Biophysical characterization of the role of synthesized toxins from Pamphobeteus verdolaga spider on voltage-gated calcium and potassium channels by the electrophysiological patch-clamp technique

Abstract

ABSTRACT: Six peptide toxins, vrdg66, vrdg69, vrdg164, vrdg172, vrdg177 and vrdg183, have been isolated and purified from the venom gland of the spider Pamphobeteus verdolaga. Five of them have disulfide bridged peptides (DBP) and vrdg66 does not (NDBP). However, vrdg66 has the higher percentage of bacterial growth inhibition. Combining different techniques, we have investigated the electrophysiological properties of these toxins. We examined the effect of vrdg66, vrdg69, vrdg164, vrdg172, vrdg177 and vrdg183, on cardiomyocytes murine cells. These toxins modify the amplitude and gating of ion currents, giving a hypothesis that some peptides could be pore blockers (PBs) (that bind to the extracellular side of the channel pore modifying the amplitude) and others could be gating modifiers (GMs), binding to the voltage-sensing domains (VSD). The effects on Ca+ and K+ currents in cardiomyocytes were studied using the patch-clamp technique in whole-cell recording configuration, voltage-clamp mode. vrdg183 decrease the current density with EC50 ∼ 0,76 μM (66.67% reduction in maximum amplitude at Cav with 1 μM). vrdg1v64 (10μM) slow the inactivation process of Ca+ currents, shift the steady-state activation and inactivation parameters to more positive potentials. vrdg172 (1 μM) mainly inhibited peak current at Kv over Cav channels. vrdg66, vrdg69 and vrdg177 (1 μM) modulate both Kv and Cav. vrdg6 had the smallest current inhibition percentage, which shows that in NDBP, the modulation of ion channels is poor. The amino acid sequences of vrdg172, vrdg177, vrdg183 are almost identical except for one or two amino acids (data not shown). In all experiments, vrdg183 was more potent than others suggesting that its conservative residues are important for the toxicity of this Pamphobeteus verdolaga toxin. The development of novel venom-peptide-based therapeutics or biomolecule requires an understanding of their effects on voltage-gated ion channels. We conclude that vrdg183 and vrdg164 are cardiotoxic and combine the classical effects of spider toxins (slowing of inactivation kinetics, shift of steady-state activation and inactivation parameters) with a striking decrease on the ionic selectivity of Ca+ channels.