Martin-Moutot_2006_Mol.Pharmacol_69_1931

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(v)1.2) 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[(125)I]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 alpha subunits. (125)I-Tx1 binding was competitively displaced by mu conotoxin GIIIB (IC(50) = 0.5 microM) but not by 1 microM tetrodotoxin. However, the inhibition of (125)I-Tx1 binding by mu conotoxin GIIIB was abrogated in the presence of tetrodotoxin (1 microM). 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 mu conotoxin but not tetrodotoxin binding sites.