Functional differences of Munc18 isoforms reflect their affinities for the exocytosis site

The SM-protein Munc18 and its binding partner syntaxin are crucial for two distinct steps of regulated exocytosis, docking of secretory granules at the release site, and priming reactions that prepare docked granules for Ca²⁺ dependent fusion. Both proteins cluster simultaneously at the docking site seconds after a granule arrives at the plasma membrane. To study the mechanisms of these separate Munc18 functions, we generated a Munc18-1 knockout (1KO) and a double knockout of both Munc18-1 and Munc18-2 (DKO) in insulin-secreting cells, and quantified granule docking, exocytosis, and protein clustering at the docking site by TIRF imaging and capacitance measurements. In the 1KO, priming and exocytosis were lost, but granule docking remained intact. In the DKO, both priming and granule docking were impaired. Expression of either Munc18-1 or -2 rescued these defects, as did expression of mutants with weakened binding to syntaxin (EA and EK mutants). Munc18 clustered at granule docking sites, with apparent affinities decreasing in the order Munc18-1 > Munc18-1EA > Munc18-1EK > Munc18-2. Clustering of syntaxin-1 and - 3 at granules was impaired in both knockout lines. Finally, single molecule and imaging fluorescence recovery after photobleaching (FRAP) revealed that Syntaxin and Munc18 molecules were bound to each other, regardless of whether the proteins were clustered at a granule or not. We conclude that that Munc18-1 and - 2 molecules co-cluster with syntaxin at the release site and support docking and priming, and different affinities of the isoforms for the granule docking site contribute to the observed functional preferences.