Figure 1. AtxA and the LW mutant, but not the enzymatically inactive AtnL, display the triphasic modulation of twitch tension characteristic of presynaptically neurotoxic sPLA₂s. The effects of recombinant AtxA (10 µg/ml; 0.725 µM), the LW mutant (6 µg/ml; 0.430 µM) and AtnL (6 µg/ml; 0.433 µM) on muscle twitch tension were determined at 37°C. (A and B). The NM preparation was partially paralyzed by a low Ca²⁺ concentration (0.38–0.50 mM) Krebs solution and stimulated indirectly. Note that, upon addition of LW, the triphasic muscle twitch response occurred within 10 min (B). (C and D) The NM preparation was stimulated indirectly (C) or directly (D) in standard Krebs solution containing 2.5 mM Ca²⁺ as described in materials and methods. Note that AtxA, but not AtnL (C), inhibited muscle contractions. Proteins were added to the bath at time 0. Twitch tension is expressed as a percentage of the control value. The number of repeats of experiments is shown in brackets. Each point on the diagrams represents the mean ± SE. Standard errors are indicated by bars unless smaller than symbols.