Abstract
In the ciliate Paramecium, a variety of well characterized processes are regulated by Ca2+, e.g. exocytosis, endocytosis and ciliary beat. Therefore, among protozoa, Paramecium is considered a model organism for Ca2+ signaling, although the molecular identity of the channels responsible for the Ca2+ signals remains largely unknown. We have cloned - for the first time in a protozoan - the full sequence of the gene encoding a putative inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) receptor from Paramecium tetraurelia cells showing molecular characteristics of higher eukaryotic cells. The homologously expressed Ins(1,4,5)P3-binding domain binds [3H]Ins(1,4,5)P3, whereas antibodies unexpectedly localize this protein to the osmoregulatory system. The level of Ins(1,4,5)P3-receptor expression was reduced, as shown on a transcriptional level and by immuno-staining, by decreasing the concentration of extracellular Ca2+ (Paramecium cells rapidly adjust their Ca2+ level to that in the outside medium). Fluorochromes reveal spontaneous fluctuations in cytosolic Ca2+ levels along the osmoregulatory system and these signals change upon activation of caged Ins(1,4,5)P3. Considering the ongoing expulsion of substantial amounts of Ca2+ by the osmoregulatory system, we propose here that Ins(1,4,5)P3 receptors serve a new function, i.e. a latent, graded reflux of Ca2+ to fine-tune [Ca2+] homeostasis.
Highlights
Increases in the concentration of intracellular Ca2+, [Ca2+]i, govern a variety of processes in response to cell stimulation, such as exocytosis and cell contraction
We propose a role of PtIP3RN in the homeostasis of cytosolic [Ca2+]i based on spontaneous [Ca2+]i fluctuations seen along the osmoregulatory system (ORS) and the effect – variable – uncaging Ins(1,4,5)P3 has on these fluctuations
Cloning of the gene encoding Pt IP3RN A partial sequence resembling that of the Ins(1,4,5)P3 receptor, M24E11u(rc), was isolated in a pilot genome project of Paramecium (Dessen et al, 2001; Sperling et al, 2002)
Summary
Increases in the concentration of intracellular Ca2+, [Ca2+]i, govern a variety of processes in response to cell stimulation, such as exocytosis and cell contraction. A rise in intracellular Ca2+ may be due to Ca2+ influx from the outside medium or the activation of stores, such as the endoplasmic or sarcoplasmic reticulum (ER or SR). Stores may comprise Ca2+-release channels of the inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] or ryanodine receptor (RyR) type (Berridge et al, 2000). I.e. non-stimulated, activity of intracellular stores, and any involvement of such channels and their potential contribution to overall cell function, would be much less amenable to analysis than stimulated responses and, has so far not been described. We have found evidence that such problems may occur in the osmoregulatory system (ORS) of Paramecium.
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