Interaction of ganglioside GM1 with the B subunit of cholera toxin modulates intracellular free calcium in sensory neurons

Abstract

The B subunit of cholera toxin, which binds specifically to GM1 ganglioside on cell surfaces, has previously been shown to modulate intracellular calcium levels and growth in several cell types. To explore a role for such changes in calcium in the growth regulatory function of cell-associated GM1 in neurons, dissociated neurons from chicken embryonic day 8 dorsal root ganglia were exposed to the B subunit. To enhance sensitivity to B subunit, some neurons were also enriched with added GM1 (100 microM) and then exposed to B subunit. Incubation of naive cultures with 1 microgram/ml of the B subunit was sufficient to produce modest increases in intracellular free calcium above basal levels in a minor percentage of cells for at least 5 min, as measured by fura-2 fluorescence imaging. Pretreatment of the cells with GM1 for 48 hr increased even further the elevations in intracellular free calcium and the percentage of responding neurons observed after B subunit exposure. These increases in intracellular calcium required the presence of external Ca2+, but were not inhibited by calcium channel blockers. Such changes in calcium were accompanied by fine alterations in morphology affecting mostly the branching of neurites and were more pronounced in the presence of GM1. However, the morphological changes did not result in altered neurofilament protein expression. Immunogold electron microscopy using anti-choleragenoid depicted extensive aggregations of immunoreactive gold particles on neuronal surfaces, which were more extensive in cells treated with GM1. The results demonstrate that cell incorporated GM1 may modulate calcium fluxes, perhaps accounting for the growth regulatory functions of GM1 in both neuronal and other cell types.

Induction of Calcium Influx through TRPC5 Channels by Cross-Linking of GM1 Ganglioside Associated with α5β1 Integrin Initiates Neurite Outgrowth

“…The proposal that CtxB induced Ca 2ϩ influx through L-type Ca 2ϩ channels, with GM1 functioning as the constitutive negative modulator (Carlson et al, 1994), was later questioned by the finding that the channel functioned in a voltageindependent manner (Fang et al, 2002). This was consistent with a previous report that CtxB-induced elevation of [Ca 2ϩ ] i in dorsal root ganglia neurons was not inhibited by L-type Ca 2ϩ channel blockers (Milani et al, 1992). The present study indicates the relevant channel is TRPC5, a member of the canonical subfamily belonging to the TRP superfamily of signaltransduction-gated ion channels first discovered in Drosophila (Montell et al, 1985).…”

“…Physiological relevance was further suggested by the fact that CtxB induced [Ca 2ϩ ] i elevation in primary cultures of dorsal root ganglia neurons with alteration of neurite morphology (Milani et al, 1992) and in CGN cultures with enhancement of cell survival (Wu et al, 1996). Anti-GM1 Abs of the cross-linking IgM type were shown to induce similar Ca 2ϩ changes (Quattrini et al, 2001) and neurite outgrowth in neuroblastoma cells (O'Hanlon et al, 2003).…”

“…This property of CtxB was observed in various non-neuronal cells (Dixon et al, 1987;Buckley et al, 1990;Gabellini et al, 1991;Guoy et al, 1994) as well as in some neuronal cell lines (Carlson et al, 1994;Fang et al, 2000) and primary neurons (Milani et al, 1992;Wu et al, 1996). The chief consequence of this induced Ca 2ϩ influx, in the case of N18 and NG108-15 neural cell lines, was outgrowth of axon-like processes (Wu et al, 1998a;Fang, 2000).…”

The multi-tasked life of GM1 ganglioside, a true factotum of nature

“…This crosslinking ligand of GM1 was shown to trigger Ca 2+ influx in N18 cells [36], the response being more robust if preceded by elevation of surface GM1 via sialidase treatment [37]. Similar CtxB effects were observed in primary neurons of both the PNS [38] and CNS [39], but not glial cells [40,41], while cells of the immune system resembled neurons in that regard (see below). Crosslinking of GM1 causes co-crosslinking of proteins associated with GM1 (e.g., integrins) and this in turn induces tyrosine autophosphorylation of an associated kinase with consequent signaling, as described below.…”

Cerebellar neurons lacking complex gangliosides degenerate in the presence of depolarizing levels of potassium

“…Possible existence of such mechanism(s) was previously suggested in the demonstration that GM1 and other gangliotetraose gangliosides protected CGN in culture against glutamate toxicity (28) and Neuro-2a cells subjected to Ca 2ϩ ionophore toxicity (17). Calcium modulatory effects of gangliosides have been observed in several other studies, involving both exogenous (29)(30)(31)(32)(33)(34)(35)(36) and endogenous (18,(37)(38)(39)(40)(41)(42)(43)(44) gangliosides. GM1 has been the predominant endogenous ganglioside to be studied from this standpoint, mainly in the context of its plasma membrane locus.…”