ENDOGENOUS GANGLIOSIDE GM1 MODULATES L-TYPE CALCIUM-CHANNEL ACTIVITY IN N18 NEUROBLASTOMA-CELLS

Digital imaging fluorescence microscopy was used to investigate the ef fect of the B subunit of cholera toxin on calcium homeostasis in neuro blastoma N18 cells. The B subunit, which binds specifically to ganglio side GM1 in the outer leaflet of the cell membrane, was found to induc e a sustained increase of intracellular calcium concentration ([Ca2+]( i)). The increase in [Ca2+](i) was not observed in the absence of extr acellular calcium, or in the presence of the calcium chelator EGTA, an d was blocked by nickel. The B subunit was also found to induce an inf lux of manganese ions, as indicated by a quench of the intracellular f ura-2 fluorescence. These data suggest that the B subunit induces an i ncrease in calcium influx in N18 cells. Potassium-induced depolarizati on also stimulated manganese influx; however, after the onset of depol arization-induced influx, the B subunit had no further effect. This oc clusion suggests involvement of voltage-dependent calcium channels. Tr eatment with BayK8644, a dihydropyridine agonist selective for L-type calcium channels, induced manganese influx that was not altered by the B subunit and apparently blocked the effect of the B subunit itself. Furthermore, the dihydropyridine L-type channel antagonists niguldipin e or nicardipine completely inhibited B subunit-induced manganese infl ux. Thus, the B subunit-induced manganese influx is likely due to acti vation of an L-type voltage-dependent calcium channel. Spontaneous inf lux of manganese ions was also inhibited by nicardipine or niguldipine and by exogenous gangliosides. Ganglioside GM1 was more potent than G M3, but globoside had no significant effect. The modulation of L-type calcium channels by endogenous ganglioside GM1 has important implicati ons for its role in neural development, differentiation, and regenerat ion and also for its potential function in the electrical excitability of neurons.