Biomedical subjects

F Couraud

Publications and source records attributed to F Couraud.

At least 55 records · Page 3Linked to original sources

PubMed1987

Photoaffinity labeling of the K+-channel-associated apamin-binding molecule in smooth muscle, liver and heart membranes.

High-affinity binding sites for mono[125I]iodoapamin were detected in membranes (Kd = 59 pM, Bmax = 24 fmol/mg protein) and cultured cells (Kd = 69 pM, Bmax = 2.8 fmol/mg protein) from rat heart and in membranes from guinea-pig ileum (Kd = 67 pM, Bmax 42 fmol/mg protein) and liver (Kd = 15 pM, Bmax = 43 fmol/mg protein). Binding was stimulated by K+ ions (K0.5 = 0.3-0.5 mM). Covalent labeling with arylazide [125I]iodoapamin derivatives showed that smooth muscle, liver and heart binding molecules are associated with a 85-87-kDa polypeptide. A second strongly labeled 57-kDa component was identified in liver membranes only.

Affinity Labels↗

PubMed1987

Detection and photoaffinity labeling of the Ca2+-activated K+ channel-associated apamin receptor in cultured astrocytes from rat brain.

Apamin, an 18-amino acid bee venom peptide, is a specific blocker of a class of Ca2+ activated K+ channels. Mono 125I-iodoapamin was used to detect the K+ channel-associated receptor site in cultured astrocytes from rat brain. Specific high-affinity binding to intact glial cells with a Kd of about 90 pM at 1 degree C and pH 7.5 was demonstrated by equilibrium and kinetic methods. The average receptor capacity was 3 fmol/mg cell protein which is 2 to 3-fold lower than in primary cultured neurons. Binding was stimulated by K+ ions, but to a lesser extent than with neuronal receptors. Photoaffinity labeling of receptor/ion channel components using an arylazide derivative of 125I-monoiodoapamin revealed the presence of the 86- and 33-kDa polypeptides, previously detected in neurones. However a 59-kDa peptide which is present in synaptic membrane preparations from adult rat brain, but not in cultured neurons, was also clearly labeled in intact astrocytes. This indicates that the 59-kDa polypeptide is not a proteolytic fragment of the 86-kDa chain but an associated subunit which is only accessible to photolabeling in certain apamin receptor preparations. Apamin-sensitive Ca2+-activated K+ channels in astrocytes may be one of the pathways by which glial cells redistribute K+ in the central nervous system (CNS).

Affinity Labels↗

PubMed1987

Early appearance of cells bearing Na+ channels in developing mouse brain. A quantitative analysis using light microscopic autoradiography.

125I-alpha-Scorpion toxin (alpha-ScTx) binds to a component of the voltage-sensitive Na+ channel. We have previously shown that receptor capacity on dissociated mouse brain cells increases between days 12 and 19 of fetal life as does the expression of neurotoxin-sensitive 22Na+ influx. In the present study we have investigated the distribution of Na+ channels at the cellular level. Quantitative analysis by light-microscopic autoradiography was carried out on dissociated brain cells labeled with 125I-alpha-ScTx at 13, 15 and 18 fetal days. We have shown that at day 13 a large population of cells (39% of total) is alpha-ScTx-labeled, providing direct confirmation for a wide-spread presence of Na+ channels at an early stage of mouse brain development. The subsequent increase in receptor number with age is due both to an increase in alpha-ScTx-labeled cells (to 53% and 97% at days 15 and 18, respectively) and to an increase in the receptor density on these cells (10.9, 12.7 and 34.5 silver grains/1000 microns2 of cell surface for the 3 stages studied).

Animals↗

PubMed1987

Solubilization of the apamin receptor associated with a calcium-activated potassium channel from rat brain.

The apamin binding protein was solubilized from rat brain synaptic membranes using sodium cholate. Receptor yield and stability depended closely on the detergent/protein ratio. In optimum conditions the receptor retained high affinity for mono 125I-iodoapamin with Kd = 40 pM at pH 7.5 and 1 degree C and a binding capacity of 17 fmol/mg protein. 125I-apamin binding was stimulated by K+ ions with a K0.5 = 0.6 mM, demonstrating that the regulatory K+ site is also part of the soluble complex. Other ions could be substituted for K+ with an affinity sequence Tl+ = K+ = Rb+ greater than Cs+ greater than NH4+ greater than Li+ or Na+. Binding was inhibited by the neuromuscular blockers gallamine and tubocurarine and by the K+ channel blockers quinidine and tetraethylammonium chloride but not by 4-aminopyridine, in agreement with known pharmacological profile for inhibition of apamin-sensitive K+ permeability. Increasing the K+ concentration did not reverse inhibition by tetraethylammonium ions demonstrating that it does not bind competitively to the regulatory cationic site. Analysis of the covalently labeled apamin binding protein/sodium cholate complex by density gradient centrifugation indicated a high molecular weight with S20,w = 20 S.

Animals↗

PubMed1987

Allosteric modulation of neurotoxin binding to voltage-sensitive sodium channels by Ptychodiscus brevis toxin 2.

The effects of Ptychodiscus brevis toxin 2 (PbTx-2) on the binding of neurotoxins at four different neurotoxin receptor sites on voltage-sensitive sodium channels in rat brain synaptosomes were examined. Binding of saxitoxin at neurotoxin receptor site 1 and Leiurus quinquestriatus alpha-scorpion toxin (LqTx) at neurotoxin receptor site 3 was unaffected. PbTx-2 enhanced binding of batrachotoxinin A 20-alpha-benzoate (BTX-B) to neurotoxin receptor site 2 and Centruroides suffusus suffusus beta-scorpion toxin (CsTx II) to site 4 on sodium channels. These results support the proposal that PbTx-2 and related toxins act at a new receptor site (site 5) that has not been previously analyzed in binding experiments. Half-maximal effects of PbTx-2 were observed in the range of 20-50 nM PbTx-2. The enhancement of BTX-B binding was reduced by depolarization. Saturating concentrations of PbTx-2 reduced KD values for binding of BTX-B and CsTx-II 2.9-fold and 2.6-fold, respectively. The effects of PbTx-2 and LqTx in enhancing BTX-B binding were synergistic. A model involving both preferential binding of BTX-B, PbTx-2, LqTx, and CsTx II to active states of sodium channels and allosteric interactions among the four receptor sites at which these toxins act accommodates these and previous results.

Allosteric Regulation↗

PubMed1986

Molecular structure of rat brain apamin receptor: differential photoaffinity labeling of putative K+ channel subunits and target size analysis.

Two photoreactive apamin derivatives were prepared with an aryl azide [[(azidonitrophenyl)amino]acetate (ANPAA)] group coupled at different positions on the neurotoxin molecule. These ligands were used to identify membrane components in the environment of the neuronal binding site that is associated with a Ca2+-activated K+ channel. 125I-[alpha-ANPAA-Cys1] apamin labeled a single Mr 86 000 chain in cultured neurons whereas two bands corresponding to Mr 86 000 and 59,000 were detected in synaptic membrane preparations, suggesting that the Mr 59,000 polypeptide may be a degradation product. 125I-[epsilon-ANPAA-Lys4]apamin however incorporated uniquely into two smaller components with Mr 33,000 and 22,000 in both cultured neurons and synaptic membranes. Randomly modified 125I-ANPAA-apamin gave a cross-linking profile equivalent to the sum of those obtained with the two defined derivatives. The apamin binding site seems to be located at the frontier between three or more putative K+ channel subunits which are only accessible from limited regions of the receptor-associated photoprobe. Irradiation of frozen rat brain membranes with high-energy electrons led to a reduction in 125I-apamin receptor capacity, yielding a target size for the functional binding unit of Mr 84,000-115,000, which could be constituted by the Mr 86,000 subunit alone or by the Mr 86,000 subunit in conjuction with one of the two smaller subunits.

Affinity Labels↗

PubMed1986

Activation of the voltage-sensitive sodium channel by a beta-scorpion toxin in rat brain nerve-ending particles.

Neurotoxins purified from scorpion venoms previously had been divided into two classes according to their binding properties in rat brain synaptosomes. However, the pharmacological action of beta-scorpion toxin (beta-ScTx) on this preparation has not yet been described. In this report we show that a beta-ScTx induced an increase in 22Na+ uptake through synaptosomal voltage-sensitive sodium channels since this stimulation was abolished by tetrodotoxin (TTX). The increase was smaller than with veratridine and no synergy was observed between beta-ScTx and veratridine, as is the case for alpha-scorpion toxin (alpha-ScTx) and veratridine. The effects of alpha- and beta-ScTx were additive and the concentration-effect curves for each type of toxin were not modified by the other, suggesting that these two types of toxins act through distinct and noninteracting receptor sites. This was confirmed by the absence of mutual modification of the equilibrium and kinetic binding properties. beta-ScTx was shown to inhibit the uptake and to stimulate the release of [3H]gamma-aminobutyric acid. These effects were blocked by TTX, and no synergy was observed with veratridine. It was concluded that all these effects are mediated by the activation of voltage-sensitive sodium channels induced by the binding of beta-ScTx to a receptor site (site 4) distinct from those for other neurotoxins acting on sodium channels.

Animals↗

PubMed1986

Neurotoxin-sensitive sodium channels in neurons developing in vivo and in vitro.

Fetal mouse brain cells were investigated by 22Na+ flux assays with the aim to determine the ontogenetic time course of appearance of functional voltage-sensitive sodium channels. Their pharmacological properties were assessed by measurement of the response to known neurotoxins, acting at site 1, 2, or 3 of the Na+ channel. Brain cell suspensions, prepared at 11-19 d of prenatal development in vivo, and fetal brain neurons in culture were explored. In vivo neurotoxin-sensitive Na+ influx becomes detectable at 12 d of gestation, in concordance with the time of appearance of saturable binding sites for alpha-scorpion toxin (alpha-ScTx) and saxitoxin. Progression in fetal age or in time in vitro is accompanied by an increase in the initial rate and in the amplitude of Na+ uptake stimulated by batrachotoxin or veratridine. The general pharmacological properties of developing Na+ channels are very similar to the known properties of voltage-dependent Na+ channels in adult nerve: Batrachotoxin acts as a full channel agonist and veratridine as a partial agonist. Their respective apparent affinities are increased in presence of alpha-ScTx, in agreement with the known positive cooperativity of toxins acting at sites 2 and 3 of the Na+ channel. alpha-ScTx alone induces a small increase in Na+ permeability; its effect is greatly amplified in the presence of batrachotoxin or veratridine. The apparent affinity of alpha-ScTx is reduced by cell depolarization. Tetrodotoxin and saxitoxin block the increase in Na+ permeability induced by batrachotoxin, veratridine, and alpha-ScTx.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Ultrastructural localization of voltage-sensitive sodium channels using [125I]alpha scorpion toxin.

The distribution of alpha scorpion toxin (alpha-ScTx) receptors was examined in differentiated mouse neuroblastoma cell cultures (N IE 115 clone) by electron microscope autoradiography using [125I]alpha-ScTx. This neurotoxin binds specifically to voltage-sensitive sodium channels, slowing down the inactivation of the sodium permeability. Quantitative analysis demonstrated that only plasma membranes were labelled. The alpha-ScTx receptors seemed to be randomly dispersed on both cell bodies and cell processes. Microvilli protruding from the cell bodies carried more sodium channels than other parts of the membrane. The specific binding site density for alpha-ScTx varied from 4 (cell body membrane) to 13 (cell process membrane) per square micrometer.

Animals↗

PubMed1985

Photoaffinity labeling of components of the apamin-sensitive K+ channel in neuronal membranes.

An azidonitrophenylaminoacetyl mono[125I]iodoapamin derivative was prepared which showed specific binding to rat neuronal membranes. UV photolysis lead to the irreversible occupation of binding sites. Photo-labeling of intact primary cultured rat neurones followed by membrane solubilization, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography revealed the covalent incorporation of radioactivity into 3 main components with Mr = 86,000, 30,000, and 23,000. Labeling was completely prevented by a competing excess of native apamin. Similar studies on purified synaptic membranes from the rat brain showed another labeling pattern with major bands corresponding to Mr = 86,000 and 59,000. Although the reasons for the partial discrepancy between cultured embryonic neurons and an adult brain membrane fraction are not yet clear, we conclude that these proteins are intimately associated with the apamin binding site and are probably components of a type of Ca2+-activated K+ channel.

Affinity Labels↗

PubMed1985

Ultrastructural visualization of Na+-channel associated [125I]alpha-scorpion toxin binding sites on fetal mouse nerve cells in culture.

Purified neurotoxin II from the scorpion Androctonus australis Hector (alpha-ScTx) has previously been shown to bind specifically to the voltage-sensitive Na+ channels of excitable cells. Recent studies, using high specific activity 125I-labeled alpha-ScTx, demonstrated specific binding to neuronal cells derived from fetal mouse brains. In the present study, 125I-labeled alpha-ScTx was used to localize the voltage-sensitive Na+ channels in cultured fetal mouse brain cells. By quantitative electron microscope autoradiography we demonstrate that specific alpha-ScTx binding sites are selectively located at the plasma membrane. Estimates of their density revealed that neurites at 13 days in vitro carry at least 6 X more specific alpha-ScTx sites than cell body membrane.

Animals↗

PubMed1984

Binding of beta-scorpion toxin: a physicochemical study.

The binding to rat brain synaptosomes of a beta-scorpion toxin, i.e., toxin II of Centruroides suffusus suffusus (Css II), was studied as a function of pH, temperature, and concentration of some monovalent and divalent cations. At 10 degrees C and pH 6.0, the specific binding of 125I-labeled Css II corresponds to a single class of noninteracting high-affinity binding sites (KD = 0.18 nM) with a capacity (4.2 pmol/mg of protein) that is almost identical with that generally accepted for saxitoxin. The equilibrium dissociation constant of beta-scorpion toxin is pH independent, but the maximum binding capacity is reduced with increasing pH. Li+, guanidinium, Ca2+, Mg2+, and Mn2+ modified the apparent KD of the 125I-labeled Css II toxin. The equilibrium dissociation constant varies markedly with the temperature. The van't Hoff plot of the data is curvilinear, corresponding to a standard free-energy change associated with an entropy-driven process. The association rate constant also varies considerably with the temperature whereas the Arrhenius plot is linear between 1 and 30 degrees C. The energy of activation determined from these data is 17.6 kcal/mol. These results support the hypothesis that a cluster of nonpolar amino acid residues present on one face of the molecule is involved in the toxin-receptor interaction.

Animals↗

PubMed1984

Interactions of the neurotoxin apamin with a Ca2+-activated K+ channel in primary neuronal cultures.

Mono[125I]iodoapamin bound to specific sites on cultured rat embryonic neurons. The dissociation constant for the receptor-neurotoxin complex measured at equilibrium was 60-120 pM at pH 7.2 and 4 degrees C, with a maximal binding capacity of 3-8 fmol/mg of cell protein. Apamin inhibited calcium ionophore-induced 86Rb+ release from cell cultures. The dose effect curve for this pharmacological test corresponded closely to the displacement of 125I-apamin by native apamin in binding experiments. Formation of the 125I-apamin receptor complex requires exogenous K+. Reduced binding in the absence of K+ was due to diminished binding capacity rather than a lower affinity. The apamin receptor seems to be associated with a cell surface K+ site which shows 50% occupancy at 1.6 mM, and which could be involved in the regulation of channel activity. Apamin sites were present at the earliest developmental stage tested and their number did not evolve during 8 days in culture. In the same period, however, alpha-scorpion toxin binding increased by a factor of 10. The ontogenesis of Ca2+-activated K+ channels does not seem to occur in parallel with that of voltage-sensitive Na+ channels.

Animals↗

PubMed1984

Interaction of scorpion toxins with the sodium channel.

Scorpion toxins are miniproteins that have been characterized with regard to their molecular properties including their pharmacological action. On these bases, toxins specific to mammals or to insects have been described and within the first category alpha- and beta-toxins identified. These toxins were chemically modified and used as molecular probes of the sodium channel. Thus, the interaction of the toxins with the pharmacological target could be followed and a better definition of this target could be obtained.

Animals↗

PubMed1983

Photoaffinity labeling of alpha- and beta- scorpion toxin receptors associated with rat brain sodium channel.

Azido nitrophenylaminoacetyl [125I]iodo derivative of toxin II from Centruroides suffusus suffusus, a beta-toxin, and azido nitrophenylaminoacetyl [125I]iodo derivative of toxin V from Leiurus quinquestriatus quinquestriatus, an alpha-toxin, have been covalently linked after binding to their receptor sites that are related to the voltage sensitive sodium channel present in rat brain synaptosomes. Both derivatives labeled two polypeptides of 253000 +/- 20000 and 35000 +/- 2000 mol. wt. Labeling was blocked for each derivative by a large excess of the corresponding native toxin but no cross inhibition was obtained. These results suggest that both alpha - and beta - scorpion toxin receptors are located on or near the same two membrane polypeptides which may be part of the voltage dependent sodium channel.

Affinity Labels↗

PubMed1983

High-affinity binding of alpha-scorpion toxin: a neuronal property.

alpha-Scorpion toxin binding to its receptor--one component of the voltage-sensitive sodium channel--was studied in an attempt to define its phenotypic specificity. To this end we investigated the ability of neuronal, glial myogenic and fibroblastic cell lines to bind alpha-toxin II, purified from venom of the scorpion Androctonus australis Hector. A single class of saturable high-affinity (Kd congruent to 1 nM) binding sites, was present only in cell lines exhibiting some of the characteristics of normal neuronal cells, such as the N18, NIE-115, NS20, BN10-10, NG108-15 and T28 cell lines. NIA-103, which is an electrically non-excitable neuronal cell, gave negative results. In glial (G26-20, TR6B, C6) myogenic (T984) or fibroblastic (L) cell lines, we were unable to detect high-affinity binding sites for alpha-scorpion toxin. Primary cultures of rat skeletal muscle cells were also negative. Thus specific binding in the nanomolar range seems to be selectively associated with the neuronal phenotype. alpha-Scorpion toxin binding was tested before and after induction of neurites: in N18, NIE-115, NS20 cell lines, the differentiation brought on an increase in the number of binding sites but had little effect on the dissociation constant; in the hybrids NG108-15 and T28 high affinity saturable binding sites were detectable after but not prior to morphological differentiation.

Animals↗

PubMed1983

Voltage-sensitive Na+ channels in the neurohypophysis of the rat as demonstrated by 125I-labelled scorpion toxin.

Fresh rat neural lobe slices were incubated in the presence of [125I] alpha-scorpion toxin (ScTX), a specific marker of Na+ channels. Quantitative electron microscope autoradiography revealed preferential, irregularly spaced labeling of the axolemma of neurosecretory axons, with a significantly higher crude specific activity than any other neuronal or non-neuronal compartment. The number of specific binding sites at the neural lobe surface was calculated to be about 23 per microns2 of axolemma.

Animals↗

PubMed1983

Alpha-scorpion neurotoxin derivatives suitable as potential markers of sodium channels. Preparation and characterization.

Modified scorpion neurotoxins, i.e. mono-biotinylated and mono-azido derivatives either on lysine 58 or lysine 60 have been characterized both at the structural level (sequence and circular dichroism) and by their pharmacological activity (toxicity to mice and ability to displace 125I-Androctonus australis Hector toxin II from its receptor sites. The results allowed us to pinpoint a region of the molecule including lysine residues 58 and 60 that is important for neurotoxin receptor interaction. Furthermore, as these derivatives retain, after 125I labeling, high binding capacities to synaptosomal membranes, they can be used as potential labels of the sodium channel either by covalent binding or using the avidin-biotin system.

Animals↗