Synaptotagmin 7 outperforms synaptotagmin 1 to open and stabilize nascent fusion pores via robust membrane penetration

Abstract Synaptotagmin-1 and synaptotagmin-7 are two prominent Ca 2+ sensors that regulate exocytosis in neuronal and neuroendocrine cells. Upon binding Ca 2+ , both proteins partially penetrate lipid bilayers that bear anionic phospholipids, but the specific underlying mechanisms that enable them to trigger exocytosis remain controversial. Here, we examined the biophysical properties of these two synaptotagmin isoforms and compared their interactions with phospholipid membranes. We discovered that synaptotagmin-1•membrane interactions are greatly influenced by membrane order; tight packing of phosphatidylserine inhibits binding due to impaired membrane penetration. In contrast, synaptotagmin-7 exhibits robust membrane binding and penetration activity regardless of phospholipid acyl chain structure. Thus, synaptotagmin-7 is a “super-penetrator”. We exploited these observations to specifically isolate and examine the role of membrane penetration in synaptotagmin function. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that membrane penetration is a critical component that underlies how synaptotagmin proteins regulate reconstituted, exocytic fusion pores in response to Ca 2+ .