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

Abstract

AbstractCa2+-triggered exocytosis is a crucial aspect of neuronal and neuroendocrine cell function, yet many of the underlying molecular mechanisms that regulate these processes are unknown. Here, we contrast the biophysical properties of two prominent neuronal Ca2+ sensors, synaptotagmin (syt) 1 and syt7. In both proteins, four Ca2+-binding loops partially penetrate bilayers that harbor anionic phospholipids, and mutagenesis studies suggest that these interactions are important for function. However, these mutations also alter the interaction of syts with the SNARE proteins that directly catalyze membrane fusion. To directly assess the role of syt membrane penetration, we took a different approach and found that Ca2+-syt1-membrane interactions are strongly influenced by membrane order; tight lateral packing of phosphatidylserine abrogates syt1 binding to lipid bilayers due to impaired membrane penetration. Function could be restored by making the membrane penetration loops more hydrophobic, or by inclusion of cholesterol. In sharp contrast, syt7 unexpectedly exhibited robust membrane binding and penetration activity, regardless of the lipid acyl chain structure. Thus, syt7 is a ‘super-penetrator’. We exploited these observations to specifically isolate and examine the role of membrane penetration in syt function. By altering bilayer composition, rather than protein structure, we disentangled the roles of syt-membrane versus syt-SNARE interactions. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that membrane penetration underlies the ability of syts to directly regulate reconstituted, exocytic fusion pores in response to Ca2+.