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 Ca(2+) influx in response to extracellular ATP and ADP (EC(50)=7.5microM and 6.1microM, respectively). Indicative of P2X receptor activation, responses were rapid reaching peak within 2.91+/-0.04s, required extracellular Ca(2+), were inhibited by Gd(3+), modified by extracellular pH and were not affected by deletion of either the single Gbeta or iplA genes. Responses also remained unaffected by disruption of p2xA or p2xE showing that these genes are not involved. Cu(2+) and Zn(2+) inhibited purine-evoked Ca(2+) influx with IC(50) values of 0.9 and 6.3microM, respectively. 300microM Zn(2+) completely abolished the initial large rapid rise in intracellular Ca(2+) 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.

Indicative of cell surface P2X ion channel activation, extracellular ATP evokes a rapid and transient calcium influx in the model eukaryote Dictyostelium discoideum. Five P2X-like proteins (dP2XA-E) are present in this organism. However, their roles in purinergic signaling are unclear, because dP2XA proved to have an intracellular localization on the contractile vacuole where it is thought to be required for osmoregulation. To determine functional properties of the remaining four dP2X-like proteins and to assess their cellular roles, we recorded membrane currents from expressed cloned receptors and generated a quintuple knock-out Dictyostelium strain devoid of dP2X receptors. ATP evoked inward currents at dP2XB and dP2XE receptors but not at dP2XC or dP2XD. beta,gamma-Imido-ATP was more potent than ATP at dP2XB but a weak partial agonist at dP2XE. Currents in dP2XB and dP2XE were strongly inhibited by Na(+) but insensitive to copper and the P2 receptor antagonists pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid and suramin. Unusual for P2X channels, dP2XA and dP2XB were also Cl(-)-permeable. The extracellular purinergic response to ATP persisted in p2xA/B/C/D/E quintuple knock-out Dictyostelium demonstrating that dP2X channels are not responsible. dP2XB, -C, -D, and -E were found to be intracellularly localized to the contractile vacuole with the ligand binding domain facing the lumen. However, quintuple p2xA/B/C/D/E null cells were still capable of regulating cell volume in water demonstrating that, contrary to previous findings, dP2X receptors are not required for osmoregulation. Responses to the calmodulin antagonist calmidazolium, however, were reduced in p2xA/B/C/D/E null cells suggesting that dP2X receptors play a role in intracellular calcium signaling.