Intracellular Ca2+ signals in Dictyostelium chemotaxis are mediated exclusively by Ca2+ influx

We measured folate- and cAMP-induced changes in cytoplasmic free calcium concentration ([Ca2+](i)) using recombinant aequorin reconstituted in living Dictyostelium cells with coelenterazine-h. The resulting semi-synthetic protein displayed increased sensitivity to Ca2+ allowing accurate measurement of chemoattractant-induced transients at low resting levels. Both folate-and cAMP-induced Ca2+ responses were developmentally regulated, exhibited remarkably similar kinetics and were dependent on the relative rather than the absolute magnitude of increases in attractant concentration, They began after a short delay of 5-10 seconds, leading to a maximum increase in cytosolic calcium concentration after similar to 25 seconds and a return to basal level within similar to 60 seconds after stimulation, Responses elicited by the two chemoattractants were dose-dependent and saturated between 4 and 20 mu M. They depended on the presence of free extracellular calcium ions and were inhibited in a concentration-dependent manner between 10(-4) and 10(-5) M. In accordance with Ca-45(2+)-uptake measurements by Milne and Coukell (J. Cell Biol. (1991) 112, 103-110), both responses were also completely inhibited by 15 mu M Ruthenium Red, 15 mu M carbonylcyanide m-chlorophenyl-hydrazone (CCCP) and 500 mu M gadolinium ions. Under conditions that prohibited influx of Ca2+ from the extracellular medium there were no detectable changes in [Ca2+](i) that could be related to a separate release of the ion from intracellular stores. Together, these results show that the Ca2+ signals involved in chemotaxis correlate temporally with actin depolymerization (not polymerization) and are mediated by Ca2+ influx, not IP3-mediated intracellular release.