recombinant atxa , atnl and its quadruple h28y / l31v / n33g / s49d mutant lw were prepared by in vitro refolding following expression in e. coli as described . all other chemicals were from sigma aldrich or gibco life technologies .
adult male balb / c mice ( 2025 g ) were maintained and humanely killed according to the guiding principles in the use of animals in toxicology ( society of toxicology , 1999 , guidelines are available at www.toxicology.org ) and the animals ( scientific procedures ) act 1986 .
hemidiaphragms and accompanying phrenic nerves were dissected and placed into 10 ml tissue baths containing krebs solution ( 118.4 mm nacl , 4.7 mm kcl , 25 mm nahco3 , 1.2 mm kh2po4 , 1.4 mm mgso4 and 2.5 mm cacl2 ) , maintained at 37c and oxygenated with a mixture 95% o2 and 5% co2 .
muscle contractions were evoked indirectly or directly . in the case of indirect neurally - evoked twitches ,
the attached phrenic nerve was stimulated at a frequency of 0.2 hz with rectangular pulses of 0.2 ms duration and a supramaximal voltage ( < 15 v ) from a grass force displacement transducer .
muscle twitches were recorded on a grass polygraph model 79 ( grass instruments ) . in order to reveal the triphasic effect of recombinant proteins on nm transmission ,
in some experiments the preparation was partially paralyzed ( to 1520% of control ) by reducing the concentration of ca to 0.380.50 mm and allowing the tissues to equilibrate for 3045 min . in these experiments
recombinant proteins were added to the tissue bath and changes in the amplitude of twitch tension responses were followed continuously .
the functionality of the muscle in the preparation at the end of the recordings was checked directly by acetylcholine stimulation or indirectly by 2.5 mm ca stimulation . in experiments with directly - evoked twitches the preparation was set up as described for the phrenic nerve - hemidiaphragm for recording neurally - evoked twitches .
in addition , one end of a bipolar stimulating electrode was sutured into the diaphragm muscle near the costal margin and the other one was attached to the base of the hemidiaphragm .
the directly - evoked twitches were recorded by supramaximal voltage stimulation ( 50 v ) at a frequency of 0.2 hz and 2 ms duration . to eliminate nm transmission in the preparation , d - tubocurarine ( 10 m ) , an antagonist of the nicotinic acetylcholine receptors ,
was added into the organ bath 5 min prior to the beginning of direct stimulation .
positive controls for the neurotoxic triphasic effect were performed by adding taipoxin ( 0.51 g / ml ) after reducing the concentration of ca in order to partially paralyze the nm preparation as described above .
recombinant atxa , atnl and its quadruple h28y / l31v / n33g / s49d mutant lw were prepared by in vitro refolding following expression in e. coli as described . all other chemicals were from sigma aldrich or gibco life technologies .
adult male balb / c mice ( 2025 g ) were maintained and humanely killed according to the guiding principles in the use of animals in toxicology ( society of toxicology , 1999 , guidelines are available at www.toxicology.org ) and the animals ( scientific procedures ) act 1986 .
hemidiaphragms and accompanying phrenic nerves were dissected and placed into 10 ml tissue baths containing krebs solution ( 118.4 mm nacl , 4.7 mm kcl , 25 mm nahco3 , 1.2 mm kh2po4 , 1.4 mm mgso4 and 2.5 mm cacl2 ) , maintained at 37c and oxygenated with a mixture 95% o2 and 5% co2 .
muscle contractions were evoked indirectly or directly . in the case of indirect neurally - evoked twitches ,
the attached phrenic nerve was stimulated at a frequency of 0.2 hz with rectangular pulses of 0.2 ms duration and a supramaximal voltage ( < 15 v ) from a grass force displacement transducer .
muscle twitches were recorded on a grass polygraph model 79 ( grass instruments ) . in order to reveal the triphasic effect of recombinant proteins on nm transmission ,
in some experiments the preparation was partially paralyzed ( to 1520% of control ) by reducing the concentration of ca to 0.380.50 mm and allowing the tissues to equilibrate for 3045 min . in these experiments
recombinant proteins were added to the tissue bath and changes in the amplitude of twitch tension responses were followed continuously .
the functionality of the muscle in the preparation at the end of the recordings was checked directly by acetylcholine stimulation or indirectly by 2.5 mm ca stimulation . in experiments with directly - evoked twitches the preparation was set up as described for the phrenic nerve - hemidiaphragm for recording neurally - evoked twitches . in addition
, one end of a bipolar stimulating electrode was sutured into the diaphragm muscle near the costal margin and the other one was attached to the base of the hemidiaphragm .
the directly - evoked twitches were recorded by supramaximal voltage stimulation ( 50 v ) at a frequency of 0.2 hz and 2 ms duration . to eliminate nm transmission in the preparation , d - tubocurarine ( 10 m ) , an antagonist of the nicotinic acetylcholine receptors ,
was added into the organ bath 5 min prior to the beginning of direct stimulation .
positive controls for the neurotoxic triphasic effect were performed by adding taipoxin ( 0.51 g / ml ) after reducing the concentration of ca in order to partially paralyze the nm preparation as described above .
 the molecular mechanism of action of presynaptically neurotoxic secreted phospholipases a2 ( spla2s ) has not been fully elucidated . 
 we have recently proposed a model to explain one of the hallmarks of their action  the reduction in endocytosis leading to synaptic vesicle depletion in nerve terminals . 
 our results speak strongly in favor of a mechanism in which both specific protein - protein interactions and enzymatic activity of the neurotoxic spla2 ammodytoxin a ( atxa ) are necessary for impairment of clathrin - dependent endocytosis in yeast cells . 
 the reduction of endocytosis was strictly dependent on the enzymatic activity of spla2s expressed ectopically in our yeast model cells and was not observed with the catalytically inactive , non - neurotoxic atxa - homolog , ammodytin l ( atnl ) . 
 here we confirm the validity of the model in mammalian cells also , by demonstrating that the enzymatically active mutant of atnl , shown to inhibit endocytosis in yeast , acts as a presynaptically neurotoxic spla2 at the mammalian neuromuscular junction . 
Materials and Methods
Recombinant proteins and chemicals
Neuromuscular activity