Capacitance measurements reveal stepwise fusion events in degranulating mast cells

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Abstract
Mast cells undergo an extensive and violent morphological transformation on stimulation. Here we describe the dynamics of fusion of the secretory granules in individual mast cells during exocytosis. The cell membrane capacitance (proportional to the cell surface area) was measured using the whole-cell patch-pipette technique, in which the intracellular space is dialysed with the solutions used to fill the patch pipette. Our results show that degranulation occurs spontaneously and reproducibly if the GTP analogue, GTP-gamma-S, and Mg-ATP are present in the pipette filling solution. Contrary to previous reports, in these conditions Ca2+ (and/or Ca2+ buffers) is not required for degranulation. Although electrogenic Ca2+ entry was not detected before or during degranulation and membrane conductance remained low, the capacitance, and by implication the area of the membrane of degranulating cells, increased sigmoidally and stepwise. We conclude that stepwise increases of capacitance are due to the fusion of individual secretory granules with the plasma membrane, and that guanine nucleotide regulatory proteins are involved in the control of this process.