Membrane depolarization in LA-N-1 cells. The effect of maitotoxin is Ca(2+)- and Na(+)-dependent.

Abstract

We investigated the influence of ion compositions on the membrane potential in LA-N-1 human neuroblastoma cells using bisoxonol as a potential-sensitive fluorescent dye. The ability of K+, ouabain, veratridine, and maitotoxin to induce membrane depolarization was evaluated. Increasing concentrations of K+ ions from 10 to 50 mM caused a dose-dependent increase of bisoxonol fluorescence, which was completely independent on Na+ and Ca2+. Ouabain (5 mM), an inhibitor of the Na+, K(+)-ATPase, failed to induce membrane depolarization. Veratridine (40 and 100 microM), a Na+ channel activator, only in the presence of 10 micrograms of Leiurus scorpion venom reduced the membrane potential. Maitotoxin (MTX) from 3 to 10 ng/mL depolarized LA-N-1 cells in a dose-dependent manner, and produced a rapid and sustained increase of intracellular free calcium monitored by means of fluorescent probe fura-2. The MTX-induced depolarization and the increase in cytosolic free calcium concentration were dependent on extracellular Ca2+ ions. On the other hand, Na+ ions also seem to be, although only partially, implicated in the MTX effects, since both the blockade of tetrodotoxin (TTX)-sensitive voltage-operated Na+ channels and the removal of Na+ ions were able to reduce the depolarization. In conclusion, our data indicate that the depolarizing action of MTX on LA-N-1 cells is Ca(2+)- and Na(+)-dependent, although the latter only partially, and that this effect is dependent on Ca2+ influx into the cells likely through a voltage-insensitive calcium-entry system.