Munc18-1 tuning of vesicle merger and fusion pore properties

Abstract

The release ofhormones and neurotransmitters, mediated byregulated exocytosis, can bemodifiedby regulationofthe fusion pore. The fusion poreisconsideredstable and narrowinitially, eventually leadingtothe complete mergerofthe vesicleandtheplasma membranes. Byusingthe high-resolutionpatch-clampcapacitancetechnique,westudiedsinglevesicles andasked whethertheSec1/Munc18 proteins, interacting with the membrane fusion-mediating SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, affect fusion pore properties. Munc18-1 mutants were transfected into lactotrophs to affect the interaction of Munc18-1 with syntaxin1 (Synt1) (R39C), Rab3A (E466K), and Mints (P242S). Compared withwild-type,Munc18-1 E466Kincreased the frequencyofthe fusion event. The latter two mutants increased the fusion pore dwell-time. All the mutants stabilized narrow fusion pores and increased the amplitude of fusion events, likely via preferential fusion of larger vesicles, since overexpression of Munc18-1 R39C did not affect the average size of vesicles, as determined by stimulated emission depletion (STED) microscopy. Single-molecule atomic force microscopy experiments revealed that wild-type Munc18-1, but not Munc18-1 R39C, abrogates the interaction between synaptobrevin2 (Syb2) and Synt1binarytrans-complexes. However, neither formofMunc18-1 affected the interactionofSyb2 withthe preformed binarycis-Synt1A-SNAP25B complexes. This indicates that Munc18-1 performs a proofing function by inhibiting tethering of Syb2-containing vesicles solely to Synt1 at the plasmalemma and favoring vesicular tethering to the preformed binary cis-complex of Synt1A-SNAP25B. The association of Munc18-1 with the ternary SNARE complex leads to tuning of fusion pores via multiple and converging mechanisms involving Munc18-1 interactions with Synt1A, Rab3A, and Mints. © 2011 the authors.