Complexin-1 and synaptotagmin-1 compete for binding sites on membranes containing PtdInsP.

Complexin-1 is an essential protein for neuronal exocytosis that acts to depress spontaneous fusion events while enhancing evoked neurotransmitter release. In addition to binding soluble N-ethylmaleimide-sensitive factor attachment protein receptors, it is well established that complexin associates with membranes in a manner that depends upon membrane curvature. In the present work, we examine the membrane binding of complexin using electron paramagnetic resonance spectroscopy, fluorescence anisotropy, and total internal reflection fluorescence microscopy. The apparent membrane affinity of complexin is found to strongly depend upon the concentration of protein used in the binding assay, and this is a result of a limited number of binding sites for complexin on the membrane interface. Although both the N- and C-terminal regions of complexin associate with the membrane interface, membrane affinity is driven by its C-terminus. Complexin prefers to bind liquid-disordered membrane phases and shows an enhanced affinity toward membranes containing phosphatidylinositol 4-5-bisphosphate (PI(4,5)P). In the presence of PI(4,5)P, complexin is displaced from the membrane surface by proteins that bind to or sequester PI(4,5)P. In particular, the neuronal calcium sensor synaptotagmin-1 displaces complexin from the membrane but only when PI(4,5)P is present. Complexin and synaptotagmin compete on the membrane interface in the presence of PI(4,5)P, and this interaction may play a role in calcium-triggered exocytosis by displacing complexin from its fusion-inhibiting state.