ATP Transport through VDAC and the VDAC–Tubulin Complex Probed by Equilibrium and Nonequilibrium MD Simulations

Voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane, serves as a principal pathway for ATP, ADP, and other respiratory substrates across this membrane. Using umbrella-sampling simulations, we established the thermodynamic and kinetic components governing ATP transport across the VDAC1 channel. We found that there are several low-affinity binding sites for ATP along the translocation pathway and that the main barrier for ATP transport is located around the center of the channel and is formed predominantly by residues in the N-terminus. The binding affinity of ATP to an open channel was found to be in the millimolar to micromolar range. However, we show that this weak binding increases the ATP translocation probability by about 10-fold compared with the VDAC pore in which attractive interactions were artificially removed. Recently, it was found that free dimeric tubulin induces a highly efficient, reversible blockage of VDAC reconstituted into planar lipid membranes. It was proposed that by blocking VDAC permeability for ATP/ADP and other mitochondrial respiratory substrates tubulin controls mitochondrial respiration. Using the Rosetta protein–protein docking algorithm, we established a tentative structure of the VDAC–tubulin complex. An extensive set of equilibrium and nonequilibrium (under applied electric field) molecular dynamics (MD) simulations was used to establish the conductance of the open and blocked channel. It was found that the presence of the unstructured C-terminal tail of tubulin in the VDAC pore decreases its conductance by more than 40% and switches its selectivity from anionic to cationic. The subsequent 1D potential of mean force (PMF) computations for the VDAC–tubulin complex show that the state renders ATP transport virtually impossible. A number of residues pivotal for tubulin binding to the channel were identified that help to clarify the molecular details of VDAC–tubulin interaction and to provide new insight into the mechanism of the control of mitochondria respiration by VDAC

AMBnTßCD blocks the single pores formed by C2IIa and Ib in planar lipid membranes.

<p><i>A</i>. Currents through single C2IIa (left) and Ib (right) channels at 50 mV applied voltage. The two topmost tracks represent the AMBnTßCD-free control experiments. The fast flickering between open and closed states (1/f noise) was mostly but not completely removed here by averaging over 100-ms time interval. In the presence of increasing AMBnTßCD, the channels were spontaneously blocked (three lower current tracks). The dashed lines represent zero current levels. <i>B</i>. Typical time histograms of AMBnTßCD-induced C2IIa (top) and Ib (bottom) current fluctuations. Original current recordings collected with 15 kHz filter and 50 kHz sampling were additionally filtered with a 300 Hz filter to exclude most of the high-frequency 1/f events. T_off represents the time channels spent in the blocked state and T_on is the time between successful blockages. Data were fitted by direct single-exponential (i.e. log probability) fitting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023927#pone.0023927-Sigworth1" target="_blank">[79]</a>. The fits were obtained using variable metrics as a search method and maximum likelihood as a minimization method. Measurements were performed in 1 M KCl solutions at pH 6 and 50 mV applied voltage. 0.4 µM and 2.3 µM AMBnTßCD concentrations were used for the C2IIa and Ib time-histograms, respectively, as statistically more represented.</p

The time point of AMBnTßCD application determines the protective effect of this compound against intoxication of Vero cells with C2 toxin.

<p>Vero cells were grown in 24-well plates and AMBnTßCD (10 µM) was applied to the cell medium either 30, 15 or 5 min before C2 toxin (200 ng/ml C2IIa+100 ng/ml C2I) was added to the cells or AMBnTßCD was added together with the toxin into the medium. In parallel, AMBnTßCD was added to the cells 5, 15, 30 or 60 min after the toxin. For a control, cells were treated with medium alone or with C2 toxin in the absence of AMBnTßCD. The cells were incubated for 3 h at 37°C and pictures were taken to determine the percentages of round cells. Values are given as mean ± S.D. (n = 3) and significance was tested for each sample treated with C2 toxin and AMBnTßCD against cells treated with C2 toxin only using the student's t-test (***p<0.0005; **p<0.005; *p<0.05; n. s. = not significant).</p

The ß-cyclodextrin derivative AMBnTßCD but not methyl-ß-cyclodextrin protects Vero cells from intoxication with C2 toxin when administered at 10 µM final concentration.

<p>Vero cells grown in 24-well plates were treated for 1 h at 37°C with either AMBnTßCD (10 µM) or methyl-ß-cyclodextrin (MßCD, 10 µM) and subsequently cells were challenged with C2 toxin (200 ng/ml C2IIa+100 ng/ml C2I). After 1.5 and 2.5 h of incubation at 37°C, pictures were taken to determine the percentages of round cells. Values are given as mean ± S.D. (n = 3) and significance was tested for each time point between toxin-treated samples and samples treated with either AMBnTßCD plus C2 toxin or MßCD plus C2 toxin by using the student's t-test (***p<0.0005; n. s. = not significant).</p

AMBnTßCD protects Vero cells from intoxication with iota toxin from <i>C. perfringens</i> and inhibits the pH-dependent membrane translocation of the toxin.

<p><i>A</i>. Time- and concentration-dependent inhibition of the intoxication of Vero cells with iota toxin. Vero cells grown in 24-well dishes to subconfluency were treated for 30 min at 37°C with 2, 5, 10 and 20 µM final concentrations of AMBnTßCD or without AMBnTßCD for control. Iota toxin (200 ng/ml Ib+100 ng/ml Ia) was added and cells were further incubated at 37°C with the toxin in the absence or presence of AMBnTßCD. Pictures were taken after the indicated incubation periods, the number of total cells and round cells were counted and the percentages of round cells calculated. Values are given as mean ± S.D. (n = 3) and significance was tested for each time point between iota toxin-treated samples without and with the respective concentration of AMBnTßCD by using the student's t-test (***p<0.0005; ***<0.005; *<0.05). <i>B</i>. The time point of AMBnTßCD application determines the protective effect against iota toxin. AMBnTßCD (10 µM) was applied to Vero cells at 30, 15 or 5 min before iota toxin (200 ng/ml Ib+100 ng/ml Ia), together with iota toxin or 5, 15 or 30 min after the toxin. As a control, cells were treated with medium or with iota toxin alone. The cells were incubated for 3 h at 37°C and pictures were taken to determine the percentages of round cells. Values are given as mean ± S.D. (n = 3) and significance was tested between cells treated with iota toxin alone and cells treated with toxin and AMBnTßCD by using the student's t-test (***p<0.0005; **p<0.005). <i>C</i>. AMBnTßCD inhibits the pH-dependent membrane translocation of iota toxin across the cytoplasmic membranes of intact Vero cells. Cells were incubated for 30 min at 37°C with 100 nM Baf A1 and subsequently for 30 min at 4°C in serum-free medium with iota toxin (1000 ng/mL Ib+500 ng/ml Ia) or without toxin for control. Then, 10 µM of AMBnTßCD were added (for control no AMBnTßCD) and the pH of the medium was adjusted to 4.5 with HCl (for control pH 7.5) and cells were exposed for 15 min to 37°C to trigger pore formation by Ib membrane translocation of Ia. Subsequently, cells were further incubated at 37°C in neutral medium containing Baf A1 and pictures were taken after 45 min and 3 h of incubation. The percentages of round (i. e. intoxicated) cells were determined, values are given as mean ± S.D. (n = 3). Significance was tested for each time point between samples treated with iota toxin under acidic condition in the absence or presence of AMBnTßCD by using the student's t-test (***p<0.0005).</p

AMBnTßCD blocks the transmembrane pores formed by C2IIa and Ib in planar lipid membranes at the multichannel level.

<p><i>A</i>. Multichannel C2II (top) and Ib (bottom)-induced conductance changed by AMBnTßCD addition. The current recordings were additionally filtered over 500-ms time interval. 0.1 M KCl solutions at pH 6 were buffered by MES. Recordings were taken at 20 mV applied voltage. <i>B</i>. Typical multichannel titration curves for C2IIa (left) and Ib (right)-modified membranes show about 15 times lower IC₅₀ values for C2II channels. All multichannel measurements were taken at 20 mV applied voltage.</p

AMBnTßCD inhibits the pH-dependent membrane translocation of C2 toxin across cytoplasmic membranes of intact cells.

<p>Vero cells were incubated for 30 min at 37°C with 100 nM Baf A1 and subsequently for 30 min at 4°C in serum-free medium with C2 toxin (400 ng/mL C2IIa+200 ng/ml C2I) or without toxin for control. Then, the medium was removed and cells were exposed to a short acidic shift with warm medium (5 min, pH 4.5, 37°C, Baf A1) to trigger pore formation by C2IIa and membrane translocation of C2I. In parallel, cells were exposed for 5 min to neutral medium (37°C, pH 7.5, Baf A1) as a control. In some samples, AMBnTßCD (10 µM) was present during acidic shift. Subsequently, the medium was changed and cells were further incubated at 37°C in neutral medium, still in the presence of Baf A1 to prevent the normal uptake of C2 toxin via acidified endosomes. Pictures were taken after 30 and 90 min of incubation. The percentages of round (i.e. intoxicated) cells were determined from the pictures, values are given as mean ± S.D. (n = 3). Significance was tested for each time point between samples, treated with C2 under acidic conditions in the absence or presence of AMBnTßCD by using the student's t-test (***p<0.0005).</p

Effect of AMBnTßCD on receptor binding of C2 toxin.

<p>Vero cells were incubated for 30 min at 4°C with C2 toxin (200 ng/ml C2IIa+100 ng/ml C2I) to enable toxin binding to the receptor on the cell surface. Then, the medium was removed and cells were washed to remove any unbound toxin. Fresh medium containing 10 µM of AMBnTßCD was added and cells were further incubated at 37°C to trigger internalization of the cell-bound C2 toxin. As a control, cells were incubated with fresh medium without AMBnTßCD or left untreated. After 3 h, pictures were taken to determine the percentages of round cells (scale bar = 100 µm) (<i>A</i>). Values are given as mean ± S.D. (n = 3) and significance was tested between toxin-treated samples with or without AMBnTßCD by using the student's t-test (***p<0.0005). <i>B</i>. Western blot detection of cell-associated C2I protein. Equal amounts of cell lysate proteins were subjected to SDS-PAGE, blotted and C2I was visualized in a Western blot with a specific antibody against the N-terminal domain of C2I. Purified C2I protein was run as a control in the same gel (not shown).</p

Pre-treatment of Vero epithelial cells and CHO-K1 fibroblasts with the ß-cyclodextrin derivative AMBnTßCD protects cells from intoxication with <i>C. botulinum</i> C2 toxin.

<p><i>A</i>. Time- and concentration-dependent inhibition of the intoxication of Vero cells by C2 toxin. Vero cells were grown in 24-well dishes to subconfluency and treated with 5, 10 and 20 µM final concentrations of AMBnTßCD for 30 min at 37°C. Subsequently C2 toxin (200 ng/ml C2IIa+100 ng/ml C2I) was added and cells were further incubated with the toxin in the presence of AMBnTßCD at 37°C. For a control, cells were left untreated or treated with C2 toxin alone or with AMBnTßCD alone. Pictures were taken after 2, 4, 6 and 24 h. The morphology of cells is shown after 4 h of C2 toxin-treatment (scale bar = 100 µm) (A). <i>B</i>. The number of total cells and round cells were counted from the pictures and the percentages of round cells calculated (lower panel). Values are given as mean ± S.D. (n = 3) and significance was tested for each time point between toxin-treated samples with or without AMBnTßCD by using the student's t-test (***p<0.0005; ***<0.005; *<0.05). <i>C</i>. AMBnTßCD inhibits the intoxication of CHO-K1 cells with C2 toxin. CHO-K1 cells were incubated with 10 µM of AMBnTßCD and after 30 min C2 toxin was applied exactly as described above. For a control, cells were treated without toxin or without AMBnTßCD or were left untreated. Pictures from the cells were taken after 3, 6 and 24 h (scale bar = 25 µm) and the percentages of round cells were determined. Values are given as mean ± S.D. (n = 3) and significance was tested for each time point between toxin-treated samples with or without AMBnTßCD by using the student's t-test (***p<0.0005).</p

Halothane Changes the Domain Structure of a Binary Lipid Membrane

X-ray and neutron diffraction studies of a binary lipid membrane demonstrate that halothane at physiological concentrations produces a pronounced redistribution of lipids between domains of different lipid types identified by different lamellar <i>d</i>-spacings and isotope composition. In contrast, dichlorohexafluorocyclobutane (F6), a halogenated nonanesthetic, does not produce such significant effects. These findings demonstrate a specific effect of inhalational anesthetics on mixing phase equilibria of a lipid mixture