SNARE Conformational Changes that Prepare Vesicles for Exocytosis

When cells release hormones and neurotransmitters through exocytosis, cytosolic Ca 2+ triggers the fusion of secretory vesicles with the plasma membrane. It is well known that this fusion requires assembly of a SNARE protein complex. However, the timing of SNARE assembly relative to vesicle fusion —essential for understanding exocytosis —has not been demonstrated. To investigate this timing, we constructed a probe that detects the assembly of two plasma membrane SNAREs, SNAP25 and syntaxin-1A, through fluorescence resonance energy transfer (FRET). With two-photon imaging, we simultaneously measured FRET signals and insulin exocytosis in β cells from the pancreatic islet of Langerhans. In some regions of the cell, we found that the SNARE complex was preassembled, which enabled rapid exocytosis. In other regions, SNARE assembly followed Ca 2+ influx, and exocytosis was slower. Thus, SNARE proteins exist in multiple stable preparatory configurations, from which Ca 2+ may trigger exocytosis through distinct mechanisms and with distinct kinetics. [Display omitted] ► A new FRET probe (SLIM) detects conformational states of SNAP25 during exocytosis ► There are regional variations in conformation of SNAP25 in resting β cells ► Conformation changes in SNAP25 could precede exocytosis in the low FRET regions ► Calcium can trigger assembling of SNAREs for exocytosis in β cells