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J Barhanin

Publications and source records attributed to J Barhanin.

103 records · Page 6Linked to original sources
PubMed1983

Molecular size of different neurotoxin receptors on the voltage-sensitive Na+ channel.

Measurements were made of the molecular sizes of two distinct receptors on the Na+ channel in rat brain synaptosomes that are specific for different neurotoxins. Radiation inactivation of the binding of radiolabeled derivatives of the toxins was consistent with Mr = 260,000 for the tetrodotoxin receptor and Mr = 266,000 for the receptor specific for two scorpion toxins, toxin II from Centruroides suffusus suffusus and toxin gamma from Tityus serrulatus serrulatus. Covalent cross-linking of the latter to its receptor similarly indicated Mr = 270,000. It seems most likely that these two distinct receptors reside on the same molecule.

Amphibian Proteins↗
PubMed1983

Electrophysiological characterization, solubilization and purification of the Tityus gamma toxin receptor associated with the gating component of the Na+ channel from rat brain.

Electrophysiological studies with neuroblastoma cells have shown that toxin gamma from the venom of the scorpion Tityus serrulatus is a new toxin specific for the gating system of the Na+ channel. The procedure which solubilizes the tetrodotoxin receptor from rat brain also solubilizes the Tityus gamma toxin receptor. Binding experiments on the solubilized receptor with a radioiodinated derivative of Tityus gamma toxin have shown: (i) that the TiTx gamma-receptor complex is very stable with a dissociation constant of 8.6 X 10(-12) M and a very slow dissociation (T 1/2 = 15 h); (ii) that the toxin recognizes a class of sites with a 1:1 stoichiometry with those for tetrodotoxin (Bmax = 1.3 pmol/mg protein). The radioiodinated Tityus gamma-receptor complex has been substantially purified by ion-exchange chromatography, lectin affinity chromatography and sucrose gradient sedimentation. A ratio of one Tityus gamma toxin binding site per tetrodotoxin binding site was found throughout the purification. The purified material exhibited a sedimentation coefficient of 10.4S and had an apparent mol. wt. of 270 000 on SDS-gel electrophoresis. No other polypeptide chains were demonstrated to be associated with this large protein in the Tityus gamma receptor. The main conclusion is that the tetrodotoxin binding site associated with the selectivity filter of the Na+ channel and the Tityus gamma toxin binding site associated with the gating component are probably carried by the same polypeptide chain.

Action Potentials↗
PubMed1983

Purification of binding protein for Tityus gamma toxin identified with the gating component of the voltage-sensitive Na+ channel.

The gating component associated with the voltage-sensitive Na+ channel from electroplax membranes of Electrophorus electricus has been purified by using toxin gamma from the venom of the scorpion Tityus serrulatus serrulatus. The toxin-binding site was efficiently solubilized with Lubrol PX, resulting in an extract of high initial specific activity. Purification was achieved by adsorption of the toxin-binding component to DEAE-Sephadex A-25 followed by desorption at high ionic strength and chromatography on either wheat germ agglutinin-Ultrogel or Sepharose 6B. Maximal final specific activities were at least 42% of the specific activity expected for a pure toxin-binding component. The purified material exhibited a Stokes radius of 85 A, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated a single polypeptide component of Mr 270,000. Furthermore, tetrodotoxin binding activity and Tityus gamma toxin binding activity copurified, suggesting that the selectivity filter and the gating component of the Na+ channel are carried by the same polypeptide chain.

Animals↗
PubMed1982

Tityus serrulatus venom contains two classes of toxins. Tityus gamma toxin is a new tool with a very high affinity for studying the Na+ channel.

The interaction of TiTx gamma, the major toxin in the venom of the scorpion Tityus serrulatus, with its receptor in excitable membranes was studied with the use of 125I-TiTx gamma. This derivative retains biological activity, and its specific binding to both brain synaptosomes and electroplaque membranes from Electrophorus electricus is characterized by a dissociation constant equal to that of the native toxin-receptor complex, about 2 to 5 pM. This very high affinity results mainly from a very slow rate of dissociation, equivalent to a half-life longer than 10 h at 4 degrees C. There is a 1:1 stoichiometry between TiTx gamma binding and tetrodotoxin binding to the membranes, but neither tetrodotoxin nor any of 7 other neurotoxins that are representative of 4 different classes of effectors of the Na+ channel interfere with TiTx gamma binding. Similarly, local anesthetics and other molecules that affect other types of ionic channels or neurotransmitter receptors have no effect on TiTx gamma binding. However, toxin II from Centruroides suffusus suffusus does compete with TiTx gamma, though its affinity for the receptor is much lower. Since the Centruroides toxin II is known to affect Na+ channel function, these two scorpion toxins must be put into a fifth class of Na+ channel effectors.

Animals↗
PubMed1982

Specific binding of toxin II from Centruroides suffusus suffusus to the sodium channel in electroplaque membranes.

The binding of toxin II from the scorpion Centruroides suffusus suffusus (CssII) to electroplaque membranes from Electrophorus electricus was studied with the use of a radiolabeled derivative of the toxin ([125I]CssII). Specific binding of the latter to the membranes required the protonation of a group, either in the membrane or in the toxin itself, with an apparent pKa value of 7.5 and also the presence of a certain minimum concentration of ions, though there was no requirement for a specific ion. At 20 degrees C and pH 6 the second-order rate constant for formation of the [125I]CssII-membrane complex was about 5 X 10(6) M-1 s-1, while the first-order constant for its dissociation was about 2 X 10(-3) s-1. Under equilibrium conditions specific binding of [125I]CssII was a simple saturable function of [125I]CssII concentration, characterized by a dissociation constant of 0.4-0.7 nM and a maximum capacity of 0.9-2.4 pmol of toxin/mg of membrane protein. The latter value was the same as the number of membrane sites that could specifically bind a radiolabeled derivative of tetrodotoxin. Unlabeled CssII displaced bound [125I]CssII with an apparent dissociation constant of about 1 nM. None of 19 other neurotoxins or local anaesthetics known to interact with Na+ channels in excitable cells affected [125I]CssII binding, but it was completely inhibited by toxin gamma from the scorpion Tityus serrulatus serrulatus. These findings suggest that the Na+ channel possesses a distinct class of binding sites to which these two scorpion toxins bind with high affinities. On the other hand, no CssII receptor was detected in crab axonal membranes, indicating that it is not a characteristic feature of all Na+ channels.

Animals↗
PubMed1982

Centruroides toxin, a selective blocker of surface Na+ channels in skeletal muscle: voltage-clamp analysis and biochemical characterization of the receptor.

This paper describes the effects of a toxin from the scorpion Centruroides suffusus suffusus on frog skeletal muscle. The main findings are the following, (i) Centruroides toxin (CssII) blocks the early phase of the inward sodium current in the muscle that arises from influx via Na+ channels in the surface membrane, but it does not affect the late phase of the inward current that represents flux through Na+ channels in the T-tubule membranes, (ii) CssII, in marked contrast to tetrodotoxin, does not affect contraction of the muscle, (iii) Measurements of the binding of 125I-labeled CssII to a partially purified membrane preparation from the muscle indicate that the Kd of the CssII--receptor complex is approximately 0.4 nM. The half-life for the dissociation of this complex is 3 min at 22 degrees C and 16 min at 2 degrees C. Binding of the radiolabeled toxin varies markedly with pH and becomes insignificant at pH greater than 8.5. Proteolytic digestion of the membrane preparation decreases its ability to bind CssII, suggesting that the receptor is a protein. (iv) The number of binding sites for a radiolabeled derivative of tetrodotoxin on the membrane preparation was similar to that for CssII. However, neither tetrodotoxin nor any of seven other neurotoxins and some local anesthetics that alter the functioning of the Na+ channel have any effect on the binding of CssII to the muscle membrane. These results therefore indicate that CssII belongs to a different class of neurotoxins that has a different receptor on the Na+ channel.

Animals↗
PubMed1981

Purification and pharmacological properties of eight sea anemone toxins from Anemonia sulcata, Anthopleura xanthogrammica, Stoichactis giganteus, and Actinodendron plumosum.

Eight different polypeptide toxins from sea anemones of four different origins (Anemonia sulcata, Anthopleura xanthogrammica, Stoichactis giganteus, and Actinodendron plumosum) have been studied. Three of these toxins are new; the purification procedure for the five other ones has been improved. Sea anemone toxins were assayed (i) for their toxicity to crabs and mice, (ii) for their affinity for the specific sea anemone toxin receptor situated on the Na+ channels of rat brain synaptosomes, and (iii) for their capacity to increase, in synergy with veratridine, the rate of 22Na+ entry into neuroblastoma cells via the Na+ channel. Some of the toxins are more active on crustaceans, whereas others are more toxic to mammals. A very good correlation exists between the toxic activity to mice, the affinity of the toxin for the Na+ channel in rat brain synaptosomes, and the stimulating effect on 22 Na+ uptake by neuroblastoma cells. The observation has also been made that the most cationic toxins are also the most active on mammals and the least active on crustaceans. Toxicities (LD50) to mice of the most active sea anemone toxins and of the most active scorpion toxins are similar, and sea anemone toxins at high enough concentrations prevent binding of scorpion toxins to their receptor. However, scorpion toxins have affinities for the Na+ channel which are approximately 60 times higher than those found for the most active sea anemone toxins. Three sea anemone toxins appear to be more interesting than toxin II from A. sulcata (the "classical" sea anemone toxin) for studies of the Na+ channel structure and mechanism when the source of the channel is of a mammalian origin. Two of these three toxins can be radiolabeled with iodine while retaining their toxic activity; they appear to be useful tools for future biochemical studies of the Na+ channel.

Animals↗
PubMed1981

Structure-function relationships of sea anemone toxin II from Anemonia sulcata.

Chemical modifications of sea anemone toxin II from Anemonia sulcata have been used to study the residues involved in its toxic action on crabs and mice and in its binding properties to the Na+ channel of rat brain synaptosomes. Guanidination of th epsilon-amino groups of lysines 35, 36, and 46 with O-methylisourea hydrogen sulfate did not change the net charge of the toxin molecule and had no effect upon its toxic and binding properties. Either acetylation or fluorescamine treatment of the toxin that destroyed the positive charges of the three epsilon-amino groups and of the alpha-amino function of Gly produced an almost complete loss of toxicity and a considerable decrease in the binding activity. Iodination of the toxin on His induced practically no loss of toxic or binding properties. Carbethoxylation of both histidines 32 and 37 with diethyl pyrocarbonate provoked an important decrease of both the toxicity and the binding activity. Modifications of the guanidine side chain of Arg with 1,2-cyclohexanedione fully destroyed both toxicity and binding of the toxin to the Na+ channel. Modification of the carboxylate functions of Asp, Asp, and of the COOH-terminal Gln with glycine ethyl ester in the presence of a soluble carbodiimide completely abolished the toxicity but left the affinity for the sea anemone toxin receptor unchanged. The antagonist character of this carboxylate-modified derivative was further confirmed by electrophysiological and Na+ flux experiments. The theoretical and practical significance of these results are discussed.

Acetic Anhydrides↗
PubMed1980

Binding of sea anemone toxin to receptor sites associated with gating system of sodium channel in synaptic nerve endings in vitro.

Iodination of toxin II from the sea anemone Anemonia sulcata gives a labeled monoiododerivative that retains 80% of the original neurotoxicity. This derivative binds specifically to rat brain synaptosomes at 20 degrees C and pH 7.4 with a second-order rate constant of association ka = 4.6 x 10(4) M-1 sec-1 and a first-order rate constant of dissociation kd = 1.1 x 10(-2) sec-1. The binding occurs on the Na+ channel at a binding site distinct from that of other gating system toxins like batrachotoxin, veratridine, grayanotoxin, aconitine, and pyrethroids. The maximal binding capacity Bmax is 3.2 pmol/mg of protein (i.e., about two sea anemone toxin binding sites per tetrodotoxin binding site) and the Kd is 240 nM for the monoiododerivative and 150 nM for the native toxin. Corresponding binding parameters for the association of a 125I-labeled derivative of toxin II from the scorpion Androctonus australis Hector are Bmax = 0.3 pmol/mg of protein and Kd = 1 nM, whereas the Kd of the unmodified scorpion toxin is 0.6 nM. Competition experiments involving scorpion toxins, sea anemone toxins, and synaptosomes demonstrate that, although the sea anemone toxin is able to displace the scorpion toxin bound to synaptosomes, the scorpion toxin does not displace the sea anemone toxin. The sea anemone toxin but not the scorpion toxin binds to depolarized synaptosomes. Differences between binding properties of the two polypeptide toxins are analyzed in the discussion.

Animals↗
PubMedDate not supplied

Cardiac K+ channels and drug-acquired long QT syndrome.

The hallmark of long QT syndromes (LQTS) is an abnormal ventricular repolarization characterized by a prolonged QT interval on the electrocardiogram and a propensity to the occurrence of syncopes resulting from polymorphic ventricular tachycardia, called torsades de pointes. They may degenerate to ventricular fibrillation, possibly causing sudden death. Congenital LQTS, which implicates at least six chromosomal loci, LQT1 to LQT6, three of them corresponding to mutations concerning the coding of K+ channel proteins, give useful information about the mechanism underlying the arrhythmia. One of the potassium channel genes implicated in congenital LQTS is HERG, which encodes the IKr current channel protein. This current has provided a relevant insight into the occurrence of drug-acquired LQTS, since all drugs associated with torsades, such as erythromycin, terfenadine, haloperidol, or cisapride, also block IKr.

Heart↗
PubMedDate not supplied

Effects of calcium channel blockers on cloned cardiac K+ channels IKr and IKs.

Cloned HERG and KvLQT1-IsK K+ channels have been expressed in mammalian cells and assayed as a target for calcium channel blockers. These channels generate the rapid and slow components of the cardiac delayed rectifier K+ current, and mutations can affect them that lead to long QT syndromes. HERG is blocked by bepridil (EC50 = 0.55 microM), verapamil (EC50 = 0.83 microM) and mibefradil (EC50 = 1.43 microM), whereas nitrendipine and diltiazem have negligible effects. Steady-state activation and inactivation parameters are shifted to more negative values in the presence of the blockers. Similarly, KvLQT1-IsK is inhibited by bepridil (EC50 = 10.0 microM) and mibefradil (EC50 = 11.8 microM), whilst being insensitive to nitrendipine, diltiazem or verapamil. This work may help to understand the mechanisms of action of verapamil in certain ventricular tachycardias as well as some of the deleterious adverse cardiac events associated with bepridil and mibefradil.

Animals↗