biostudies-literatureMakke MDeutsche ForschungsgemeinschaftRP92438https://www.ebi.ac.uk/biostudies/studies/S-EPMC11589869biostudies-literatureUnknown12Complexin determines magnitude and kinetics of synchronized secretion, but the underlying molecular mechanisms remained unclear. Here, we show that the hydrophobic face of the amphipathic helix at the C-terminus of Complexin II (CpxII, amino acids 115-134) binds to fusion-promoting SNARE proteins, prevents premature secretion, and allows vesicles to accumulate in a release-ready state in mouse chromaffin cells. Specifically, we demonstrate that an unrelated amphipathic helix functionally substitutes for the C-terminal domain (CTD) of CpxII and that amino acid substitutions on the hydrophobic side compromise the arrest of the pre-fusion intermediate. To facilitate synchronous vesicle fusion, the N-terminal domain (NTD) of CpxII (amino acids 1-27) specifically cooperates with synaptotagmin IeLifeKey determinants of the dual clamp/activator function of Complexin.PMC11589869CRC 1027Pastor-Ruiz AGaya SMakke MBruns DYarzagaray AFrisch WZimmer MfalseKey determinants of the dual clamp/activator function of Complexin.Complexin determines magnitude and kinetics of synchronized secretion, but the underlying molecular mechanisms remained unclear. Here, we show that the hydrophobic face of the amphipathic helix at the C-terminus of Complexin II (CpxII, amino acids 115-134) binds to fusion-promoting SNARE proteins, prevents premature secretion, and allows vesicles to accumulate in a release-ready state in mouse chromaffin cells. Specifically, we demonstrate that an unrelated amphipathic helix functionally substitutes for the C-terminal domain (CTD) of CpxII and that amino acid substitutions on the hydrophobic side compromise the arrest of the pre-fusion intermediate. To facilitate synchronous vesicle fusion, the N-terminal domain (NTD) of CpxII (amino acids 1-27) specifically cooperates with synaptotagmin I2024-01-01T00:00:00Z2024 Nov2026-06-03T07:25:47.379Z2025-04-04T02:33:25.32ZS-EPMC115898693958532610.7554/eLife.92438