Characterization of cholera toxin B subunit‐induced Ca 2+ influx in neuroblastoma cells: Evidence for a voltage‐independent GM1 ganglioside‐associated Ca 2+ channel

The role of endogenous GM1 ganglioside in neurite outgrowth has been studied in N18 and NG108‐15 neuroblastoma cells with the GM1‐specific ligand cholera toxin B subunit (Ctx B), which stimulates Ca 2+ influx together with neuritogenesis. Our primary goal has been to identify the nature of the calcium channel that is modulated by GM1. An L‐type voltage‐operated Ca 2+ channel (VOCC) was previously proposed as the mediator of this phenomenon. This investigation, employing fura‐2 fluorescent measurements and specific channel blockers and other agents, revealed that GM1 modulates a hitherto unidentified Ca 2+ channel not of the L type. It was opened by Ctx B; was permeable to Ca 2+ and Ba 2+ but not Mn 2+ ; and was blocked by Ni 2+ , Cd 2+ , and La 3+ . Although most dihydropyridines inhibited Ctx B‐induced Ca 2+ influx as well as neurite outgrowth at higher concentrations, they and other VOCC blockers at normally employed concentrations failed to do so, suggesting uninvolvement of VOCC. In addition, Ca 2+ influx induced by Ctx B was not mediated by cGMP‐dependent or G‐protein‐coupled nonselective cation channels, as demonstrated by the cGMP antagonist Rp‐cGMPS or the G‐protein/receptor uncoupling agent suramin, respectively. Finally, Ca 2+ influx was unlikely to be due to inhibition or reversal of Na + ‐Ca 2+ exchanger via Ctx B induction of Na + uptake, insofar as no effect was seen on blocking Na + channels, inhibiting Na + ‐K + ‐ATPase, or eliminating extracellular Na + . The results suggest that this novel channel is gated by interaction with GM1, which, when associated with the channel and bound by appropriate ligand, promotes Ca 2+ influx. This in turn induces signaling for the onset of neuritogenesis. © 2002 Wiley‐Liss, Inc.