Inhibition of neuronal acetylcholine sensitivity by alpha-toxins from Bungarus multicinctus venom

Bungarus multicinctus venom contains several alpha-toxins in addition to the widely used alpha-bungarotoxin (Bgt 2.2). We have found that two of the alpha-toxins (Bgt 3.1 and 3.3) inhibit neuronal acetylcholine (AcCho) sensitivity when tested on ciliary ganglion neurons in cell culture. Over 90% of the AcCho sensitivity recorded in response to iontophoretic application of AcCho was blocked when the neurons were incubated with either of the toxins at 10(-7) M for 1 hr at 37 degrees C. The blockade could be partially reversed by incubating the neurons for 1-2 hr in medium lacking the toxins. The neurons also had a high-affinity binding site for Bgt 2.2, as indicated by binding studies with rhodamine-labeled Bgt 2.2. Concentrations of Bgt 2.2(10(-7) M) that should be nearly adequate to saturate the high-affinity site, however, had no detectable effect on AcCho sensitivity of the neurons. Higher concentrations of Bgt 2.2(10(-5) M) produced a partial inhibition of AcCho sensitivity, suggesting either that the neurons had two classes of binding sites for Bgt 2.2 (with the low-affinity site affecting AcCho sensitivity) or that the preparation of Bgt 2.2 contained minor components (e.g., Bgt 3.1 or 3.3) that were responsible for the blockade. The mechanisms by which Bgt 3.1 and 3.3 inhibit neuronal AcCho sensitivity remain unknown. If they bind specifically to the AcCho receptor, they will be useful agents for studying the distribution and regulation of this membrane component.