Refilling of cortical calcium stores in Paramecium cells: in situ analysis in correlation with store-operated calcium influx.

This is the first thorough study of refilling of a cortical calcium store in a secretory cell after stimulation in which we combined widely different methodologies. Stimulation of dense-core vesicle (“trichocysts”) exocytosis in Paramecium involves a Ca2+-influx” superimposed to Ca2+-release from cortical stores (“alveolar sacs” (ASs)). In quenched-flow experiments, membrane fusion frequency rose with increasing [Ca2+]o in the medium, from ∼20–25% at [Ca2+]o ≤0.25μM to 100% at [Ca2+]o between 2 and 10μM, i.e. close to the range of estimated local intracellular [Ca2+] during membrane fusion. Next, we analyzed Ca2+-specific fluorochrome signals during stimulation under different conditions. Treatment with actin-reactive drugs had no effect on Ca2+-signaling. In double trigger experiments, with BAPTA in the second secretagogue application (BAPTA only for stimulation and analysis), the cortical Ca2+-signal (due solely to Ca2+ released from cortical stores) recovered with t1/2 ∼65min. When ASs were analyzed in situ by X-ray microanalysis after different trigger times (+Ca2+o), t1/2 for store refilling was similar, ∼60min. These values are similar to previously measured 45Ca2+-uptake by isolated ASs. In sum we find, (i) exogenous Ca2+ increases exocytosis/membrane fusion performance with EC50=0.7μM, (ii) Ca2+-signaling in this system is not sensitive to actin-reactive drugs, and (iii) refilling of these cortical calcium stores goes on over hours and thus is much slower than expected.