Poster 141 - Tx1, a toxin from the venom of the spider Phoneutria Nigriventer, blocks L-type calcium

Poster 141. Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil. Abstracts... J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.500.

Tx1, A Toxin From The Venom Of The Spider Phoneutria Nigriventer, Blocks L-Type Calcium Channels In GH3 Cells.

¹Lomeo, R.; ⁴Gouvêa dos Santos, R.; ³Cruz, J.S.; ³Mafra, R.; ⁴Soares, M.A.; ⁵Cordeiro M.N.; ^1,2Pimenta, A.M.C. and ^1,2De Lima, M.E.

1 Lab. Venenos e Toxinas Animais, 2 Núcleo de Biomoléculas, 3 Lab.Membranas Excitáveis - Depto.Bioquímica e Imunologia - ICB, UFMG, Belo Horizonte; 4 Lab Radiobiologia, TR4, CDTN/CNEN, Belo Horizonte; 5 Centro de Pesquisa Prof.Carlos Ribeiro Diniz - FUNED, Belo Horizonte – MG, Brasil.

Clonal GH3 cell lines are derived from a primary culture from pituitary tumor cells. Previous reports have demonstrated that this cell line exhibits voltage-dependent K⁺, Ca²⁺ and large conductance Ca²⁺-activated K⁺ channels (BKCa). The venom of the South American spider Phoneutria nigriventer has many neurotoxic peptides which act on neuronal ion channels. Tx1, one of these toxins, induces tail elevation, ileum contraction, excitation and spastic paralysis of the hind legs. Although we have shown that ¹²⁵I-Tx1 binds to myenteric plexus-longitudinal muscle membranes preparation from guinea pig ileum with high affinity, the exact target of Tx1 is still unknown. The aim of this work was to shed more light into the action mechanism of Tx1 using GH3 cells as a model for binding and eletrophysiological assays.

Tx1 highly purified was radiolabelled with ¹²⁵I and used as a probe for binding assays. ¹²⁵I-Tx1 bound to specific sites on GH3 membranes (~60% specific binding). Native Tx1 competed with the ¹²⁵I-Tx1 bound to the specific sites in a dose-dependent manner (IC50 ~ 0.2 nM). Nifedipine (1nM), a dihidropiridine, competed with ~20% of the ¹²⁵I-Tx1 binding sites. In order to address the Tx1 target identity ³H-PN200-110 was used as a specific L-type calcium channel radiotracer. Native Tx1 (~10nM) inhibited 40% of the specific interaction of ³H-PN200-110 with L-type calcium channels. In eletrophysiological assays Tx1 (~1mM) inhibited 50% of the L-type calcium current endogenously expressed in GH3 cells.

In this work we showed that GH3 cells have specific sites for ¹²⁵I-Tx1 which are sensitive to nifedipine. It has been shown that up to 80% of the calcium influx in these cells is attributed to be due to L-type calcium channels. The reverse competition assay with ³H-PN200-110 and eletrophysiological data confirmed that L-type calcium channels are target sites for this toxin. To summarize, Tx1 binds to specific sites on GH3 cells and this interaction results in L-type calcium channel blockade.