Reversal of BoNT/A-mediated inhibition of muscle paralysis by 3,4-diaminopyridine and roscovitine in mouse phrenic nerve-hemidiaphragm preparations.

Botulinum neurotoxins (BoNTs) comprise a family of neurotoxic proteins synthesized by anaerobic bacteria of the genus Clostridium. Each neurotoxin consists of two polypeptide chains: a 100kDa heavy chain, responsible for binding and internalization into the nerve terminal of cholinergic motoneurons and a 50kDa light chain that mediates cleavage of specific synaptic proteins in the host nerve terminal. Exposure to BoNT leads to cessation of voltage- and Ca(2+)-dependent acetylcholine (ACh) release, resulting in flaccid paralysis which may be protracted and potentially fatal. There are no approved therapies for BoNT intoxication once symptoms appear, and specific inhibitors of the light chain developed to date have not been able to reverse the consequences of BoNT intoxication. An alternative approach for treatment of botulism is to focus on compounds that act by enhancing ACh release. To this end, we examined the action of the K(+) channel blocker 3,4-diaminopyridine (3,4-DAP) in isolated mouse hemidiaphragm muscles intoxicated with 5pM BoNT/A. 3,4-DAP restored tension within 1-3min of application, and was effective even in totally paralyzed muscle. The Ca(2+) channel activator (R)-roscovitine (Ros) potentiated the action of 3,4-DAP, allowing for use of lower concentrations of the K(+) channel blocker. In the absence of 3,4-DAP, Ros was unable to augment tension in BoNT/A-intoxicated muscle. This is the first report demonstrating the efficacy of the combination of 3,4-DAP and Ros for the potential treatment of BoNT/A-mediated muscle paralysis.