Search PubMed⌕ Search

Biomedical subjects

I Szabò

Publications and source records attributed to I Szabò.

At least 19 recordsLinked to original sources
PubMed2004

Ion channels and membrane rafts in apoptosis.

Ion channels have been demonstrated to be a central element in the induction and the execution of apoptosis. In particular, mitochondrial ion channels, including not only the permeability transition pore but also a mitochondrial, ATP-sensitive (mKATP) channel as well as a mitochondrial calcium-activated potassium channel are involved critically in apoptotic changes in mitochondria. Ion channels in the cell membrane that are altered by induction of apoptosis include potassium, chloride and calcium channels. The Kv1.3 potassium channel belongs to the best-characterized ion channels involved in apoptosis and a genetic model of cells deficient for Kv1.3 has indicated a critical role for Kv1.3, at least in some forms of apoptosis. The mechanisms regulating ion channels during apoptosis are, however, still poorly defined. Recent studies have suggested a function for distinct membrane domains, termed rafts, in the cell membrane for the regulation of ion channels during apoptosis. Small sphingolipid- and cholesterol-enriched membrane domains are modified by many apoptotic stimuli to form large ceramide-enriched membrane platforms. These platforms serve to cluster receptor molecules, to re-organize intracellular signalling molecules including ion channels, to bring ion channels into close contact with their regulators and/or to separate proteins from a specific ion channel. Finally, the lipid composition of the cell membrane might be involved directly in ion channel regulation.

Animals↗
PubMed2001

Determination of photosystem II subunits by matrix-assisted laser desorption/ionization mass spectrometry.

Photosystem II of higher plants and cyanobacteria is composed of more than 20 polypeptide subunits. The pronounced hydrophobicity of these proteins hinders their purification and subsequent analysis by mass spectrometry. This paper reports the results obtained by application of matrix-assisted laser desorption/ionization mass spectrometry directly to isolated complexes and thylakoid membranes prepared from cyanobacteria and spinach. Changes in protein contents following physiopathological stimuli are also described. Good correlations between expected and measured molecular masses allowed the identification of the main, as well as most of the minor, low molecular weight components of photosystem II. These results open up new perspectives for clarifying the functional role of the various polypeptide components of photosystems and other supramolecular integral membrane complexes.

Chromatography, High Pressure Liquid↗
PubMed2001

Isolation and characterization of photosystem II subcomplexes from cyanobacteria lacking photosystem I.

A photosystem II (PSII) core complex lacking the internal antenna CP43 protein was isolated from the photosystem II of Synechocystis PCC6803, which lacks photosystem I (PSI). CP47-RC and reaction centre (RCII) complexes were also obtained in a single procedure by direct solubilization of whole thylakoid membranes. The CP47-RC subcore complex was characterized by SDS/PAGE, immunoblotting, MALDI MS, visible and fluorescence spectroscopy, and absorption detected magnetic resonance. The purity and functionality of RCII was also assayed. These preparations may be useful for mutational analysis of PSII RC and CP47-RC in studying primary reactions of oxygenic photosynthesis.

Blotting, Western↗
PubMed2000

KDC1, a novel carrot root hair K+ channel. Cloning, characterization, and expression in mammalian cells.

Potassium is an essential nutrient which plays an important role in many aspects of plant growth and development. Plants have developed a number of highly specific mechanisms to take up potassium from the soil; these include the expression of K(+) transporters and potassium channels in root cells. Despite the fact that root epidermal and hair cells are in direct contact with the soil, the role of these tissues in K(+) uptake is not well understood. Here we report the molecular cloning and functional characterization of a novel potassium channel KDC1 which forms part of a new subfamily of plant K(in) channels. Kdc1 was isolated from carrot root RNA and in situ hybridization experiments show Kdc1 to be highly expressed in root hair cells. Expressing the KDC1 protein in Chinese hamster ovary cells identified it as a voltage and pH-dependent inwardly rectifying potassium channel. An electrophysiological analysis of carrot root hair protoplasts confirmed the biophysical properties of the Kdc1 gene product (KDC1) in the heterologous expression system. KDC1 thus represents a major K(+) uptake channel in carrot root hair cells.

Amino Acid Sequence↗
PubMed2000

Ca2+-reversible inhibition of the mitochondrial megachannel by ubiquinone analogues.

Ubiquinone 0 and decylubiquinone have been reported to inhibit the mitochondrial permeability transition pore (PTP) [Fontaine, E., Ichas, F. and Bernardi, P. (1998) J. Biol. Chem. 273, 25734-257401, offering a new clue to its molecular composition. In patch-clamp experiments on rat liver mitochondria we have observed that these compounds also inhibit the previously described mitochondrial megachannel (MMC), confirming its identification as the PTP. Inhibition can be reversed by increasing [Ca2+], in analogy to the behavior observed with several other disparate PTP/MMC inhibitors. To rationalize the ability of Ca2+ to overcome inhibition by various quite different compounds we propose that it acts via the phospholipid bilayer.

Animals↗
PubMed2000

Temperature-dependent functional expression of a plant K(+) channel in mammalian cells.

The Arabidopsis thaliana potassium channel KAT1 was expressed and characterized in Chinese hamster ovary cells. KAT1-GFP fusion protein was successfully targeted to the plasma membrane and electrophysiological analysis revealed functional expression of KAT1 only in cells cultured at 30 degrees C. The main biophysical characteristics of KAT1 are similar to those described for the channel expressed in other systems. CHO cells represent an advantageous expression system and may be the system of choice to study the expression, assembly, function, and regulation of plant potassium channels in general.

Animals↗
PubMed2000

Ultraviolet B exposure of whole leaves of barley affects structure and functional organization of photosystem II.

This study examines the effects of ecologically important levels of ultraviolet B radiation on protein D1 turnover and stability and lateral redistribution of photosystem II. It is shown that ultraviolet B light supported only limited synthesis of protein D1, one of the most important components of photosystem II, whereas it promoted significant degradation of proteins D1 and D2. Furthermore, dephosphorylation of photosystem II subunits was specifically elicited upon exposure to ultraviolet B light. Structural modifications of photosystem II and changes in its lateral distribution between granum membranes and stroma-exposed lamellae were found to be different from those observed after photoinhibition by strong visible light. In particular, more complete dismantling of photosystem II cores was observed. Altogether, the data reported here suggest that ultraviolet B radiation alone fails to activate the photosystem II repair cycle, as hypothesized for visible light. This failure may contribute to the toxic effect of ultraviolet B radiation, which is increasing as a consequence of depletion of stratospheric ozone.

Hordeum↗
PubMed2000

Tyrosine kinases open lymphocyte chloride channels.

Osmotic swelling of lymphocytes opens outwardly rectifying Cl(-) channels (ORCC) through the src-like kinase p56(lck). The central role of this tyrosine protein kinase has been shown by genetic and pharmacologic manipulation of the enzyme. Furthermore, p56(lck) activates ORCC independently of cell volume increase. ORCC in lymphocytes and epithelial cells from cystic fibrosis (CF) patients are resistant to activation by cAMP. However, osmotic swelling as well as intracellular purified p56(lck) can activate ORCC in CF lymphocytes. In non-CF lymphocytes ORCC is opened by either, intracellular cAMP, p56(lck) or by osmotic swelling. Osmotic activation of ORCC can be blocked by the tyrosine kinase inhibitor lavendustin in both cell types. Regulation of ORCC by p56(lck) thus represents an alternative pathway of stimulating membrane chloride conductance that is left functional in cystic fibrosis. In addition to osmoregulation these mechanisms could play a major role when cells actively change their volume, i.e. during proliferation and apoptosis. Activation of the tyrosine kinase p56(lck) is an important regulatory step for opening of chloride channels in lymphocytes.

Animals↗
PubMed1999

Inhibition of the vacuolating and anion channel activities of the VacA toxin of Helicobacter pylori.

VacA, the vacuolating cytotoxin secreted by Helicobacter pylori, is believed to be a major causative factor in the development of gastroduodenal ulcers. This toxin causes vacuolation of cultured cells and it has recently been found to form anion-selective channels upon insertion into planar bilayers as well as in the plasma membrane of HeLa cells. Here, we identify a series of inhibitors of VacA channels and we compare their effectiveness as channel blockers and as inhibitors of VacA-induced vacuolation, confirming that the two phenomena are linked. This characterization opens the way to studies in other experimental systems and to the search for a specific inhibitor of VacA action.

Bacterial Proteins↗
PubMed1999

Porin is present in the plasma membrane where it is concentrated in caveolae and caveolae-related domains.

Mitochondrial porin, or voltage-dependent anion channel, is a pore-forming protein first discovered in the outer mitochondrial membrane. Later investigations have provided indications for its presence also in other cellular membranes, including the plasma membrane, and in caveolae. This extra-mitochondrial localization is debated and no clear-cut conclusion has been reached up to now. In this work, we used biochemical and electrophysiological techniques to detect and characterize porin within isolated caveolae and caveolae-like domains (low density Triton-insoluble fractions). A new procedure was used to isolate porin from plasma membrane. The outer surface of cultured CEM cells was biotinylated by an impermeable reagent. Low density Triton-insoluble fractions were prepared from the labeled cells and used as starting material to purify a biotinylated protein with the same electrophoretic mobility and immunoreactivity of mitochondrial porin. In planar bilayers, the porin from these sources formed slightly anion-selective pores with properties indistinguishable from those of mitochondrial porin. This work thus provides a strong indication of the presence of porin in the plasma membrane, and specifically in caveolae and caveolae-like domains.

Animals↗
PubMed1999

Formation of anion-selective channels in the cell plasma membrane by the toxin VacA of Helicobacter pylori is required for its biological activity.

The vacuolating toxin VacA, a major determinant of Helicobacter pylori-associated gastric diseases, forms anion-selective channels in artificial planar lipid bilayers. Here we show that VacA increases the anion permeability of the HeLa cell plasma membrane and determines membrane depolarization. Electrophysiological and pharmacological approaches indicated that this effect is due to the formation of low-conductance VacA pores in the cell plasma membrane and not to the opening of Ca(2+)- or volume-activated chloride channels. VacA-dependent increase of current conduction both in artificial planar lipid bilayers and in the cellular system was effectively inhibited by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), while2-[(2-cyclopentenyl-6,7dichloro-2, 3-dihydro-2-methyl-1-oxo-1H-inden-5-yl)oxy]acetic acid (IAA-94) was less effective. NPPB inhibited and partially reversed the vacuolation of HeLa cells and the increase of ion conductivity of polarized Madine Darby canine kidney cell monolayers induced by VacA, while IAA-94 had a weaker effect. We conclude that pore formation by VacA accounts for plasma membrane permeabilization and is required for both cell vacuolation and increase of trans-epithelial conductivity.

Animals↗
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

The tyrosine kinase p56lck mediates activation of swelling-induced chloride channels in lymphocytes.

Osmotic cell swelling activates Cl- channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56lck, a src-like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (ICl-swell) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of ICl-swell is defective in p56lck-deficient cells. Retransfection of p56lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of ICl-swell. Addition of purified p56lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56lck washed into the cytoplasm activates ICl-swell in native and p56lck-deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56lck, slow single-channel gating events can be observed revealing a unitary conductance of 25-28 pS. In accordance with our patch-clamp data, osmotic swelling increases activity of immunoprecipitated p56lck. We conclude that osmotic swelling activates ICl-swell in lymphocytes via the tyrosine kinase p56lck.

Benzoquinones↗
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

Tyrosine phosphorylation-dependent suppression of a voltage-gated K+ channel in T lymphocytes upon Fas stimulation.

Selective cell death plays a critical role in the development of the immune system and in the elimination of target cells expressing foreign antigens. Most of programmed cell death occurs by apoptosis. Apoptotic cell death of lymphocytes can be triggered by ligation of APO-1/Fas (CD95) antigen (Suda, T., and Nagata, S. (1994) J. Exp. Med. 179, 873-879; Nagata, S., and Golstein, P. (1995) Science 267, 1449-1456). We find that activation of Fas leads to the inhibition of the voltage-dependent n-type K+ channels (Kv1.3) studied by patch clamp technique in Jurkat T lymphocytes. Tyrosine kinases have been shown to be crucial in Fas-induced cell death (Eischen, C. M., Dick, C. J., and Leibson, P. J. (1994) J. Immunol. 153, 1947-1954). The inhibition of the current is correlated with the tyrosine phosphorylation of immunoprecipitated and blotted K+ channel protein. We show, that the Src-like protein-tyrosine kinase inhibitor herbimycin A and the deficiency of the p56(lck) tyrosine kinase in mutant Jurkat cells abolished the channel inhibition and phosphorylation by anti-Fas antibody, while reconstitution of the p56(lck) kinase partly restored these effects of Fas receptor triggering. These results suggest a regulation of n-type K+ channels by tyrosine kinases upon Fas receptor triggering, which might be important for apoptosis.

Apoptosis↗