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Phoneutria nigriventer Toxin 1: A Novel, State-Dependent Inhibitor of Neuronal Sodium Channels That Interacts with µ Conotoxin Binding Sites

Institut National de la Santé et de la Recherche Médicale U641 (N.M.-M., G.A., M.S., C.V.R.), Centre National de la Recherche Scientifique Formation de Recherche en Evolution 2738 (P.M.), and Université de la Méditerranée, Institut Fédératif de Recherches 11 (N.M.-M., P.M., G.A., M.S., C.V.R.), Marseille, France; and Laboratório de Radiobiologia, Comissão Nacional de Energia Nuclear–CNEN/CDTN (R.G.D.S.), Fundação Ezequiel Dias (M.N.C.), and Departamento de Bioquímica e Imunologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais (M.E.D.L.), Belo Horizonte, Brazil

A toxin was purified to homogeneity from the venom of the South American armed spider Phoneutria nigriventer and found to have a molecular mass of 8600 Da and a C-terminally amidated glycine residue. It appears to be identical to Toxin 1 (Tx1) isolated previously from this venom. Tx1 reversibly inhibited sodium currents in Chinese hamster ovary cells expressing recombinant sodium (Na_v1.2) channels without affecting their fast biophysical properties. The kinetics of inhibition of peak sodium current varied with membrane potential, with on-rates increasing and off-rates decreasing with more depolarized holding potentials in the –100 to –50 mV range. Thus, the apparent affinity of Tx1 for the channel increases as the membrane is depolarized. A mono[¹²⁵I]iodo-Tx1 derivative displayed high-affinity binding to a single class of sites (K_D = 80 pM, B_max = 0.43 pmol/mg protein) in rat brain membranes. Solubilized binding sites were immunoprecipitated by antibodies directed against a conserved motif in sodium channel subunits. ¹²⁵I-Tx1 binding was competitively displaced by µ conotoxin GIIIB (IC₅₀ = 0.5 µM) but not by 1 µM tetrodotoxin. However, the inhibition of ¹²⁵I-Tx1 binding by µ conotoxin GIIIB was abrogated in the presence of tetrodotoxin (1 µM). Patch-clamp and binding data indicate that P. nigriventer Tx1 is a novel, state-dependent sodium-channel blocker that binds to a site in proximity to pharmacological site 1, overlapping µ conotoxin but not tetrodotoxin binding sites.

Address correspondence to: Dr. C. Van Renterghem, INSERM U 641, Faculté de Médecine Secteur Nord, Bd Pierre Dramard, F-13916 Marseille, cedex 20, France. E-mail: vanr.c{at}jean-roche.univ-mrs.fr