Phoneutria nigriventer Toxin 1: A Novel, State-Dependent Inhibitor of Neuronal Sodium Channels That Interacts with μ Conotoxin Binding Sites
2006
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 α subunits. 125 I-Tx1 binding was competitively displaced by μ conotoxin GIIIB (IC 50 = 0.5 μM) but not by 1 μM tetrodotoxin. However, the inhibition of 125 I-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.
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