Promiscuous and Reversible Blocker of Presynaptic Calcium Channels in Frog and Crayfish Neuromuscular Junctions FromPhoneutria nigriventerSpider Venom

Abstract

Peptide channel blockers found in venoms of many predators are useful pharmacological tools and potential therapeutic agents. The venom of the Brazilian spider Phoneutria nigriventer contains a fraction, ω-phonetoxin-IIA (ω-Ptx-IIA, 8360 MW), which blocks Ca2+channels. At frog neuromuscular junctions (NMJ) bathed in normal Ca2+(1.8 mM) saline, ω-Ptx IIA did not affect spontaneous transmitter release but reversibly reduced evoked transmitter release by 75 and 95% at 12 and 24 nM, respectively. In contrast, toxin effects were irreversible in low-Ca2+(0.5 mM) saline. Ca2+imaging in normal-Ca2+saline showed that ω-Ptx-IIA partially blocked stimulus-dependent presynaptic Ca2+signals, and the blockade was almost completely reversible. Increases in spontaneous release frequency induced by high extracellular K+were blocked by ω-Ptx-IIA. Therefore ω-Ptx-IIA blocks N-type Ca2+channels, which admit Ca2+that triggers transmitter release at the frog NMJ. Additional evidence predicts that ω-Ptx-IIA binds to N-type Ca2+channels at a different site from that of ω-Conotoxin-GVIA. ω-Ptx-IIA also gave a low-affinity partial blockade of transmitter release and presynaptic Ca2+signals at crayfish NMJs where P-type channels are blocked by ω-agatoxin-IVA. The Ca2+-dependent reversibility and promiscuity of this toxin may make it highly useful experimentally and therapeutically.