Relationship between fusion pore opening and release during mast cell exocytosis studied with patch amperometry

We have studied exocytosis in rat peritoneal mast cells by cell-attached patch amperometry. Step increases in capacitance were accompanied by typical amperometric spikes due to the release of 5-hydroxytryptamine (serotonin), indicating exocytosis of typical mast cell granules. We have measured the time course of fusion pore expansion, and correlated it with release from the granule matrix. The fusion pore of mast cell granules grows in three stages. The initial expansion of the pore occurred at a rate of 5 nS/s, and in many cases an observable amperometric foot was detected. A second, rapid expansion phase occurred with a rate as high as 1000 nS/s, coinciding with the upstroke of the amperometric spike. A third, slower phase, with a rate of 5 nS/s, completed the final expansion of the fusion pore. These data reveal the very late stages in the exocytotic process, and demonstrate that the size of the fusion pore does not limit release during the upstroke of the amperometric spike or during the final, slow expansion that occurs during for the decay of the amperometric spike.