INTERACTIONS BETWEEN HEAVY-METAL CHELATORS AND BOTULINUM NEUROTOXINS AT THE MOUSE NEUROMUSCULAR-JUNCTION

Exposure of isolated mouse hemidiaphragms to botulinum neurotoxins, 0. 1 nM BoNT-A or BoNT-B, at 36 degrees C reduced nerve-elicited peak iso metric twitch tension to 50% of control values at 55 min (BoNT-A) to 6 8 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, dela yed the onset of muscle failure in a dose-dependent manner by up to fi ve-fold, TPEN doses between 2 and 10 mu M were required to antagonize significantly the muscle block produced by BoNT, and the delay in onse t was maximal between 10 and 50 mu M TPEN. Treatment of muscles with a Zn2+-TPEN coordination complex, rather than TPEN alone, eliminated an y beneficial effects of TPEN on BoNT intoxication, indicating that the se effects were mediated by chelation of Zn2+. Other metal chelators t hat 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 mu M. There was no corresponding change in twitches from direct electr ical stimulation of the muscle. After BoNT (A or B serotype) had reduc ed the muscle twitch by 20 to 70%, however, subsequent application of TPEN rapidly depressed nerve-elicited twitches. The shift from potenti ation to depression after BoNT treatment suggests that presynaptic ves icle mobilization and/or release involve Zn2+-dependent enzymes and th at BoNTs interact with these enzyme pathways.