Extension of helix 12 in munc18-1 induces vesicle priming

Anders S. Munch, Girish H. Kedar, Jan R.T. van Weering, Sonia Vazquez-Sanchez, Enqi He, Timon André, Thimo Braun, Thomas H. Söllner, Matthijs Verhage, Jakob B. Sørensen*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Munc18-1 is essential for vesicle fusion and participates in the docking of large dense-core vesicles to the plasma membrane. Recent structural data suggest that conformational changes in the 12th helix of the Munc18-1 domain 3a within the Munc18-1:syntaxin complex result in an additional interaction with synaptobrevin-2/VAMP2 (vesicle-associated membrane protein 2), leading to SNARE complex formation. To test this hypothesis in living cells, we examined secretion from Munc18-1-null mouse adrenal chromaffin cells expressing Munc18-1 mutants designed to either perturb the extension of helix 12 (Δ324 – 339), block its interaction with synaptobrevin-2 (L348R), or extend the helix to promote coil– coil interactions with other proteins (P335A). The mutants rescued vesicle docking and syntaxin-1 targeting to the plasma membrane, with the exception of P335A that only supported partial syntaxin-1 targeting. Disruptive mutations (L348R or Δ324 –339) lowered the secretory amplitude by decreasing vesicle priming, whereas P335A markedly increased priming and secretory amplitude. The mutants displayed unchanged kinetics and Ca2+ dependence of fusion, indicating that the mutations specifically affect the vesicle priming step. Mutation of a nearby tyrosine (Y337A), which interacts with closed syntaxin-1, mildly increased secretory amplitude. This correlated with results from an in vitro fusion assay probing the functions of Munc18-1, indicating an easier transition to the extended state in the mutant. Our findings support the notion that a conformational transition within the Munc18-1 domain 3a helix 12 leads to opening of a closed Munc18-1:syntaxin complex, followed by productive SNARE complex assembly and vesicle priming.

Original languageEnglish
Pages (from-to)6881-6891
Number of pages11
JournalJournal of Neuroscience
Volume36
Issue number26
DOIs
Publication statusPublished - 29 Jun 2016

Cite this