Novel Types of Ca²⁺ Release Channels Participate in the Secretory Cycle of Paramecium Cells

Abstract

A database search of the Paramecium genome reveals 34 genes related to Ca 2؉ -release channels of the inositol-1,4,5-trisphosphate (IP 3 ) or ryanodine receptor type (IP 3 R, RyR). Phylogenetic analyses show that these Ca 2؉ release channels (CRCs) can be subdivided into six groups (Paramecium tetraurelia CRC-I to CRC-VI), each one with features in part reminiscent of IP 3 Rs and RyRs. We characterize here the P. tetraurelia CRC-IV-1 gene family, whose relationship to IP 3 Rs and RyRs is restricted to their C-terminal channel domain. CRC-IV-1 channels localize to cortical Ca 2؉ stores (alveolar sacs) and also to the endoplasmic reticulum. This is in contrast to a recently described true IP 3 channel, a group II member (P. tetraurelia IP 3 R N -1), found associated with the contractile vacuole system. Silencing of either one of these CRCs results in reduced exocytosis of dense core vesicles (trichocysts), although for different reasons. Knockdown of P. tetraurelia IP 3 R N affects trichocyst biogenesis, while CRC-IV-1 channels are involved in signal transduction since silenced cells show an impaired release of Ca 2؉ from cortical stores in response to exocytotic stimuli. Our discovery of a range of CRCs in Paramecium indicates that protozoans already have evolved multiple ways for the use of Ca 2؉ as signaling molecule.Ca 2ϩ is an important component of cell activity in all organisms, from protozoa to mammals. Thereby Ca 2ϩ may originate from the outside medium and/or from internal stores (7, 18). Ca 2ϩ release from internal stores is mediated by various Ca 2ϩ release channels (CRCs), of which the inositol-1,4,5-trisphosphate receptor (IP 3 R) and ryanodine receptor (RyR) families have been studied most extensively (8,9,29,63 (14). IP 3 generates and maintains a Ca 2ϩ gradient in the hyphal tip of Neurospora crassa and the IP 3 -sensitive channels have been reconstituted and characterized with the planar bilayer method (87). In summary, these publications suggest that IP 3 -dependent signaling pathways are conserved among unicellular organisms, including protozoa.Despite these data, the molecular characterization of IP 3 or ryanodine receptors in low eukaryotes is currently a challenge since the identification of orthologues has not been possible thus far, probably because of evolutionary sequence divergence (66). Traynor et al. (96) identified an IP 3 receptor-like protein, IplA, in Dictyostelium discoideum, which possesses regions related to IP 3 R sequences, but thus far no evidence for IP 3 interaction exists. We have recently described an IP 3 R in the ciliated protozoa Paramecium tetraurelia (referred to here as P. tetraurelia IP 3 R N ) (53), with features characteristic of mammalian IP 3 Rs in terms of topology and ability for IP 3 binding. The expression level of P. tetraurelia IP 3 R N is modulated by extracellular Ca 2ϩ concentrations ([Ca 2ϩ ] o ) and immunofluorescence studies reveal an unexpected localization to the contractile vacuole complex (CVC), the major organelle involved in osmoregulation...

Ca2+ signalling early in evolution – all but primitive

“…Most recent computational analysis of the mammalian RyR also came to the conclusion of six transmembrane domains (Ramachandran et al, 2013), just as we have shown for the homologous molecule CRC-IV in P. tetraurelia Ladenburger et al, 2009). .…”

“…By using the P. tetraurelia genomic database (Aury et al, 2006;Arnaiz et al, 2007), genomic sequences that are indicative of CRCs with characteristic domain structures have been identified (Ladenburger et al, 2006;Ladenburger et al, 2009;Ladenburger and Plattner, 2011) (Fig. 1).…”

“…In panel C, the repetitive arrangement of the cortical Ca 2+ stores can be clearly identified. B was previously published in Journal of Cell Science (Ladenburger et al, 2006); C was reproduced from Ladenburger et al, 2009 with permission from American Society for Microbiology . (Ladenburger and Plattner, 2011).…”

“…There is, however, no conclusive evidence that this protein acts as an intracellular Ca 2+ -release channel. Hitherto, CRCs have been extensively characterised only in the ciliate Paramecium tetraurelia (Box 1) -and this was through detailed domain analysis rather than analysis of overall similarities, gene silencing and monitoring physiological effects, including Ca 2+ dynamics (Ladenburger et al, 2006;Ladenburger et al, 2009;Ladenburger and Plattner, 2011). The CRCs family in P. tetraurelia is represented by 34 members (in contrast to only six members in vertebrates), which are surprisingly diverse in their molecular structure, subcellular distribution and physiology.…”

Biochemical and Genetic Evidence for the Presence of Multiple Phosphatidylinositol- and Phosphatidylinositol 4,5-Bisphosphate-Specific Phospholipases C in Tetrahymena

“…In this evolutionary context, ciliates like Tetrahymena and Paramecium represent unique cases. Besides the expansion of PI-PLC genes, which are likely to have nonredundant roles, at least in Tetrahymena (this study), they apparently express members of an expanded family of IP3R-related Ca 2ϩ channels, the functionality of which has been previously demonstrated, at least in Paramecium studies (27,28). Collectively, these findings imply that the regulation of Ca 2ϩ homeostasis via a PI-PLCIns(1,4,5)P 3 -IP3R axis is likely to be evolutionarily conserved and elaborated in ciliates; accordingly, future studies aimed at unraveling the role of Tetrahymena PLC1-PLC2 (and PRIP) may focus primarily on well-established Ca 2ϩ -regulated processes.…”