Differential regulation of exocytotic fusion and granule-granule fusion in eosinophils by Ca2+ and GTP analogs

Dynamics of degranulation was studied in horse eosinophils by patch clamp capacitance measurements. Degranulation was stimulated by intracellular application of calcium, and GTPgammaS or guanosine 5'-(beta,gamma-imido)triphosphate at different concentrations via the patch pipette. Degranulation was quantified by measuring the delay time between the beginning of intracellular perfusion and the first exocytotic event, determining the distribution of time intervals between fusion events and the capacitance step size distributions under the different conditions. The degranulation dynamics could be well reproduced using a computer model assuming three independent rate constants for granule-plasma membrane fusion, granule fusion with already exocytosed granules, and intracellular granule-granule fusion. The rate of granule-plasma membrane fusion is sensitive to both, the GTP analog and [Ca2+]i. The rate of granule-exocytosed granule fusion is sensitive to [Ca2+]i but insensitive to the GTP analogs, and the rate of granule-to-granule fusion is sensitive to the GTP analog but insensitive to [Ca2+]i. Granule fusions with the three different target compartments thus involve different regulatory mechanisms.