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, omega-phonetoxin-IIA (omega-Ptx-IIA, 8360 MW), which blocks Ca2+ channels. At frog neuromuscular junctions (NMJ) bathed in normal Ca2+ (1.8 mM) saline, omega-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 omega-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 omega-Ptx-IIA. Therefore omega-Ptx-IIA blocks N-type Ca2+ channels, which admit Ca2+ that triggers transmitter release at the frog NMJ. Additional evidence predicts that omega-Ptx-IIA binds to N-type Ca2+ channels at a different site from that of omega-Conotoxin-GVIA. omega-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 omega-agatoxin-IVA. The Ca2+-dependent reversibility and promiscuity of this toxin may make it highly useful experimentally and therapeutically.

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

“…However, the use of the dextran conjugate reduces dye compartmentalization and handling by the nerve and NMJs. Calcium Green 1-3,000 MW dextran conjugate has a good diffusion rate and demonstrates reduced compartmentalization 38 .…”

Phoneutria nigriventer venom: A pharmacological treasure

“…PnTx3-4 affects the neurotransmission by blocking presynaptic calcium channels associated with exocytosis in mammals, lower vertebrates and arthropods (de Lima et al, 2016;Troncone et al, 2003). PnTx3-4 potently inhibits high-voltage-activated Cav channels in the sensory neurons of dorsal root.…”

Relative Distribution of Ca2+Channels at the Crayfish Inhibitory Neuromuscular Junction

“…Multiple classes of Ca 2ϩ channels are found in many preparations, such as the rat cerebellum (Mintz et al 1995;Regehr and Mintz 1994), calyx of Held (Wu et al 1999), mouse hippocampus (Qian and Noebels 2001), mossy fiber terminals (Castillo et al 1994;Honda et al 2000), crayfish excitor (Troncone et al 2003), and the crab slow and fast closer excitor muscles (Rathmayer et al 2002). Similar to the results presented here, P-type channels found in these preparations control a majority of release (Ͼ80%), despite the fact that non-Ptype channels contribute a sizable fraction of Ca 2ϩ influx (50 -70%).…”