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J Barhanin

Publications and source records attributed to J Barhanin.

At least 91 records · Page 5Linked to original sources
PubMed1987

Identification and affinity labeling of very high affinity binding sites for the phenylalkylamine series of Ca+ channel blockers in the Drosophila nervous system.

The interaction of putative Ca2+ channels of Drosophila head membranes with molecules of the phenylalkylamine series was studied from binding experiments using (-)-[3H]D888 and (+/-)-[3H]verapamil. These ligands recognize a single class (Kd = 0.1-0.4 nM; Bmax = 1600-1800 fmol/mg of protein) of very high affinity binding sites. The most potent molecule in the phenylalkylamine series was (-)-verapamil with a Kd value as exceptionally low as 4.7 pM. Molecules in the benzothiazepine and diphenylbutylpiperidine series of Ca2+ channel blockers as well as bepridil inhibited (-)-[3H]D888 binding in a competitive way with Kd values between 12 and 190 nM, suggesting a close correlation, as in the mammalian system, between these receptor sites and those recognizing phenylalkylamines. A tritiated (arylazido)phenylalkylamine with high affinity for the Drosophila head membranes, phenylalkylamine receptor Kd = 0.24 nM), was used in photoaffinity experiments. A protein of Mr 135,000 +/- 5,000 was specifically labeled after ultraviolet irradiation.

Affinity Labels↗
PubMed1987

Photoaffinity labelling and phosphorylation of a 165 kilodalton peptide associated with dihydropyridine and phenylalkylamine-sensitive calcium channels.

Partially purified fractions of dihydropyridine and phenylalkylamine receptors associated with voltage-dependent calcium channels in rabbit skeletal muscle were found to contain two glycopeptides of similar molecular weight. A peptide of approximately 165 kDa was photoaffinity labelled with an arylazido-phenylalkylamine Ca channel inhibitor and also was phosphorylated with cAMP-dependent protein kinase. Another peptide of 170 kDa could be distinguished from the 165 kDa peptide by peptide mapping and differences in electrophoretic mobility. The results suggest that the 165 kDa peptide contains the sites responsible for regulation of calcium channel activity by calcium channel inhibitors as well as by neurotransmitters that regulate its activity in a cAMP-dependent manner.

Affinity Labels↗
PubMed1987

The calcium channel antagonists receptor from rabbit skeletal muscle. Reconstitution after purification and subunit characterization.

The Ca2+ channel antagonists receptor from rabbit skeletal muscle was purified to homogeneity. Following reconstitution into phosphatidylcholine vesicles, binding experiments with (+)[3H]PN 200-110, (-)[3H]D888 and d-cis-[3H]diltiazem demonstrated that receptor sites for the three most common Ca2+ channel markers copurified with binding stoichiometries close to 1:1:1. Sodium dodecyl sulfate gel analysis of the purified receptor showed that it is composed of only one protein of Mr 170,000 under non-reducing conditions and of two polypeptides of Mr 140,000 and 32,000 under disulfide-reducing conditions. Iodination of the protein of Mr 170,000 and immunoblots experiments with antisera directed against the different components demonstrated that the Ca2+ channel antagonists receptor is a complex of Mr 170,000 composed of a polypeptide chain of Mr 140,000 associated to one polypeptide chain of Mr 32,000 by disulfide bridges. One of the problems concerning this subunit structure of the putative Ca2+ channel was the presence of smaller polypeptide chains of Mr 29,000 and 25,000. Peptide mapping of these polypeptide chains and analysis of their cross-reactivity with sera directed against the proteins of Mr 170,000 and 32,000 demonstrated that they were degradative products of the Mr 32,000 component. Both the large (140 kDa) and the small (32 kDa) component of the putative Ca2+ channel are heavily glycosylated. At least 20-22% of their mass were removed by enzymatic deglycosylation. Finally the possibility that both the 140-kDa and 32-kDa components originate from a single polypeptide chain of Mr 170,000 which is cleaved by proteolysis upon purification is discussed.

Animals↗
PubMed1987

Purification and characterization of the dihydropyridine-sensitive voltage-dependent calcium channel from cardiac tissue.

The dihydropyridine-sensitive voltage-dependent Ca2+ channel from cardiac tissue was purified 900-fold using DEAE-Sephadex A-25, concanavalin A-Sepharose, and wheat germ agglutinin-Sepharose. The purified preparation was highly enriched in a peptide of 140,000 daltons when electrophoresed on sodium dodecyl sulfate gels in the presence of 2-mercaptoethanol, or 170,000 when electrophoresed in the presence of iodoacetamide. Polyclonal antibodies raised against the purified subunits of the rabbit skeletal muscle Ca2+ channel recognized the 170-kDa protein in preparations electrophoresed under nonreducing conditions, and the large peptide of 140 kDa and smaller peptides of 29-32 kDa in preparations analyzed under reducing conditions. Monoclonal antibodies, which were raised against the native Ca2+ channel from skeletal muscle, immunoprecipitated [3H]PN 200-110 binding activity from solubilized cardiac membranes and immunoprecipitated 125I-labeled peptides (from the purified cardiac Ca2+ channel preparation) which migrated as a single species of 170 kDa under nonreducing conditions, or as 140, 32, and 29 kDa under reducing conditions. The results show that the purified cardiac Ca2+ channel, like that previously purified from skeletal muscle, consists of a major component of 170 kDa which is comprised of a 140-kDa peptide linked by disulfide bonds to smaller peptides of 32-29 kDa. Peptide maps of the 140-kDa peptide purified from cardiac and skeletal muscle preparations were strikingly similar, suggesting a high degree of homology in their primary sequence.

Animals↗
PubMed1986

Antibodies reveal the cytolocalization and subunit structure of the 1,4-dihydropyridine component of the neuronal Ca2+ channel.

Rabbit brain synaptosomes bind the 1,4-dihydropyridine derivative (+)[3H]-PN 200-110 with an equilibrium dissociation constant of 0.04 nM and a maximal binding capacity of 400 fmol/mg of protein. Using polyclonal antibodies raised against the different components of the skeletal muscle 1,4-dihydropyridine receptor, we have demonstrated that the brain and muscle receptors share the same subunit composition comprising a large polypeptide chain of Mr 140,000 associated by disulfide bridges with a smaller peptide of Mr 32,000. These antibodies have been used in immunofluorescence staining of brain sections. They reveal a distribution of the Ca2+ channel protein similar to that of 1,4-dihydropyridine binding sites with (+)[3H]PN 200-110 by the autoradiographic technique.

Animals↗
PubMed1986

The voltage-dependent sodium channel is co-localized with the acetylcholine receptor at the vertebrate neuromuscular junction.

Isolated motor endplates from mouse intercostal muscles can be obtained after subcellular fractionation. On these motor endplates, localization of the nicotinic receptor and of the voltage-dependent Na+ channel coincides as demonstrated by double labeling with rhodamine alpha-bungarotoxin and a specific anti-Na+ channel monoclonal antibody. High density of Na+ channel at the motor endplate is confirmed by the enrichment in TTX binding sites as compared to the crude homogenate. In contrast isolated motor endplates are almost completely devoid of Ca2+ channel antagonist binding sites.

Animals↗
PubMed1986

Immunochemical analysis of subunit structures of 1,4-dihydropyridine receptors associated with voltage-dependent Ca2+ channels in skeletal, cardiac, and smooth muscles.

Previous purification studies of the 1,4-dihydropyridine receptor associated with the calcium channel of rabbit skeletal muscle had shown that it is composed of a large glycoprotein of Mr 140,000-145,000 associated with a smaller component of Mr 32,000-34,000. Specific antisera have now been prepared against the larger component (anti-140 serum) and the smaller one (anti-32 serum). The specificity of these two antisera has been analyzed by immunoblot assays with microsomal preparations of rabbit skeletal muscle. Under disulfide-reducing conditions the anti-140 serum specifically labeled a polypeptide of Mr 140,000 while the anti-32 serum labeled three polypeptides of Mr 32,000, 29,000, and 26,000. Under nonreducing conditions both the anti-140 and the anti-32 sera specifically recognized a single large polypeptide of Mr 170,000. The same type of approach showed that the dihydropyridine receptor in cardiac and smooth muscles had a polypeptide composition similar to that found in skeletal muscle with a large polypeptide of Mr 170,000-176,000 made of two different chains of about Mr 140,000 and 34,000-32,000 associated by disulfide bridges.

Animals↗
PubMed1986

Characterization and photoaffinity labeling of receptor sites for the Ca2+ channel inhibitors d-cis-diltiazem, (+/-)-bepridil, desmethoxyverapamil, and (+)-PN 200-110 in skeletal muscle transverse tubule membranes.

In order to further understand the molecular nature of the voltage-sensitive Ca2+ channel in skeletal muscle, we have performed classical radioligand binding studies and photoaffinity labeling with different types of tritiated inhibitors of the Ca2+ channel. The equilibrium dissociation constants (KD) for (-)-[3H]desmethoxyverapamil, d-cis-[3H]diltiazem, and (+/-)-[3H]bepridil at their receptor sites in skeletal muscle transverse tubule membranes are: 1.5 +/- 0.5, 50 +/- 5, and 20 +/- 5 nM, respectively. Maximum binding capacities in picomoles/milligram of protein were: 70 +/- 10 for (-)-[3H]desmethoxyverapamil, 50 +/- 15 for d-cis-[3H]diltiazem, and 75 +/- 15 for (+/-)-[3H]bepridil. The kinetics of association at 10 degrees C for the three types of tritiated compounds were relatively slow (3 X 10(5) M-1 S-1 for (-)-[3H]desmethoxyverapamil, 8 X 10(3) M-1 S-1 for d-cis-[3H]diltiazem, and 4.2 X 10(5) M-1 S-1 for (+/-)-[3H]bepridil). The dissociation of (-)-[3H]desmethoxyverapamil and d-cis-[3H]diltiazem from their receptor sites was also a slow process with half-lives of dissociation of 33 and 36 min, respectively. Competition studies using the three tritiated ligands suggest that they bind to the same receptor site which appears to be in a 1:1 stoichiometry with the dihydropyridine receptor. Photoaffinity labeling with high intensity ultraviolet light in the presence of (+/-)-[3H]bepridil or d-cis[3H]diltiazem resulted in the specific covalent incorporation of radioactivity into a polypeptide of Mr 170,000 +/- 10,000. A polypeptide of Mr 170,000 was also specifically labeled in photoaffinity labeling experiments using the high affinity dihydropyridine derivative (+)-[3H]PN 200-100.

Affinity Labels↗
PubMed1986

Dihydropyridine-sensitive Ca2+ channels: molecular properties of interaction with Ca2+ channel blockers, purification, subunit structure, and differentiation.

[3H]Desmethoxyverapamil, (S/-)[3H]bepridil, and d-cis-[3H]diltiazem bind to skeletal muscle transverse-tubule (T-tubule) membranes with affinity constants in the range of 2-50 nM and with Bmax values of the order of 50-80 pmol/mg of protein. These drugs are mutually competitive and modulate allosterically the 1,4-dihydropyridine binding. The molecular size of the native [3H]nitrendipine receptor of T-tubule membranes has been determined using the radiation inactivation technique. The [3H]nitrendipine receptor was found to have a Mr of 210,000 +/- 20,000. Photoaffinity labelling using (+)[3H]PN 200-110 indicates the presence in the dihydropyridine receptor of a polypeptide (or polypeptides) of Mr 170,000 +/- 5,000. The 3-[(3-cholamidopropyl)-dimethylammonio]-1 propane sulfonate (CHAPS) solubilization of the T-tubule membranes preserved the binding properties of the different drugs to the calcium channel. The dihydropyridine-sensitive calcium channel has been substantially purified by a combination of ion exchange chromatography, lectin affinity chromatography, and gel filtration. The purified material contained polypeptides of apparent molecular weights of 142,000, 32,000, and 33,000, which copurified with (+)[3H]PN 200-110 binding activity. Two stages in which there is an increased binding of [3H]nitrendipine have been observed during chick myogenesis in ovo. The first one occurs during embryonic life and has the same properties as in the in vitro development. The second stage occurs near hatching and corresponds to a large increase in the number of nitrendipine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1985

The 1,4-dihydropyridine receptor associated with the skeletal muscle voltage-dependent Ca2+ channel. Purification and subunit composition.

The dihydropyridine receptor associated with the voltage-dependent Ca2+ channel from rabbit skeletal muscle has been purified using the tritiated derivative of (+)-PN 200-110. The drug was used not only as a marker associated with the solubilized receptor but also in direct binding experiments performed after each purification step. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilization of a microsomal preparation resulted in an extract with a specific binding activity of 10 pmol/mg of protein. A combination of chromatographic steps utilizing anion exchange, lectin affinity, and gel filtration resulted in an 80-fold purification to a specific binding activity of 800 pmol/mg of protein. The affinity of (+)-[3H]PN 200-110 for the solubilized receptor was only slightly altered after the purification procedure. The KD values were 0.7 and 1.8 nM on the starting material and the most purified fractions, respectively. The subunit composition of the dihydropyridine receptor was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was consistent with three polypeptides of Mr 142,000, 33,000, and 32,000. The last two small components were not covalently associated with the larger one. In spite of a careful investigation of the conditions which improved the stability of the dihydropyridine receptor, a partial denaturation could not be prevented during purification. This resulted in an underestimation of receptor purity when calculated from the maximal specific binding activity as compared to the enrichment in the three polypeptides observed after polyacrylamide gel electrophoresis. Finally, application of the same purification procedure to solubilized microsomal preparations of chick and frog skeletal muscle demonstrated the presence of a large polypeptide component of Mr 135,000-141,000 associated with the Ca2+ channel from these sources. The doublet of small molecular weight was not found with the frog muscle.

Animals↗
PubMed1985

The voltage-dependent Na+ channel of insect nervous system identified by receptor sites for tetrodotoxin, and scorpion and sea anemone toxins.

Receptor sites for some of the most important toxins known to be specific for voltage-sensitive Na+ channel in the mammalian nervous system have been identified in a purified membrane preparation of house fly brain. Very high affinities have been found for the association of tetrodotoxin or tetrodotoxin derivatives with the insect Na+ channel (Kd = 0.03 - 0.08 nM). The gamma toxin from the Brazilian scorpion Tityus serrulatus forms a complex with the Na+ channel having a Kd of 6.1 pM. The Kd value for toxin II from the sea anemone Anemonia sulcata is 0.12 microM. These results show a high degree of conservation of the pharmacological properties of the brain Na+ channels between insects and mammals.

Amphibian Proteins↗
PubMed1985

A monoclonal immunotoxin acting on the Na+ channel, with properties similar to those of a scorpion toxin.

We describe the properties of a monoclonal antibody against the Na+ channel. The antibody, 72.38, competitively inhibited (Ki = 1.5 X 10(-9) M) the binding of an 125I-labeled toxin from the Brazilian scorpion Tityus serrulatus (125I-TiTX gamma) to Na+ channels of rat brain membranes. No significant inhibition of binding of a number of other Na+ channel toxins was observed. The inhibition of 125I-TiTX gamma binding also was observed with the solubilized Na+ channel from rat brain membranes (Ki = 2 X 10(-9) M). Antibody 72.38 antagonized 125I-TiTX gamma binding to Na+ channels from different animal species (fish, avian, and mammalian) and from different tissues (electroplax, brain, heart, and muscle). Moreover, 72.38 has been used for immunofluorescence labeling of Na+ channels in rat sciatic nodes of Ranvier and cultured dorsal root ganglion cells. Electrophysiological experiments on rat muscle cells fully confirmed the similarity between TiTX gamma and 72.38 seen in binding experiments. Both produce slow oscillations of the membrane potential accompanied by bursts of action potentials which are due to a selective action on the Na+ channel. TiTX gamma and 72.38 are without effect on the ion selectivity of the Na+ channel, but they both drastically change the voltage-dependence of activation and inactivation of the Na+ channel.

Animals↗
PubMed1984

Purification of the dihydropyridine receptor of the voltage-dependent Ca2+ channel from skeletal muscle transverse tubules using (+) [3H]PN 200-110.

The rabbit skeletal muscle T-tubule membranes preparation is the richest source of organic Ca2+ blocker receptor associated with the voltage-dependent Ca2+ channel. Solubilization by 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (CHAPS) in the presence of glycerol leads to a 52% recovery of active receptors as determined by (+)[3H]PN 200-110 binding experiments. The dissociation constant of the (+) [3H]PN 200-110 solubilized-receptor complex was 0.4 +/- 0.2 nM by equilibrium binding and 0.13 nM from the rate constants of association (k1 = 0.116 nM-1 min-1) and dissociation (k-1 = 1.5 10(-2) min-1). The (+) [3H]PN 200-110 receptor has been substantially purified by a combination of filtration of Ultrogel A2 column and lectin affinity chromatography in the presence of trace amount of specifically bound (+) [3H]PN 200-110. The purified material contained polypeptides of apparent molecular weights of 142 000, 32 000 and 33 000. These three components copurified with (+)[3H]PN 200-110 binding activity.

Animals↗
PubMed1984

Reconstitution of highly purified saxitoxin-sensitive Na+-channels into planar lipid bilayers.

Highly purified Na+-channels isolated from rat brain have been reconstituted into virtually solvent-free planar lipid bilayer membranes. Two different types of electrically excitable channels were detected in the absence of any neurotoxins. The activity of both channels was blocked by saxitoxin. The first channel type is highly selective for Na+ over K+ (approximately 10:1), it shows a bursting behavior, a conductance of 25 pS in Na+-Ringer and undergoes continuous opening and closing events for periods of minutes within a defined range of negative membranes voltages. The second channel type has a conductance of 150 pS and a lower selectivity for Na+ and K+ (2.2:1); only a few opening and closing events are observed with this channel after one voltage jump. The latter type of channel is also found with highly purified Na+-channel from Electrophorus electricus electroplax. A qualitative analysis of the physicochemical and pharmacological properties of the high conductance channel has been carried out. Channel properties are affected not only by saxitoxin but also by a scorpion (Centruroides suffusus suffusus) toxin and a sea anemone (Anemonia sulcata) toxin both known to be selective for the Na+-channel. The spontaneous transformation of the large conductance channel type into the small one has been considered; the two channel types may represent the expression of activity of different conformational states of the same protein.

Animals↗
PubMed1984

Tityus gamma toxin, a high affinity effector of the Na+ channel in muscle, with a selectivity for channels in the surface membrane.

Toxin gamma from the venom of Tityus serrulatus scorpion produces a partial block of the surface Na+ channel in frog muscle. This block occurs with no change in the voltage-dependence or in the kinetics of the remaining surface Na+ current. The partial blockade of Na+ channel activity occurs with no change in tubular Na+ currents nor in twitch tension. The maximum effect of the toxin is attained at concentrations as low as 3 X 10(-10) M. Hyperpolarization to potentials more negative than the resting potential (E = -90 mV) reduces or abolishes the effect of the toxin. Radioiodinated toxin gamma binds to frog muscle membranes with a very high affinity corresponding to a dissociation constant of about 1 X 10(-11) M. Data obtained with both rabbit and frog muscle indicate that toxin gamma is specific for Na+ channels in surface membranes. Toxin gamma does not seem to bind to Na+ channels in T-tubule membranes. The biochemical data are in good agreement with electrophysiological studies and data on contraction. There is one Tityus gamma toxin binding site per tetrodotoxin binding site in surface membranes. Competition experiments have confirmed that Tityus gamma toxin binds to a new toxin receptor site on the Na+ channel structure. This site is the same that the toxin II from Centruroides suffusus binding site, but this toxin has 100 times less affinity for the Na+ channel than Tityus gamma toxin.

Animals↗
PubMed1984

New scorpion toxins with a very high affinity for Na+ channels. Biochemical characterization and use for the purification of Na+ channels.

Biochemical characterization of the Tityus gamma toxin receptor associated with the voltage-sensitive Na+ channel was carried out in different tissue preparations with the use of an iodinated toxin derivative. The affinity of the toxin for the receptor is high with a dissociation constant of 4 X 10(-12) M for rat synaptosomes. The density of binding sites is in the range of 0.3 to 2 pmol/mg of protein. Toxin gamma does not seem to bind to Na+ channels located on transverse-tubule membranes of skeletal muscle, but only to Na+ channels located on the sarcolemma. Both affinity labelling and radiation inactivation analysis indicate a molecular weight for the toxin receptor of 270 000 daltons. The same molecular weight is found using the tetrodotoxin. Only one single major protein component of the Na+ channel was purified from Electrophorus electroplax, rat brain membranes and chick heart membrane using the toxin gamma as a marker. The molecular weight of this component is 230 000-270 000 daltons. Reconstitution of the purified Na+ channel into planar lipid bilayers has been carried out. Two different types of electrically excitable channels with conductances of 150 and 25 pS were detected. The activity of both channels is blocked by saxitoxin.

Animals↗