Regulation of neuronal SNARE complexes by complexin and calcium(+2)-synaptotagmin
McNew, James A.
Doctor of Philosophy
Synaptic transmission relies on an exquisitely orchestrated series of protein-protein interactions that ultimately results in neurotransmitter release into the synaptic cleft. The final step in this reaction is membrane fusion of the synaptic vesicle with the presynaptic plasma membrane catalyzed by SNARE proteins. Several extrinsic protein factors impinge on the cycle of SNARE complex assembly to regulate vesicle release in space and time. These include general regulators such as the Rab family of GTPases and the Munc18/Sect (SM) proteins, as well as specific regulators of synaptic transmission including complexin and synaptotagmin. The interplay of these regulatory factors and their precise mechanisms of action remain an area of intense investigation. This work shows that fusion driven by a t-SNARE complex of Syntaxin1A/SNAP25 and the v-SNARE VAMP2, but not their yeast equivalents, is inhibited by complexin. Furthermore, inner leaflet mixing is strongly impaired relative to total lipid mixing indicating that inhibition by complexin arrests fusion at a hemifusion intermediate. When the calcium sensor synaptotagmin is added in the absence of calcium to the complexin-inhibited reaction, the arrest persists. However, when calcium is introduced, complexin inhibition is relieved and full fusion rapidly proceeds as evidenced by restoration of inner leaflet mixing. These results suggest that the combination of complexin and synaptotagmin provides a strong calcium-dependent clamp to inhibit full fusion at hemifusion until released by calcium influx.
Neurosciences; Cell biology; Biochemistry