Internalization and Membrane Translocation

Since the L chain of TeTx and BoNTs is responsible for the cytosolic activity of the CNTs, at least this domain of the toxin molecule must reach the cytosol. Pharmacological and morphologic evidence indicates that the CNTs enter the cell by endocytosis (Black and Dolly, 1986 b) and that TeTx and BoNTs have to pass through a low pH step for neuron intoxication to occur (Williamson and Neale, 1992 Simpson et at., 1994). Acidic pH does not activate the toxin directly via a structural change, since the direct introduction of the L chain in the neutral pH environment of the cytosol is sufficient to block exocytosis (Penner et at., 1986 Anhert-Hilger et al., 1989 b Bittner et al., 1989 a, b Mochida et al., 1989 Weller et al., 1991). Hence, low pH is necessary for the process of L chain membrane translocation from the vesicle lumen into the cytosol, by analogy with the other bacterial protein toxins with a three-domain structure (Montecucco et al., 1994). 

The interaction of CNTs with membrane bilayers has been studied mainly with model membrane systems, and limited data have been obtained in vivo. Available evidence indicates that at low pH TeTx and BoNTs undergo a conformational change from a water soluble neutral form to an acidic form, the latter characterized by the exposure of hydrophobic segments. This increase in hydrophobicity allows penetration of both the H and L chains into the hydrocarbon core of the lipid bilayer (Montecucco etal., 1994). Following this low pH-induced membrane insertion, TeTx and BoNTs form ion channels in planar lipid bilayers (Beise et al., 1994 Montecucco et al., 1994). These channels are cation-selective, have few tens of pS conductance and are per- 

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Future important discoveries will be the identification of the neuron-specific receptors of CNTs and of the mode of internalization and membrane translocation of the neurotoxins. Another important line of research is aimed at finding specific inhibitors of these metallo-proteinases. Inhibitors which can cross the neuronal plasmalemma into the cytosol would be potential therapeutic agents in the treatment of tetanus and botulism. The modification of BoNTs to prolong their life time inside the NMJs would be an important research goal to improve the treatment of dystonias. The determination of the three-dimensional structure of these neurotoxins will greatly accelerate the research on these fronts. 

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