Analysis of the structure and electrophysiological actions of halitoxins: 1,3 alkyl-pyridinium salts from Callyspongia ridleyi

We have chemically characterized a preparation of halitoxins, (1,3 alkyl-py ridinium salts) isolated from the marine sponge Callyspongia ridleyi. At co ncentrations of 50 and 5 mu g/ml the halitoxin preparation caused irreversi ble membrane potential depolarization, decreased input resistance and inhib ited evoked action potentials when applied to cultured dorsal root ganglion neurones. Under whale cell voltage clamp the halitoxins produced an increa se in cation conductance that was attenuated by replacing sodium with N-met hyl-D-glucamine. Fura-2 fluorescence ratiometric calcium imaging was used t o directly measure calcium flux into neurones after exposure to halitoxins. Calcium influx, evoked by the halitoxins, persisted when the neurones were bathed in medium containing the voltage-activated calcium channel antagoni sts cadmium and nickel. Experiments on undifferentiated F-11 cells showed l ittle or no calcium influx in response to depolarizing concentrations of po tassium and indicated that halitoxins evoked massive calcium influx in the absence of voltage-activated calcium channels. The halitoxins also produced transient increases in intracellular calcium when F-11 cells were bathed i n calcium-free medium suggesting that the toxins could release calcium from intracellular stores. The port-forming action of the halitoxins was identi fied when the toxins were applied to artificial lipid bilayers composed of phosphatidylcholine and cholesterol. Halitoxins evoked channel-like activit y in the lipid bilayers, with estimated unitary conductances of between 145 pS and 2280pS, possibly indicating that distinct channels could be produced by the different components in the preparation of halitoxins.