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.
Fang
Yu
Y
Department of Neurosciences, New Jersey Medical School, UMDNJ, Newark, New Jersey 07103, USA.
Xie
Xin
X
Ledeen
Robert W
RW
Wu
Gusheng
G
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002772:Cholera Toxin / Q000494:pharmacology* / Q000633:toxicity; D016257:Fura-2; D005677:G(M1) Ganglioside / Q000378:metabolism; D008012:Lidocaine / Q000494:pharmacology; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology; D013498:Suramin / Q000494:pharmacology / Q000633:toxicity; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D005677:G(M1) Ganglioside; D013498:Suramin; D000255:Adenosine Triphosphate; D002772:Cholera Toxin; D008012:Lidocaine; D002118:Calcium; D016257:Fura-2