RESUMEN
Four different isoforms of the Voltage-Dependent Anion Channel (VDAC) have been identified in Arabidopsis plant cells. The electrophysiological characteristics of several VDAC channels from animal as well as plant cells are well documented, but those of this model plant are unknown. One isoform, AtVDAC-3 was obtained either directly by cell-free synthesis or produced in Escherichia coli, as inclusion bodies, and re-natured. An electrophysiological study of the purified proteins in planar lipid bilayers showed that both methods yielded proteins with similar channel activity. The characteristics of AtVDAC-3 are that of a bona fide VDAC-like channel.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Ingeniería de Proteínas/métodos , Canales Aniónicos Dependientes del Voltaje/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Sistema Libre de Células , Fenómenos Electrofisiológicos , Escherichia coli/genética , Membrana Dobles de Lípidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Canales Aniónicos Dependientes del Voltaje/genética , Canales Aniónicos Dependientes del Voltaje/aislamiento & purificaciónRESUMEN
Bacteriophage lambda that binds to liposomes bears its receptor maltoporin (LamB) and is able to inject its DNA into the internal space. During this process, the liposomes are permeabilized, suggesting that a transmembrane channel has formed (Roessner and Ihler (1986) J. Biol. Chem. 261, 386-390). This pore possibly constitutes the pathway used by lambda DNA to cross the membrane. We reconstituted purified LamB from Shigella in liposomes that were incubated with lambda phages. Addition of this mixture to a bilayer chamber resulted in the incorporation in planar bilayers of high-conductance channels whose conductance, kinetics and voltage dependence were totally different from those of maltoporin channels.
Asunto(s)
Bacteriófago lambda/metabolismo , Canales Iónicos/metabolismo , Porinas/metabolismo , Receptores Virales/metabolismo , Proteínas de la Membrana Bacteriana Externa , Bacteriófago lambda/patogenicidad , Conductividad Eléctrica , Canales Iónicos/efectos de los fármacos , Cinética , Liposomas , Potenciales de la Membrana , Porinas/efectos de los fármacos , Receptores Virales/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Shigella/metabolismo , Shigella/virología , Trisacáridos/farmacologíaRESUMEN
Bid is a proapoptotic, BH3-domain-only member of the Bcl-2 family. In Fas-induced apoptosis, Bid is activated through cleavage by caspase 8 into a 15.5-kDa C-terminal fragment (t(c)Bid) and a 6.5 kDa N-terminal fragment (t(n)Bid). Following the cleavage, t(c)Bid translocates to the mitochondria and promotes the release of cytochrome c into the cytosol by a mechanism that is not understood. Here we report that recombinant t(c)Bid can act as a membrane destabilizing agent. t(c)Bid induces destabilization and breaking of planar lipid bilayers without appearance of ionic channels; its destabilizing activity is comparable with that of Bax and at least 30-fold higher than that of full-length Bid. Consistently, t(c)Bid, but not full-length Bid, permeabilizes liposomes at physiological pH. The destabilizing effect of t(c)Bid on liposomes and planar bilayers is independent of the BH3 domain. In contrast, mutations in the BH3 domain impair t(c)Bid ability to induce cytochrome c release from mitochondria. The permeabilizing effect of t(c)Bid on planar bilayers, liposomes, and mitochondria can be inhibited by t(n)Bid. In conclusion, our results suggest a dual role for Bid: BH3-independent membrane destabilization and BH3-dependent interaction with other proteins. Moreover, the dissociation of Bid after cleavage by caspase 8 represents an additional step at which apoptosis may be regulated.
Asunto(s)
Proteínas Portadoras/metabolismo , Caspasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Proteínas Portadoras/genética , Caspasa 8 , Caspasa 9 , Hidrólisis , Membrana Dobles de Lípidos , Liposomas , Ratones , Mitocondrias/metabolismo , Mutación , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
MscL is a mechanosensitive channel that is gated by tension in the membrane bilayer alone. It is a homo-oligomer of a protein comprising two transmembrane segments connected by an external loop, with the NH(2) and COOH termini located in the cytoplasm. The contributions of the extramembranous domains of the channel to its activity were investigated by specific proteolysis during patch-clamp experiments. Limited proteolysis of the COOH terminus or the NH(2) terminus increased the mechanosensitivity of the channel without changing its conductance. Strikingly, after cleavage of the external loop of each monomer, the channel was still functional, and its mechanosensitivity was increased dramatically, indicating that the loop acts as a spring that resists the opening of the channel and promotes its closure when it is open. These results indicate that the integrity of most of the extramembranous domains is not essential for mechanosensitivity. They suggest that these domains counteract the movement of the transmembrane helices to which they are connected, thus setting the level of sensitivity of the channel to tension.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/fisiología , Activación del Canal Iónico/fisiología , Canales Iónicos/química , Canales Iónicos/fisiología , Proteolípidos/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Membrana Celular/fisiología , Quimotripsina/metabolismo , Quimotripsina/farmacología , Activación del Canal Iónico/efectos de los fármacos , Membrana Dobles de Lípidos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Protoplastos/fisiología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tripsina/metabolismo , Tripsina/farmacologíaRESUMEN
Upon osmotic downshock, a few cytoplasmic proteins, including thioredoxin, elongation factor Tu (EF-Tu), and DnaK, are released from Tris-EDTA-treated Escherichia coli cells by an unknown mechanism. We have shown previously that deletion of mscL, the gene coding for the mechanosensitive channel of the plasma membrane with the highest conductance, prevents the release of thioredoxin. We confirm and extend the implication of MscL in this process by showing that the release of EF-Tu and DnaK is severely impaired in MscL-deficient strains. Release of these proteins is not observed in the absence of a Tris-EDTA treatment which disrupts the outer membrane, indicating that, in intact cells, they are transferred to the periplasm upon shock, presumably through the MscL channel.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Canales Iónicos/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , Transporte Biológico , Citoplasma/metabolismo , Escherichia coli/fisiología , Canales Iónicos/genética , Presión Osmótica , Periplasma/metabolismoRESUMEN
Patch-clamp experiments performed on membrane fragments of Corynebacterium glutamicum fused into giant liposomes revealed the presence of two different stretch-activated conductances, 600 to 700 pS and 1,200 to 1,400 pS in 0.1 M KCl, that exhibited the same characteristics in terms of kinetics, ion selectivity, and voltage dependence.
Asunto(s)
Corynebacterium/fisiología , Canales Iónicos/fisiología , Proteínas Bacterianas/fisiología , Membrana Celular/fisiología , Canales Iónicos/efectos de los fármacos , Cinética , Liposomas , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp , Cloruro de Potasio/farmacología , Estrés MecánicoRESUMEN
Mechanosensitive channels are ion channels whose activity is dependent on a mechanical stress on the membrane. They are believed to play a central role in mechanotransduction, the process by which mechanical energy is converted into electrical or chemical signals in biological cells. Recent progress, which has been made at the molecular level, is presented, and the two current models of activation of these channels are discussed.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/metabolismo , Electrofisiología/métodos , Canales Iónicos/fisiología , Proteínas de la Membrana , Modelos Biológicos , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Canales Epiteliales de Sodio , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Canales de Sodio/genética , Canales de Sodio/metabolismoRESUMEN
Escherichia coli cells possess several mechanosensitive ion channels but only MscL, the channel with the highest conductance, which is activated at the highest membrane tension, has been cloned. We investigated the putative involvement of MscL in the effluxes caused by osmotic downshock. Osmotic shock caused the release of potassium glutamate, trehalose, and glycine betaine from wild type cells and cells lacking MscL. There was no difference between the two strains, but the extreme rapidity of the efflux process, as shown herein for glycine betaine, suggests that it is channel-mediated. Osmotic downshock also induces the release of some cytosolic proteins from EDTA-treated cells. We investigated the release of thioredoxin. This protein was totally released from wild type cells but was retained by MscL- cells. Release was restored by expression of the gene coding for MscL. Thus MscL is not necessary for the excretion of osmoprotectants, but it does open in vivo during shock and catalyzes the efflux of thioredoxin and possibly other small cytosolic proteins. It follows that the other mechanosensitive channels, which are known to be activated at lower tension, must also open during osmotic shock. Their opening and that of MscL could account for the rapid release of osmolytes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Canales Iónicos/metabolismo , Tiorredoxinas/metabolismo , Betaína/metabolismo , Transporte Biológico , Ácido Glutámico/metabolismo , Presión Osmótica , Potasio/metabolismo , Trehalosa/metabolismoRESUMEN
Purified PhoE porins from Escherichia coli were reconstituted in giant proteoliposomes obtained by dehydration-rehydration, and studied by the patch-clamp technique. The following electrophysiological characteristics were observed. (i) The channels for which the probability of opening is maximum around 0 mV, closed at positive and negative potentials, at voltages higher than +/-120 mV. (ii) The channels behaved asymmetrically in response to positive and negative potentials. (iii) The channels exhibited two types of kinetics (fast and slow) on very different time scales. (iv) The channels had several closed states including a reversible inactivated state and a large number of substates. Similar characteristics have been described for channels other than bacterial porins, in particular mitochondrial porins and maxi-chloride channels of the plasma membrane of animal cells. These characteristics might constitute the electrophysiological fingerprint of a superfamily of ion channels for which the basic structure, rather than sequence, would have been conserved during evolution.
Asunto(s)
Proteínas Bacterianas/fisiología , Escherichia coli/química , Activación del Canal Iónico , Canales Iónicos/clasificación , Porinas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/clasificación , Proteínas de Escherichia coli , Potenciales de la Membrana , Familia de Multigenes , Técnicas de Placa-Clamp , Porinas/química , Porinas/clasificación , ProteolípidosRESUMEN
Mechanosensitive ion channels from Escherichia coli were studied in giant proteoliposomes reconstituted from an inner membrane fraction, or in giant round cells in which the outer membrane and the cell wall had been disrupted by a lysozyme-EDTA treatment and a mild osmotic shock. Patch-clamp experiments revealed the presence in these two preparations of an array of different conductances (100 to 2,300 pS in 0.1 M KCl) activated by stretch. The electrical activity induced by stretch in the native membrane was complex, due to the activation of several different conductances. In contrast, patches of proteoliposomes generally contained clusters of identical conductances, which differed from patch to patch. These experiments are consistent with the notion that these different conductances correspond to different proteins in the plasma membrane of E. coli, which segregate into clusters of identical channels on dilution involved in reconstitution in proteoliposomes. These conductances could be grouped into three subfamilies of poorly selective channels. In both preparations, the higher the conductance, the higher was the negative pressure needed for activation. We discuss the putative role of these channels as parts of a multicomponent osmoregulatory system.
Asunto(s)
Escherichia coli/fisiología , Canales Iónicos/fisiología , Mecanorreceptores/fisiología , Presión , Equilibrio Hidroelectrolítico/fisiología , Membrana Celular/efectos de los fármacos , Tamaño de la Célula , Quelantes/farmacología , Ácido Edético/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/ultraestructura , Muramidasa/farmacología , Técnicas de Placa-Clamp , Proteolípidos , Umbral Sensorial , Estrés MecánicoRESUMEN
A fraction highly enriched with inner membranes of E. coli was fused with liposomes, using the dehydration-rehydration technique, to produce giant liposomes amenable to patch-clamp recordings. Among the several channels present in this type of preparation, one was further characterized. The channel has a conductance of some 200 pS (in 0.1 M KCl) and is weakly selective for cations (PK/PCl = 4). The channel stays open at negative and low positive membrane potentials and shows an increasing probability of closure with increasing voltage. High positive membrane potentials favor transitions to a long-lived inactivated state, following slow kinetics. Voltage-dependent rapid flickerings of the same amplitude, between open state and other short-lived closed states, are superposed on these kinetics. The channel is presumed to be localized in the inner membrane, but its characteristics are also compatible with those of porins of the outer membrane. However, the major porins OmpF and OmpC, purified and reconstituted into giant liposomes, exhibited a marked different behavior.
Asunto(s)
Escherichia coli/metabolismo , Canales Iónicos/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Cationes/metabolismo , Conductividad Eléctrica , Cinética , Liposomas , Potenciales de la Membrana , PorinasRESUMEN
E. coli porins (OmpF and OmpC) were purified and reconstituted into liposomes which were enlarged to giant proteoliposomes by dehydration-rehydration and studied by patch-clamp. The porins could be closed by voltage pulses under -100 mV. The kinetics of closure was slow, with closure events of about 200 pS in 0.1 M KCl. Rapid fluctuations (in the millisecond range) of about one third (60-70 pS) of the large closure steps were also observed. The data are interpreted as follows: an increase in membrane potential favours the cooperation transition of multimers towards an inactivated state, while monomers which have not been inactivated can flicker rapidly between an open and a short-lived closed state.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Escherichia coli/metabolismo , Liposomas/metabolismo , Cinética , Potenciales de la MembranaRESUMEN
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.
Asunto(s)
Bacillus subtilis/efectos de los fármacos , Enterococcus faecalis/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Gadolinio/farmacología , Adenosina Trifosfato/metabolismo , Bacillus subtilis/metabolismo , Cationes , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/fisiología , Enterococcus faecalis/metabolismo , Escherichia coli/metabolismo , Glutamatos/metabolismo , Ácido Glutámico , Lactosa/metabolismo , Potenciales de la Membrana , Ósmosis , Potasio/metabolismo , Rubidio/metabolismoRESUMEN
The visualization of serotonin, 5-methoxytryptamine, and tryptamine in the rat midbrain has been made possible by the development of antibodies raised against these conjugated molecules. It has been suggested that 6-hydroxytryptamine (6-HT) might also be a neurotransmitter in this region. To test this hypothesis, 6-HT was synthesized and antibodies were raised in the rabbit. The high avidity (IC50 = 5 x 10(-9) M) and specificity [cross-reactivity ratio between 6-HT-glutaraldehyde (G)-bovine serum albumin (BSA) and 5-HT-G-BSA, the most immunoreactive compound, was 1,500] rendered these antibodies reliable tools for specific molecular detection of 6-HT in the G-fixed tissues. In the dopaminergic region, 6-HT immunoreactivity was noted in the substantia nigra but was particularly intense in the red nuclei, where it seems to be localized in the magnocellular division in the form of large 6-HT neurons. In contrast, there were few 6-HT neurons in the raphe nuclei. Thus, 6-HT may be a new putative neurotransmitter existing in the red nuclei, in addition to the other neurotransmitters already described in this region, in the nigro-rubral pathway, and in the rubral projection from the dorsal raphe nuclei. 6-HT is possibly implicated in motor control and might exert hallucinogenic properties as do other 6-hydroxylated indoleamines.
Asunto(s)
Mesencéfalo/metabolismo , Serotonina/análogos & derivados , Animales , Anticuerpos/inmunología , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunohistoquímica , Masculino , Ratas , Ratas Endogámicas , Serotonina/metabolismo , Coloración y Etiquetado , Distribución TisularRESUMEN
Inner and outer membranes of Escherichia coli and contact zones were isolated and fused separately with giant liposomes amenable to patch-clamp recording. Different types of large pressure-activated channels were localized in the inner membrane fraction which also contained smaller, pressure-insensitive channels. The outer membrane contained pressure-insensitive channels with large conductances and long opening and closing times which are likely to be porins. Large channels were also observed in contact zones.