FUSION PORE CLOSURE DETERMINES SUBQUANTAL KISS-AND-RUN SEROTONIN RELEASE FROM FULLY LOADED DENSE-CORE VESICLES

During kiss-and-run exocytosis, a transient fusion pore opens, releases part of the vesicular quantum, and closes without full fusion. This report investigates two questions: first, whether dense-core vesicles undergoing kiss-and-run contain the same number of molecules as those undergoing full fusion; second, whether subquantal release results from pore closure or vesicle depletion. We analyzed amperometric recordings of extrasynaptic serotonin release from dense-core vesicles in cultured leech Retzius neurons. Full-fusion events released 91,585±34,428 molecules (n=14), whereas kiss-and-run events released 58,344±20,759 molecules, 64% of the quantum. We analyzed the biexponential dynamics of the filtered amperometric current using Ohmic equations for cylindrical and toroidal pores. Hydrated flux packing (HFP) independently estimated the minimum peak pore diameter from the number of hydrated serotonin molecules crossing the pore. Kiss-and-run peak diameters were 2.1 to 3.2 nm, corresponding to the passage of 4 to 10 hydrated molecules. The vesicular serotonin content of individual kiss-and-run events was estimated by a frozen pore extrapolation (FPE), in which pores were kept at the peak HFP diameter. FPE yielded 102,650±10,989 molecules per vesicle (n=15), similar to the quantum obtained by integrating full-fusion events. Therefore, kiss-and-run subquantal release did not reflect a smaller quantum. We then compared the decay of serotonin flux with that estimated through the frozen pore at its peak diameter. In every event, the flux decayed faster, reaching half its peak in 0.59 (0.23 to 0.95) of the time taken by the frozen pore flux. We conclude that subquantal release during kiss-and-run exocytosis is governed by fusion-pore closure rather than vesicle depletion.