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.     

Large unselective pore in lipid bilayer membrane formed by positively charged peptides containing a sequence of gramicidin A

Ion-channel activity of a series of gramicidin A analogues carrying charged amino-acid sequences on the C-terminus of the peptide was studied on planar bilayer lipid membranes and liposomes. It was found that the analogue with the positively charged sequence GSGRRRRSQS forms classical cationic pores at low concentrations and large unselective pores at high concentrations. The peptide was predominantly in the right-handed beta(6.3)-helical conformation in liposomes as shown by circular dichroism spectroscopy. The single-channel conductance of the large pore was estimated to be 320pS in 100mM choline chloride as judged from the fluctuation analysis of the multi-channel current. The analogue with the negatively charged sequence GSGEEEESQS exhibited solely classical cationic channel activity. The ability of a peptide to form different type of channels can be used in the search for broad-spectrum antibiotics.     

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

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

Effect of Membrane Composition on Antimicrobial Peptides Aurein 2.2 and 2.3 From Australian Southern Bell Frogs

The effects of hydrophobic thickness and the molar phosphatidylglycerol (PG) content of lipid bilayers on the structure and membrane interaction of three cationic antimicrobial peptides were examined: aurein 2.2, aurein 2.3 (almost identical to aurein 2.2, except for a point mutation at residue 13), and a carboxy C-terminal analog of aurein 2.3. Circular dichroism results indicated that all three peptides adopt an α-helical structure in the presence of a 3:1 molar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPC/DMPG), and 1:1 and 3:1 molar mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG). Oriented circular dichroism data for three different lipid compositions showed that all three peptides were surface-adsorbed at low peptide concentrations, but were inserted into the membrane at higher peptide concentrations. The ³¹P solid-state NMR data of the three peptides in the DMPC/DMPG and POPC/POPG bilayers showed that all three peptides significantly perturbed lipid headgroups, in a peptide or lipid composition-dependent manner. Differential scanning calorimetry results demonstrated that both amidated aurein peptides perturbed the overall phase structure of DMPC/DMPG bilayers, but perturbed the POPC/POPG chains less. The nature of the perturbation of DMPC/DMPG bilayers was most likely micellization, and for the POPC/POPG bilayers, distorted toroidal pores or localized membrane aggregate formation. Calcein release assay results showed that aurein peptide-induced membrane leakage was more severe in DMPC/DMPG liposomes than in POPC/POPG liposomes, and that aurein 2.2 induced higher calcein release than aurein 2.3 and aurein 2.3-COOH from 1:1 and 3:1 POPC/POPG liposomes. Finally, DiSC₃5 assay data further delineated aurein 2.2 from the others by showing that it perturbed the lipid membranes of intact S. aureus C622 most efficiently, whereas aurein 2.3 had the same efficiency as gramicidin S, and aurein 2.3-COOH was the least efficient. Taken together, these data show that the membrane interactions of aurein peptides are affected by the hydrophobic thickness of the lipid bilayers and the PG content.     

Structural Polymorphism of Gramicidin A Channels: Ion Conductivity and Spectral Studies

The relation between the various spatial structures of the gramicidin A channels and their ionic conductance has been studied. For this aim, various conformations of the peptide were pre-formed in liposomal bilayer and after subsequent fusion of liposomes with planar lipid bilayer the measured channel conductance was correlated with gramicidin structures established in liposomes. To form the single-stranded π6.3π 6.3 helix the peptide and lipid were co-dissolved in TFE prior to liposome preparation. THF and other solvents were used to form parallel (↑ ↑ π π) and antiparallel (↑ ↓ π π) double helices. Conformation of gramicidin in liposomes made by various phosphatidylcholines was monitored by CD spectroscopy, and computer analysis of the spectra obtained was performed. After fusion of gramicidin containing liposomes with planar bilayer membranes from asolectin, the histograms of single-channel conductance were obtained. The histograms had one or three distinct peaks depending on the liposome preparation. Assignment of the structure of the channel to conductance levels was made by correlation of CD data with conductance histograms. The channel-forming analogue, des(Trp-Leu)₂-gramicidin A, has been studied by the same protocol. The channel conductances of gramicidin A and the shortened analogue increase in the following order: ↑ ↓ π π 2 ↑ ↑ π π < π 6.3π6.3. Single-channels formed by double helices have higher dispersity of conductance than the π6.3π6.3 helical channel. Lifetimes of the double helical and the π6.3π6.3 helical channels are very close to each other. The data obtained were compared with theoretically predicted properties of double helices [1].     

Formation of Two Different Types of Ion Channels by Amphotericin B in Human Erythrocyte Membranes

The polyene antibiotic amphotericin B (AmB) is known to form aqueous pores in lipid membranes and biological membranes. Here, membrane potential and ion permeability measurements were used to demonstrate that AmB can form two types of selective ion channels in human erythrocytes, differing in their interaction with cholesterol. We show that AmB induced a cation efflux (negative membrane polarization) across cholesterol-containing liposomes and erythrocytes at low concentrations (≤1.0 × 10⁻⁶ M), but a sharp reversal of such polarization was observed at concentrations greater than 1.0 × 10⁻⁶ M AmB, an indication that aqueous pores are formed. Cation-selective AmB channels are also formed across sterol-free liposomes, but aqueous pores are only formed at AmB concentrations 10 times greater. The effect of temperature on the AmB-mediated K⁺ efflux across erythrocytes revealed that the energies of activation for channel formation are negative and positive at AmB concentrations that lead predominantly to the formation of cation-selective channels and aqueous pores, respectively. These findings support the conclusion that the two types of AmB channels formed in human erythrocytes differ in their interactions with cholesterol and other membrane components. In effect, a membrane lipid reorganization, as induced by incubation of erythrocytes with tetrathionate, a cross-linking agent of the lipid raft–associated protein spectrin, led to differential changes in the activation parameters for the formation of both types of channels, reflecting the different lipid environments in which such structures are formed.     

The biosynthesis of gramicidin S in a cell-free system

1. A cell-free system prepared from Bacillus brevis cells, harvested in the late phase of growth and consisting of the 11000g supernatant, has been shown to incorporate into gramicidin S the five constituent amino acids added in labelled form. The results are consistent with complete synthesis and not merely a completion of pre-existing intermediate peptides. 2. The incorporation of ¹⁴C-labelled amino acids by the 11000g supernatant into gramicidin S requires an energy source. Omission of phosphoenolpyruvate and pyruvate kinase from the incubation mixture prevents incorporation into gramicidin S. The cell-free system incorporates [¹⁴C]-leucine, -proline and -phenylalanine over a period of 4hr. With [¹⁴C]leucine, incorporation into gramicidin S takes place in the range pH6–9 with maximum incorporation at pH7·0. High concentrations of chloramphenicol or puromycin decreased the incorporation into gramicidin S by only about 20%. 3. The 50000g supernatant exhibited no decrease in ability of incorporating [¹⁴C]valine into gramicidin S as compared with the 11000g supernatant. About 40% of the incorporating ability remained in the 105000g supernatant after 3hr. centrifugation. When recombining the 105000g sediment with the 105000g supernatant, some increase in incorporation over that obtained with the supernatant alone was obtained. The findings tend to support the view that gramicidin S is synthesized in a different manner from that of proteins.     

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

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

Peptide-induced membrane leakage by lysine derivatives of gramicidin A in liposomes,planar bilayers,and erythrocytes

Introducing a charged group near the N-terminus of gramicidin A (gA) is supposed to suppress its ability to form ion channels by restricting its head-to-head dimerization. The present study dealt with the activity of [Lys1]gA, [Lys3]gA, [Glu1]gA, [Glu3]gA, [Lys2]gA, and [Lys5]gA in model membrane systems (planar lipid bilayers and liposomes) and erythrocytes. In contrast to the Glu-substituted peptides, the lysine derivatives of gA caused non-specific liposomal leakage monitored by fluorescence dequenching of lipid vesicles loaded with carboxyfluorescein or other fluorescent dyes. Measurements of electrical current through a planar lipid membrane revealed formation of giant pores by Lys-substituted analogs, which depended on the presence of solvent in the bilayer lipid membrane. The efficacy of unselective pore formation in liposomes depended on the position of the lysine residue in the amino acid sequence, increasing in the row: [Lys2]gA < [Lys5]gA < [Lys1]gA < [Lys3]gA. The similar series of potency was exhibited by the Lys-substituted gA analogs in facilitating erythrocyte hemolysis, whereas the Glu-substituted analogs showed negligible hemolytic activity. Oligomerization of the Lys-substituted peptides is suggested to be involved in the process of nonselective pore formation.     

Oligotryptophan-tagged antimicrobial peptides and the role of the cationic sequence

The effects of varying the cationic sequence of oligotryptophan-tagged antimicrobial peptides were investigated in terms of peptide adsorption to model lipid membranes, liposome leakage induction, and antibacterial potency. Heptamers of lysine (K7) and arginine (R7) were lytic against Escherichia coli bacteria at low ionic strength. In parallel, both peptides adsorbed on to bilayers formed by E. coli phospholipids, and caused leakage in the corresponding liposomes. K7 was the more potent of the two peptides in causing liposome leakage, although the adsorption of this peptide on E. coli membranes was lower than that of R7. The bactericidal effect, liposome lysis, and membrane adsorption were all substantially reduced at physiological ionic strength. When a tryptophan pentamer tag was linked to the C-terminal end of these peptides, substantial peptide adsorption, membrane lysis, and bacterial killing were observed also at high ionic strength, and also for a peptide of lower cationic charge density (KNKGKKN-W5). Strikingly, the order of membrane lytic potential of the cationic peptides investigated was reversed when tagged. This and other aspects of peptide behavior and adsorption, in conjunction with effects on liposomes and bacteria, suggest that tagged and untagged peptides act by different lytic mechanisms, which to some extent counterbalance each other. Thus, while the untagged peptides act by generating negative curvature strain in the phospholipid membrane, the tagged peptides cause positive curvature strain. The tagged heptamer of arginine, R7W5, was the best candidate for E. coli membrane lysis at physiological salt conditions and proved to be an efficient antibacterial agent.     

Porous membranes for reconstitution of ion channels

Functional biological synthetic composite (BSC) membranes were made using phospholipids, biological membrane proteins and permeable synthetic supports or membranes. Lipid bilayers were formed on porous polycarbonate (PC), polyethylene terephthalate (PETE) and poly (l-lactic acid) (PLLA) membranes and in 10-100 μm laser-drilled pores in a 96-well plastic plate as measured by increased resistance or decreased currents. Bilayers in 50 μm and smaller pores were stable for up to 4 h as measured by resistance changes or a current after gramicidin D reconstitution. Biological membrane transport reconstitution was then carried out. Using vesicles containing Kv1.5 K⁺ channels, K⁺ currents and decreased resistance were measured across bilayers in 50 μm pores in the plastic plate and PLLA membranes, respectively, which were inhibited by compound B, a Kv1.5 K⁺ channel inhibitor. Functional reconstitution of Kv1.5 K⁺ channels was successful. Incorporation of membrane proteins in functional form in stable permeable membrane-supported lipid bilayers is a simple technology to create BSC membranes that mimic biological function which is readily adaptable for high throughput screening.     

Membrane-disruptive abilities of β-hairpin antimicrobial peptides correlate with conformation and activity: A P and H NMR study

The membrane interaction and solution conformation of two mutants of the β-hairpin antimicrobial peptide, protegrin-1 (PG-1), are investigated to understand the structural determinants of antimicrobial potency. One mutant, [A_6,8,13,15] PG-1, does not have the two disulfide bonds in wild-type PG-1, while the other, [Δ_4,18 G₁₀] PG-1, has only half the number of cationic residues. ³¹P solid-state NMR lineshapes of uniaxially aligned membranes indicate that the membrane disorder induced by the three peptides decreases in the order of PG-1>[Δ_4,18 G₁₀] PG-1?[A_6,8,13,15] PG-1. Solution NMR studies of the two mutant peptides indicate that [Δ_4,18 G₁₀] PG-1 preserves the β-hairpin fold of the wild-type peptide while [A_6,8,13,15] PG-1 adopts a random coil conformation. These NMR results correlate well with the known activities of these peptides. Thus, for this class of peptides, the presence of a β-hairpin fold is more essential than the number of cationic charges for antimicrobial activity. This study indicates that ³¹P NMR lineshapes of uniaxially aligned membranes are well correlated with antimicrobial activity, and can be used as a diagnostic tool to understand the peptide-lipid interactions of these antimicrobial peptides.     

Reversible sheet-turn conformational change of a cell-penetrating peptide in lipid bilayers studied by solid-state NMR

The membrane-bound conformation of a cell-penetrating peptide, penetratin, is investigated using solid-state NMR spectroscopy. The ¹³C chemical shifts of ¹³C, ¹⁵N-labeled residues in the peptide indicate a reversible conformational change from β-sheet at low temperature to coil-like at high temperature. This conformational change occurs for all residues examined between positions 3 and 13, at peptide/lipid molar ratios of 1:15 and 1:30, in membranes with 25-50% anionic lipids, and in both saturated DMPC/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylchloline/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) membranes and unsaturated POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol) membranes. Thus, it is an intrinsic property of penetratin. The coil state of the peptide has C-H order parameters of 0.23-0.52 for C^α and C^β sites, indicating that the peptide backbone is unstructured. Moreover, chemical shift anisotropy lineshapes are uniaxially averaged, suggesting that the peptide backbone undergoes uniaxial rotation around the bilayer normal. These observations suggest that the dynamic state of penetratin at high temperature is a structured turn instead of an isotropic random coil. The thermodynamic parameters of this sheet-turn transition are extracted and compared to other membrane peptides reported to exhibit conformational changes. We suggest that the function of this turn conformation may be to reduce hydrophobic interactions with the lipid chains and facilitate penetratin translocation across the bilayer without causing permanent membrane damage.     

Preparation and conformational analysis of C-glycosyl β- and β/β-peptides

Ten C-glycosyl β²- and β/β²-peptides with three to eight amino acid residues have been prepared. Solution and solid-phase peptide syntheses were employed to assemble β²-amino acids in which C-glycosylic substituents are attached to the C-2 position of β-amino acids. Conformational analysis of the C-glycosyl β²-peptides using NMR and CD spectra indicates that the tripeptide can form a helical secondary structure. Besides, helix directions of the C-glycosyl β/β²-peptides are governed by the configuration at the α-carbon of the peptide backbone that originates from the stereocenter of the C-glycosyl β²-amino acids.     

Insight into early events in the aggregation of the prion protein on lipid membranes

The key molecular event underlying prion diseases is the conversion of the monomeric and α-helical cellular form of the prion protein (PrP^C) to the disease-associated state, which is aggregated and rich in β-sheet (PrP^Sc). The molecular details associated with the conversion of PrP^C into PrP^Sc are not fully understood. The prion protein is attached to the cell membrane via a GPI lipid anchor and evidence suggests that the lipid environment plays an important role in prion conversion and propagation. We have previously shown that the interaction of the prion protein with anionic lipid membranes induces β-sheet structure and promotes prion aggregation, whereas zwitterionic membranes stabilize the α-helical form of the protein. Here, we report on the interaction of recombinant sheep prion protein with planar lipid membranes in real-time, using dual polarization interferometry (DPI). Using this technique, the simultaneous evaluation of multiple physical properties of PrP layers on membranes was achieved. The deposition of prion on membranes of POPC and POPC/POPS mixtures was studied. The properties of the resulting protein layers were found to depend on the lipid composition of the membranes. Denser and thicker protein deposits formed on lipid membranes containing POPS compared to those formed on POPC. DPI thus provides a further insight on the organization of PrP at the surface of lipid membranes.     

The relationship between the binding to and permeabilization of phospholipid bilayer membranes by GS14dK4, a designed analog of the antimicrobial peptide gramicidin S

The cationic β-sheet cyclic tetradecapeptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK₄) is a diastereomeric lysine ring-size analog of the potent naturally occurring antimicrobial peptide gramicidin S (GS) which exhibits enhanced antimicrobial but markedly reduced hemolytic activity compared to GS itself. We have previously studied the binding of GS14dK₄ to various phospholipid bilayer model membranes using isothermal titration calorimetry [Abraham, T. et al. (2005) Biochemistry 44, 2103-2112]. In the present study, we compare the ability of GS14dK₄ to bind to and disrupt these same phospholipid model membranes by employing a fluorescent dye leakage assay to determine the ability of this peptide to permeabilize large unilamellar vesicles. We find that in general, the ability of GS14dK₄ to bind to and to permeabilize phospholipid bilayers of different compositions are not well correlated. In particular, the binding affinity of GS14dK₄ varies markedly with the charge and to some extent with the polar headgroup structure of the phospholipid and with the cholesterol content of the model membrane. Specifically, this peptide binds much more tightly to anionic than to zwitterionic phospholipids and much less tightly to cholesterol-containing than to cholesterol-free model membranes. In addition, the maximum extent of binding of GS14dK₄ can also vary considerably with phospholipid composition in a parallel fashion. In contrast, the ability of this peptide to permeabilize phospholipid vesicles is only weakly dependent on phospholipid charge, polar headgroup structure or cholesterol content. We provide tentative explanations for the observed lack of a correlation between the affinity and extent of GS14dK₄ binding to, and degree of disruption of the structure and integrity of, phospholipid bilayers membranes. We also present evidence that the lack of correlation between these two parameters may be a general phenomenon among antimicrobial peptides. Finally, we demonstrate that the affinity of binding of GS14dK4 to various phospholipid bilayer membranes is much more strongly correlated with the antimicrobial and hemolytic activities of this peptide than with its effect on the rate and extent of dye leakage in these model membrane systems.     

Structure-activity relationship of gramicidin S analogues on membrane permeability

The previous study of the action of gramicidin S on bacteria (Katsu, T., Kobayashi, H. and Fujita, Y. (1986) Biochim. Biophys. Acta 860, 608-619) prompted us to investigate further the structure-activity relationship of the gramicidin S analogues on membrane permeability. Two types of the gramicidin S analogues were used in the present study: (1) cyclo(-X-D-Leu-D-Lys-D-Leu-L-Pro-)2, where X = Gly, D-Leu and D-cyclohexylalanine (D-cHxAla); (2) N,N'-diacetyl derivative of gramicidin S (diacetyl-gramicidin S) which lacks a cationic moiety of gramicidin S. All the analogues have a beta-sheet conformation as gramicidin S. The following cellular systems were used: Staphylococcus aureus as Gram-positive bacteria, Escherichia coli as Gram-negative bacteria, human erythrocytes, rat liver mitochondria and artificial liposomal membranes. It was found that gramicidin S and one of the type 1 analogues having X = D-cHxAla induced the efflux of K+ through the cytoplasmic membrane of all types of the cells. In addition, these two peptides had the ability to lower the phase transition temperature of dipalmitoylphosphatidylcholine. Accordingly, it was concluded that, if peptides can expand greatly the membrane structure of neutral lipids which constitute main parts of the biological membrane, they can stimulate the permeability of cells without any selectivity. The action of the type 2 peptide, diacetyl-gramicidin S, was strongly cell dependent. Although this peptide stimulated the efflux of K+ from mitochondria, it did not do so efficiently, if at all, from S. aureus, E. coli and erythrocytes. In experiments using liposomes, diacetyl-gramicidin S increased markedly the permeability of liposomes composed of egg phosphatidylcholine. The presence of egg phosphatidylethanolamine or cholesterol reduced its activity. These results on liposomes explained well the low sensitivity of diacetyl-gramicidin S against E. coli and erythrocytes in terms of lipid constituents of the membranes. The mechanism of action of diacetyl-gramicidin S was discussed from the formation of a boundary lipid induced by this peptide.     

Probing human proteins for short encrypted antimicrobial peptides reveals Hs10, a peptide with selective activity for gram-negative bacteria

BackgroundSome cationic and amphiphilic α-helical segments of proteins adsorb to prokaryotic membranes when synthesized as individual polypeptide sequences, resulting in broad and potent antimicrobial activity. However, amphiphilicity, a determinant physicochemical property for peptide-membrane interactions, can also be observed in some β-sheets.MethodsThe software Kamal was used to scan the human reference proteome for short (7–11 amino acid residues) cationic and amphiphilic protein segments with the characteristic periodicity of β-sheets. Some of the uncovered peptides were chemically synthesized, and antimicrobial assays were conducted. Biophysical techniques were used to probe the molecular interaction of one peptide with phospholipid vesicles, lipopolysaccharides (LPS) and the bacterium Escherichia coli.ResultsThousands of compatible segments were found in human proteins, five were synthesized, and three presented antimicrobial activity in the micromolar range. Hs10, a nonapeptide fragment of the Complement C3 protein, could inhibit only the growth of tested Gram-negative microorganisms, presenting also little cytotoxicity to human fibroblasts. Hs10 interacted with LPS while transitioning from an unstructured segment to a β-sheet and increased the hydrodynamic radius of LPS particles. This peptide also promoted morphological alterations in E. coli cells. Conclusions: Data presented herein introduce yet another molecular template to probe proteins in search for encrypted membrane-active segments and demonstrates that, using this approach, short peptides with low cytotoxicity and high selectivity to prokaryotic cells might be obtained.General SignificanceThis work widens the biotechnological potential of the human proteome as a source of antimicrobial peptides with application in human health.     

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.