Presynaptic protein interactions in vivo: evidence from botulinum A, C, D and E action at frog neuromuscular junction.
The present study examines the paralytic action of botulinum neurotoxins at their natural target, the neuromuscular junction. We asked whether syntaxin, synaptosome-associated protein of 25 kDa (SNAP-25) and vesicle-associated membrane protein (VAMP/synaptobrevin), the proteins proteolysed by botulinum, are susceptible to cleavage in frog nerve terminals, and whether they form complexes in vivo. In control terminals, the three SNAREs were distributed in broad bands at 1 micrometer intervals, at sites consistent with presynaptic Ca2+ channels. Within 3 h, botulinum A, C, D and E (BoNT/A/C/D/E) blocked nerve-evoked muscle contractions but their effects on substrate immunoreactivity varied. The effect of BoNT/A on either C-terminus or N-terminus immunoreactivity of SNAP-25 was undetectable after 3-h incubation, although C-terminus immunoreactivity was reduced after 24 h; N-terminus immunoreactivity was not affected even after 36 h. BoNT/E reduced C-terminus immunoreactivity of SNAP-25 1.5 h after toxin application when transmitter release was blocked, but required 24 h to reduce N-terminus immunoreactivity. BoNT/C reduced syntaxin immunoreactivity after 24-h incubation but did not affect SNAP-25. BoNT/D reduced VAMP immunoreactivity at 3 h while it increased SNAP-25 C-terminal staining fourfold. BoNT/A and BoNT/C applied together for 24 h reduced syntaxin immunoreactivity and that of both C- and N-terminus of SNAP-25, indicating that retention of SNAP-25 N-terminus after cleavage by BoNT/A depended on intact syntaxin. Therefore, we infer that SNAP-25 interacts with VAMP and with syntaxin in vivo. Neurotoxin action abolished only 40-60% of SNAP-25, VAMP or syntaxin immunoreactivity suggesting that distinct pools of these proteins, not immediately involved in triggered exocytosis, are resistant to proteolysis.
University of Toronto, Department of Physiology, Medical Sciences Building, Toronto, Ontario, Canada, M5S 1A8.
D A
DA
Raciborska
W S
WS
Trimble
M P
MP
Charlton
D000818:Animals; D001905:Botulinum Toxins / Q000494:pharmacology*; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000187:drug effects / Q000378:metabolism; D009469:Neuromuscular Junction / Q000187:drug effects* / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism; D017729:Presynaptic Terminals / Q000187:drug effects* / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D011894:Rana pipiens; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D013997:Time Factors; D033921:Vesicular Transport Proteins
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D001905:Botulinum Toxins