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First published online 15 August 2006
doi: 10.1242/jcs.03075

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An Ins(1,4,5)P₃ receptor in Paramecium is associated with the osmoregulatory system

Department of Biology, University of Konstanz, 78457 Konstanz, Germany

View larger version (34K): [in a new window]   Fig. 1. Molecular characterization of PtIP3RN. (A) Schematic representation of macronuclear sequences of the PtIP3RN gene from P. tetraurelia (Pt): The PtIP3RN gene is flanked upstream by a gene (PtHG) homologous to hemoglobin of Paramecium triaurelia (Yamauchi et al., 1995), accession number S60032, and downstream by a gene (PtSNF1 PK) homologous to a putative SNF1-related protein kinase [(Zagulski et al., 2004), accession No YP_054292]. Start (+1) and stop codons (+8825) of IP3RN were determined by RT-PCR, likewise introns, which are shown as triangles. The positions of the introns are indicated at nucleotide level at 5' (intron 5') and 3' end (intron 3'). (B) Domain structure of the Paramecium Ins(1,4,5)P3 receptor IP3RN. Results of sequence analysis of single domains are summarized in the table. (C) Modeling of the Ins(1,4,5)P3-binding domain using the Swiss-Model homology-modeling server (Peitsch and Jongeneel, 1993). (Right) Published structure of the Ins(1,4,5)P3-binding region of mouse Ins(1,4,5)P3 receptor type 1 (Bosanac et al., 2002); (left) model of the Paramecium Ins(1,4,5)P3-binding region. Areas that were aligned are shown in green, red regions are not compatible to the given structure. The arrow indicates the Ins(1,4,5)P3-binding site. (D) Hydrophobicity analysis of the channel domain of IP3RN reveals six transmembrane regions. (E) Evolutionary relationship of the Paramecium IP3RN protein. Predictions from multiple sequence alignments are shown in a neighbor-joining tree with 1000 bootstrap replicates generated with the MEGA version 3.0 program. Sequences representing the three different types of mammalian Ins(1,4,5)P3 receptors were from Mus musculus (MmIP3R type 1, P11881), Homo sapiens (HsIP3R type 2, Q14571) and Rattus norvergicus (RnIP3R type 3, AAA41446). Other metazoan IP3R sequences were from Aplysia californica (AcIP3R, ABD62080), Oikopleura dioica (OcIP3R, AAT47836), Caenorhabditis elegans (CeIP3R1-IFa, AAW30668), Xenopus laevis (XlIP3R, BAA03304), Drosophila melanogaster (DmIP3R, BAA14399), Panulirus argus (PaIP3R, AAC61691) and Asterina pectinifera (ApIP3R, BAB84088). Bootstrap support values are given above the branches and evolutionary distances are indicated by the scale bar below.

View larger version (99K): [in a new window]   Fig. 2. Sequence analysis of IP3RN. Alignment of PtIP3RN and rat Ins(1,4,5)P3R3 (RnIP3R3) using the `Blast 2 Sequences' tool (Tatusova and Madden, 1999). Sequences are shown in single-letter code and are numbered on the left side. Residues that are identical are shaded black, similar residues are shaded gray. The putative binding region for Ins(1,4,5)P3 is boxed in yellow. Amino acids shaded blue are involved in Ins(1,4,5)P3-binding, those bordered in dark blue are essential for Ins(1,4,5)P3-binding (Yoshikawa et al., 1996). The homology domain for Ins(1,4,5)P3Rs and RyRs (RIH-domain) is boxed in green, the channel domain in light blue. The six transmembrane regions are highlighted in red, the pore region in yellow. The antigenic region used to raise a polyclonal Ab is boxed in gray. Rat Ins(1,4,5)P3R3 sequences (accession number L06096) are published by Blondel et al. (Blondel et al., 1993).

View larger version (28K): [in a new window]   Fig. 3. Expression and [3H]Ins(1,4,5)P3-binding activity of the putative Ins(1,4,5)P3-binding domain of PtIP3RN. (A) Overexpression of GFP alone (top) and GFP fused to the putative Ins(1,4,5)P3-binding domain (IP3BD) of IP3RN (bottom) in Paramecium 7S cells. (B) Western blot analysis of immuno-precipitated GFP-IP3BD fusion protein or GFP alone with GFP-specific Ab. (C) [3H]Ins(1,4,5)P3-([3H] IP3) binding assay using agarose beads coupled to protein A either with GFP alone or with GFP-IP3BD fusion protein. Inhibition of specific [3H]Ins(1,4,5)P3-binding was measured in the presence of 10 µM cold Ins(1,4,5)P3. The graph represents one out of five experiments.

View larger version (35K): [in a new window]   Fig. 4. Characterization of polyclonal antibodies against IP3RN. (A) Affinity-purified anti-IP3RN Abs recognize the polypeptide corresponding to IP3RN residues R896-Q1001 (AG) with high affinity in immuno-blots (second and third lanes), whereas the preimmunserum (PIS) does not show any interaction (fourth lane). The first lane, C, shows 2 µg of the purified AG (Coomassie Blue-stained) used for immunization. (B) Western blot analysis using anti-IP3RN Abs. 100,000-g pellet of whole Paramecium cell homogenate (left lane) was extracted with 2% Triton X-100 and insoluble proteins (middle lane) were separated from soluble proteins (right lane). (C) Subcellular distribution of IP3RN in Paramecium cells. Immunofluorescence analysis shows that Abs against IP3RN stain the ORS.

View larger version (94K): [in a new window]   Fig. 5. Immuno-gold EM localization of PtIP3RN. (A,B) Dense labeling (gold grains) occurs in the layer around the collecting canals (CC) and represents the smooth spongiome (SS) (A), whereas the decorated spongiome (DS) shows only few gold grains (B). (C) Labeling also occurs directly adjacent to the lumen of the collecting canal. Bars, 0.1 µm.

View larger version (57K): [in a new window]   Fig. 6. Influence of extracellular [Ca2+]o on IP3RN gene expression. (A) Immuno-fluorescence images using IP3RN-specific Abs (upper panels, -IP3RN) or Abs against V-type H+-ATPase (lower panels, -vATPase). Cells were exposed for 24 hours to different levels of [Ca2+]o as indicated. IP3RN seems to be downregulated with decreasing [Ca2+]o. Images were acquired and processed under strictly identical conditions. (B) RNA prepared from cells, which were incubated for 24 hours with different [Ca2+]o, was analyzed by RT-PCR using primers against PtIP3RN or against Ptactin8 (control). (C) Quantification of fluorescence confirms downregulation of IP3RN mRNA compared with actin-8.

View larger version (40K): [in a new window]   Fig. 7. Li+ affects subcellular distribution of IP3RN. (A) Immuno-fluorescence analysis of cells grown in media with 1 µM [Ca2+]o and incubated with 25 mM LiCl for the times indicated, followed by immuno-labeling with IP3RN-specific Abs (left panels, -IP3RN) or Abs against V-type H+-ATPase (right panels, -vATPase). The IP3RN is selectively affected, with a maximal outcome after 3 hours, resulting in reduced ORS-staining and increased diffuse background fluorescence. As a control, cells were treated with NaCl for 3 hours (bottom panels) with no remarkable effect. (B) Contraction periods of contractile vacuoles of cells treated with LiCl for 3 hours are significantly prolonged (black bar) when compared to control cells (white bar, P<0.001) or to cells treated with NaCl (light gray bar, P<0.001). Contraction periods of cells incubated with NaCl are only slightly prolonged in comparison to control cells, with weak significance (P=0.014 for NaCl to untreated control). Three hours after treatment with LiCl, contraction periods return to control levels when cells are transferred to culture medium (dark gray bar).

View larger version (82K): [in a new window]   Fig. 8. Recordings of Ca2+ signals in close proximity to the ORS. (A) Elevated by a large Ca2+ signal traveling through the whole cell, small Ca2+ sparks localized close to the ampullae and radial arms (RA) can be observed (arrows) These spontaneous subcellular Ca2+ signals move along the ORS (CV, contractile vacuole). (B) Line tracings of the different spots marked by colored arrows in A. Trace d represents the large Ca2+ signal which elevates the small additional Ca2+ sparks visualized in traces a-c.

View larger version (59K): [in a new window]   Fig. 9. Effect of Ins(1,4,5)P3-uncaging on local Ca2+ signaling at different distances from the ORS. Recordings of Ca2+ signals before (-0.36 seconds) and directly after the release of caged Ins(1,4,5)P3 by UV light (+0.36 seconds, +0.72 seconds) show an increase in fluorescence after the activation of Ins(1,4,5)P3 in close proximity to the ORS (arrow) (bar, 10 µm). Graph shows Ca2+ traces analyzed from different areas in the cell, which were adjusted to the minimal fluorescence value before UV illumination (Fmin). During baseline recordings, spontaneous Ca2+ signals are observed. Illumination with UV light for 1 second (gray bar), i.e. uncaging of Ins(1,4,5)P3, results in an increase of the Ca2+ signal from the area close to the upper vacuole (red) which is not seen to such an extent in the anterior (green) or posterior (blue) part of the cell.

View larger version (16K): [in a new window]   Fig. 10. Effects of Ins(1,4,5)P3-uncaging on Ca2+ oscillations over large areas; anterior (left) and posterior cell poles (right). Recordings of Ca2+ signals (red) before and after the release of caged Ins(1,4,5)P3 by UV light compared to the contraction period (black) of the contractile vacuole. Ca2+ signals were recorded on the anterior or posterior part of the same cell, away from contraction sites. Peaks of black curves correspond to the contracted state of the vacuole. During baseline recordings spontaneous Ca2+ oscillations are observed. Illumination with UV light (gray bar) for uncaging of Ins(1,4,5)P3 does not allow to recognize a global increase of Ca2+ signals, but affects the periodicity of the oscillating Ca2+-signal.

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