Cation binding by valinomycin and trinactin at the air-water interface: Cooperativity in cation binding by valinomycin

A previous communication reported the uptake of monovalent cations by a valinomycin monolayer at the air-water interface (Colacicco, G., Gordon, E. E. and Berchenko, G. (1968) Biophys. J. 8,22a). A similar study has been done with trinactin. As in the case of valinomycin, an elevated surface potential is obtained when the cation-ionophore complex is formed. A surface potential of 0.82 V was obtained for the trinactin-cation complex, as compared with 0.54 V for uncomplexed trinactin. The observed cation selectivity NH₄⁺ > K⁺ > Rb⁺ > Cs⁺, Na⁺ and Li⁺ is in agreement with partition and bilayer conductance experiments.A minimum packing area of 130 Ų obtained for the trinactin-cation complex was in excellent agreement with the 125 Ų predicted from space filling models, reinforcing the suggestion that area-per-molecule calculations obtained at the air-wate interface can provide useful information on the molecular dimensions of these hydrophobic, relatively low molecular weight transport antibiotics.Comparison of the data obtained previously with valinomycin and with trinactin revealed two striking differences: (1) a large inflection in the force-area curve concurrent with cation binding and indicative of a conformational change was obtained with valinomycin,, but no evidence was found with trinactin; (2) the uptake of cations by trinactin could be predicted by simple equilibrium expressions, but the uptake of cations by valinomycin was strongly cooperative. Possible mechanisms for this cooperative association fo cations are discussed.     

Characterization of the binding of valinomycin to bacteriorhodopsin

Circular dichroism spectroscopy has been used to investigate the binding of valinomycin to bacteriorhodopsin in purple membrane suspensions. Addition of valinomycin to purple membrane suspensions obtained from Halobacterium halobium causes the circular dichroism spectrum to shift from an aggregate spectrum to one resembling a monomer spectrum, indicating a loss of chromophore-chromophore interactions. By observing the spectral change upon titration of valinomycin, an apparent dissociation constant of 30–40 M for valinomycin binding was determined. Kinetics of dark adaptation for valinomycin-treated purple membrane are comparable to those for monomeric bacteriorhodopsin. Centrifugation studies demonstrate that valinomycin-treated purple membrane sediments the same as untreated purple membrane suspensions. These results are consistent with a model in which valinomycin binds specifically to bacteriorhodopsin without disrupting the purple membrane fragments.Abbreviations BR bacteriorhodopsin - CD circular dichroism - Tricine N-[tris-(hydroxymethyl) methyl] glycine     

Structure of valinomycin by molecular dynamics studies.

Valinomycin is an important ionophore which exhibits a high conformational flexibility. The study of various conformations adopted by this molecule together with the study of flexibility in a given conformation can throw light on the ion transport by the ionophore across the membrane. Molecular dynamics (MD) studies are ideal to characterize the flexibility in different parts of the molecule and can also give an idea of various conformations adopted by the molecule at a given temperature. Hence MD studies at 100K have been carried out on the minimized crystal structure of the molecule to scan the possible conformations in the neighbourhood of the well known 'bracelet' like structure of uncomplexed Valinomycin, Properties, like the flexibility, average values, r.m.s. fluctuations of the various intramolecular hydrogen bonds are discussed. Energy minimization has been carried out on selected MD simulated points to analyze the characteristics of the unique conformation adopted by this molecule at this temperature.     

Inhibition by valinomycin of atractyloside binding to the membrane-bound ADP/ATP carrier: Counteracting effect of cations

The atractyloside binding capacity of rat heart mitochondria, but not the binding affinity, was markedly decreased by preincubation of the mitochondria with valinomycin in isotonic KCl medium. Maximum inhibition was attained with 5 ng of valinomycin per mg of mitochondrial protein; it corresponded to a 40% decrease of the atractyloside binding capacity. The inhibitory effect of valinomycin was maximal between pH 7.0 and 7.5. It was more marked for heart mitochondria than for liver mitochondria. Valinomycin inhibition of atractyloside binding to heart mitochondria was counteracted by nigericin and FCCP, by sublytic concentrations of cationic surfactants such as cetyltrimethylammonium bromide, and by low concentrations of trivalent and divalent metal ions at acidic pH's still compatible with atractyloside binding, i.e., down to pH 5.5; trivalent metal ions were more effective than divalent metal ions. The effect of valinomycin was also counteracted by exceedingly high concentrations of K+ (more than 300 mM), resulting in a substantial increase in the ionic strength. These results were discussed in terms of the relation between the atractyloside binding capacity of the inner mitochondrial membrane and the surface potential of this membrane.     

Toxicity of valinomycin on insects

Valinomycin was administered to Musca domestica, Periplaneta americana, Aedes aegypti, and mice. Although topical application to house flies did not cause appreciable toxicity, the substance was highly toxic when injected or administered per os, characteristic nervous symptoms occurring in intoxicated flies. Injected valinomycin was almost equally toxic to cockroaches. Aedes aegypti larvae, on the other hand, were hardly affected by the substance.On the cockroach semi-isolated heart preparation, heart block occurred also at low concentrations.Preliminary trials showed that valinomycin abolished the response of leg muscles to both direct and indirect stimulation and interrupted conduction of stimulus on the nervous cord connectives.     

Permeability transition-independent release of mitochondrial cytochrome c induced by valinomycin.

To examine whether valinomycin induces a mitochondrial permeability transition (PT), we investigated its effects on mitochondrial functions under various conditions. The acceleration of mitochondrial respiration and swelling, induced by valinomycin, were found to be insensitive to inhibitors of the ordinary PT, indicating that valinomycin does not induce the ordinary PT. Results of experiments using mitochondria isolated from transgenic mice expressing human bcl-2 also supported this conclusion. Furthermore, evidence for induction of PT pores by valinomycin was not obtained by either electron microscopic analysis of mitochondrial configurations or by measurement of the permeability of the inner mitochondrial membrane by use of polyethylene glycol. However, valinomycin did induce a significant release of cytochrome c, and thus it may be a nice tool to study the processes of mitochondrial cytochrome c release.     

Increase in fluorescence energy transfer across lipid bilayers induced by valinomycin

The translocator antibiotic, valinomycin, increases the energy transfer between fluorophores across a lipid bilayer membrane, contrary to the effect of an inert protein adsorbate. The distance separating the fluorophores is reduced, suggesting that this translocator provokes a perturbation in the palisade arrangement of lipid molecules in the bilayer.     

Location of valinomycin in lipid vesicles

The location of the cyclododecadepsipeptide, valinomycin in vesicles formed from two synthetic lipids is studied by differential scanning calorimetry, spin-label partitioning electron paramagnetic resonance and [¹H]-nuclear magnetic resonance. The results show that valinomycin is located near the head group region of dipalmitoyl phosphatidyl choline vesicles and in the hydrophobic core of the dimyristoyl phosphatidyl choline vesicles in the liquid crystalline phase.     

Neurite formation and membrane changes of mouse neuroblastoma cells induced by valinomycin

A clonal cell line of mouse neuroblastoma cells was found to undergo morphological differentiation in the presence of a K⁺ ionophore, valinomycin, in the assay medium. This effect was blocked by increasing the concentration of KCl of the medium, suggesting that the changes in resting membrane potential and ion fluxes may be involved in the mechanism of the formation of neurites. No enhancement of the neurite formation was observed in salines containing high concentrations of KCl in the absence of valinomycin. Depolarizing agents including veratridine, gramicidin and ouabain did not stimulate the outgrowth of neurites. Neither electrophoretic mobility of the cells nor molecular anisotropy of fluorescence probes in the membranes was modified by the treatment of valinomycin. Instead, it modified the slow binding phase in kinetics of the interaction of 1-anilinonaphthalene-8-sulfonate (ANS) with the cells, which is related to the penetration process of the probe into membranes. Valinomycin also enhanced the fluorescence intensity of ANS by increasing the binding sites in neuroblastoma cells.     

Induction of apoptosis by valinomycin: mitochondrial permeability transition causes intracellular acidification

In order to determine whether disruption of mitochondrial function could trigger apoptosis in murine haematopoietic cells, we used the potassium ionophore valinomycin. Valinomycin induces apoptosis in the murine pre-B cell line BAF3, which cannot be inhibited by interleukin-3 addition or Bcl-2 over-expression. Valinomycin triggers rapid loss of mitochondrial membrane potential. This precedes cytoplasmic acidification, which leads to cysteine-active-site protease activation, DNA fragmentation and cell death. Bongkrekic acid, an inhibitor of the mitochondrial permeability transition, prevents acidification and subsequent induction of apoptosis by valinomycin.     

Neurite formation and membrane changes of mouse neurobalstoma cells induced by valinomycin

A clonal cell line of mouse neuroblastoma cells was found to undergo morphological differentiation in the presence of a K+ ionophore, valinomycin, in the assay medium. This effect was blocked by increasing the concentration of KCl of the medium, suggesting that the changes in resting membrane potential and ion fluxes may be involved in the mechanism of the formation of neurites. No enhancement of the neurite formation was observed in salines containing high concentrations of KCl in the absence of valinomycin. Depolarizing agents including veratridine, gramicidin and ouabain did not stimulate the outgrowth of neurites. Neither electrophoretic mobility of the cells nor molecular anisotropy of fluorescence probes in the membranes was modified by the treatment of valinomycin. Instead, it modified the slow binding phase in kinetics of the interaction of 1-anilinonaphthalene-8-sulfonate (ANS) with the cells, which is related to the penetration process of the probe into membranes. Valinomycin also enhanced the fluorescence intensity of ANS by increasing the binding sites in neuroblastoma cells.     

Mutual inactivation of valinomycin and protonophores by complex formation in liposomal membranes

The stimulation presence of a protonophore [3,5-di(ter-butyl)-4-hydroxybenzylidenemalononitrile or carbonyl cyanide m-chlorophenylhydrazone] and valinomycin in a liposome suspension results in time-dependent inactivation of ion transport by both the protonophore and valinomycin. Correlation of the inactivation with spectrophotometric observations on the formation of a complex between the protonophore and valinomycin strongly suggests that the complex observed has no (or very low) activity for the transport of either H+ or K+. The stoichiometry of valinomycin and the protonophore in the inactive complex is shown to be 1:1.     

Entry of poliovirus into cells is blocked by valinomycin and concanamycin A

Poliovirus contains a virus particle devoid of a lipid envelope that does not require an intact pH to enter into susceptible cells. Thus, the blockade of pH gradient generated in endosomes is not sufficient to impede the translocation of poliovirus particles to the cytoplasm, suggesting that translocation takes place at the plasma membrane. Measuring both viral protein synthesis and eIF4G-1 cleavage mediated by poliovirus protease 2A has been used to monitor productive entry of poliovirus into cells. Translation of the input poliovirus RNA produces enough 2A(pro) to cleave eIF4G-1, providing a sensitive assay to estimate poliovirus RNA delivery to the cytoplasm followed by its translation. Combination of concanamycin A, a vacuolar proton-ATPase inhibitor, and valinomycin, an ionophore that promotes K(+) efflux from cells, powerfully prevented poliovirus infection. Moreover, modifying the ionic conditions of the culture medium (increasing the concentration of K(+) and decreasing the concentration of Na(+)), together with concanamycin A, also significantly interfered with poliovirus entry. These findings suggest that poliovirus RNA requires an intact concentration of K(+) ions inside the cells to be uncoated and to gain access to the cytoplasm. In addition, the actual contribution of concanamycin A (as well as other inhibitors of endocytosis) to the total inhibition of productive poliovirus entry points to the idea that at least some percentage of polioviral subparticles translocates from the endosomes.     

Impedance analysis of valinomycin activity in nano-BLMs

Nano-black lipid membranes (nano-BLMs) were obtained by functionalization of highly ordered porous alumina substrates with an average pore diameter of 60 nm based on a self-assembled alkanethiol submonolayer followed by spreading of 1,2-diphytanoyl-sn-glycero-3-phosphocholine dissolved in n-decane on the hydrophobic substrate. By means of impedance spectroscopy, we analyzed the influence of the self-assembled alkanethiol submonolayer on the electrical properties of the nano-BLMs as well as their long-term stability. We were able to stably integrate nano-BLMs into a flow through system, which allowed us to readily exchange buffer solutions several times and accounts for mass transport phenomena. The ionophore valinomycin was successfully inserted into nano-BLMs and its transport activity monitored as a function of different potassium and sodium ion concentrations reflecting the specificity of valinomycin for potassium ions.