Die Funktion von Munc-18 in der Exozytose sekretorischer Granulen

Calcium-dependent neurotransmitter release from secretory vesicles is a highly regulated process involving several evolutionary conserved protein families. These include SNARE-proteins (soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor), Rab GTPases as well as the family of cytosolic SM proteins. SNARE proteins are membrane-anchored proteins possessing a characteristic stretch of 60-70 amino acids termed the “SNARE motif”. Specific combinations of SNARE proteins are able to assemble into highly stable complexes of four parallel ?-helices via their SNARE motives, forming the so-called “core complex”. The assembly of SNAREs of vesicular and plasma membranes pulls the membranes in close apposition, a prerequisite for subsequent membrane fusion. The importance of SNARE proteins for membrane fusion becomes obvious when cells are poisoned with clostridial neurotoxins as Tetanus toxin or Botulinum toxin that cleave SNARE proteins proteolytically, eventually leading to the inhibition of neurotransmission. The synaptic SM protein munc-18-1 has been originally identified by its high affinity interaction with the plasma membrane SNARE protein syntaxin1. The observation that binding of munc-18-1 to syntaxin1 precludes the formation of the core complex contributed substantially to the idea that munc-18 acts as an inhibitor of exocytosis by regulating the availability of syntaxin1 for SNARE complexes. Still, this does not explain why munc-18-1 is essential for membrane fusion. Thus, although munc-18-1 has a central role in membrane fusion, its precise function is little understood. In this study, the role of the munc-18-1/syntaxin1 interaction in exocytosis of secretory granules in neuroendocrine cells was investigated. To this end, a combined biochemical and electrophysiological approach was chosen. The munc-18-1/syntaxin1 interaction was perturbed by overexpressing wildtype munc-18-1 protein and by introducing point mutations into the syntaxin-binding region of munc-18-1. Syntaxin-binding of the mutants was analysed biochemically in several binding assays followed by overexpression of the mutant proteins as well as the wildtype-protein in the neuroendocrine cell line PC12. The effects of overexpression were subsequently characterised by carbon fibre amperometry. Overexpression of munc-18-WT protein did not affect exocytosis, making an inhibitory function of munc-18-1 in exocytosis unlikely. The point mutants used (munc-18-D34N and –R39C) showed a differentially reduced syntaxin1 binding. R39C still showed binding to syntaxin1, while D34N could not be detected in a complex with syntaxin1. Surprisingly, although both mutants had a reduced syntaxin1 binding ability, their effects on exocytosis were opposite: R39C caused a decrease in the number of exocytotic events, while D34N caused an increase. This pointed towards the involvement of a further munc-18-1 binding partner. Thus, we characterised the interaction of munc-18-WT, -D34N, and –R39C with Mint1. Mint1 is a synaptic multidomain protein possessing a phosphotyrosine binding domain (PTB domain) as well as two PDZ domains that bind to further presynaptic proteins connecting Mint1 to numerous presynaptic protein complexes. The observation that in the presence of syntaxin1 and Mint1, the preferred protein complex of D34N is different from the one preferred by R39C (Mint1/Munc-18 complex and Munc-18/syntaxin1 complex, respectively) led to the hypothesis that the stimulatory effect of the D34N mutant is caused by the more frequent interactions with Mint1 that were observed in this study. The electrophysiological characterization of Mint1 overexpression in PC12 cells showed an inhibitory effect. Thus, the stimulating effect of the D34N mutant may be explained by more frequent interactions with inhibitory Mint1 causing a disinhibition. In contrast, the R39C-mutant presumably exerts its effects via its perturbed syntaxin1-binding. This study shows that munc-18-1 is not an inhibitory factor in exocytosis. Munc- 18-1’s function in exocytosis is not limited to its interaction with syntaxin1. It can be involved in numerous protein interactions at the synapse via its interaction with Mint1 and may represent an important link between the membrane fusion machinery via syntaxin1 and proteins organising the release site via Mint1.