The Mechanism Of Complexin In Regulating The Vesicle Exocytosis

One of the most controversial protein complexin(Cpx, also known as synaphin) is a kind of small soluble molecule, mainly exsits in the eukaryotic nervous system, which can be involved in the synaptic vesicle exocytosis. Cpx binds to the partly assembled SNARE complexes, clamps the SNARE complexes not fully assembled. In the presence of Ca2+, the vesicle protein synaptotagmin will displace Cpx, allowing the SNARE protein complexes to bind the vesicle to the presynaptic membrane, promote the fusion pore opening. In this paper, we studied the molecular mechanisms of Cpx in the synaptic vesicle exocytosis in vitro and in vivo. The results help us to better understand the whole process of synaptic vesicle fusion.In synaptic terminals, Cpx can suppress spontaneous mini release and promote Ca2+-triggered synchronized neurotransmitter release. A large amount of researches have been done for identifying the functions and molecular mechanisms of Cpx, however the functions of C-terminal sequence of Cpx, which contains almost half of Cpx in length, are still mainly uncovered. In this paper, we identify the effects of Cpx C-terminal on the plasma membrane vesicle( PM)synaptic vesicle( SV)association, spontaneous vesicle fusion and Ca2+-triggered fusion.We found that wild type Cpx-1(CpxWT) showed a strong effect in promoting vesicle association, synchronizing Ca2+-triggered fusion, and inhibiting spontaneous fusion. Cpx-1 4M mutant(Cpx4M), which impairs the interaction between Cpx and ternary SNARE complex, lost its effects in all membrane fusion processes. In the absence of C-terminal domain, the effect of Cpx on vesicle association and spontaneous fusion was vanished, while little effect in synchronizing Ca2+-triggered fusion was observed. Further study showed the inhibitory effect of C-terminal domain truncated Cpx-1(Cpx1-86) on vesicle association was significant even at a concentration below what was required for membrane interaction( 30 n M). No significant difference in spontaneous fusion was observed for Cpx1-86 ranging. Cpx1-86 could promote of Ca2+-triggered synchronized fusion even at the concentration between 100 and 500 n M. Next we found pre-incubate Cpx-1 with PM vesicles resulted in a phenotype similar to C-terminal domain deletion(Cpx1-86). Vesicle association was blocked, and suppression of spontaneous fusion was failed, while negligible effect on synchronizing Ca2+-triggered fusion was observed. While pre-incubate Cpx-1 with SV vesicles resulted in a phenotype similar to CpxWT. In conclusion we infer Cpx C-terminal sequence was binding to synaptic vesicle membranes preferentially.Then we wanted to confirm this hypothesis in vivo. First, we suppressed the endogenous Cpx expression via RNA interference, introduced different mutants of Cpx-1 to the primary cultured hippocampal neurons. Then we recorded the spontaneous miniature excitatory postsynaptic currents(m EPSC) and evoked-EPSC. The results showed that Cpx knockdown increases the frequency of spontaneous fusion, but decreases the Ca2+-triggered release. Vesicle anchored mutant Cpx-11-86-CSP? but not the plasma membrane anchored mutant Cpx-11-86-CAIM rescued the suppression function of Cpx. Both Cpx-11-86-CSP? and Cpx-11-86-CAIM could rescue the impairments induced by Cpx suppression in Ca2+-triggered fusion. These in vivo results verified our hypothesis, Cpx C-terminal sequence was required for suppressing spontaneous fusion by binding to synaptic vesicle membrane preferentially.