Ca(2+ )regulation in the absence of the iplA gene product in Dictyostelium discoideum

BACKGROUND: Stimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca(2+ )concentration ([Ca(2+)](i)). The [Ca(2+)](i)-change is composed of liberation of stored Ca(2+ )and extracellular Ca(2+)-entry. The significance of the [Ca(2+)](i)-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca(2+)-buffers in the cytosol indicates that a [Ca(2+)](i)-increase is required for chemotaxis. Yet, the iplA(- )mutant disrupted in a gene bearing similarity to IP(3)-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca(2+)](i)-transient which favours the view that [Ca(2+)](i)-changes are insignificant for chemotaxis. RESULTS: We investigated Ca(2+)-fluxes and the effect of their disturbance on chemotaxis and development of iplA(- )cells. Differentiation was altered as compared to wild type amoebae and sensitive towards manipulation of the level of stored Ca(2+). Chemotaxis was impaired when [Ca(2+)](i)-transients were suppressed by the presence of a Ca(2+)-chelator in the cytosol of the cells. Analysis of ion fluxes revealed that capacitative Ca(2+)-entry was fully operative in the mutant. In suspensions of intact and permeabilized cells cAMP elicited extracellular Ca(2+)-influx and liberation of stored Ca(2+), respectively, yet to a lesser extent than in wild type. In suspensions of partially purified storage vesicles ATP-induced Ca(2+)-uptake and Ca(2+)-release activated by fatty acids or Ca(2+)-ATPase inhibitors were similar to wild type. Mn(2+)-quenching of fura2 fluorescence allows to study Ca(2+)-influx indirectly and revealed that the responsiveness of mutant cells was shifted to higher concentrations: roughly 100 times more Mn(2+ )was necessary to observe agonist-induced Mn(2+)-influx. cAMP evoked a [Ca(2+)](i)-elevation when stores were strongly loaded with Ca(2+), again with a similar shift in sensitivity in the mutant. In addition, basal [Ca(2+)](i )was significantly lower in iplA(- )than in wild type amoebae. CONCLUSION: These results support the view that [Ca(2+)](i)-transients are essential for chemotaxis and differentiation. Moreover, capacitative and agonist-activated ion fluxes are regulated by separate pathways that are mediated either by two types of channels in the plasma membrane or by distinct mechanisms coupling Ca(2+)-release from stores to Ca(2+)-entry in Dictyostelium. The iplA(- )strain retains the capacitative Ca(2+)-entry pathway and an impaired agonist-activated pathway that operates with reduced efficiency or at higher ionic pressure.