gramicidin-a and cesium-chloride

We demonstrate that the cation-selective channel formed by gramicidin A can be used as a reliable sensor for studying the multivalent ion accumulation at the surfaces of charged lipid membranes and the "charge inversion" phenomenon. In asymmetrically charged membranes with the individual leaflets formed from pure negative and positive lipids bathed by 0.1 M CsCl solutions the channel exhibits current rectification, which is comparable to that of a typical n/p semiconductor diode. We show that even at these highly asymmetrical conditions the channel conductance can be satisfactorily described by the electrodiffusion equation in the constant field approximation but, due to predictable limitations, only when the applied voltages do not exceed 50 mV. Analysis of the changes in the voltage-dependent channel conductance upon addition of trivalent cations allows us to gauge their interactions with the membrane surface. The inversion of the sign of the effective surface charge takes place at the concentrations, which correlate with the cation size. Specifically, these concentrations are close to 0.05 mM for lanthanum, 0.25 mM for hexaamminecobalt, and 4 mM for spermidine.

Topics: Cations; Cesium; Chlorides; Electric Conductivity; Gramicidin; Lipid Bilayers; Semiconductors; Solutions; Surface Properties

The direct role of the dioxolane group on the gating and single-channel conductance of different stereoisomers of the dioxolane-linked gramicidin A (gA) channels reconstituted in planar lipid bilayers was investigated. Four different covalently linked gA dimers were synthesized. In two of them, the linker was the conventional dioxolane described previously (SS and RR channels). Two gAs were covalently linked with a novel modified dioxolane group containing a retinal attachment (ret-SS and ret-RR gA dimers). These proteins also formed ion channels in lipid bilayers and were selective for monovalent cations. The presence of the bulky and hydrophobic retinal group immobilizes the dioxolane linker in the bilayer core preventing its rotation into the hydrophilic lumen of the pore. In 1 M HCl the gating kinetics of the SS or RR dimers were indistinguishable from their retinal counterparts; the dwell-time distributions of the open and closed states in the SS and ret-SS were basically the same. In particular, the inactivation of the RR was not prevented by the presence of the retinal group. It is concluded that neither the fast closing events in the SS or RR dimers nor the inactivation of the RR are likely to be a functional consequence of the flipping of the dioxolane inside the pore of the channel. On the other hand, the inactivation of the RR dimer was entirely eliminated when alkaline metals (Cs(+) or K(+)) were the permeating cations in the channel. In fact, the open state of the RR channel became extremely stable, and the gating characteristics of both the SS and RR channels were different from what was seen before with permeating protons. As in HCl, the presence of a retinal in the dioxolane linker did not affect the gating behavior of the SS and RR in Cs(+)- or K(+)-containing solutions. Alternative hypotheses concerning the gating of linked gA dimers are discussed.

Topics: Anti-Bacterial Agents; Cations; Cesium; Chlorides; Chromatography; Dimerization; Gramicidin; Kinetics; Lipid Bilayers; Magnetic Resonance Spectroscopy; Metals; Models, Chemical; Phospholipids; Potassium Chloride; Protein Binding; Retina; Time Factors

The hydrophobic channel-forming polypeptide gramicidin adopts a left-handed antiparallel double helix conformation with 6.4 residues per turn when in complex with monovalent cation salts in a methanol environment. The crystal structure of the gramicidin/potassium thiocyanate complex (a = 32.06 A, b = 51.80 A, and c = 31.04 A; space group P2(1)2(1)2(1)) has been solved to 2.5 A with an R-factor of 0.193. In the structure, binding sites for the cations are formed by the polypeptide backbone carbonyl groups tilting away from the helix axis toward the ions located in the central lumen. The polypeptide backbone conformations and the side-chain orientations in this potassium complex are significantly different from those in the previously solved gramicidin/caesium chloride crystal complex, due to the requirements for interactions with the smaller sized potassium cation. The locations and numbers of potassium binding sites also differ considerably from the locations and numbers of caesium binding sites in the other structure. Combining information from all the cation binding sites in the two gramicidin/ion complexes produces different views of the three-dimensional structures of a cation as it is transported along a transmembrane pore, and provides an experimental structural basis for modeling the dynamics of peptide-ion binding and ion transport.

Topics: Anions; Anti-Bacterial Agents; Binding Sites; Cations; Cesium; Chlorides; Crystallography, X-Ray; Dimerization; Gramicidin; Hydrogen Bonding; Ion Channels; Methanol; Models, Molecular; Protein Structure, Secondary; Thiocyanates; Tryptophan

One topic of this study is the comparison of different preparation techniques to build up solid supported lipid bilayers onto gold substrates. The deposited lipid bilayers were investigated by a.c. impedance spectroscopy. Three different strategies were applied: (1) The gold surface was initially covered with a chemisorbed monolayer of octadecanethiol or 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). The second monolayer consisting of phospholipids was then deposited onto this hydrophobic surface by (i) the Langmuir-Schaefer-technique, (ii) from lipid solution in n-decane/isobutanol, (iii) by the lipid/detergent dilution technique or (iv) by fusion of vesicles. (2) Charged molecules carrying thiol-anchors for attachment to the gold surface by chemisorption were used. Negatively charged surfaces of 3-mercaptopropionic acid were found to be excellent substrates that allow the attachment of planar lipid bilayers by applying positively charged dimethyldioctadecylammoniumbromide (DODAB) vesicles or negatively charged 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol vesicles in the presence of chelating Ca2+-ions. If positively charged first monolayers of mercaptoethylammoniumhydrochloride were used we were able to attach mixed 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol/1,2-dimyristoyl-sn-glycero- 3-phosphoethanolamine vesicles to form planar lipid bilayers via electrostatic interaction. (3) Direct deposition of lipid bilayers is possible from vesicles containing 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). A critical amount of more than 50 mol% of DMPTE was found to be necessary to form a solid supported lipid bilayer. Bilayers obtained with these different preparation techniques were scrutinized with respect to their capacitances, kinetics of formation and their long-term stabilities by impedance spectroscopy. The second feature of this paper is the application of the supported bilayers to study ion transport through channel-forming peptides. We used a DODAB-bilayer for the reconstitution of gramicidin D channels. By circular dichroism measurements we verified that the peptide is in its channel conformation. The ion transport of Cs+-ions through the channels was recorded by impedance analysis.

Topics: Cesium; Chlorides; Electric Impedance; Electrochemistry; Gold; Gramicidin; Ion Transport; Lipid Bilayers; Phosphatidic Acids; Quaternary Ammonium Compounds; Signal Processing, Computer-Assisted

Topics: Binding Sites; Bromides; Cesium; Chlorides; Crystallography, X-Ray; Gramicidin; Ligands; Models, Structural; Protein Conformation; Thiocyanates

Recently we reported that rapid fluctuations of ion currents flowing through open gramicidin A channels exceed the expected level of pure transport noise at low ion concentrations (Heinemann, S. H. and F. J. Sigworth. 1990. Biophys. J. 57:499-514). Based on comparisons with kinetic ion transport models we concluded that this excess noise is likely caused by current interruptions lasting approximately 1 microsecond. Here we introduce a method using the higher-order cumulants of the amplitude distribution to estimate the kinetics of channel closing events far below the actual time resolution of the recording system. Using this method on data recorded with 10 kHz bandwidth, estimates for gap time constants on the order of 1 microsecond were obtained, similar to the earlier predictions.

Topics: Cesium; Chlorides; Electric Conductivity; Gramicidin; Ion Channels; Kinetics; Lipid Bilayers; Mathematics; Models, Biological

Single-wavelength Cu K alpha anomalous scattering has been used to determine the structure of a crystalline complex of gramicidin A and caesium chloride. The asymmetric unit in these crystals, with space group P2(1)2(1)2(1) and a = 32.118 (6), b = 52.103 (12), c = 31.174 (7) A, contains four independent monomers (two dimers) of the pentadecapeptide. This structure falls in an intermediate size range for which direct methods and multiple isomorphous replacement are generally not successful for obtaining phase information. However, using the Bijvoet differences and the partial structure of the caesium atoms which have been incorporated in the crystals, it has been possible to obtain information on this crystal form. Because the caesium atoms dominate the scattering of these crystals, inclusion of the Friedel mate information in the restrained least-squares refinement has been essential. These studies extend the utility of single-wavelength anomalous-scattering phase determination to a macromolecular structure in which the partial structure of the anomalous scatterer is large.

Topics: Cesium; Chlorides; Crystallization; Crystallography; Gramicidin; Least-Squares Analysis; Models, Molecular; Scattering, Radiation

Current-voltage relations have been measured for the fluxes of caesium ions through pores formed by gramicidin in lipid bilayer membranes. The ionic currents have been separated from capacitative currents using a bridge circuit with an integrator as null-detector. The conductances during brief voltage pulses were small enough to avoid the effects of diffusion polarization and the ionic strength was raised using choline chloride or magnesium sulfate to reduce the effects of double-layer polarization. Under these conditions the current-voltage relations have the same shape at 0.1 and 1 mM, but different shapes for higher concentrations. These data demonstrate that the fluxes do not obey independence for concentrations above 10 mM, but they cannot be used in isolation to support a particular value of the binding constant. The shape observed at low concentrations suggests that entry of ions into the pore remains weakly potential dependent even at 300 mV.

Topics: Cesium; Chlorides; Electric Conductivity; Gramicidin; Ion Channels; Models, Biological; Permeability