The Munc 18-1 domain 3a hinge-loop controls syntaxin-1A nanodomain assembly and engagement with the SNARE complex during secretory vesicle priming

Munc 18-1 and syntaxin-1A control SNARE-dependent neuroexocytosis and are organized in nanodomains on the plasma membrane of neurons and neurosecretory cells. Deciphering the intra- and intermolecular steps via which they prepare secretory vesicles (SVs) for fusion is key to understanding neuronal and hormonal communication. Here, we demonstrate that expression of a priming-deficient mutant lacking 17 residues of the domain 3a hinge-loop (Munc 18-1(Delta 317-333)) in PC12 cells engineered to knockdown Munc 18-1/2 markedly prolonged SV docking. Single-molecule analysis revealed nonhomogeneous diffusion of Muncl 8-1 and syntaxin-1A in and out of partially overlapping nanodomains. Whereas Munc 18-1(WT) mobility increased in response to stimulation, syntaxin-1A became less mobile. These Munc 18-1 and syntaxin-1A diffusional switches were blocked by the expression of Munc 18-1(Delta 317-333), suggesting that a conformational change in the Munc 18-1 hinge-loop controls syntaxin-1A and subsequent SNARE complex assembly. Accordingly, syntaxin-1A confinement was prevented by expression of botulinum neurotoxin type E. The Munc 18-1 domain 3a hinge-loop therefore controls syntaxin-1A engagement into SNA RE complex formation during priming.