We developed a method for measuring the efflux of 5-hydroxytryptamine (5-HT, serotonin) from isolated intact granules of the mast cell of the beige mouse. This method combines electroporation of the vesicle membrane with amperometric detection of 5-HT. A single secretory granule is placed between two platinum electrodes (distance ~100 μm and positioned adjacent (<1 μm) to a carbon fiber microelectrode. A short (~30 μs) high-intensity voltage pulse (electric field of ~5 kV/cm) is delivered to the electrodes to trigger the mechanical breakdown of the granule membrane, which activates the release of 5-HT. We observed concurrent swelling of the granule matrix with the oxidation of 5-HT at the carbon fiber electrode (overpotential + 650 mV). Similar to the release of secretory products during exocytosis, the oxidation current exhibits a spike-like time course with a noninstantaneous rising phase (time between onset of currant and maximum flux, t(max)) with ~25% of the molecules released during this period. When the current reaches its maximum, the granule matrix attains its maximum swollen state. We found that the rising phase depends on the initial cross-sectional area of the granule (t(max) ≃ 21r2) and reflects the time required for membrane rupture. The average t 1/4 /(spike) of the amperometric spikes was found to be ≃150 ms, which is 3-7 times faster than the t 1/4 measured during cellular exocytosis.
Marszalek, P. E., Farrell, B., Verdugo, P., & Fernandez, J. M. (1997). Kinetics of release of serotonin from isolated secretory granules I. Amperometric detection of serotonin from electroporated granules. Biophysical Journal, 73(3), 1160–1168. https://doi.org/10.1016/S0006-3495(97)78148-7