ELECTROPHYSIOLOGICAL STUDIES OF NEPHILA CLAVATA VENOM AND TOXIN INFLUENCE ON HIPPOCAMPAL NEURONAL MEMBRANES AFTER PRETREATMENT WITH ASPERGILLUS ORYZAE PROTEASES

Abstract

Background. The use of Arthropodae toxins for electrophysiological experiments is very important, but experimental data in this regard are scarce. The aim of this study was to obtain experimental data about the influence of Nephila clavata spider venom and its main active component – toxin JSTX-3 on the glutamate channel-receptor complex.
Methods. Kainate was used as agonist of glutamate channel-receptor complex because it initiated non-inactivated transmembrane electric currents in rat hippocampal membranes in electrophysiological experiments were used for the study. All chemicals were applied to perfused hippocampal pyramidal neuronal membranes using ’concentration-clamp’ technique and voltage-clamp recording.
Results and discussion. The studied substances – integral venom and toxin JSTX-3 – demonstrated the properties of glutamate channel-receptor complex antagonists. The amplitudes of electric transmembrane currents activated by glutamate, kaina­te, and quisqualate decreased (sometimes to zero) after the application of glutamate channel-receptor complex antagonists to the rat hippocampal membrane under the voltage-clamp conditions. The kinetics of activation and desensitization (in case of glutamate and quisqualate) of transmembrane electric currents were not affected by these antagonists.
The effects of Nephila clavata integral venom were studied the in concentrations of 10^-8–10^-4 units/µL, the effects of JSTX-3 – in the concentrations of 10^-6–10^-5 mol/L. Integral venom did not block the studied currents completely, but it reduced their amplitudes to a certain level. Integral venom blocked glutamate-activated currents up to 36±15 % of the initial values, kainate-activated – up to 34±16 %. In contrast, JSTX-3 almost completely blocked ion currents activated by these agonists at the holding potential of -100 mV: the amplitudes of kainate-activated currents under the action of this blocker decreased to 6±3 % of the initial values. Integral venom blocking effects were irreversible in contrast to partially reversible JSTX-3 action. The differences between antagonists were also revealed in the quantitative characteristics of blocking action.
The following effects were studied under the antagonists’ influence: the degree of currents suppression and their removal by “washing”, “dose–effect” dependency, the antagonists’ influence on activated and inactivated receptors; kinetics of the antagonists’ action and removal, dissociation constants for blockers with reversible action.
Conclusions about the mechanisms of the antagonists’ influence on the glutamate channel-receptor complex, the physiological role of integral venom and JSTX-3 as well as comparison of the caused effects were made. The influence of Aspergillus oryzae proteases pretreatment during hippocampal neurons preparation for experiments, and the creation of a physical model of the molecular system “glutamate channel-receptor complex – glutamate – antagonist – proteases” are discussed.