Biomedical subjects

M Thieffry

Publications and source records attributed to M Thieffry.

At least 19 recordsLinked to original sources

PubMed1995

Involvement of the peptide sensitive channel in the translocation of basic peptides into mitochondria.

The Peptide Sensitive Channel (PSC), a cationic channel of the mitochondrial outer membrane, is blocked by several highly basic peptides. Among these peptides, the most active are pCOX IV (1-12)Y, a mitochondrial addressing peptide and dynorphin B (1-13), a peptide unrelated to mitochondrial physiology. The voltage-dependent characteristics of the block duration of the PSC induced by these peptides and the fact that these peptides are imported into mitochondria in an in vitro assay suggest the involvement of the PSC in peptide translocation into mitochondria. We have analyzed the interaction of Mast Cell Degranulating peptide (MCD), a disulfide rich basic peptide, with yeast and mammalian mitochondria. Electrophysiological experiments with native and reduced forms of this peptide (nMCD and rMCD) showed an interaction of both forms with the yeast PSC. On the other hand, only rMCD blocked the electrical activity of the bovine adrenal cortex PSC. Similarly, although both forms inhibited the import of dynorphin B (1-13) into yeast mitochondria, only rMCD inhibited this import in bovine mitochondria. The correlation between electrophysiological and biochemical data strongly suggest that dynorphin B is translocated across the outer membrane at the level of the PSC.

Adrenal Cortex↗

PubMed1994

Reversible and irreversible effects of basic peptides on the mitochondrial cationic channel.

We have previously shown that a 13-residue basic peptide, derived from the presequence of a mitochondrial precursor, blocked the cationic channel of the outer mitochondrial membrane. The properties of the blockade suggested that the peptide could go through the pore in the presence of a sufficient driving force. In an attempt to evaluate more precisely the relevance of such an interpretation, we have examined the effect on the same channel of basic peptides from 16 to 34 residues, most of which are parts of or derive from mitochondrial presequences. Two peptides were found to induce a reversible voltage-dependent blockade, the properties of which were the same as those of the blockade induced by the 13-residue peptide. The others had a similar effect, but triggered in addition a modification of the voltage gating that persisted after washing the peptide out. The modification was in turn abolished by trypsin added to the side of the channel previously exposed to the peptide. The protease acted on the bound peptide and not on the channel itself. The irreversible modification of the voltage gating, the mechanism of which remains obscure, was not specific for mitochondrial-addressing sequences.

Adrenal Cortex↗

PubMed1993

Solubilization and reconstitution of the mitochondrial peptide-sensitive channel.

In addition to the voltage-dependent anion channel (VDAC), mitochondrial outer membranes contain a cationic channel of large conductance, which is blocked by a mitochondrial addressing peptide (peptide-sensitive channel, PSC). Bovine adrenal cortex mitochondria were solubilized in 1.5% octyl beta-glucoside, and membrane vesicles were reconstituted by slow dilution with a low ionic strength buffer. The reconstituted vesicles contained a functional channel possessing the electrical characteristics of the cationic channel, including its sensitivity to the mitochondrial addressing peptide. Important features of the described protocol are the nature of the detergent, its concentration, and the addition of glycerol during the whole procedure. No solubilization could be observed in the presence of cholate.

Adrenal Cortex↗

PubMed1992

Properties of the mitochondrial peptide-sensitive cationic channel studied in planar bilayers and patches of giant liposomes.

A voltage-dependent cationic channel of large conductance is observed in phospholipid bilayers formed by the tip-dip method from proteoliposomes derived from mitochondrial membranes. It is blocked by peptide M, a 13 residue peptide having the properties of a mitochondrial signal sequence. To verify the reliability of the experimental approach, mitochondrial membranes from bovine adrenal cortex or porin-deficient mutant yeast were either fused to planar bilayers or incorporated in giant liposomes which were studied by patch clamp. Cationic channels were found with both techniques. They had the same conductance levels and voltage-dependence as those which have been described using the tip-dip method. Moreover, they were similarly blocked by peptide M. The voltage-dependence of block duration was analyzed in planar bilayer and tip-dip records. Results strengthen the idea that peptide M might cross the channel. Other mitochondrial channels were observed in planar bilayers and patch clamp of giant liposomes. Because they were never detected in tip-dip records, they are likely to be inactivated at the surface monolayer used to form the bilayer in this type of experiment.

Adrenal Cortex↗

PubMed1991

A peptide-sensitive channel of large conductance is localized on mitochondrial outer membrane.

Applying the technique of 'tip-dip' to mitochondria, we have shown the existence in this organelle of a cationic channel of large conductance, which is blocked by a 13-residue peptide possessing the sequence of the N-terminal extremity of the cytochrome c oxidase subunit IV precursor. To study the submitochondrial localization of the channel, the effect of trypsin on isolated channels and on entire mitochondria were compared. One side of isolated channels is sensitive to trypsin, which eliminates the voltage dependence. Channels isolated from trypsinized mitochondria were devoid of voltage dependence and were blocked by the peptide. This suggests a localization of the channel on the outer membrane. Consistent with this hypothesis, the channel was observed with the highest frequency in outer membrane fractions purified by different procedures, either from bovine adrenal cortex or from rat liver mitochondria. Such a localization is also consistent with digitonin solubilization experiments. The channel was solubilized before the inner membrane marker, cytochrome c oxidase. The orientation of the channel was inferred from its trypsin sensitivity and its potential dependence: a transmembrane potential (inside negative) will close the channel.

Animals↗

PubMed1991

A 28 kDa mitochondrial protein is radiolabelled by crosslinking with a 125I-labelled presequence.

A 13-residue peptide containing the first 12 amino acids of the N-terminal part of the signal sequence of yeast cytochrome c oxidase subunit IV is shown by chemical crosslinking to interact with a mitochondrial protein. This result is obtained with mitochondria from four different origins. Submitochondrial localization experiments suggest that the 28 kDa labelled component is present on the outer face of the inner membrane. Since such addressing peptides are imported into mitochondria through the same machinery as protein precursors, the 28 kDa protein might be a component of the translocation apparatus.

Amino Acid Sequence↗

PubMed1990

Comparison of mitochondrial cationic channels in wild-type and porin-deficient mutant yeast.

Bilayers were formed at the tip of microelectrodes from a suspension of proteoliposomes derived from wild-type and porin-deficient mutant yeast mitochondria. In both preparations, identical cationic channels of large conductance were recorded. This result rules out any relationship between this channel and the outer membrane voltage-dependent anion channel, the activity of which is carried by porin. The ionic selectivity and the voltage-dependence of the yeast cationic channel suggest that it is related to that recently described in mammalian mitochondria. This hypothesis is further supported by the fact that both channels are blocked by a mitochondrial addressing peptide.

Amino Acid Sequence↗

PubMed1989

Blockade of a mitochondrial cationic channel by an addressing peptide: an electrophysiological study.

A voltage-dependent cationic channel of large conductance is observed in phospholipid bilayers formed at the tip of microelectrodes from proteoliposomes derived from mitochondrial membranes. This channel was blocked by a 13-residue peptide with the sequence of the amino terminal extremity of the nuclear-coded subunit IV of cytochrome c oxidase. The blockade was reversible, voltage- and dose-dependent. The peptide did not affect the activity of a Torpedo chloride channel observed under the same conditions. From experiments with phospholipid monolayers, it is unlikely that the peptide inserts into bilayers under the experimental conditions used. The blockade was observed from both sides of the membrane, being characterized by more frequent transitions to the lower conductance states, and a maximum effect was observed around 0 mV. Channels, the gating mechanism of which had been eliminated by exposure to trypsin, were also blocked by the peptide. For trypsinized channels, the duration of the closure decreased and the blockade saturated at potentials below -30 mV. These observations are consistent with a translocation of the peptide through the channel. Dynorphin B, which has the same length and charge as the peptide, had some blocking activity. Introduction of negative charges in the peptide by succinylation suppressed the activity.

Amino Acid Sequence↗

PubMed1989

Ionic mitochondrial channels: characteristics and possible role in protein translocation.

Most of the mitochondrial proteins are synthesized in the cytoplasm as precursors which are then translocated into the organelle. These precursors have a NH2-terminal extension which functions as a mitochondrial targeting signal. The import process through mitochondrial membranes is voltage-dependent; its mechanism is still unknown. Translocation has been proposed to occur through specific channels, thus, indicating the interest of the study of mitochondrial ionic channels. Two anion channels with different electrical characteristics have been described in the outer and the inner membranes. Using the technique of "Tip-Dip", we have shown the existence of a cation channel of large conductance in mitochondria. The characteristics of this channel differ from that of the other mitochondrial anion channels. A positively charged 13-residue synthetic peptide, with the sequence of the amino terminal extremity of the nuclear-coded subunit IV of yeast cytochrome C oxidase, induces a blockade of the cationic channel. From the characteristics of the blockade, it is likely that the channel could be permeable to the peptide. The specificity of this effect suggests that this channel might be involved in protein translocation.

Amino Acid Sequence↗

PubMed1989

[Blockage of a mitochondrial cationic channel by a mitochondrial addressing peptide].

A 13-residue peptide, including the sequence of the amino terminal end of cytochrome c oxidase subunit IV precursor, blocks a cationic channel from mitochondrial membranes. The effect is reversible and voltage-dependent. The blocking properties suggest that the peptide plugs the pore. Furthermore, when the transmembrane potential favours transfer, the peptide appears to be able to cross the channel.

Amino Acid Sequence↗

PubMed1988

Incorporation in lipid bilayers of a large conductance cationic channel from mitochondrial membranes.

Membranes from subcellular fractions of adrenal medulla were incorporated in phospholipid bilayers formed at the tip of microelectrodes. Current fluctuations recorded in the presence of a transmembrane potential revealed the existence of a voltage-dependent channel of large conductance. This channel is characterized by fast kinetics and four conductance levels separated by jumps of 100, 220 and 220 pS in 150 mM NaCl. It is permeant to Na+,K+, tetraethylammonium, Cl- and acetate and has some cation selectivity. Exposure to trypsin or pronase abolished the voltage-dependence. Upon subcellular fractionation, the activity was found to be associated with mitochondria. A similar activity was observed in mitochondrial fractions from other organs. By its kinetics, its selectivity and its potential-dependence, this channel differs from the voltage-dependent anion channel of outer mitochondrial membranes.

Adrenal Medulla↗

PubMed1987

[Demonstration of a large-conduction ion channel in subcellular fractions of the adrenal medulla].

The "tip-dip" technique (formation of a lipid bilayer at the tip of a microelectrode) allows the electrophysiological study of organelle membranes. An ionic channel of large conductance has been found by this technique in membrane preparations from adrenal medulla. This channel is voltage-sensitive and it has 4 levels of conductance. Its subcellular origin is to be determined; it is borne by a structure which sediments between 1,500 and 25,000 X g.

Adrenal Medulla↗

PubMed1984

The effect of calcium ions on the glutamate response and its desensitization in crayfish muscle fibres.

The responses of crayfish muscle fibres to bath application or long ionophoresis of L-glutamate were studied in normal and low Ca2+ solutions. The smaller responses recorded in low Ca2+ solutions have characteristics suggesting a faster desensitization. Desensitization and recovery have complex kinetics. Desensitization is faster and recovery slower when external Ca2+ concentration is reduced. Both components of the recovery phase, which can be fitted by the sum of two exponentials, are affected by the external Ca2+ concentration. Recovery can be accelerated by external Ca2+ ionophoresis onto desensitized glutamate receptors. Responses to brief glutamate pulses of low intensity are not affected by Ca2+ reduction. For higher intensities, signs of desensitization are detectable early in the rising phase of the response. Concanavalin A (Con A) blocks both desensitization and Ca2+ dependence with similar time courses. Whether or not the preparation has been treated with Con A, the slowly rising responses recorded in isotonic Ca2+ do not show signs of desensitization. Con A causes a partial blockade of the glutamate response. The Ca2+ dependence of the glutamate response can be explained by the Ca2+ dependence of the desensitization process, the cation acting at ectocellular sites of the muscle membrane.

Animals↗

PubMed1984

Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminals triggers a Ca-dependent acetylcholine release.

The venom glands of the annelid Glycera convoluta contain a neurotoxin which triggers ACh release from frog motor terminals and Torpedo synaptosomes. This neurotoxin binds to presynaptic, but not postsynaptic plasma membranes prepared from Torpedo electric organ. The binding site is an ectocellularly oriented protein. The binding does not require Ca. It is inhibited by pretreatment of the membrane by Concanavalin A. The toxin induced ACh release is Ca-dependent and inhibited by D 600.

Acetylcholine↗

PubMed1983

Binding of a Glycera convoluta neurotoxin to cholinergic nerve terminal plasma membranes.

The crude extract of venom glands of the polychaete annelid Glycera convoluta triggers a large Ca2+-dependent acetylcholine release from both frog motor nerve terminals and Torpedo electric organ synaptosomes. This extract was partially purified by Concanavalin A affinity chromatography. The biological activity was correlated in both preparations to a 300,000-dalton band, as shown by gel electrophoresis. This confirmed previous determinations obtained with chromatographic methods. This glycoprotein binds to presynaptic but not postsynaptic plasma membranes isolated from Torpedo electric organ. Pretreatment of intact synaptosomes by pronase abolished both the binding and the venom-induced acetylcholine release without impairing the high K+-induced acetylcholine release. Pretreatment of nerve terminal membranes by Concanavalin A similarly prevented the binding and the biological response. Binding to Torpedo membranes was still observed in the presence of EGTA. An antiserum directed to venom glycoproteins inhibited the neurotoxin so we could directly follow its binding to the presynaptic membrane. Glycera convoluta neurotoxin has to bind to a ectocellularly oriented protein of the presynaptic terminal to induce transmitter release.

Acetylcholine↗

PubMed1982

Concanavalin A blocks the Ca2+ -dependence of crayfish muscle fiber responses to glutamate.

(1) The response of crayfish muscle fibers to bath-applied glutamate is strongly inhibited when the Ca concentration of the physiological solution is reduced. Other divalent cations cannot substitute for Ca. The trivalent impermeant cation La can at low concentration replace Ca. Moreover, decreasing the Ca concentration in the presence of La potentiates the glutamate response. (2) The time course of responses to ionophoretically applied glutamate suggests a faster desensitization in low Ca solutions. The lectin concanavalin A, which blocks desensitization, also eliminates the decrease of the glutamate response in low Ca solutions. (3) The above results are compared to available data concerning Ca-dependence, desensitization and effects of concanavalin A.

Animals↗

PubMed1982

Partial purification of the Glycera convoluta venom components responsible for its presynaptic effects.

The crude extract of glands appended to the jaws of the polychaete annelid Glycera convoluta induces an important increase in the spontaneous quantal transmitter release on frog and crayfish neuromuscular junctions and on Torpedo nerve-electroplaque junctions. The venom similarly triggers acetylcholine (ACh) release from synaptosomes purified from Torpedo electric organ. At the frog neuromuscular junction, the reproducibility, the reversibility and the dose-dependence of the venom action permit a quantitative evaluation of the effect. The crude venom extract has been fractionated by gel-filtration. The effect on transmitter release has been found in a high molecular weight fraction distinct from those which contain the protease and phospholipase activities.

Acetylcholine↗

PubMed1980

Effects of high calcium solutions on glutamate sensitivity of crayfish muscle fibres.

Crayfish neuromuscular preparations were studied after 18--36 h exposure to high calcium solutions. As previously reported for frog neuromuscular preparations the treatment damaged the nerve terminals and decreased junctional potentials. The resting potentials and input resistances of the muscle fibres were not affected; but their sensitivity to glutamate was significantly decreased when compared to that of control muscles. After exposure to high calcium, the sensitivity to gamma-aminobutyric acid, the putative transmitter at inhibitory synapses, was increased. Apparently normal twitches were elicited by direct stimulation, and calcium spikes could still be observed in the fibres. A decreased sensitivity to glutamate was also noted in experiments carried out on denervated muscles 8 months after section of the motor axons. Possible relations between nerve terminal damage and the decrease in sensitivity to glutamate are discussed.

Animals↗