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Botulinum neurotoxins (BoNT) cause muscle paralysis by inhibiting the exocytosis at motor nerve terminals. Initially, they bind to abundant complex polysialogangliosides via a conserved pocket within the C-terminal half of their cell binding domain (Hcc-domain). Engineering of this binding site in BoNT/A leads to mutants with >3-fold increased potency, which can be exploited as new therapeutics with decreased dosage and thus might prolong the period until immune response in patients. Additionally, protein receptors are required for productive neurotoxin uptake. While BoNT/B and G share the synaptic vesicle proteins synaptotagmin (Syt) I and Syt-II as protein receptors, BoNT/A uniquely binds SV2. Recently, the interaction site of Syt I and Syt-II was located near the ganglioside binding pocket within the Hcc-domain of BoNT/B and G. Furthermore, the BoNT/B–Syt II interaction could be characterised at the atomic level. None of the so-far identified protein receptors interacts with BoNT/C, D, E, F and TeNT. Employing the ex vivo mouse phrenic nerve-hemidiaphragm as a physiological target, the type and number of unknown neurotoxin receptor candidates were narrowed down. The productive uptake of BoNT/C, D, E and F is stimulation dependent, which points towards receptors being part of synaptic vesicles. Moreover, the endocytosis of BoNT/C, E and F, is inhibited by addition of any Hc-fragment derived from BoNT/C, E and F, indicating a binding to the same receptor structures.

The main object of this work is to deliver the catalytically active portion of neurotoxins (light chain) into the cytosol of mast cells to inhibit the specific release of inflammatory mediators. Mast cells play a significant role in the pathophysiology of many different diseases. In most of these diseases there is increased recruitment of mast cells and sustained secretion of many proinflammatory mediators, including autacoids, cytokines and proteases. We use the human mast cell line 1 (HMC-1), a leukaemia line expressing several features of mature human mast cells that has proved a valuable model for studying human mast cell biology. In order to stimulate fusion among secretory granules, we use intracellular application of GTPgammaS and extracellular stimulation with concanavalin A. By patch-clamp technique, we determine the changes in cell membrane capacitance, thereby studying exocytosis. The whole-cell configuration of patch-clamp is used. By electroporation, the light chains of TeNT and BoNT/C & D will be delivered into the cytoplasm of mast cells to estimate their effects on mast cell activity. Cells treated with neurotoxins will be subjected to stimulation tests “in vitro”, recording of membrane capacitance by patch-clamp and detection of cytokine release to estimate effects on mast cell activity.