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The Journal of Neuroscience, November 7, 2007, 27(45):12147-12155; doi:10.1523/JNEUROSCI.3655-07.2007

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Cellular/Molecular
Dual Modes of Munc18-1/SNARE Interactions Are Coupled by Functionally Critical Binding to Syntaxin-1 N Terminus

Mikhail Khvotchev,¹ Irina Dulubova,^3,4 Jianyuan Sun,¹ Han Dai,^3,4 Josep Rizo,^3,4 and Thomas C. Südhof^1,2,5

Departments of ¹Neuroscience, ²Molecular Genetics, ³Biochemistry, and ⁴Pharmacology and ⁵Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Correspondence should be addressed to Dr. Mikhail Khvotchev, Department of Neuroscience, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390. Email: mkhvoc{at}mednet.swmed.edu

The SM (Sec1/Munc18-like) protein Munc18-1 and the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins syntaxin-1, SNAP-25, and synaptobrevin/VAMP (vesicle-associated membrane protein) constitute the core fusion machinery for synaptic vesicle exocytosis. Strikingly, Munc18-1 interacts with neuronal SNARE proteins in two distinct modes (i.e., with isolated syntaxin-1 alone in a "closed" conformation and with assembled SNARE complexes containing syntaxin-1 in an "open" conformation). However, it is unclear whether the two modes of Munc18/SNARE interactions are linked. We now show that both Munc18/SNARE interaction modes involve the same low-affinity binding of the extreme syntaxin-1 N terminus to Munc18-1, suggesting that this binding connects the two Munc18/SNARE interaction modes to each other. Using transfected cells as an in vitro assay system, we demonstrate that truncated syntaxins lacking a transmembrane region universally block exocytosis, but only if they contain a free intact N terminus. This block is enhanced by coexpression of either Munc18-1 or SNAP-25, suggesting that truncated syntaxins block exocytosis by forming an untethered inhibitory SNARE complex/Munc18-1 assembly in which the N-terminal syntaxin/Munc18 interaction is essential. Introduction of an N-terminal syntaxin peptide that disrupts this assembly blocks neurotransmitter release in the calyx of Held synapse, whereas a mutant peptide that does not disrupt the SNARE complex/Munc18 assembly has no effect. Viewed together, our data indicate that binding of Munc18 to the syntaxin N terminus unites different modes of Munc18/SNARE interactions and is essential for exocytic membrane fusion.

Key words: neurotransmitter release; synaptic vesicle exocytosis; SNARE proteins; membrane fusion; FRET; calyx of Held; Munc18; SM proteins

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Received May 1, 2007; revised Sept. 10, 2007; accepted Sept. 16, 2007.

Correspondence should be addressed to Dr. Mikhail Khvotchev, Department of Neuroscience, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390. Email: mkhvoc{at}mednet.swmed.edu

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