Synaptotagmin 7 outperforms synaptotagmin 1 to promote the formation of large, stable fusion pores via robust membrane penetration

Abstract

Ca2+-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+.

Synaptotagmin 7 C2 domains induce membrane curvature stress via electrostatic interactions and the wedge mechanism

“…Analogously, cholesterol both thickens membranes and favors curvature. We (AHB and AJS) recently found that the thickening preference of cholesterol outweighed its curvature preference, leading to cholesterol depletion at the pore neck (43). Proteins can have a much broader range of membrane interactions compared to cholesterol, and how proteins and lipids work together in the context of a fusion pore is not clear.…”

“…Furthermore, experiments with Syt-1 have demonstrated that the tandem C2AB (but particularly C2B) disorders and demixes phosphatidylserine and PI(4,5)P2 chains (12, 42). Recently, Syt-7 was shown to penetrate more deeply into model bilayers than Syt-1, and that this penetration was critical for its function stabilizing fusion pore open states (43). Given that Syt-7 domains interact with strongly curved membranes during fusion, we seek to understand the interplay of induced membrane disordering (thinning), demixing, and curvature stabilization.…”