Effects of gramicidin and tryptophan-N-formylated gramicidin on the sodium and potassium content of human erythrocytes

Summary

In order to get a better understanding in the mechanism by which tryptophan-N-formylated gramicidin (NFG) and gramicidin kill the malaria parasite Plasmodium falciparum in vitro, we studied the capacity of these peptides to change the potassium, as well as the sodium, composition of normal human erythrocytes, and their ability to cause cell lysis. It is shown that both peptides are able to induce potassium leakage from, and sodium flux into, erythrocytes in such a manner that it is most likely that they are able to form cation channels in the membrane of these cells. For both peptides, potassium efflux proceeds at a faster rate than sodium influx, but this difference is greater for NFG than for gramicidin. This explains the observation that gramicidin is more lytic than NFG is, even when comparing concentrations that show the same antimalarial activity. The finding that gramicidin is approximately 10 times more active than NFG in causing potassium efflux from normal erythrocytes, as well as in killing the malaria parasite, supports the hypothesis that peptideinduced parasite death is related to their capacity to induce potassium leakage from infected erythrocytes. Finally, the observation that erythrocytes are able to restore their normal ion contents after losing more than 50% of their potassium content by incubation with NFG or gramicidin, suggests that, in vivo, and upon treatment with drug concentrations that cause full inhibition of parasite growth, these cells would not be irreversibly damaged by action of the drugs.     

Study of interaction between cation fluxes in gramicidin A-modified bilayer phospholipid membranes. Determination of the number of ion-binding sites and their position in the gramicidin channel

Simultaneous studies were carried out of isotope and electric parameters of spheric bilayer membranes modified with gramicidin A and its analog O-pyromellithylgramicidin (PG) having three negative charges on the N-end of the molecule. The relationship between the electric coefficients of permeability and the isotope ones PG/P* = n was determined by two independent methods. It has been found that for the membranes modified with gramicidin A in RbCl concentrations from 2.2 x 10(-3) to 10(-1) M the value n is constant and approximates 2 and with RbCl concentration 1 M, n = 1.6. For the membranes modified with PG in 0.1 M solutions of PbCl n = 2. The results obtained in terms of the model of unilinear ion diffusion in a narrow pore indicate that in a gramicidin channel there are two sites of cation binding which are located near the channel mouth.     

Raman and infrared spectroscopic study of gramicidin A conformations

Raman scattering and infrared spectroscopic techniques were used to study the vibrational spectrum and conformation of the membrane channel protein gramicidin A in the solid state, in organic solutions and, using Raman scattering only, in a phospholipid environment. The investigation also includes measurements on head- and tail-group-modifled gramicidin A and a potassium thiocyanate-gramicidin A complex. Tentative identification of the molecular vibrations is proposed on the basis of the data on model compounds. The existence of four distinct conformations of the gramicidin A chain is established: conformation I present in the solid state, and CH₃OH and CD₃OD solutions; conformation II present in films cast from CHCl₃ solution; conformation III present in (CH₃)₂SO and (CD₃)₂SO solutions at concentrations below 0.5 m gramicidin A; and conformation IV present in the potassium thiocyanate-gramicidin A complex. The data obtainable on a gramicidin A-phospholipid suspension indicate a gramicidin A conformation in this environment corresponding either to the conformation I or II. The details of the spectra in the amide I region are shown to be consistent with a β-parallel hydrogen-bonded π_LD helix for conformational I, in terms of the polypeptide vibrational calculations of Nevskaya and co-workers. Conformation II is found to be consistent with an antiparallel double-stranded π_LD helix, while conformations III and IV probably have π-helical structures with larger channel diameters. The data on head- and tail-modified gramicidin A molecules indicate that their conformations are only slightly different from that of gramicidin A in conformation I.     

Electrogenic behavior of synaptic vesicles from Torpedo californica.

Electrical potential changes in pure synaptic vesicles from Torpedo californica were monitored with the fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide. Vesicles resuspended in variable external sodium ion in the presence of gramicidin established sodium ion membrane diffusion potentials. Vesicles resuspended in choline or acetylcholine chloride became hyperpolarized upon addition of gramicidin. Hyperpolarization was subsequently partially reversed spontaneously by choline or acetylcholine influx, which was confirmed by gel filtration, to yield a new, less negative, stable membrane potential. Thus, acetylcholine and choline are taken up electrogenically by synaptic vesicles.     

Simulation of voltage-driven hydrated cation transport through narrow transmembrane channels.

Molecular dynamics studies for the voltage-driven transport of the alkali metal ions lithium, sodium, and potassium through gramicidin A-type channels filled with water molecules are presented. The number of water molecules in the channel is obtained from a previous study (Skerra, A., and J. Brickmann, 1987, Biophys. J., 51:969-976). It is shown that the selectivity of the intrachannel ion diffusion through our model pore conforms to the experimentally observed selectivity of the gramicidin A channel. It is demonstrated that the number of water molecules in the channel plays a key role for the selectivity.     

The supply of nutrient ions by diffusion to plant roots in soil

Summary A single-root technique is used to measure the rate of supply of potassium by diffusion to 1-cm portions of cylindrical roots of onion and leek plants growing in soils containing different levels of exchangeable potassium. The relation between uptake and characteristics of the plant and soil is interpreted on the basis of a diffusion supply model. Uptake is accounted for in terms of the geometry of the absorbing root surface, the physiologically controlled absorbing power of the root, and the diffusion through the soil. The different uptakes of potassium by roots of comparable absorbing power from different soils can be predicted with some success from calculations using the root dimensions and either diffusion coefficients of potassium in soil, derived from flux to a cation exchange resin paper, or the form of the potassium scorption isotherm relating the concentration of labile ions to those in the soil solution. It is calculated that diffusion through the soil has reduced potassium uptake by the roots to between 87 and 39 per cent of that expected for roots of the same absorbing power in a stirred culture solution at the same initial soil solution concentration.     

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

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

Effect of gramicidin A and thallium ions on cation effluxes in frog sartorius muscle

The effluxes of potassium, rubidium, sodium and lithium from the sartorius muscle of Rana temporaria in magnesium-Ringer solution free of sodium and potassium have been studied with the flame-emission technique. The channel-forming antibiotic gramicidin A (2.5 X X10(-7)-1 X 10(-6) mol/l) enhanced the efflux of potassium and rubidium and increased the rate constants of these effluxes. Gramicidin had small if any effect on sodium and lithium effluxes and rate constants. After 60-100 min in a gramicidin-containing medium, the potassium efflux and the corresponding rate constant reached a steady-state level. This steady-state value depended on gramicidin concentration. Effect of gramicidin on both the potassium efflux and the rate constant was partially reversible. Thallium ions (2.5 X 10(-3) and 5 X 10(-3) mol/l) in sodium- and potassium- free magnesium Ringer solution caused a large increase in effluxes of all the cations examined (K+, Rb+ and Na+) both in presence and absence of gramicidin. Possible mechanisms of gramicidin and thallium effects are discussed.     

All-trans retinol in rod photoreceptor outer segments moves unrestrictedly by passive diffusion

The visual pigment protein of vertebrate rod photoreceptors, rhodopsin, contains an 11-cis retinyl moiety that is isomerized to all-trans upon light absorption. Subsequently, all-trans retinal is released from the protein and reduced to all-trans retinol, the first step in the recycling of rhodopsin's chromophore group through the series of reactions that constitute the visual cycle. The concentration of all-trans retinol in photoreceptor outer segments can be monitored from its fluorescence. We have used two-photon excitation (720 nm) of retinol fluorescence and fluorescence recovery after photobleaching to characterize the mobility of all-trans retinol in frog photoreceptor outer segments. Retinol produced after rhodopsin bleaching moved laterally in the disk membrane bilayer with an apparent diffusion coefficient of 2.5 +/- 0.3 micro m(2) s(-1). The diffusion coefficient of exogenously added retinol was 3.2 +/- 0.5 micro m(2) s(-1). These diffusion coefficients are in close agreement with those reported for lipids, suggesting that retinol is not tightly bound to protein sites that would be diffusing much more slowly in the plane of the membrane. In agreement with this interpretation, a fluorescent-labeled C-16 fatty acid diffused laterally with a similar diffusion coefficient, 2.2 +/- 0.2 micro m(2) s(-1). Retinol also moved along the length of the rod outer segment, with an apparent diffusion coefficient of 0.07 +/- 0.01 micro m(2) s(-1), again suggesting that it is not tightly bound to proteins that would confine it to the disks. The axial diffusion coefficient of exogenously added retinol was 0.05 +/- 0.01 micro m(2) s(-1). In agreement with passive diffusion, the rate of axial movement was inversely proportional to the square of the length of the rod outer segment. Diffusion of retinol on the plasma membrane of the outer segment can readily account for the measured value of the axial diffusion coefficient, as the plasma membrane comprises approximately 1% of the total outer-segment membrane. The values of both the lateral and axial diffusion coefficients are consistent with most of the all-trans retinol in the outer segments moving unrestricted and not being bound to carrier proteins. Therefore, and in contrast to other steps of the visual cycle, there does not appear to be any specialized processing for all-trans retinol within the rod outer segment.     

Ion fluxes in giant excised cardiac membrane patches detected and quantified with ion-selective microelectrodes

We have used ion-selective electrodes (ISEs) to quantify ion fluxes across giant membrane patches by measuring and simulating ion gradients on both membrane sides. Experimental conditions are selected with low concentrations of the ions detected on the membrane side being monitored. For detection from the cytoplasmic (bath) side, the patch pipette is oscillated laterally in front of an ISE. For detection on the extracellular (pipette) side, ISEs are fabricated from flexible quartz capillary tubing (tip diameters, 2-3 microns), and an ISE is positioned carefully within the patch pipette with the tip at a controlled distance from the mouth of the patch pipette. Transport activity is then manipulated by solution changes on the cytoplasmic side. Ion fluxes can be quantified by simulating the ion gradients with appropriate diffusion models. For extracellular (intrapatch pipette) recordings, ion diffusion coefficients can be determined from the time courses of concentration changes. The sensitivity and utility of the methods are demonstrated with cardiac membrane patches by measuring (a) potassium fluxes via ion channels, valinomycin, and Na/K pumps; (b) calcium fluxes mediated by Na/Ca exchangers; (c) sodium fluxes mediated by gramicidin and Na/K pumps; and (d) proton fluxes mediated by an unknown electrogenic mechanism. The potassium flux-to-current ratio for the Na/K pump is approximately twice that determined for potassium channels and valinomycin, as expected for a 3Na/2K pump stoichiometery (i.e., 2K/charge moved). For valinomycin-mediated potassium currents and gramicidin-mediated sodium currents, the ion fluxes calculated from diffusion models are typically 10-15% smaller than expected from the membrane currents. As presently implemented, the ISE methods allow reliable detection of calcium and proton fluxes equivalent to monovalent cation currents <1 pA in magnitude, and they allow detection of sodium and potassium fluxes equivalent to <5 pA currents. The capability to monitor ion fluxes, independent of membrane currents, should facilitate studies of both electrogenic and electroneutral ion-coupled transporters in giant patches.     

Measuring oxygen diffusion coefficients with polarographic oxygen electrodes. II. Fermentation Media

Fermentation media consist of a large number of chemicals which composition undergoes alteration during the course of fermentations. In consequence, the conventional methods and correlations for gas diffusion coefficient measurement and prediction cannot be easily applied to such systems. Oxygen diffusion coefficients have been measured in simulated chemical systems as well as in complex solutions of nutrient broth, using the polarographic technique introduced in a previous article. It is identified that sugars and salts are the major factors influencing oxygen diffusion coefficients in these aqueous fermentation media. The effect of salts on oxygen diffusion coefficients in electrolyte solutions has been found to be well correlated with the square root of total ionic strength of electrolyte solutions. The individual effect of glucose and its combined effect with salts are explored in order to reach rational correlations capable of predicting oxygen diffusion coefficients in synthetic fermentation media. For aqueous solutions of glucose plus salts, it is observed that the log-additive relationship can be used to account for the combined effect. Finally, a linear correlation has been established in measuring oxygen diffusion coefficients in aqueous solutions having different concentrations of nutrient broth.     

The permeability of liposomes to nonelectrolytes. II. The effect of nystatin and gramicidin A.

The process of selective permeation of nonelectrolytes across liposomes of different lipid composition and amount of cholesterol has been studied. The extent of the selectivity for diffusion within the membranes has been found to be related to the physical state of the hydrocarbon chains. It has been also found that incorporation of cholesterol into egg-lecithin membranes decreases the overall permeability by affecting the dehydration step more than the subsequent diffusion of the solute. The incorpporation into liposomes of the antibiotics nystatin and gramicidin A produces changes in the selective permeation of nonelectrolytes that are consistent with the formation by these molecules of aqueous pores of fixed dimensions. Finally, comparisons are made between the process of permeation in biological membranes and in liposomes with and without antibiotics.     

Solvent history dependence of gramicidin A conformations in hydrated lipid bilayers.

Reconstituted lipid bilayers of dimyristoylphosphatidylcholine (DMPC) and gramicidin A' have been prepared by cosolubilizing gramicidin and DMPC in one of three organic solvent systems followed by vacuum drying and hydration. The conformational state of gramicidin as characterized by 23Na NMR, circular dichroism, and solid state 15N NMR is dependent upon the cosolubilizing solvent system. In particular, two conformational states are described; a state in which Na+ has minimal interactions with the polypeptide, referred to as a nonchannel state, and a state in which Na+ interacts very strongly with the polypeptide, referred to as the channel state. Both of these conformations are intimately associated with the hydrophobic core of the lipid bilayer. Furthermore, both of these states are stable in the bilayer at neutral pH and at a temperature above the bilayer phase transition temperature. These results with gramicidin suggest that the conformation of membrane proteins may be dictated by the conformation before membrane insertion and may be dependent upon the mechanism by which the insertion is accomplished.     

Vibrational analysis of peptides, polypeptides and proteins. XXVII. Structure of gramicidin S from normal mode analyses of low-energy conformations

Normal mode calculations have been carried out on three low-energy structures of gramicidin S obtained from conformational energy calculations. When the results on the amide modes are compared with observed bands in the infrared and Raman spectra of crystalline gramicidin S and its N-deuterated derivative, one of the structures is clearly disfavored. Of the other two, one is slightly favored, and it corresponds to the lowest-energy structure obtained from the energy calculations. Spectra from solutions in DMSO and CH3 OH suggest that the molecular conformation is essentially retained in these solvents.     

Molecular dynamics estimates of ion diffusion in model hydrophobic and KcsA potassium channels

Molecular dynamics simulations are carried out to obtain estimates of diffusion coefficients of biologically important Na+, K+, Ca2+ and Cl- ions in hydrophobic cylindrical channels with varying radii and large reservoirs. Calculations for the cylindrical channels are compared to those for the KcsA potassium channel, for which the protein structure has recently been determined from X-ray diffraction experiments. Our results show that ion diffusion is maintained at reasonably high levels even within narrow channels, and does not support the very small diffusion coefficients used in some continuum models in order to fit experimental data. The present estimates of ion diffusion coefficients are useful in the calculation of channel conductance using the Poisson-Nernst-Planck theory, or Brownian dynamics.     

Rotating wave solutions of the FitzHugh–Nagumo equations

This paper will treat the bifurcation and numerical simulation of rotating wave (RW) solutions of the FitzHugh-Nagumo (FHN) equations. These equations are often used as a simple mathematical model of excitable media. The dependence of the solutions on a uniformly applied current, and also on the diffusion coefficients or domain size will be studied. Ranges of applied current and/or diffusion coefficients in which RW solutions are observed will be described using bifurcation theory and continuation methods. The bifurcation of time-periodic solutions of these FHN equations without diffusion is described first. Similar methods are then used to find RW solutions on a circular ring and numerical simulations are described. These results are then extended to investigate RW solutions on annular rings of finite cross-section. Scaling arguments are used to show how the existence of solutions depends on either the diffusion coefficient or on the size of the region.     

Diffusion of acetophenone and phenethyl alcohol in the calcium-alginate-bakers' yeast-hexane system

The intrabead diffusion coefficients of acetophenone and phenethyl alcohol were measured at 30 degrees C in the triphasic immobilized yeast-water-hexane system. Saccharomyces cerevisiae cells were deactivated with hydrochloric acid and entrapped in calcium-alginate beads. Measurements of dry cell loss during deactivation, shrinkage of the beads during deactivation and the final porosity of the beads were made for various cell loadings. Final concentrations of wet cells in the beads ranged from approximately 0.25 to 0.30 g/mL. Mass transfer in the hexane phase, external to the beads, was eliminated experimentally. The estimated error of 5% to 10% in the diffusion coefficients is within the experimental error associated with the bead method. The effect of significant sampling volumes on the diffusivities was estimated theoretically and accounted for experimentally. The intrabead concentration of acetophenone and phenethyl alcohol was 150 to 800 ppm. The deactivated cells were shown to be impervious to acetophenone so that the measured diffusivities are extracellular parameters. The cell volume fraction in the beads ranged from 0.70 to 0.90, significantly higher than previously reported data. The effective diffusion coefficients conform to the random pore model. No diffusional interaction between acetophenone and phenethyl alcohol was observed. The addition of 2 vol% ethanol or methanol slightly increased the diffusivities. The thermodynamic partition coefficients were measured in the bead-free water-organic system and found to be an order of magnitude lower than the values calculated from the analysis of the diffusion data for the organic-bead system, suggesting that bead-free equilibrium data cannot be used in triphasic systems. (c) 1994 John Wiley & Sons, Inc.     

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.     

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.