Bafilomycin A1 inhibits the action of tetanus toxin in spinal cord neurons in cell culture.
Tetanus toxin (TeNT) is one of the clostridial neurotoxins that act intracellularly to block neurotransmitter release. However, neither the route of entry nor the mechanism by which these toxins gain access to the neuronal cytoplasm has been established definitively. In murine spinal cord cell cultures, release of the neurotransmitter glycine is particularly sensitive to blockade by TeNT. To test whether TeNT enters neurons through acidic endosomes or is routed through the Golgi apparatus, toxin action on potassium-evoked glycine release was assayed in cultures pretreated with bafilomycin A1 (baf A1) or brefeldin A (BFA). baf A1, which inhibits the vacuolar-type H(+)-ATPase responsible for endosome acidification, diminishes the staining of acidic compartments and interferes with the action of TeNT in a dose-dependent manner. TeNT blockade of evoked glycine release is inhibited by 50 and 90% in cultures pretreated with 50 and 100 nM baf A1, respectively, compared with cultures treated with the inhibitor alone. The effects of baf A1 are fully reversible. In contrast, BFA, which disrupts Golgi function, has no effect on TeNT action. These findings provide evidence that TeNT enters the neuronal cytoplasm through baf A1-sensitive acidic compartments and that TeNT is not trafficked through the Golgi apparatus before its translocation into the neuronal cytosol.
Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
L C
LC
Williamson
E A
EA
Neale
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004136:Dinitrobenzenes / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D018942:Macrolides; D051379:Mice; D008985:Monensin / Q000494:pharmacology; D009474:Neurons / Q000502:physiology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013116:Spinal Cord / Q000502:physiology*; D013569:Synapses / Q000502:physiology; D013744:Tetanus Toxin / Q000037:antagonists & inhibitors*
D000900:Anti-Bacterial Agents; D004136:Dinitrobenzenes; D007202:Indicators and Reagents; D018942:Macrolides; D013744:Tetanus Toxin; C040929:bafilomycin A1; D008985:Monensin; C042432:3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine; D000254:Sodium-Potassium-Exchanging ATPase; D005998:Glycine