Purinergic-mediated Ca2+ influx in Dictyostelium discoideum

The presence of five P2X-like genes (p2xA-E) in Dictyostelium suggests that nucleotides other than cAMP may act as extracellular signalling molecules in this model eukaryote. However, p2xA was found to have an exclusively intracellular localisation making it unclear whether Dictyostelium utilise P2 receptors in a manner analogous to vertebrates. Using an apoaequorin expressing strain we show here that Dictyostelium do possess cell surface P2 receptors that facilitate Ca2+ influx in response to extracellular ATP and ADP (EC50 = 7.5 μM and 6.1 μM, respectively). Indicative of P2X receptor activation, responses were rapid reaching peak within 2.91 ± 0.04 s, required extracellular Ca2+, were inhibited by Gd3+, modified by extracellular pH and were not affected by deletion of either the single Gβ or iplA genes. Responses also remained unaffected by disruption of p2xA or p2xE showing that these genes are not involved. Cu2+ and Zn2+ inhibited purine-evoked Ca2+ influx with IC50 values of 0.9 and 6.3 μM, respectively. 300 μM Zn2+ completely abolished the initial large rapid rise in intracellular Ca2+ revealing the presence of an additional smaller, slower P2Y-like response. The existence of P2 receptors in Dictyostelium makes this organism a valuable model to explore fundamental aspects of purinergic signalling.