valinomycin and 1-2-diphytanoylphosphatidylcholine

The study of ion channel activity and the screening of possible inhibitor molecules require reliable methods for production of active channel proteins, their insertion into artificial membranes and for the measurement of their activity. Here we report on cell-free expression of soluble and active K

Topics: Elapid Venoms; Escherichia coli; Fluorescent Dyes; Gene Expression; Genetic Vectors; Humans; Isoxazoles; Kv1.1 Potassium Channel; Kv1.3 Potassium Channel; Membrane Potentials; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Proteolipids; Recombinant Proteins; Subcellular Fractions; Valinomycin

ortho-Carborane (1,2-C2B10H12) was found to be a carrier of protons in both mitochondrial and artificial lipid membranes, suggesting that this dicarborane can reversibly release hydrogen ions and diffuse through the membranes in neutral and anionic forms. Similar to conventional uncouplers (e.g. 2,4-dinitrophenol), o-carborane stimulated mitochondrial respiration and decreased the membrane potential at concentrations of tens of micromoles. Protonophoric activity of o-carborane was observed both by a fluorometric assay using pyranine-loaded liposomes and electrical current measurements across planar lipid bilayers. Substantial contribution of the proton flux to the o-carborane-mediated current was proved by a shift of the zero current voltage upon imposing a pH gradient across the membrane. Meta-carborane (1,7-C2B10H12) lacked the protonophoric activity in line with its reduced C-H acidity. The results suggest that weak C-H acids can exhibit protonophoric activity in the biological environment. The finding of a new class of protonophoric compounds is of substantial interest due to promising anti-obesity and anti-diabetic properties of uncouplers.

Topics: Animals; Arylsulfonates; Boranes; Boron Compounds; Hydrogen-Ion Concentration; Kinetics; Lewis Acids; Lipid Bilayers; Liposomes; Membrane Potential, Mitochondrial; Mitochondria, Liver; Mitochondrial Membranes; Phosphatidylcholines; Rats; Uncoupling Agents; Valinomycin

Gramicidin D was incorporated in a biomimetic membrane consisting of a lipid bilayer tethered to a mercury electrode via a hydrophilic spacer, and its behavior was investigated in aqueous 0.1 M KCl by potential-step chronocoulometry and electrochemical impedance spectroscopy. The impedance spectra, recorded from 0.1 to 1 x 10(5) Hz over a potential range of 0.7 V, were fitted to a series of RC meshes, which were related to the different substructural elements of the biomimetic membrane. These impedance spectra were compared with those obtained by incorporating valinomycin, under otherwise identical conditions. The potential dependence of the stationary currents reported on bilayer lipid membranes by Bamberg and Läuger (Bamberg, E.; Läuger, P. J. Membrane Biol. 1973, 11, 177-194) as well as those extracted from potential-step chronocoulometric measurements was interpreted by relating the increase in gramicidin dimerization to a progressive increase in single-file K+ flux along the dimeric channels. An analogous approach was adopted in explaining the difference between the impedance spectra obtained with gramicidin D and those obtained with valinomycin. It is concluded that gramicidin has a low tendency to form dimers in the absence of ionic flux.

Topics: Algorithms; Biomimetics; Electrochemistry; Gramicidin; Lipid Bilayers; Metals; Phosphatidylcholines; Potassium Chloride; Spectrum Analysis; Valinomycin

Tethered membranes have been proven during recent years to be a powerful and flexible biomimetic platform. We reported in a previous article on the design of a new architecture based on the self-assembly of a thiolipid on ultrasmooth gold substrates, which shows extremely good electrical sealing properties as well as functionality of a bilayer membrane. Here, we describe the synthesis of lipids for a more modular design and the adaptation of the linker part to silane chemistry. We were able to form a functional tethered bilayer lipid membrane with good electrical sealing properties covering a silicon oxide surface. We demonstrate the functional incorporation of the ion carrier valinomycin and of the ion channel gramicidin.

Topics: Biophysics; Electric Impedance; Electrochemistry; Gold; Gramicidin; Ion Channels; Ionophores; Ions; Lipid Bilayers; Lipids; Microscopy, Atomic Force; Models, Chemical; Phosphatidylcholines; Phytol; Protein Array Analysis; Silanes; Silicon; Silicon Dioxide; Spectrophotometry; Substrate Specificity; Temperature; Time Factors; Valinomycin