ABSTRACT: The mechanism of the action of β-bungarotoxin (β-BuTx) in the facilitation of spontaneous transmitter release at neuromuscular synapse was investigated in Xenopus cell culture using whole-cell patch clamp recording. Exposure of the culture to β-BuTx dose-dependently enhances the frequency of spontaneous synaptic currents (SSCs). Buffering the rise of intracellular Ca with BAPTA-AM hampered the facilitation of SSC frequency induced by β-BuTx. The β-BuTx-enhanced SSC frequency was reduced when the pharmacological Ca-ATPase inhibitor thapsigargin was used to deplete intracellular Ca store. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate (IP3) but not ryanodine receptors effectively occluded the increase of SSC frequency elicited by β-BuTx. Treating cells with either wortmannin or LY294002, two structurally different inhibitors of phosphatidylinositol 3-kinase (PI3K) and with phospholipase C (PLC) inhibitor U73122, abolished the β-BuTx-induced facilitation of synaptic transmission. The β-BuTx-induced synaptic facilitation was completely abolished while there was presynaptic loading of the motoneuron with GDPβS, a non-hydrolyzable GDP analogue and inhibitor of G protein. Taken collectively, these results suggest that β-BuTx elicits Ca release from the IP3 sensitive intracellular Ca stores of the presynaptic nerve terminal. This is done via PI3K/PLC signaling cascades and G protein activation, leading to an enhancement of spontaneous transmitter release.