Contribution of endoplasmic reticulum to Ca(2+) signals in Dictyostelium depends on extracellular Ca(2+).

We recently reported the first molecular genetic evidence that Dictyostelium Ca(2+) responses to chemoattractants include a contribution from the endoplasmic reticulum (ER) - responses are enhanced in mutants lacking calreticulin or calnexin, two major Ca(2+)-binding proteins in the ER, even though the influx of Ca(2+) into the mutants is reduced. Compared with wild-type cells, the ER in the mutants contributes at least 30-70 nM additional Ca(2+) to the responses. Here we report that this additional ER contribution to the cytosolic Ca(2+) signal depends upon extracellular Ca(2+)- it does not occur in the absence of extracellular Ca(2+), increases to a maximum as the extracellular Ca(2+) levels rise to 10 microM and then remains constant at extracellular Ca(2+) concentrations up to at least 250 microM. These results suggest that Ca(2+) influx causes the intracellular release, in the simplest scenario by a mechanism involving Ca(2+)-induced Ca(2+) release from the ER. By way of contrast, we show that Ca(2+) responses to mechanical stimulation are reduced, but still occur in the absence of extracellular Ca(2+). Unlike the responses to chemoattractants, mechanoresponses thus include contributions from the ER that are independent of extracellular Ca(2+).

Release of Ca2+ from the endoplasmic reticulum contributes to Ca2+ signaling in Dictyostelium discoideum.

Ca2+ responses to two chemoattractants, folate and cyclic AMP (cAMP), were assayed in Dictyostelium D. discoideum mutants deficient in one or both of two abundant Ca2+-binding proteins of the endoplasmic reticulum (ER), calreticulin and calnexin. Mutants deficient in either or both proteins exhibited enhanced cytosolic Ca2+ responses to both attractants. Not only were the mutant responses greater in amplitude, but they also exhibited earlier onsets, faster rise rates, earlier peaks, and faster fall rates. Correlations among these kinetic parameters and the response amplitudes suggested that key events in the Ca2+ response are autoregulated by the magnitude of the response itself, i.e., by cytosolic Ca2+ levels. This autoregulation was sufficient to explain the altered kinetics of the mutant responses: larger responses are faster in both mutant and wild-type cells in response to both folate (vegetative cells) and cAMP (differentiated cells). Searches of the predicted D. discoideum proteome revealed three putative Ca2+ pumps and four putative Ca2+ channels. All but one contained sequence motifs for Ca2+- or calmodulin-binding sites, consistent with Ca2+ signals being autoregulatory. Although cytosolic Ca2+ responses in the calnexin and calreticulin mutants are enhanced, the influx of Ca2+ from the extracellular medium into the mutant cells was smaller. Compared to wild-type cells, Ca2+ release from the ER in the mutants thus contributes more to the total cytosolic Ca2+ response while influx from the extracellular medium contributes less. These results provide the first molecular genetic evidence that release of Ca2+ from the ER contributes to cytosolic Ca2+ responses in D. discoideum.