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Biomedical subjects

M Zoratti

Publications and source records attributed to M Zoratti.

At least 19 recordsLinked to original sources
PubMed1999

Molecular and cellular activities of Helicobacter pylori pathogenic factors.

Stomach infection with pathogenic strains of Helicobacter pylori causes in some patients severe gastroduodenal diseases. These bacteria produce various virulence factors and, here, we review the recent acquisition on the biochemical mode of action of three major factors. We discuss the role of urease both as buffer of the stomach pH and as source of ammonia. The vacuolating toxin alters the endocytic pathway of non-polarized cells, inducing the release of acid hydrolases, the depression of extracellular ligand degradation and of antigen processing and, in the presence of ammonia, swelling of late-prelysosomal compartments. In polarized epithelial monolayers, vacuolating toxin induces an increase of the paracellular permeability, independent of vacuolation. The neutrophil activating protein induces the production of oxygen radicals in human neutrophils and could contribute to the damage of the stomach mucosa. The activities of these factors are discussed in terms of the need of the bacterium of increasing the supply of nutrients from the stomach lumen and from the mucosa.

Bacterial Proteins↗
PubMed1999

Helicobacter pylori vacuolating toxin forms anion-selective channels in planar lipid bilayers: possible implications for the mechanism of cellular vacuolation.

The Helicobacter pylori VacA toxin plays a major role in the gastric pathologies associated with this bacterium. When added to cultured cells, VacA induces vacuolation, an effect potentiated by preexposure of the toxin to low pH. Its mechanism of action is unknown. We report here that VacA forms anion-selective, voltage-dependent pores in artificial membranes. Channel formation was greatly potentiated by acidic conditions or by pretreatment of VacA at low pH. No requirement for particular lipid(s) was identified. Selectivity studies showed that anion selectivity was maintained over the pH range 4.8-12, with the following permeability sequence: Cl- approximately HCO3- > pyruvate > gluconate > K+ approximately Li+ approximately Ba2+ > NH4+. Membrane permeabilization was due to the incorporation of channels with a voltage-dependent conductance in the 10-30 pS range (2 M KCl), displaying a voltage-independent high open probability. Deletion of the NH2 terminus domain (p37) or chemical modification of VacA by diethylpyrocarbonate inhibited both channel activity and vacuolation of HeLa cells without affecting toxin internalization by the cells. Collectively, these observations strongly suggest that VacA channel formation is needed to induce cellular vacuolation, possibly by inducing an osmotic imbalance of intracellular acidic compartments.

Bacterial Proteins↗
PubMed1998

Tyrosine kinase-dependent activation of a chloride channel in CD95-induced apoptosis in T lymphocytes.

CD95/Fas/APO-1 mediated apoptosis is an important mechanism in the regulation of the immune response. Here, we show that CD95 receptor triggering activates an outwardly rectifying chloride channel (ORCC) in Jurkat T lymphocytes. Ceramide, a lipid metabolite synthesized upon CD95 receptor triggering, also induces activation of ORCC in cell-attached patch clamp experiments. Activation is mediated by Src-like tyrosine kinases, because it is abolished by the tyrosine kinase inhibitor herbimycin A or by genetic deficiency of p56lck. In vitro incubation of excised patches with purified p56lck results in activation of ORCC, which is partially reversed upon addition of anti-phosphotyrosine antibody. Inhibition of ORCC by four different drugs correlates with a 30-65% inhibition of apoptosis. Intracellular acidification observed upon CD95 triggering is abolished by inhibition of either ORCC or p56lck. The results suggest that tyrosine kinase-mediated activation of ORCC may play a role in CD95-induced cell death in T lymphocytes.

Apoptosis↗
PubMed1998

Novel aspects of the electrophysiology of mitochondrial porin.

The recent findings that mitochondrial porin, VDAC, participates in supramolecular complexes and is present in the plasmamembrane need to be reconciled with its biophysical properties. We report here that VDAC often displays previously unobserved or unappreciated behaviors. Reconstituted VDAC can: a) exhibit fast gating when in any of many conductance substates; b) close completely, although briefly, on its own; c) close for a long periods, in the presence of König's polyanion; d) take several milliseconds to re-open when an applied transmembrane potential is switched off; e) be desensitized by prolonged exposure to high voltages, so that it will not re-open to the full conductance state upon subsequent return to zero voltage; f) display polarity-dependent voltage-induced closure. These behaviors are especially noticeable when the observations are conducted on a single reincorporated channel, suggesting that interactions between copies of VDAC may play a role in determining its electrophysiological properties. Any model of VDAC's structure, gating and function should take these observations into account.

Animals↗
PubMed1998

Double-stranded DNA can be translocated across a planar membrane containing purified mitochondrial porin.

The transport of genetic material across biomembranes is a process of great relevance for several fields of study. However, much remains to be learned about the mechanisms underlying transport, one of which implies the involvement of proteic DNA-conducting pores. Entry of genetic material into mitochondria has been observed under both physiological and pathological conditions. We report here that double-stranded DNA can move through a planar bilayer membrane containing isolated mitochondrial porin (voltage-dependent anion channel). The transport is driven by the applied electrical field, and the presence of DNA is associated with a decrease of current conduction by the pores. The passage of DNA does not take place if the bilayer has not been doped with any protein or in the presence of both reconstituted porin and anti-porin antibody. Translocation does not occur if the bilayer contains Shigella sonnei maltoporin, gramicidin A channels, or a 30 pS anion-selective channel plus other proteins. These results show that mitochondrial porin is capable of mediating the transport of genetic material, revealing a new property of this molecule and further confirming the idea that DNA can move through proteic pores.

Biological Transport↗
PubMed1997

DNA translocation across planar bilayers containing Bacillus subtilis ion channels.

The mechanisms by which genetic material crosses prokaryotic membranes are incompletely understood. We have developed a new methodology to study the translocation of genetic material via pores in a reconstituted system, using techniques from electrophysiology and molecular biology. We report here that planar bilayer membranes become permeable to double-stranded DNA (kilobase range) if Bacillus subtilis membrane vesicles containing high conductance channels have been fused into them. The translocation is an electrophoretic process, since it does not occur if a transmembrane electrical field opposing the movement of DNA, a polyanion, is applied. It is not an aspecific permeation through the phospholipid bilayer, since it does not take place if no proteins have been incorporated into the membrane. The transport is also not due simply to the presence of polypeptides in the membrane, since it does not occur if the latter contains gramicidin A or a eukaryotic, multi-protein vesicle fraction exhibiting 30-picosiemens anion-selective channel activity. The presence of DNA alters the behavior of the bacterial channels, indicating that it interacts with the pores and may travel through their lumen. These results support the idea that DNA translocation may take place through proteic pores and suggest that some of the high conductance bacterial channels observed in electrophysiological experiments may be constituents of the DNA translocating machinery in these organisms.

Aequorin↗
PubMed1996

The high-conductance channels of yeast mitochondrial outer membranes: a planar bilayer study.

The high-conductance channels present in the outer membranes of wild-type and porin-less yeast mitochondria have been characterized electrophysiologically after incorporation in planar bilayer membranes. The most prominent activity was ascribed to a voltage-dependent, substate-rich, cationic channel which generally inactivated at potentials positive in the cis compartment, in agreement with the observations from patch-clamp experiments on porin-less mitoplasts. This channel has been identified as the so-called "peptide-sensitive channel" (PSC). We also observed similar channels displaying either no inactivation, or inactivation at both positive and negative potentials. These latter properties match those already described for mammalian and yeast PSC, respectively. These different behaviors are tentatively explained as arising from the presence, or lack of, peptides bound to the PSC. Very high conductances, apparently due to cooperative gating, were frequently displayed. In wild-type membranes, activity ascribable to the porin was also observed.

Electric Conductivity↗
PubMed1995

The high-conductance channel of porin-less yeast mitochondria.

Patch-clamp and planar bilayer experiments on porin-less yeast mitochondria have allowed the characterization of a cationic channel activated at matrix-side positive (unphysiological) potentials. In voltage-pulse experiments, inactivation was a faster process than activation and the time constant for inactivation was more steeply dependent on voltage than the one for activation. The channel exhibited various conductance states whose occupancy depended on the applied transmembrane potential. In bilayer experiments, the presence of the pCOx-IV leader peptide induced fast gating in a voltage-dependent manner. A comparison with previously described activities suggests that the pore may coincide with the peptide-sensitive channel (PSC) (Thieffry et al. (1988) EMBO J. 7, 1449-1454) as well as with two other activities (Dihanich et al. (1989) Eur. J. Biochem. 181, 703-708; Tedeschi et al. (1987) J. Membr. Biol. 97, 21-29) assigned to the mitochondrial outer membrane. The possible relationship of this channel to the mitochondrial megachannel is discussed.

Ion Channels↗
PubMed1994

Electrophysiology of the inner mitochondrial membrane.

The application of electrophysiological techniques to mitochondrial membranes has allowed the observation and partial characterization of several ion channels, including an ATP-sensitive K(+)-selective one, a high-conductance "megachannel", a 107 pS anionic channel and three others studied at alkaline pH's. A reliable correlation with the results of non-electrophysiological studies has been obtained so far only for the first two cases. Activities presumed to be associated with the Ca2+ uniporter and with the adenine nucleotide translocator, as well as the presence of various other conductances have also been reported. The review summarizes the main properties of these pores and their possible relationship to permeation pathways identified in biochemical studies.

Adenosine Triphosphate↗
PubMed1993

The mitochondrial permeability transition pore may comprise VDAC molecules. I. Binary structure and voltage dependence of the pore.

Electrophysiological records suggest that the pore responsible for the mitochondrial Ca(2+)-dependent permeability transition (PTP), identified as the mitochondrial megachannel (MMC) observed in patch-clamp experiments, may comprise two cooperating porin (VDAC) molecules. We have re-investigated the voltage dependence of the megachannel, which favors the closed state(s) at negative (physiological) transmembrane potentials. This behavior confirms that MMC corresponds to the permeabilization pore. As detailed in the accompanying paper [(1993) FEBS Lett. 330, 206-210] this voltage dependence resembles that of VDAC. Alpidem, a ligand of the mitochondrial benzodiazepine receptor, which reportedly comprises VDAC, the adenine nucleotide carrier and a third component, elicited currents from silent mitoplast patches, suggesting that the benzodiazepine receptor may be identical to the PTP/MMC.

Imidazoles↗
PubMed1993

The mitochondrial permeability transition pore may comprise VDAC molecules. II. The electrophysiological properties of VDAC are compatible with those of the mitochondrial megachannel.

The electrophysiological properties of isolated mitochondrial porin (VDAC), reconstituted in planar bilayers or proteoliposomes, resemble those of the mitochondrial megachannel believed to be the permeability transition pore. In particular, a correspondence was found with regard to the voltage dependence: VDAC was driven to closed states by potentials of either sign, but the effect was not symmetrical; voltages negative in the compartment to which VDAC was added were more effective. The results are consistent with the hypothesis that the PTP may consist of two cooperating VDAC channels, plus presumably an adenine nucleotide carrier dimer and a third component known to be part of the mitochondrial benzodiazepine receptor.

Animals↗
PubMed1993

A patch-clamp investigation of the Streptococcus faecalis cell membrane.

The patch-clamp technique was used to study the membrane of giant protoplasts from the gram-positive bacterium Streptococcus faecalis, demonstrating the presence of ion-conducting pores in the cytoplasmic membrane of procaryotes. The single channel recordings were characterized by a variety of conductances, ranging up to a few nanoSiemens, arising from stretch-activated, voltage-modulated, cooperative channels. Activation by stretch and voltage took place via both a decrease of the mean closed time and an increase of the mean open time of the channels, which are strictly controlled in intact cells, where they might constitute parts of a membrane apparatus or transport system.

Cell Membrane↗
PubMed1992

A patch-clamp study of Bacillus subtilis.

In patch-clamp experiments on giant protoplasts of the Gram-positive bacterium Bacillus subtilis, membrane stretch resulted in an initial transient collapse of the membrane resistance, after which stretch-activated, voltage modulated, high-conductance channels could be observed. The channel open probability increased exponentially with applied suction and positive voltage, as a result of variations of both the mean open and the mean closed times. The substate structure and other characteristics of the electrical activity suggested the presence of a family of pores exhibiting cooperative behavior. A role in osmotic protection is suggested. In the intact bacteria, the pores may be part of an unidentified envelope apparatus, having other functions as well.

Bacillus subtilis↗
PubMed1992

Gadolinium ion inhibits loss of metabolites induced by osmotic shock and large stretch-activated channels in bacteria.

Bacteria subjected to a hypotonic osmotic shock lose internal ions and also metabolites, without lysis of the cells. We show that the presence in the shock medium, at submillimolar concentrations, of the ion gadolinium, recently shown to block stretch-activated channels in Xenopus oocytes [Yang, X.-C. & Sachs, F. (1989) Science 243, 1068-1071], was sufficient to inhibit shock-induced release of metabolites such as lactose and ATP in Escherichia coli and ATP in Streptococcus faecalis. Moreover, gadolinium was observed, in patch-clamp experiments, to inhibit the giant stretch-activated channels of E. coli, S. faecalis. and Bacillus subtilis. Taken together, these data suggest that stretch-activated channels are localized in the cytoplasmic membrane of Gram-negative and Gram-positive bacteria, where they control the efflux of osmotic solutes, thus probably playing a major role in the response to hypotonic osmotic shock.

Adenosine Triphosphate↗
PubMed1992

Modulation of the mitochondrial megachannel by divalent cations and protons.

In patch-clamp experiments on rat liver mitoplasts, the 1.3 nanosiemens (in 150 mM KCl) mitochondrial megachannel was activated by Ca2+ and competitively inhibited by Mg2+, Mn2+, Ba2+, and Sr2+. Cyclosporin A, which inhibits the megachannel, also showed a competitive behavior versus Ca2+. The pore is regulated by pH in the physiological range; lower pH values cause its closure in a Ca(2+)-reversible manner. The modulating sites involved in these effects are located on the matrix side of the membrane. As illustrated in the companion paper (Bernardi, P., Vassanelli, S., Veronese, P., Colonna, R., Szabó, I., and Zoratti, M. (1992) J. Biol. Chem. 267, 2934-2939), the calcium-induced permeability transition of mitochondria is affected by these various agents in a similar manner. The results support the identification of the megachannel with the pore believed to be involved in the permeabilization process. The kinetic characteristics of the single channel events support the idea that the megachannel is composed of cooperating subunits.

Animals↗
PubMed1992

Modulation of the mitochondrial permeability transition pore. Effect of protons and divalent cations.

We have studied the induction of the mitochondrial cyclosporin A-sensitive permeability transition pore (PTP) by the bifunctional SH group reagent phenylarsine oxide (PhAsO). Addition of nanomolar concentrations of the electroneutral H(+)-K+ ionophore nigericin to nonrespiring mitochondria in sucrose medium determines a dramatic increase of the time required for PTP induction by PhAsO, while no effect of nigericin is apparent in KCl medium. Using mitochondria loaded with the internal pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, we show that the effect of nigericin is mediated by the ionophore-induced acidification of matrix pH. Indeed, experimental manipulation of pHi by a number of treatments indicates that PTP induction is directly related to matrix pH, in that the PTP induction process becomes slower as pHi decreases at constant pHo. PTP induction by PhAsO in respiration-inhibited mitochondria is stimulated by Ca2+ and inhibited by a series of divalent cations. Since PhAsO induces the PTP even in the presence of excess EGTA and in the absence of respiration (Lenartowicz, E., Bernardi, P., and Azzone, G.F. (1991) J. Bioenerg. Biomembr. 23, 679-688), we have been able to study the Ca2+ dependence of the induction process. We show that the apparent Km for Ca2+ activation is about 10(-5) M and that Ca2+, cyclosporin A, and inhibitory Me2+ ions behave as if they were competing for the same binding site(s) on the pore. Since similar results are obtained from patch-clamp experiments on the mitochondrial megachannel (Szabó, I., Bernardi, P., and Zoratti, M. (1992) J. Biol. Chem. 267, 2940-2946), we suggest that (i) the PTP and the mitochondrial megachannel are the same molecular structures and (ii) the same factors affect both the process of pore induction and its open-closed orientation.

Arsenicals↗