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Eukaryotic Cell, September 2005, p. 1513-1525, Vol. 4, No. 9
1535-9778/05/$08.00+0     doi:10.1128/EC.4.9.1513-1525.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Release of Ca²⁺ from the Endoplasmic Reticulum Contributes to Ca²⁺ Signaling in Dictyostelium discoideum

Zofia Wilczynska,¹ Kathrin Happle,² Annette Müller-Taubenberger,³ Christina Schlatterer,² Dieter Malchow,² and Paul R. Fisher^1*

Department of Microbiology, La Trobe University, Victoria 3086, Australia,¹ Universität Konstanz, 78464 Konstanz,² Max-Planck-Institut für Biochemie, 82152 Martinsried bei München, Federal Republic of Germany³

Received 19 April 2005/ Accepted 17 June 2005

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.

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* Corresponding author. Mailing address: Department of Microbiology, La Trobe University, Victoria 3086, Australia. Phone: 61 3 9479 2229. Fax: 61 3 9479 1222. E-mail: P.Fisher{at}latrobe.edu.au.

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Eukaryotic Cell, September 2005, p. 1513-1525, Vol. 4, No. 9
1535-9778/05/$08.00+0     doi:10.1128/EC.4.9.1513-1525.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.