Tracing the Evolutionary History of Inositol, 1, 4, 5-Trisphosphate Receptor: Insights from Analyses of Capsaspora owczarzaki Ca2+ Release Channel Orthologs

Abstract

Cellular Ca(2+) homeostasis is tightly regulated and is pivotal to life. Inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) are the major ion channels that regulate Ca(2+) release from intracellular stores. Although these channels have been extensively investigated in multicellular organisms, an appreciation of their evolution and the biology of orthologs in unicellular organisms is largely lacking. Extensive phylogenetic analyses reveal that the IP3R gene superfamily is ancient and diverged into two subfamilies, IP3R-A and IP3R-B/RyR, at the dawn of Opisthokonta. IP3R-B/RyR further diversified into IP3R-B and RyR at the stem of Filozoa. Subsequent evolution and speciation of Holozoa is associated with duplication of IP3R-A and RyR genes, and loss of IP3R-B in the vertebrate lineages. To gain insight into the properties of IP3R important for the challenges of multicellularity, the IP3R-A and IP3R-B family orthologs were cloned from Capsaspora owczarzaki, a close unicellular relative to Metazoa (designated as CO.IP3R-A and CO.IP3R-B). Both proteins were targeted to the endoplasmic reticulum. However, CO.IP3R-A, but strikingly not CO.IP3R-B, bound IP3, exhibited robust Ca(2+) release activity and associated with mammalian IP3Rs. These data indicate strongly that CO.IP3R-A as an exemplar of ancestral IP3R-A orthologs forms bona fide IP3-gated channels. Notably, however, CO.IP3R-A appears not to be regulated by Ca(2+), ATP or Protein kinase A-phosphorylation. Collectively, our findings explore the origin, conservation, and diversification of IP3R gene families and provide insight into the functionality of ancestral IP3Rs and the added specialization of these proteins in Metazoa.

Keywords: 4; 5-trisphosphate receptor; Capsaspora owczarzaki; calcium release channels; inositol 1.

Fig. 1.

Reconstruction of IP₃R/RyR evolution in eukaryotes. Color lines represent the origin and presence of a particular gene and its diversification into paralog subfamilies. Red crosses indicate secondary losses. The consensus domain architectures of IP₃Rs and RyR are shown in the upper left. The tree represented eukaryotic tree is a consensus from the studies (Derelle and Lang 2012; He, et al. 2014). ¹Lost in vertebrates. ²Lost in most fungi, except two Zygomyceta species (Mucor circinelloides and Phycomyces blakesleeanus).

Fig. 2.

Expression and modulation of Capsaspora owczarzaki CO.IP₃R-A, CO.IP₃R-B, and RyR. (A) Scanning electron microscopy micrograph of C. owczarzaki filopodial stage amoeba. (B) Expression of COIP₃R-A, CO.IP₃R-B, RyR, and GAPDH genes in C. owczarzaki determined by RT-PCR. (C) Representative semiquantitative RT-PCR of CO.IP₃R-A, CO.IP₃R-B, and RyR in C. owczarzaki growing under control (C) or starvation (S) conditions. GAPDH served as a loading control. (D) Histograms generated from densitometric quantification of DNA gels as shown in (C). Values were normalized to the controls. Data represent mean ± SD of ≥3 independent experiments. (E) A schematic diagram depicting the three stages of C. owczarzaki life cycle: Aggregative, filopodial, and cystic stages. (F) Expression of COIP₃R-A, CO.IP₃R-B, and RyR genes in different life stages of C. owczarzaki. Barplots indicate the FPKM values of each gene in the different stages color-coded as in (E). Asterisks indicate that the gene is significantly differentially expressed in both (two asterisks) or only one (one asterisk) pairwise comparison (aggregative vs. fillopodial and aggregative vs. cystic). Bars show standard error.

Fig. 3.

Generation of stable cells and assessing ligand binding, functionality and subcellular localization of CO.IP₃R-A and CO.IP₃R-B. (A) Blot: Lysates prepared from DT40-3KO or DT40-3KO cells stably expressing HA-tagged CO.IP₃R-A or CO.IP₃R-B were fractionated on 5% SDS-PAGE and processed in immunoblots with HA11 antibodies. Representative experiment is shown. Histograms: HA-tagged CO.IP₃R-A and CO.IP₃R-B were immunopurified and incubated with tritiated IP₃ without or with cold IP₃. Bound radioactivity as CPM was measured by scintillation. Protein A/G agarose beads were used as negative control. Total specific binding was determined by subtracting CPM in the presence of cold IP₃ from CPM in its absence. (B) HA-tagged CO.IP₃R-A was immunopurified and incubated with tritiated IP₃ and increasing concentrations of cold IP₃. Specific binding is determined by subtracting CPM values obtained in the presence of 50 μM cold from the CPM values obtained with other conditions. Normalized specific binding from three to four experiments were averaged and used for nonlinear curve fitting. (C) DT40-3KO or DT40-3KO stably expressing various IP₃R constructs were loaded with Fura-2AM, and stimulated with 500 nM trypsin to induce IP₃ formation. Ca²⁺ release was measured as a change in the 340/380 fluorescence ratios. Ratio values were normalized to the initial baseline. Shown are representative Ca²⁺ traces from the indicated cell lines. (D) Bar graphs depict the average maximum change over basal 340/380 fluorescence ratios resulting from trypsin stimulation of cells expressing the indicated constructs. Experiments were repeated at least three times with greater than 40 cells analyzed in each experiment. Data are presented as mean ± SE. HEK293 cells coexpressing Venus-UBC6 along with CO.IP₃R-A-mCherry (E) or CO.IP₃R-B-mCherry (F) were grown on glass coverslips, fixed with methanol, mounted on a slide. Images were captured using two-photon confocal microscopy.

Fig. 4.

Oligomerization of CO.IP₃R-A and CO.IP₃R-B. HEK293T was transfected with flag-tagged CO.IP₃R-A and HA-tagged CO.IP₃R-B (A), HA-tagged CO.IP₃R-B and rIP₃R1 (B) or HA-tagged CO.IP₃R-A and rIP₃R1 (C). Lysates were prepared from transfected cells and immunoprecipitated with the indicated antibodies. Mock-immunoprecipitates were carried out identically but with the omission of the immunoprecipitating antibody. The input and immunoprecipitates were processed in immunoblots and visualized by LI-COR Odyssey infrared imaging system. Representative experiments are shown. (D) Histograms depicting quantification of coimmuoprecipitated proteins shown in (A)–(C). The relative immunoreactivities of proteins were quantified and expressed as the ratios of coimmunoprecipitated proteins normalized to the amounts of the coimmunoprecipitated proteins in the input and divided by the immunoprecipitated proteins. Data are presented as mean ± SD of ≥3 independent experiments.

Fig. 5.

Modulation of CO.IP₃R-A by Ca²⁺. DT40.3KO cells expressing rIP₃R1 (A) or CO.IP₃R-A (B) were loaded with Mag-Flou4 and permeabilized. 1.5 mM ATP was added to activate SERCA pumps and fill intracellular calcium stores. Cells were then stimulated with the indicated concentrations of IP₃ in the absence (black traces) or presence of BAPTA (red traces). Fluorescence was normalized to the initial fluorescence intensity prior to release by IP₃. (C) Histograms depict maximum calcium release normalized and expressed as a percentage of control from experiments in (A) and (B).

Fig. 6.

Modulation of CO.IP₃R-A by ATP. (A, B) DT40.3KO expressing CO.IP₃R-A were loaded with Mag-Flou4 and permeabilized. 100 μM ATP was added to activate SERCA pumps and fill intracellular calcium stores. Cells were then stimulated with the indicated concentrations of IP₃ in the presence of 100 μM (A) or 1 mM ATP (B). Fluorescence was normalized to the initial fluorescence intensity prior to release by IP₃. (C) Rate of calcium release calculated from experiments in (A) and (B) by fitting the average time courses from the first 30 s of IP₃ addition. (D) Rate of calcium release calculated from experiments using cells expressing rIP₃R1 performed as in (A)–(C). Rates were normalized to the maximum release rate. Three independent plates were used. (E) DT40-3KO or DT40-3KO stably expressing wild-type CO.IP₃R-A and CO.IP₃R-AΔATPB were loaded with Fura-2AM, and stimulated with 500 nM trypsin. Ca²⁺ release was measured as a change in the 340/380 fluorescence ratios. Ratio values were normalized to the initial baseline. Shown are representative traces.

Fig. 7.

Modulation of CO.IP₃R-A by cyclic AMP-dependent protein kinase. (A) DT40.3KO expressing rIP₃R1 were loaded with Fura-2AM and plated into a 0.1% (w/v) polylysine coated 96-well plate. Cells were preincubated with 5 µM forskolin or DMSO followed by the addition of the indicated amounts of PAR peptide to activate PAR receptor. Ca²⁺ traces were acquired and analyzed using SoftMax Pro Microplate Software. Peak fluorescence for each well was normalized to the baseline fluorescence and was expressed as a percentage of the control maximum. Representative traces are shown. (B) Quantification of Ca²⁺ release expressed as percent of maximum release in cells expressing rIP₃R1 in response to different PAR peptide concentrations. (C) Quantification of Ca²⁺ release expressed as percent of maximum release in cells expressing CO.IP₃R-A treated as in the (A) and (B).