PHARMACOLOGICAL STUDIES OF TOXINS ISOLATED FROM THE VENOM OF THE SPIDER Phoneutria nigriventer

LIMA, M.E.(1), SANTOS, R.G.(1), FIGUEIREDO, S.G.(2), CORDEIRO, M.N.(3), RICHARDSON, M.(3), PELHATE, M.(4), SEAGAR, M.(5), DINIZ, C.R.(1,3)

(1)Laboratório de Venenos e Toxinas Animais, Departamento de Bioquímica e Imunologia, ICB.UFMG, (2)Departamento de Ciências Fisiológicas, UFES, (3)Fundação Ezequiel Dias, BH-MG, (4)Université D’Angers, France, (5)Université de laMediterranée, France.

The P. nigriventer venom contains many toxins acting on ionic channels. We examined the pharmacological properties of different toxins purified from this venom, including TX4(6-1) with insecticidal activity and w-PhonetoxinIIA which is active on mice. The experimental approach included binding experiments, with the radiolabeled derivatives of toxins, on cockroach (Periplaneta americana) nerve cord synaptosomes, for insect-toxin and both rat brain synaptosomes and BHK cell line membranes, for mammal-toxins. Electrophysiological recordings on cockroach isolated giant axon under current-clamp and voltage-clamp conditions were performed using the double oil-gap single fiber technique. Electrophysiological whole cell currents were also recorded from isolated stable BHK cells expressing Cav2.1, Cav2.2 and Cav2.3 channels (P/Q, N and R-type currents, respectively). Tx4(6-1) is ineffective on mammalian Na_v channels and competes with the a-like toxin ¹²⁵I-Bom IV from scorpion Buthus occitanusmardochei, for binding on the site 3 of insect Na_v channel (IC₅₀~25 nM). Physiological consequences of this binding to the insect Na_v channel were shown by electrophysiology: TX4(6-1) prolongs evoked axonal action potentials due to a slowing down of sodium current inactivation. w-Phone IIA inhibits high threshold voltage-dependent calcium currents in neurons. Calcium currents generated by Ca_v2.1, Ca_v2.2  wereblocked almost irreversibly by 3 nMwPhone IIA, whereas Ca_v2.3 showed partial and readily reversible inhibition. Binding assays with mono [¹²⁵I-wPhone IIA] indicated that membranes expressing recombinant Ca_v2.1 or Ca_v2.2 channels showed a single class of sites with similar affinity (KD~50 pM) whereas low affintiy interactions were detectable with Ca_v2.3. Binding assays demonstrated that rat brain synaptosomes display multiple classes of binding sites for ¹²⁵I-wPhone IIA. High affinity binding of ¹²⁵I-wPhone IIA was totally displaced by wPhone IIA (Ki=100 pM), but only partially by w-conotoxinGVIA (25% inhibition) and w-conotoxinMVIIC(50% inhibition at 0.3 mM). TX4(6-1) and wPhone IIA, among other Phoneutria toxins, exemplify the complex action of this venom on different ionic channels, in both mammals and insects.

Supported by CNPq, CAPES/COFECUB and FAPEMIG. 

CORRESPONDENCE TO:

Maria Elena de Lima Perez Garcia, Avenida Cel. José Dias Bicalho, 516, apto. 101, Belo Horizonte, MG, CEP: 31.275.050, Brasil, E-mail: delima@icb.ufmg.br