We had previously shown that an influx of extracellular Ca2+ (Ca2+ e ), though it occurs, is not strictly required for aminoethyldextran (AED)-triggered exocytotic membrane fusion in Paramecium. We now analyze, by quenched-flow/freeze-fracture, to what extent Ca2+ e contributes to exocytotic and exocytosis-coupled endocytotic membrane fusion, as well as to detachment of ``ghosts'' — a process difficult to analyze by any other method or in any other system. Maximal exocytotic membrane fusion (analyzed within 80 msec) occurs readily in the presence of [Ca2+] e ≥ 5 × 10−6 m, while normally a [Ca2+] e = 0.5 mm is in the medium. A new finding is that exocytosis and endocytosis is significantly stimulated by increasing [Ca2+] e even beyond levels usually available to cells. Quenching of [Ca2+] e by EGTA application to levels of resting [Ca2+] i or slightly below does reduce (by ∼50%) but not block AED-triggered exocytosis (again tested with 80 msec AED application). This effect can be overridden either by increasing stimulation time or by readdition of an excess of Ca2+ e . Our data are compatible with the assumption that normally exocytotic membrane fusion will include a step of rapid Ca2+-mobilization from subplasmalemmal pools (``alveolar sacs'') and, as a superimposed step, a Ca2+-influx, since exocytotic membrane fusion can occur at [Ca2+] e even slightly below resting [Ca2+] i . The other important conclusion is that increasing [Ca2+] e facilitates exocytotic and endocytotic membrane fusion, i.e., membrane resealing. In addition, we show for the first time that increasing [Ca2+] e also drives detachment of ``ghosts'' — a novel aspect not analyzed so far in any other system. According to our pilot calculations, a flush of Ca2+, orders of magnitude larger than stationary values assumed to drive membrane dynamics, from internal and external sources, drives the different steps of the exo-endocytosis cycle.
The Journal of Membrane Biology – Springer Journals
Published: Aug 1, 1997
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