Deuterium NMR of Val1...(2-2H)Ala3...gramicidin A in oriented DMPC bilayers.

Deuterium NMR is used to study the selectively labeled Val1...(2-2H)Ala3...gramicidin A molecule to investigate the structure and dynamics of the C alpha-2H bond in the Ala3 residue of gramicidin. Val1...(2-2H)Ala3...gramicidin A is synthesized, purified, and characterized and then incorporated into oriented bilayers of dimyristoylphosphatidylcholine sandwiched between glass coverslips. Phosphorus NMR line shapes obtained from this sample are consistent with the presence of the bilayer phase and indicate that no nonbilayer phases are present in significant amounts. Deuterium NMR line shapes obtained from this sample indicate that the motional axis of the gramicidin Ala3 residue is parallel to the coverslip normal, that the distribution of motional axis orientations has a width of 2 degrees, and that only one major conformational and dynamical state of the Ala3 C alpha-2H bond is observed on the NMR time scale. Furthermore, the Ala3 C alpha-2H bond angle relative to the motional axis is 19-20 degrees if fast axial rotation is assumed to be the only motion present but is less than or equal to 19-20 degrees in the absence of such an assumption. This result indicates that various double-stranded, helical dimer models are very unlikely to represent the structure of gramicidin in the sample studied but that the single-stranded, beta 6.3 helical dimer models are consistent with the experimental data. However, a definitive distinction between the left-handed, single-stranded, beta 6.3 helical dimer model and the right-handed, single-stranded, beta 6.3 helical dimer model cannot be made on the basis of the experimental data obtained in this study.     

2H nuclear magnetic resonance of the gramicidin A backbone in a phospholipid bilayer.

Solid-state 2H NMR spectroscopy has been employed to study the channel conformation of gramicidin A (GA) in unoriented 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) multilayers. Quadrupolar echo spectra were obtained at 44 degrees C and 53 degrees C, from gramicidin A labels in which the proton attached to the alpha carbon of residue 3, 4, 5, 10, 12, or 14 was replaced with deuterium. Because of the nearly axially symmetric electric field gradient tensor, the quadrupolar splittings obtained from an unoriented multilamellar dispersion of DMPC and singly labeled GA directly yield unambiguous orientational constraints on the C-2H bonds. The average of the ratios of the quadrupolar splittings of the left-handed amino acids to those of the right-handed amino acids, (delta vQL/delta vQD), is expected to be 0.97 +/- 0.04 for a relaxed right-handed beta 6.3LD helix, while a ratio of 0.904 +/- 0.003 is expected for a left-handed beta LD6.3 helix. Since we have experimentally determined this ratio to be 1.01 +/- 0.04, we conclude that that the helix sense of the channel conformation of GA is right-handed. Assuming that the dominant motions are fast axial diffusion of the gramicidin molecule and reorientation of the diffusion axis with respect to the local bilayer normal, then the theoretical splittings may all be scaled down by a constant motional narrowing factor. In this case, a relaxed right-handed beta LD6.3 helix, whose axis of motional averaging is roughly along the presumed helix axis, gave the best fit to experimental results. The reasonably uniform correspondence between the splittings predicted by the relaxed right-handed beta LD6.3 helix and the observed splittings, for labels from both the inner and outer turn of GA, did not reflect a peptide backbone flexibility gradient, since an outer turn (i.e., the turn of the helix closest to the interface with water) with greater flexibility would show additional motional narrowing for labels located there.     

The pore dimensions of gramicidin A.

The ion channel forming peptide gramicidin A adopts a number of distinct conformations in different environments. We have developed a new method to analyze and display the pore dimensions of ion channels. The procedure is applied to two x-ray crystal structures of gramicidin that adopt distinct antiparallel double helical dimer conformations and a nuclear magnetic resonance (NMR) structure for the beta6.3 NH2-terminal to NH2-terminal dimer. The results are discussed with reference to ion conductance properties and dependence of pore dimensions on the environment.     

Orientation of the valine-1 side chain of the gramicidin transmembrane channel and implications for channel functioning. A 2H NMR study.

The orientation of the valine-1 side chain of gramicidin was determined by solid-state 2H NMR using valine-1-deuterated (d8) gramicidin. The peptide was incorporated into DMPC bilayers that were oriented between glass plates. When the plates were oriented with their normal perpendicular to the magnetic field, four quadrupolar splittings were observed of 106, 68, 9.7, and 2.0 kHz. These resonances were assigned to C alpha D, C beta D, and the deuterons of each of the C gamma D3 methyl groups, respectively. The average orientation of the various C-D bonds was calculated with respect to the helix axis. The angle obtained for the C alpha-D resonance was consistent with a single-stranded beta 6.3-helical model for the backbone but not with double-helical models. The angles of the side chain were then fitted to a model for the right-handed beta 6.3-helix. Rotation of the valine-1 side chain yielded a set of torsion angles that matched the angles as determined from the 2H NMR measurements. The corresponding orientation of the valine-1 side chain (chi 1 = -5 degrees) was found to be quite unusual, but it explains well the importance of a branched side chain at position 1 for channel formation and stability. A van der Waals interaction between valine-1 of one monomer and alanine-5 of the other helps to stabilize the gramicidin dimer.     

Structure of gramicidin A.

Gramicidin A, a hydrophobic linear polypeptide, forms channels in phospholipid membranes that are specific for monovalent cations. Nuclear Magnetic Resonance (NMR) spectroscopy provided the first direct physical evidence that the channel conformation in membranes is an amino terminal-to-amino terminal helical dimer, and circular dichroism (CD) spectroscopy has shown the sensitivity of its conformation to different environments and the structural consequences of ion binding. The three-dimensional structure of a gramicidin/cesium complex has been determined by x-ray diffraction of single crystals using single wavelength anomalous scattering for phasing. The left-handed double helix in this crystal form corresponds to one of the intermediates in the process of folding and insertion into membranes. Co-crystals of gramicidin and lipid that appear to have gramicidin in their membrane channel conformation have also been formed and are presently under investigation. Hence, we have used a combination of spectroscopic and diffraction techniques to examine the conformation and functionally-related structural features of gramicidin A.     

The effects of calcium ions on double helical forms of gramicidin

The effects of binding calcium ions to the double helical forms of gramicidin present in methanol solution were examined using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. It was found that calcium ions principally alter the relative composition of the equilibrium mixture of gramicidin conformers present in the solvent. In the absence of calcium, both parallel and antiparallel double helices are present. However, the addition of small amounts of Ca²⁺ shifts the equilibrium towards the left-handed parallel double helical form. This conformational change prevents monovalent cations (caesiums) from binding to the gramicidin double helix, and even converts the shorter, wider anti-parallel double helical form normally produced in the presence of caesium into the longer, narrower parallel double helical form. Furthermore, a temperature study showed that calcium ions tend to stabilize this form relative to the ion-free forms. The conformation of gramicidin is further changed, becoming a disordered structure, when the concentration of Ca²⁺ is raised. Thus, the binding of divalent calcium ions has a number of dramatic effects on the conformations of gramicidin present in solution.     

The structure of an integral membrane peptide: a deuterium NMR study of gramicidin.

Solid state deuterium NMR was employed on oriented multilamellar dispersions consisting of 1,2-dilauryl-sn-glycero-3-phosphatidylcholine and deuterium (2H) exchange-labeled gramicidin D, at a lipid to protein molar ratio (L/P) of 15:1, in order to study the dynamic structure of the channel conformation of gramicidin in a liquid crystalline phase. The corresponding spectra were used to discriminate between several structural models for the channel structure of gramicidin (based on the left- and right-handed beta 6.3 LD helix) and other models based on a structure obtained from high resolution NMR. The oriented spectrum is complicated by the fact that many of the doublets, corresponding to the 20 exchangeable sites, partially overlap. Furthermore, the asymmetry parameter, eta, of the electric field gradient tensor of the amide deuterons is large (approximately 0.2) and many of the amide groups are involved in hydrogen bonding, which is known to affect the quadrupole coupling constant. In order to account for these complications in simulating the spectra in the fast motional regime, an ab initio program called Gaussian 90 was employed, which permitted us to calculate, by quantum mechanical means, the complete electric field gradient tensor for each residue in gramicidin (using two structural models). Our results indicated that the left-handed helical models were inconsistent with our observed spectra, whereas a model based on the high-resolution structure derived by Arseniev and coworkers, but relaxed by a simple energy minimization procedure, was consistent with our observed spectra. The molecular order parameter was then estimated from the motional narrowing assuming the relaxed (right-handed) Arseniev structure. Our resultant order parameter of SZZ = 0.91 translates into an rms angle of 14 degrees, formed by the helix axis and the local bilayer normal. The strong resemblance between our spectra (and also those reported for gramicidin in 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) multilayers) and the spectra of the same peptide incorporated in a lyotropic nematic phase, suggests that the lyotropic nematic phase simulates the local environment of the lipid bilayer.     

The membrane interface dictates different anchor roles for "inner pair" and "outer pair" tryptophan indole rings in gramicidin A channels

We investigated the effects of substituting two of the four tryptophans (the "inner pair" Trp(9) and Trp(11) or the "outer pair" Trp(13) and Trp(15)) in gramicidin A (gA) channels. The conformational preferences of the doubly substituted gA analogues were assessed using circular dichroism spectroscopy and size-exclusion chromatography, which show that the inner tryptophans 9 and 11 are critical for the gA's conformational preference in lipid bilayer membranes. [Phe(13,15)]gA largely retains the single-stranded helical channel structure, whereas [Phe(9,11)]gA exists primarily as double-stranded conformers. Within this context, the (2)H NMR spectra from labeled tryptophans were used to examine the changes in average indole ring orientations, induced by the Phe substitutions and by the shift in conformational preference. Using a method for deuterium labeling of already synthesized gAs, we introduced deuterium selectively onto positions C2 and C5 of the remaining tryptophan indole rings in the substituted gA analogues for solid-state (2)H NMR spectroscopy. The (least possible) changes in orientation and overall motion of each indole ring were estimated from the experimental spectra. Regardless of the mixture of backbone folds, the indole ring orientations observed in the analogues are similar to those found previously for gA channels. Both Phe-substituted analogues form single-stranded channels, as judged from the formation of heterodimeric channels with the native gA. [Phe(13,15)]gA channels have Na(+) currents that are ~50% and lifetimes that are ~80% of those of native gA channels. The double-stranded conformer(s) of [Phe(9,11)]gA do not form detectable channels. The minor single-stranded population of [Phe(9,11)]gA forms channels with Na(+) currents that are ~25% and single-channel lifetimes that are ~300% of those of native gA channels. Our results suggest that Trp(9) and Trp(11), when "reaching" for the interface, tend to drive both monomer folding (to "open" a channel) and dimer dissociation (to "close" a channel). Furthermore, the dipoles of Trp(9) and Trp(11) are relatively more important for the single-channel conductance than are the dipoles of Trp(13) and Trp(15).     

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.     

A conformational rearrangement in gramicidin A: from a double-stranded left-handed to a single-stranded right-handed helix.

A conformational transition is described for the polypeptide, gramicidin A, in which a dimer that forms a left-handed intertwined antiparallel helix is converted to a single-stranded amino terminus to amino terminus right-handed helix. The starting structure is determined here by solution NMR methods while reference is made to the well-established folding motif of gramicidin in a lipid bilayer for the ultimate conformation of this transition. Furthermore, an organic solvent system of benzene and ethanol in which gramicidin has a unique conformation is identified. This conformation is shown to be very similar to that derived from X-ray diffraction of crystals prepared from a similar solvent system.     

Determination of the structure of a membrane-incorporated ion channel. Solid-state nuclear magnetic resonance studies of gramicidin A.

Solid-state nuclear magnetic resonance (NMR) measurements on 13C-labeled analogues of the ion channel-forming peptide, gramicidin A, have been used to directly determine the structure of this peptide in lipid membranes. Seven gramicidin analogues, each labeled in a single carbonyl group of gly2, L-ala3, D-leu4, L-val7, D-leu10, D-leu12, or D-leu14 were synthesized by the solid-phase method. These gramicidin analogues were incorporated into aligned multilayers of dimyristoylphosphatidylcholine, or diether lipid bearing 14- or 16-carbon chains, at a 1:15 peptide:lipid mole ratio. Proton-enhanced, 13C, solid-state spectra were obtained at several temperatures and over a range of sample orientations with respect to the spectrometer magnetic field to permit accurate measurement of the chemical shift anisotropies. The observed anisotropies indicate that all of the labeled carbonyl bonds are oriented almost parallel to the molecular long axis and perpendicular to the lipid bilayer plane. These orientations are consistent with gramicidin forming a beta 6.3 single-strand helix that is oriented parallel to the methylene chains of the lipid molecules. Comparison of the linewidths from labeled residues that are in the innermost turn of the helix (gly2, ala3, and D-leu4), in the center of the molecule (val7), and in the turn nearest the lipid bilayer surface (D-leu10, D-leu12, and D-leu14) suggests that although the peptide behaves largely as a rigid barrel, segments of the peptide close to the membrane surface possess greater motional freedom.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H-NMR study of gramicidin A transmembrane ion channel. Head-to-head right-handed, single-stranded helices

The structure of [Val1]gramicidin A incorporated into sodium dodecyl-d25 sulphate micelles has been studied by two-dimensional proton NMR spectroscopy. Analysis of nuclear Overhauser effects, spin-spin couplings and solvent accessibility of NH groups show that the conformation of the Na+ complex of gramicidin A in detergent micelles, which in many ways mimic the phospholipid bilayer of biomembranes, is an N-terminal to N-terminal (head-to-head) dimer (Formula: see text) formed by two right-handed, single-stranded beta 6.3 helices with 6.3 residues per turn, differing from Urry's structure by handedness of the helices.     

13C solid-state NMR of gramicidin A in a lipid membrane.

The natural-abundance 13C NMR spectrum of gramicidin A in a lipid membrane was acquired under magic-angle spinning conditions. With fast sample spinning (15 kHz) at approximately 65 degrees C the peaks from several of the aliphatic, beta-, alpha-, aromatic, and carbonyl carbons in the peptide could be resolved. The resolution in the 13C spectrum was superior that observed with 1H NMR under similar conditions. The 13C linewidths were in the range 30-100 Hz, except for the alpha- and beta-carbons, the widths of which were approximately 350 Hz. The beta-sheet-like local structure of gramicidin A was observed as an upfield shift of the gramicidin alpha and carbonyl resonances. Under slow sample spinning (500 Hz), the intensity of the spinning sidebands from 13C in the backbone carbonyls was used to determine the residual chemical shift tensor. As expected, the elements of the residual chemical shift tensor were consistent with the single-stranded, right-handed beta6.3 helix structure proposed for gramicidin A in lipid membranes.     

The conformation of gramicidin A in dimethylsulphoxide solution. A full analysis of the one- and two-dimensional 1H, 13C, and 15N nuclear-magnetic-resonance spectra

The combined application of one- and two-dimensional high-field NMR techniques has led to the first assignment of the 1H, 13C, and 15N spectra of the pentadecapeptide gramicidin A in dimethylsulphoxide solution. The 62.9-MHz and 100.6-MHz 13C spin-lattice relaxation times and 13C-[1H] NOE factors for the backbone alpha carbons have been analysed in the 'model-free' approach to give a single correlation time (tau m) for isotropic overall molecular motion and an order parameter and internal correlation time for each C alpha H group in the backbone. The relatively high and constant values for the order parameter along the backbone indicate a degree of ordering of the structure, while the internal correlation times show that internal motions are progressively more rapid towards the N terminus. The average values of the vicinal HNC alpha H couplings are 7.4 Hz and 8.4 Hz respectively for the alternate L- and D-amino acid residues. The values are not consistent with either a ribbon conformation for the backbone or a right-handed beta 6.3 helix; they are consistent with the model proposed by Glickson et al. [Glickson, J. D., Mayers, D. F., Settine, J. M. & Urry, D. W. (1972) Biochemistry 11, 477-486] in which there is a rapid conformational order in equilibrium disorder equilibrium, the ordered structure being the left-handed beta 6.3 helix and the disordered state having local random-coil character.     

Conformation of gramicidin in relation to its ability to form bilayers with lysophosphatidylcholine

The ability of gramicidin to induce bilayer formation in lysophosphatidylcholine (LPC) systems was investigated as a function of the conformation of the peptide. The conformation was varied by using different solvents to cosolubilize gramicidin and lipid. Using circular dichroism (CD), it was found that when codissolved in trifluoroethanol (TFE), after drying and subsequent hydration, gramicidin is mainly present in the single-stranded beta 6.3-helical configuration, whereas when using chloroform/methanol or ethanol as the solvent, it is proposed that the dominant conformation of gramicidin in the membrane is that of the double-stranded antiparallel dimer. Employing 31P NMR, the stoichiometry for bilayer formation was found to be 6 to 7 lipid molecules per gramicidin monomer, when samples were prepared from TFE, whereas a stoichiometry of 4 was found when chloroform/methanol or ethanol was the solvent. Upon heating the latter samples, a conversion was observed in the CD pattern toward that indicative of the beta 6.3-helical configuration. This change was accompanied by an increase in the extent of bilayer formation. Next, it was investigated whether the conformation of gramicidin and its ability to induce bilayer formation were dependent on the lipid acyl chain length. CD measurements of samples prepared from TFE indicated that gramicidin, independent of acyl chain length, was present in the beta 6.3-helical configuration but the intensity of the ellipticities at 218 nm increased with the length of the acyl chain. The extent of bilayer formation in these samples was found to be largely chain length independent.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Phase equilibria and molecular packing in the N,N-dimethyldodecylamine oxide/gramicidin D/water system studied by 2H nuclear magnetic resonance spectroscopy.

A partial phase diagram of the system N,N-dimethyldodecylamine oxide (DDAO)/water/gramicidin D was determined by 2H-NMR. Both 2H2O and perdeuterated DDAO (DDAO-d31) were studied by solid state NMR techniques. Addition of gramicidin D to the micellar (L1), normal hexagonal (HI) and cubic (I) phases of DDAO induces phase separations, giving two-phase regions, which all contain a lamellar (L alpha) phase. The L alpha phase containing gramicidin is characterized by larger order parameters for DDAO-d31 compared with the corresponding order parameters in the L alpha and HI phases of DDAO-d31/H2O. The L alpha phase may stay in equilibrium with any other phase in the phase diagram. The DDAO exchange between the coexisting phases is slow on the NMR timescale, which is why the recorded NMR spectrum consists of superimposed spectra from the different phases occurring in the sample. Gramicidin D can be solubilized in appreciable quantities only in the lamellar phase of DDAO-d31. Increasing amounts of gramicidin in the liquid crystalline phases result in a continuous increase in the molecular ordering up to about 5 mol% gramicidin, where a plateau is reached. This is consistent with a recent theoretical model describing the influence on the ordering of lipids by a membrane protein with larger hydrophobic thickness than the lipid bilayer. The solvent used for dissolving gramicidin at the incorporation of the peptide in the lipid aggregates has no effect on the 2H-NMR lineshapes of DDAO-d31. It is concluded that gramicidin is solubilized in the L alpha phase and that it always adopts the channel conformation independent of a particular solvent. The channel conformation is also supported by CD studies. In some of the samples, macroscopic orientation of the lipid aggregates is observed. It is concluded that DDAO-d31 in the binary system favors an orientation with the long axis of the hydrocarbon chain perpendicular to the magnetic field, whereas when gramicidin D is present the hydrocarbon chain orients parallel to the magnetic field. This is explained by the fact that gramicidin aligns with its helical axis parallel to the magnetic field, thereby forcing also the DDAO-d31 molecules to obtain such an orientation.     

Conformation of the gramicidin A channel in phospholipid vesicles: a fluorine-19 nuclear magnetic resonance study

The membrane conformation of the peptide ionophore gramicidin A is shown by 19F NMR to be described by the N-terminal to N-terminal beta LD helical dimer model proposed by Urry [Urry, D.W. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 672-676]. Fully active analogues of gramicidin with 19F labels at both the N- and C-termini are prepared synthetically. Labeled peptides are incorporated into small unilamellar vesicles of dimyristoylphosphatidylcholine. Measurements of the accessibility of the labels to either aqueous or lipophilic paramagnetic probes show that the N-terminus of gramicidin is located in the membrane interior and the C-terminus is at the membrane surface. Of the specific models proposed for the structure of gramicidin, these data are consistent only with that of Urry. The C-terminal 19F NMR peak in vesicles actually consists of three overlapping peaks. Experiments with the aqueous shift reagent Tm3+ show that C-terminal 19F nuclei in the inner and in the outer leaflets of vesicles resonate at different frequencies. The outer leaflet peak in turn consists of two overlapping peaks, possibly due to a local rearrangement of the C-terminal label.     

Stimulation of cation transport in mitochondria by gramicidin and truncated derivatives

Gramicidin and the truncated derivatives desformylgramicidin (desfor) and des(formylvalyl)gramicidin (desval) stimulate monovalent cation transport in rat liver mitochondria. Cation fluxes were compared indirectly from the effect of cations on the membrane potential at steady state (state 4) or from the associated stimulation of electron transport. Rb+ transport was measured directly from the uptake of 86Rb. The truncated gramicidins show enhanced selectivity for K+ and Rb+ when compared to gramicidin. Moreover, the pattern of selectivity within the alkali cation series is altered, i.e., Rb+ greater than K+ greater than Cs+ greater than Na+ greater than Li+ for desfor and desval as compared to Cs+ greater than Rb+ greater than K+ = Na+ greater than Li+ for gramicidin. The cation fluxes through the truncated derivatives are more strongly dependent on the cation concentration. The presence of high concentrations of permeating cation enhances the transport of other cations through the truncated derivative channels, suggesting that cations are required for stabilizing the channel structure. In high concentrations of KCl, desfor and desval are nearly as effective as gramicidin in collapsing the mitochondrial membrane potential, and, consequently, in the uncoupling of oxidative phosphorylation and enhancement of ATP hydrolysis. Preliminary experiments with liposomes show that 86Rb exchange is stimulated by desfor and desval almost to the same extent as gramicidin. These results strongly suggest that the truncated gramicidins form a novel conducting channel which differs from the gramicidin head-to-head, single-stranded beta 6.3-helical dimer ("channel") in its conductance characteristic and its structure.(ABSTRACT TRUNCATED AT 250 WORDS)