectifier and gramicidin A-induced channels in the membrane of frog skeletal muscle fibres

The conductance of frog skeletal muscle fibres in isotonic K2SO4 solution has been measured. Experiments were carried out under current-clamp conditions using a double sucrose-gap technique. The potassium conductances of the inward rectifier and the gramicidin channel in the same muscle fibre were compared. Potassium conductance of the inward rectifier increased with the temperature, with a value of Q10 1.55 +/- 0.09 (n = 8) under hyperpolarization, and Q10 2.38 +/- 0.23 (n = 6) for the depolarizing stimulus, the difference between Q10 of potassium and gramicidin channels being statistically insignificant.     

Tryptophan contributions to the empirical free-energy profile in gramicidin A/M heterodimer channels

Gramicidin A/gramicidin M heterodimer conductances were measured in planar lipid bilayers and found to form two distinguishable populations about halfway between the gramicidin A and gramicidin M homodimer conductances. This implies that the principle difference in the gramicidin A and gramicidin M transport free-energy profiles occurs at the channel center, where it would produce similar effects on the rate-limiting barrier for the two heterodimers. Kinetic analysis based on this and nearly all previously published homodimer conductance data for both gramicidin A and gramicidin M channels confirms this conclusion, indicating that the translocation step is approximately 100-fold slower in gramicidin M homodimers than in gramicidin A homodimers and that first- and second-ion exit-rate constants are higher by factors of 24 and 10, respectively. Assuming that the ratios of rate constants are related to the free-energy difference between gramicidin A and gramicidin M, we construct an effective ion-Trp free-energy interaction profile that has a minimum at the channel center.     

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.     

Shortened analog of the gramicidin A channel argues for the doubly occupied channel as the dominant conducting state

A shortened analog of the gramicidin A transmembrane channel has been synthesized and its transport characterized in planar lipid bilayer membranes. General considerations of a shorter diffusional length and a shorter distance over which the voltage drop occurs (i.e., an increased electric field) would contribute to an increase in single-channel conductance. The finding of a decreased single-channel conductance supports the perspective that the dominant conducting state is the doubly occupied channel wherein distance-dependent repulsion due to the first ion in the channel impedes entry of the second ion in the shorter channel.     

Interaction of ions and water in gramicidin A channels: streaming potentials across lipid bilayer membranes

For very narrow channels in which ions and water cannot overtake one another (single-file transport), electrokinetic measurements provide information about the number of water molecules within a channel. Gramicidin A is believed to form such narrow channels in lipid bilayer membranes. In 0.01 and 0.1 M solutions of CsCl, KCL, and NaCl, streaming potentials of 3.0 mV per osmolal osmotic pressure difference (created by urea, glycerol, or glucose) appear across gramicidin A-treated membranes. This implies that there are six to seven water molecules within a gramicidin channel. Electroosmotic experiments, in which the water flux assoicated with current flow across gramicidin-treated membranes is measured, corroborate this result. In 1 M salt solutions, streaming potentials are 2.35 mV per osmolal osmotic pressure difference instead of 3.0 mV. The smaller value may indicate multiple ion occupancy of the gramicidin channel at high salt concentrations. Apparent deviations from ideal cationic selectivity observed while attempting to measure single-salt dilution potentials across gramicidin-treated membranes result from streaming potential effects.     

Number of water molecules coupled to the transport of sodium, potassium and hydrogen ions via gramicidin, nonactin or valinomycin.

The number of water molecules (n) coupled to the transport of cations across lipid membranes was determined in two different wats: directly from the electro-osmotic volume flux per ion, and by the use of Onsager's relation, from the open circuit streaming potential produced by an osmotic pressure difference. The results of the two approaches were in general agreement. Monoolein membranes were formed on the ends of polyethylene or Teflon tubing connected to a microliter syringe and the volume change necessary to keep the membrane at a fixed position was measured. It was necessary to make corrections for unstirred layer effects. The results for gramicidin were: n approximately 12 for 0.15 M KCl and NaCl, n approximately 6 for 3.0 M KCl and NaCl, and n approximately 0 for 0.01 M HCl. For nonactin, n approximately 4 for both 0.15 and 3.0 M KCl and NaCl. Valinomycin (for 0.15 M KCl) behaved like nonactin. It is shown that for a channel mechanism, in general, n is less than or equal to the number of water molecules in a channel that does not contain any cations. Thus, the n of 12 for the 0.15 M salts implies that the gramicidin channel can hold at least 12 water molecules. This places an important constraint on models of the channel structure. The n of 0 for HCl is consistent with a process in which protons jump along a continuous row of water molecules. The decrease of n with the 3.0 M salts may indicate that the channel becomes multiply occupied at high salt concentrations. The n of 4 for nonactin and valinomycin means that at least four water molecules are associated with the carrier . cation complex, probably in the interstices between the complex and the disordered lipid.     

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.     

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 dimeric nature of the gramicidin A transmembrane channel: Conductance and fluorescence energy transfer studies of hybrid channels

Gramicidin A, a linear peptide antibiotic, makes membranes permeable to alkali cations and hydrogen ions by forming transmembrane channels. We report here conductance and fluorescence energy transfer studies of channels containing two kinds of gramicidin. These studies of hybrid channels were designed to determine the number of molecules in a channel. The gramicidins studied were gramicidin A, dansyl gramicidin C, the p-phenylazobenzene sulfonyl derivative of gramicidin C (PABS⁴ gramicidin C), and the 4-(diethylamino)-phenylazobenzene-4-sulfonyl chloride derivative of gramicidin C (DPBS gramicidin C). The dansyl, PABS and DPBS groups were linked to the hydroxyl group of tyrosine 11 in gramicidin C. The single-channel conductance of PABS gramicidin C in planar bilayer membranes is 0.68 that of gramicidin A. Membranes containing both PABS gramicidin C and gramicidin A exhibit three kinds of channels: a pure gramicidin A, a pure PABS gramicidin C channel, and a hybrid channel with an intermediate conductance (0.82 that of gramicidin A). The dependence of the frequencies of these three kinds of channels on the mole fractions of gramicidin A and PABS gramicidin C in the membrane-forming solution fits a dimer model. Fluorescence energy transfer was used as a complementary means of ascertaining the frequency of hybrid channels. Dansyl gramicidin C was the fluorescent energy donor and DPBS gramicidin C was the energy acceptor. The efficiency of energy transfer between these chromophores in hybrid channels in liposomes was 75%. The relative quantum yield of the dansyl fluorescence was measured as a function of the mole fraction of DPBS gramicidin C. These fluorescence studies, like the single-channel conductance measurements, showed that there are two molecules of gramicidin in a 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.