Botulinum neurotoxin A blocks synaptic vesicle exocytosis but not endocytosis at the nerve terminal.
The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K(+)-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K(+) depolarization, in the presence of Ca(2+), triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A-blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca(2+) is required for synaptic vesicle membrane retrieval.
Neale
E A
EA
Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA. eneale@codon.nih.gov
Bowers
L M
LM
Jia
M
M
Bateman
K E
KE
Williamson
L C
LC
D000818:Animals; D019274:Botulinum Toxins, Type A / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D004705:Endocytosis / Q000187:drug effects*; D005089:Exocytosis / Q000187:drug effects*; D005260:Female; D005998:Glycine / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000032:analysis; D008666:Metalloendopeptidases / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000032:analysis; D009474:Neurons / Q000166:cytology / Q000187:drug effects; D011188:Potassium / Q000494:pharmacology; D017729:Presynaptic Terminals / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D011726:Pyridinium Compounds / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D050683:R-SNARE Proteins; D013116:Spinal Cord / Q000166:cytology / Q000196:embryology; D013572:Synaptic Vesicles / Q000187:drug effects* / Q000378:metabolism* / Q000648:ultrastructure; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin / Q000494:pharmacology; D013779:Tetrodotoxin / Q000494:pharmacology
C073804:FM1 43; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D050683:R-SNARE Proteins; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin; C035979:tetanospasmin; D013779:Tetrodotoxin; D006735:Horseradish Peroxidase; D008666:Metalloendopeptidases; D019274:Botulinum Toxins, Type A; D011188:Potassium; D002118:Calcium; D005998:Glycine