[The Biological Activity of the Sevanol and Its Analogues].

Previously, from the plant Thymus armeniacus a new lignan sevanol was isolated, it's structure was elucidated and was shown that it effectively inhibits the acid-sensing channel ASIC3 and also exhibits a pronounced analgesic and anti-inflammatory effect. In this work biological activity of the sevanol analog obtained by chemical synthesis from simple precursors, the stereoisomer of sevanol and a precursor molecule represents a half of sevanol was measured in electrophysiological experiments on human ASIC3 channels expressed in Xenopus laevis oocytes. Measured inhibitory activity of a synthetic analogue coincided with the activity ofthe natural molecule. Stereoisomer showed inhibitory activity drop by about a third part, and the precursor molecule showed much less significant activity. In result the significance of functional groups and a spatial configuration of sevanol in order to biological activity was shown that is important to take into account for the optimal synthesis design as well as for new drugs development on its base.

[Polypeptide toxin from sea anemone inhibiting proton-sensitive channel ASIC3].

Polypeptide toxin pi-AnmTX Hcr 1b-1 with a molecular weight 4537 Da was isolated from the whole body extract of sea anemone by a multistage liquid chromatography. The BLAST search algorithm revealed homology of the novel toxin amino acid sequence to the group of the known sea anemone toxins including BDS and APETx with similarity less then 50%. The toxin pi-AnmTX Hcr 1b-1 inhibited the amplitude of the fast component of integral ASIC3 current in electrophysiological studies on receptors expressed in Xenopus laevis oocytes. The calculated IC50 value was 5.5 +/- 1.0 microM. Among the known polypeptide toxins interacted with ASICs channels, the micro-AnmTX Hcr 1b-1 toxin is the least potent inhibitor that in our opinion correlates with a small amount of charged amino acid residues in its structure.

OsK2, a new selective inhibitor of Kv1.2 potassium channels purified from the venom of the scorpion Orthochirus scrobiculosus.

A novel inhibitor of voltage-gated K(+) channels has been purified to homogeneity from the venom of the black scorpion Orthochirus scrobiculosus. This toxin, named OsK2, has been characterized as a 28-residue peptide, containing six conserved cysteine residues and was shown to be a potent and selective blocker of Kv1.2 channels (K(d) = 97 nM). OsK2 is the second member of the 13th subfamily of short-chain K(+) channel-blocking peptides known thus far and is therefore called alpha-KTx 13.2.

Probing of NMDA channels with fast blockers.

Using whole-cell patch-clamp techniques, we studied the interaction of open NMDA channels with tetraalkylammonium compounds: tetraethylammonium (TEA), tetrapropylammonium (TPA), tetrabutylammonium (TBA), and tetrapentylammonium (TPentA). Analysis of the blocking kinetics, concentration, and agonist dependencies using a set of kinetic models allowed us to create the criteria distinguishing the effects of these blockers on the channel closure, desensitization, and agonist dissociation. Thus, it was found that TPentA prohibited, TBA partly prevented, and TPA and TEA did not prevent either the channel closure or the agonist dissociation. TPentA and TBA prohibited, TPA slightly prevented, and TEA did not affect the channel desensitization. These data along with the voltage dependence of the stationary current inhibition led us to hypothesize that: (1) there are activation and desensitization gates in the NMDA channel; (2) these gates are distinct structures located in the external channel vestibule, the desensitization gate being located deeper than the activation gate. The size of the blocker plays a key role in its interaction with the NMDA channel gating machinery: small blockers (TEA and TPA) bind in the depth of the channel pore and permit the closure of both gates, whereas larger blockers (TBA) allow the closure of the activation gate but prohibit the closure of the desensitization gate; finally, the largest blockers (TPentA) prohibit the closure of both activation and desensitization gates. The mean diameter of the NMDA channel pore in the region of the activation gate localization was estimated to be approximately 11 A.

Molecular size and hydrophobicity as factors which determine the efficacy of the blocking action of amino-adamantane derivatives on NMDA channels.

Neurons isolated from the CA-1 region of rat hippocampal slices by the "vibrodissociation" method were voltage-clamped in the whole cell configuration. The currents through NMDA channels were recorded in response to rapid application (solution exchange time <30 ms) of 100 microM aspartate (ASP) in a Mg2+-free solution in the presence of 3 microM glycine. When added to the ASP solution, amantadine as well as other amino-adamantane derivatives (AAD) produced an open-channel blockade of NMDA channels. Membrane hyperpolarization enhanced the AAD block. The affinity between NMDA channels and AAD was different for various AAD. The analysis of the experimental data led us to conclude that this affinity depended both on the molecular size of the blocker (calculated using HyperChem molecular modeling program) and on the blocker's hydrophobicity (calculated according to Hansch and Leo, 1979). The affinity between NMDA channels and AAD diminished with an increase in molecular size and raised with an increase in blocker's hydrophobicity. We propose an empirical equation which describes the dependence of affinity on the size and hydrophobicity of the blocker. The estimated critical diameter of the NMDA channel pore where the AAD blocking site is located proved to be about 17 A.

Interaction of memantine and amantadine with agonist-unbound NMDA-receptor channels in acutely isolated rat hippocampal neurons.

1. Using whole-cell patch-clamp techniques, the mechanisms of NMDA channel blockade by amino-adamantane derivatives (AADs) memantine (3, 5-dimethyl-aminoadamantane, MEM) and amantadine (1-aminoadamantane, AM) have been studied in rat hippocampal neurons acutely isolated by the vibrodissociation method. A rapid concentration-jump technique was used to replace superfusing solutions. 2. The aspartate (Asp)-induced channel opening greatly accelerated but was not a prerequisite for the recovery from the block by MEM: it was able to leave the channel without agonist assistance. The co-agonist (glycine) as well as the competitive NMDA antagonist DL-2-amino-7-phosphonoheptanoic acid (APV), did not affect this recovery. Membrane depolarization accelerated it, strongly suggesting that this process proceeded via the hydrophilic pathway of the channel. 3. A comparison of the kinetics of the recovery from the block by AADs in the presence and absence of the agonist prompted a hypothesis that the blocker trapped in the channel increased the probability of its transition to the open state. 4. Both MEM and AM were able to block NMDA channels not only in the presence but also in the absence of Asp, although in the latter case the effective blocking concentrations were much higher and the rate of the block development was much smaller than in the former case. The extent of the block increased with the duration of the blocker application. Glycine enhanced this block, while APV attenuated it. The MEM-induced blockade of agonist-unbound channels was enhanced by membrane hyperpolarization and weakened by external Mg2+. These findings strongly suggested that the blocker reached its binding sites via the same hydrophilic pathway both in the presence and absence of the agonist. 5. A comparative analysis of the channel unblocking kinetics in the presence of Asp after their blockade with or without the agonist assistance led us to conclude that in the two cases AADs were bound to the same blocking sites in the channel.

Effect of a prolonged glutamate challenge on plasmalemmal calcium permeability in mammalian central neurones. Mn2+ as a tool to study calcium influx pathways.

The rate of Mn(2+)-induced fluorescence quenching (RFQ) was used as a relative measure of plasma membrane Ca2+ permeability (PCa) in fura-2-loaded cultured hippocampal neurons and cerebellar granule cells during and after protracted (15-30 min) glutamate (GLU) treatment. Some limitations of this method were evaluated using a kinetic model of a competitive binding of Mn2+ and Ca2+ to fura-2 in the cell. In parallel experiment a contribution of Ca2+ influx to the cytoplasmic Ca2+ ([Ca2+]i) was repeatedly examined during and following a prolonged GLU challenge by short-duration "low-Ca2+ trials" (50 microM EGTA) and by measurements of 45Ca2+ uptake. Experiments failed to reveal a putative persistent increase in PCa that earlier was thought to underlie Ca2+ overload of the neuron caused by its toxic GLU treatment. By contrast, a sustained increase of [Ca2+]i was found to be associated with a progressive decrease in PCa and Ca2+ influx both in the period of GLU application and after its termination. These findings give new evidence in favour of the hypothesis that the GLU-induced Ca2+ overload of the neuron mainly from an impairment of its Ca2+ extrusion systems.

[Self stimulation against a background of action of blockers of the synthesis of proteins and oligopeptides]. / Samostimuliatsiia na fone deistviia blokatorov sinteza belkov i oligopeptidov.

The experiments on rabbits have demonstrated that blockade of protein synthesis by the administration of cycloheximide and actinomycin D abolishes self-stimulation in the central nervous system. The treatment with ACTH fragment (ACTH4-10) restored the self-stimulation. Unlike ACTH, injections of pentagastrin, Met-enkephalin, Leu-enkephalin and cholecystokinin were ineffective. The present study shows that ACTH4-10 plays an important role in genetic determination of self-stimulation behaviour.