Exposure of isolated mouse hemidiaphragms to
botulinum neurotoxins, 0.1 nM
BoNT-A or BoNT-B, at 36 degrees C reduced nerve-elicited peak isometric twitch tension to 50% of control values at 55 min (
BoNT-A) to 68 min (BoNT-B) after application. Either coincubation of BoNT with the
heavy metal chelator TPEN, preincubation with
TPEN followed by BoNT, or application of
TPEN after BoNT but before neuromuscular block, delayed the onset of muscle failure in a dose-dependent manner by up to five-fold.
TPEN doses between 2 and 10 microM were required to antagonize significantly the muscle block produced by BoNT, and the delay in onset was maximal between 10 and 50 microM
TPEN. Treatment of muscles with a Zn(2+)-
TPEN coordination complex, rather than
TPEN alone, eliminated any beneficial effects of
TPEN on BoNT intoxication, indicating that these effects were mediated by chelation of Zn2+. Other
metal chelators that were not as membrane permeant as
TPEN were ineffective in delaying BoNT
paralysis, suggesting that
TPEN acts by chelating intraterminal Zn2+. In the absence of BoNT,
TPEN caused a dose-dependent increase in nerve-elicited twitch tension with a half-maximal concentration at 8 microM. There was no corresponding change in twitches from direct electrical stimulation of the muscle. After
BoNT (A or B serotype) had reduced the muscle twitch by 20 to 70%, however, subsequent application of
TPEN rapidly depressed nerve-elicited twitches. The shift from potentiation to depression after BoNT treatment suggests that presynaptic vesicle mobilization and/or release involve Zn(2+)-dependent
enzymes and that BoNTs interact with these
enzyme pathways.