Research On The Interaction Between Complexin And SNARE Complex And The Function Of Complexin

Complexin is a high hydrophilic and small cytoplastic protein that plays an important role during the fast exocytosis. Previous researches indicate that complexin functions in the process of neurotransmitter release by interaction with SNARE complex. However, what roles do the flanking regeins of complexin play on the interaction between complexin and SNARE complex? What is the role of complexin in the assembly process of SNARE complex? And it is still unclear what the mechanism of exocytosis inhibited by high concentrations of complexin. In this dissertation, we have investigated the characteristics of the interaction between complexin and SNARE complex and the mechanism underlying the inhibition by high concentrations of complexin.Firstly, we investigated the interaction between complexin and SNARE complex by pull-down and FRET assay. We demonstrated that the fragment 71-77 of complexin is necessary for its binding to SNARE complex. Moreover, abolishing the three positive charged residues in this fragment inhibited the interaction between complexin and SNARE complex. These results indicate that fragment 71-77 may affect the binding of complexin to SNARE complex by electrostatic interactions.Then, to analyze the role complexin on the assembly of SNARE complex, we established a series of SNARE complex mutants to mimic the partially assembly SNARE complex. The interaction assay showed that complexin can bind to the partially assembly SNARE complex. However, the binding ability of complexin is positively correlated with the extent to which SNARE complex is assembled. There results suggest that complexin can bind to SNARE complexin before its complete formation. The more completely SNARE complex form, the more tightly complexin binds to SNARE complex.Lastly, we established and screened a PC12 cell line that stably expresses complexin to investigate the mechanism underlying the inhibition by high concentrations of complexin. Here, we demonstrated that when overexpressed in PC12 cells, complexin prevented the recycling of SNARE complex, which in turn leads to a reduction of RRP of large dense core vesicle. Hence, in addition to its putative role arresting the hemifusion, complexin may take part in the recycling of SNARE complex by displacing NSF and alpha-SNAP from the SNARE complex.The above results reveal that how complexin interacts with SNARE complex and the mechanism underling the inhibition by high concentration of complexin, which is helpful to understand the function of complexin during the exocytosis.