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M Zoratti

Publications and source records attributed to M Zoratti.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1992

The mitochondrial megachannel is the permeability transition pore.

Single-channel electrophysiological recordings from rat liver mitoplast membranes showed that the 1.3-nS mitochondrial megachannel was activated by Ca++ and inhibited by Mg++, Cyclosporin A, and ADP, probably acting at matrix-side sites. These agents are known to modulate the so-called mitochondrial permeability transition pore (Gunter, T. E., and Pfeiffer, D. R. (1990) Am. J. Physiol. 258, C755-C786) in the same manner. Furthermore, the megachannel is unselective, and the minimum pore size calculated from its conductance is in agreement with independent estimates of the minimum size of the permeabilization pore. The results support the tentative identification of the megachannel with the pore believed to be involved in the permeabilization process.

Adenosine Diphosphate↗
PubMed1991

Flow-force relationships during energy transfer between mitochondrial proton pumps.

The effect of inhibitors of proton pumps, of uncouplers and of permeant ions on the relationship between input force, delta mu H+, and output flows of the ATPase, redox and transhydrogenase H(+)-pumps in submitochondrial particles was investigated. It is concluded that: (1) The decrease of output flow of the transhydrogenase proton pump, defined as the rate of reduction of NADP+ by NADH, is linearily correlated with the decrease of input force, delta mu H+, in an extended range of delta mu H+, independently of whether the H(+)-generating pump is the ATPase or a redox pump, or whether delta mu H+ is depressed by inhibitors of the H(+)-generating pump such as oligomycin or malonate, or by uncouplers. (2) The output flows of the ATPase and of the site I redox H(+)-pumps exhibit a steep dependence on delta mu H+. The flow-force relationships differ depending on whether the depression of delta mu H+ is induced by inhibitors of the H(+)-generating pump, by uncouplers or by lipophilic anions. (3) With the ATPase as H(+)-consuming pump, at equivalent delta mu H+ values, the output flow is more markedly inhibited by malonate than by uncouplers; the latter, however, are more inhibitory than lipophilic anions such as ClO4-. With redox site I as proton-consuming pump, at equivalent delta mu H+ values, the output flow is more markedly inhibited by oligomycin than by uncouplers; again, uncouplers are more inhibitory than ClO4-. (4) The results provide further support for a delocalized interaction of transhydrogenase with other H(+)-pumps.

Adenosine Triphosphate↗
PubMed1991

The giant channel of the inner mitochondrial membrane is inhibited by cyclosporin A.

In patch-clamp experiments on rat liver mitoplasts, cyclosporin A inhibited the activity of the recently described (Petronilli, V., Szabó, I., and Zoratti, M. (1989) FEBS Lett. 259, 137-143) 1.3-nanosiemens channel of the inner mitochondrial membrane at concentrations in the 10(-8)-10(-7) M range. The inhibitor acts when present on the matrix side of membrane. The Ca2(+)-dependent "permeability transition channel" of mitochondria is inhibited by cyclosporin A in the same concentration range. The results suggest therefore that the same pore is responsible for the permeabilization of the inner mitochondrial membrane and for the conduction of the high currents observed in electrophysiological experiments.

Animals↗
PubMed1990

Cooperative mechanosensitive ion channels in Escherichia coli.

A patch-clamp investigation was carried out on giant Escherichia coli spheroplasts. The membrane exhibited stretch-induced as well as "spontaneous" activity, with similar characteristics, i.e., a large number of conductance values arising from the cooperative behavior of channels in functional clusters. It appears likely that the same molecular species are responsible for both stretch-induced and "spontaneous" current conduction; the channel multiplexes can either respond to membrane stretch or function in an activate state, presumably brought about by the previous application of the mechanical stimulus.

Cell Membrane↗
PubMed1990

Stretch-activated composite ion channels in Bacillus subtilis.

The presence of ion-conducting pores in the membrane of Bacillus subtilis giant protoplasts was discovered using the patch-clamp technique. Membrane stretch caused the activation of several conductances with values in the nS range. The observations indicate the presence of substate levels and of aggregates of channels behaving in a cooperative manner. Following repeated stretch cycles, the channels exhibited spontaneous activity. The characteristics of the electrical phenomena afterwards changed in time in a manner suggesting the decay of the giant channels into lower-conductance species, presumably corresponding to building blocks of the giant stretch-activated channels.

Bacillus subtilis↗