The new vrdg183 toxin from the Colombian spider Pamphobeteus verdolaga inhibits L-type Ca2+ currents through an allosteric mechanism

Spider venoms are enriched in toxins that modulate mammalian voltage-gated ion channels (VGIC) with pharmacological potential. However, the inventory of toxins of American spiders with effects on voltage-gated Ca 2+ channels has lagged the number of toxins interacting with other channels. Here, we aimed at identifying new Ca 2+ channel-modulating toxins of the Colombian spider Pamphobeteus verdolaga . The sequences of five novel short, positively charged, disulfide-bridged peptides, and one more short, non-bridged control peptide, were selected from the transcriptomic database of the venom gland of P. verdolaga . Peptides were synthesized and screened at 1 μM for their effects on L-type Ca 2+ currents (I CaL ) in mouse cardiomyocytes using patch-clamp. I K were also evaluated to gain information about the selectivity of the peptides. 3D modeling, docking and molecular dynamics simulations were likewise performed. vrdg 183 (19 amino acid residues, four cysteines, charge +7, 2.33 kDa), followed by vrdg 69 and vrdg 177, inhibited over 60% of I CaL . vrdg 172 and vrdg 164 showed a moderate effect of less than 40%. vrdg 183 had a half-maximal inhibitory concentration for I CaL of 858.28 nM and was the only toxin lacking effect on I K . vrdg 183, consisting of two antiparallel β-strands, was successfully docked to the extracellular L6 IV loop at the outer pore region of the human cardiac Ca v 1.2 with a binding energy of -9.5 kJ/mol. Conclusion : This is the first report of toxins of P. verdolaga with effects on VGIC. vrdg 183 highlights as a very low molecular weight, moderately potent I CaL inhibitor through an allosteric mechanism, without effect on I K . • Five novel toxins of P. verdolaga were synthesized and tested against I CaL and I K • vrdg 69, vrdg 164, vrdg 172 and vrdg 177 unspecifically inhibited I CaL and I K • vrdg 183 (19 aa, 2.33 KDa) specifically inhibited I CaL with an IC 50 of 858.28 nM • Molecular docking identified the binding site of vrdg 183 to the human Ca v 1.2 • We report the first P. verdolaga toxins with effects on voltage-gated ion channels