Subplasmalemmal Ca-stores in Paramecium tetraurelia : Iientification and characterisation of a sarco(endo)plasmic reticulum-like Ca2+-ATPase by phosphoenzyme intermediate formation and its inhibition by caffeine

Considering increasing interest in calcium stores in protozoa, including parasitic forms, and specifically in subplasmalemmal stores in higher eukaryotes, we have isolated subplasmalemmal calcium stores (alveolar sacs) from the ciliated protozoan, Paramecium tetraurelia. Using antibodies against established sarco(endo)plasmic reticulum Caz+-ATPases (SERCAs) we detected in Western blots of subcellular fractions a band of -106 kDa size selectively in alveolar sacs but not, for example, in plasma membranes and concomitant restriction of immunofluorecence labelling to the cell cortex of permeabilised cells. These results are the same as with ABs against a peptide derived from a cloned SERCA-like gene from Paramecium [Hauser K., Pavlovic N., Kissmehl R., Plattner H. Molecular characterization of a sarco(endo)plasmic reticulum Ca2+-ATPase gene from Paramecium tefraurelia and localisation of its gene product to subplasmalemmal calcium stores. Biochem J 1998; 334: 31-381. When such isolated alveolar sacs were now tested for phosphoenzyme intermediate (EP) formation, a phosphoprotein of the same apparent molecular mass (-106 kDa) as in blots could be identified in gel autoradiograms. This EP corresponds to that formed in the reaction cycle of different SERCA-types, with dependency on Caz+ and Mg*+, sensitivity to La3+ or insensitivity towards calmodulin, calmodulin antagonists and vanadate. However, EP formation in alveolar sacs is not inhibited by established SERCA inhibitors (e.g. thapsigargi[ci]n tested up to 100 PM). Surprisingly, caffeine, which is frequently used to mobilise Ca2+ from intracellular stores, strongly inhibits EP formation. In parallel experiments, we did not find any similar effect with sarcoplasmic reticulum isolated from skeletal muscle. We conclude that the -106 kDa protein of alveolar sacs in Paramecium may represent a SERCA-like Ca*+-ATPase with some unorthodox features, which might be relevant also for some other protozoan systems. In this case, the established Ca*+-mobilizing effect of caffeine may be amplified by inhibiting store refilling.