The neurotoxic phospholipase A(2), beta-bungarotoxin (beta-BuTx), is a component of the snake venom from the Taiwanese banded krait Bungarus multicinctus. beta-BuTx affects presynaptic nerve terminal function of the neuromuscular junction and induces widespread neuronal cell death throughout the mammalian and avian CNS. To analyse the initial events of beta-BuTx-mediated cell death, the toxin was applied to cultured rat hippocampal neurons where it induced neuronal cell death in a concentration-dependent manner (EC(50) approximately equal to 5 x 10(-13) M) within 24 h. Fluorescence labelled beta-BuTx was completely incorporated by neurons within < 10 min. Binding and uptake of beta-BuTx, as well as induction of cell death, were efficiently antagonized by preincubation with dendrotoxin I, a blocker of voltage-gated potassium channels devoid of phospholipase activity. Binding of beta-BuTx was selective for neurofilament-positive cells. As evident from intense annexin-V and TUNEL stainings, application of beta-BuTx induced apoptotic cell death exclusively in neurons, leaving astrocytes unaffected. No evidence was obtained for any contribution of either caspases or calpains to beta-BuTx-induced apoptosis, consistent with the inability of the inhibitors Z-Asp-DCB and calpeptin, respectively, to protect neurons from beta-BuTx-induced cell death. These observations indicate that induction of cell death by beta-BuTx comprises several successive phases: (i) binding to neuronal potassium channels is the initial event, followed by (ii) internalization and (iii) induction of apoptotic cell death via a caspase-independent pathway.