Biochemical characterization, distribution and phylogenetic analysis of Drosophila melanogaster ryanodine and IP₃ receptors, and thapsigargin-sensitive Ca²⁺ ATPase

Intracellular Ca²⁺ dynamics is a key factor in cellular signaling and physiology (for a review, see Bootman et al., 2001). In particular, the system of endomembranes that forms the sarco(endo)plasmic reticulum plays a vital role in Ca²⁺ handling in most eukaryots(Carafoli and Klee, 1999). In this compartment two families of intracellular Ca²⁺ release channels have been characterized: the ryanodine receptors (RyR) and the inositol 1,4,5-trisphosphate receptors (IP₃R)(Nori et al., 1993). Besides these, there is evidence for two more intracellular Ca²⁺ releasing channels: the NAAPD and the sphingolipid receptors(Petersen and Cancela, 1999; Cancela, 2001). Ca²⁺re-uptake by the sarco(endo)plasmic reticulum is mediated by the thapsigargin-sensitive sarco(endo)plasmic reticulum Ca²⁺ ATPase(SERCA), whose structure and function has been studied extensively in mammalian muscle systems (MacLennan,1990).

The development of the fruit fly Drosophila melanogaster is amenable to multidisciplinary analyses (for a review, see Campos-Ortega and Hartenstein,1997) and is thus a powerful system in which to examine the role of these proteins in intracellular Ca²⁺ homeostasis. In this organism, a single RyR gene with 26 exons, dry, and a single IP₃R gene with 12 exons, dip, exist and have been genetically characterized (Takeshima et al., 1994; Sinha and Hasan,1999). In contrast, RyR and IP₃R in vertebrates are coded by at least three different genes that, due to alternative splicing,present a large number of isoforms(Rubtsov and Batrukova, 1997; Marks, 1997). A similar situation occurs with the thapsigargin-sensitive Ca²⁺ ATPase. In D. melanogaster only one gene coding for this P-type ATPase has been detected, CaP60A (Magyar et al.,1995); whereas in vertebrates, at least three different isoforms of this Ca²⁺ ATPase have been reported(Misquitta et al., 1999).

Despite extensive genetic and molecular biology data for these proteins,there is a dearth of basic biochemical information on the DrosophilaRyR, IP₃R and SERCA proteins. In order to reap the benefit from a genetic and molecular biology tractable model organism with single RyR,IP₃R and SERCA proteins, we characterize here these important molecules using Drosophila native endomembranes.

1. To characterize the proteins biochemically and pharmacologically we performed radioligand-binding assays for the Drosophila ryanodine and IP₃ receptors, and a coupled enzymatic determination for the thapsigargin-sensitive Ca²⁺ ATPase. These studies used subcellular fractions of native membranes of this insect, allowing us to compare the biochemical properties of these Drosophila proteins with their vertebrate homologs.

2. We complemented these data with a detailed evaluation of the anatomical localization of these proteins in embryos and adults using fluorescent probes,confocal microscopy and image analysis. In particular, we noted widespread and high levels of expression and co-expression in the digestive tract.

3. We performed a comprehensive comparison of the amino acid sequences of the two calcium release channels and the endoplasmic reticulum Ca²⁺pump from D. melanogaster with similar proteins from other species. Our results show that these Drosophila proteins share many characteristics with their vertebrate cognates. However, differences in[³H]-ryanodine binding and SERCA activity compared with vertebrate isoforms, imply also unique properties of the Drosophila RyR and SERCA.

Wild type Oregon R (WT) and mutant yellow white (lighter cuticle color and white-eyed) (YW) stocks of D. melanogaster were used for the experiments. YW flies were included to ask whether the Ca²⁺-binding capability of the pigment present in WT flies could influence the biochemical assays or the fluorescent images.

Membrane preparation started with 5-7 g of adult flies, and was performed according to Damiani et al. (Damiani et al., 1991), as modified by Martinez-Merlos et al.(Martinez-Merlos et al.,1997). Briefly, three fractions: total membranes (TM), low-speed pellet (LSP), and soluble fraction (SF) were obtained. Flies were homogenized with a Politron in 10 volumes of 10 mM HEPES, pH 7.4, 20 mM KCl, 0.5% CHAPS, 1 mM EGTA and one pill of the peptidase-inhibitors Complete Inhibitors (Roche,Basel, Switzerland). LSP was obtained after centrifugation of the homogenate at 650 g for 10 minutes and resuspension in 10 mM HEPES, pH 7.4, 0.5 M NaCl. The supernatants were then centrifuged at 120,000 g for 90 minutes, to obtain the SF (supernatant) and the TM(pellet) fractions. TM, including mitochondrial and microsomal membranes, was resuspended in 0.3 M sucrose, 10 mM imidazole, pH 7.4 with the peptidases-inhibitor Complete Inhibitors. This method allows suitable membrane preparations for [³H]-ryanodine and [³H]-IP₃binding using small portions of tissue(Damiani et al., 1991).

The protocol for the microsomal fraction was similar to the one mentioned above, except for the centrifugation cycles(Aguilar-Delfín et al.,1996): (1) 1000 g for 10 minutes; (2) supernatant then centrifuged at 9500 g for 30 minutes; (3) second supernatant ultracentrifuged at 110,000 g for 90 minutes; and finally (4) the pellet containing the crude microsomal fraction was resuspended and stored as above. Protein concentration was quantified following Lowry et al. (Lowry et al.,1951) with bovine serum albumin as standard.

[³H]-ryanodine was incubated for 16 hours at room temperature with 100 μg of the Drosophila TM or the microsomal fraction in 0.25 ml of binding buffer containing 200 mM MOPS, pH 7.4, 1 mM CaCl₂, 0.3 M KCl, 10 mg/ml bovine serum albumin (BSA) and 3 nM[³H]-ryanodine (Chu et al.,1990). Non-specific binding was defined using 10 μM unlabeled ryanodine. At the end of the incubation time, the samples were filtered through Whatman GF/F glass fiber filters using a multifilter harvester(Brandel, Gaithersburg, MD). The filters were washed with five 5 ml aliquots of cold 0.3 M KCl and counted in a liquid scintillation counter, after the addition of 5 ml of Tritosol (Fricke,1975). Free Ca²⁺ concentrations in the samples were calculated with the program Chelator(Schoenmakers et al.,1992).

[³H]-IP₃ was incubated for 30 minutes at 0°C with 100 μg of Drosophila TM or microsomal fraction in 120 μl of binding buffer containing 25 mM Tris-HCl, pH 8.5, 5 mM NaHCO₃, 1 mM EDTA, 0.25 mM DTT and 4 nM [³H]-IP₃ following Furiuchi et al. (Furiuchi et al., 1993). Non-specific binding was defined with 10 μM of unlabeled IP₃. The samples were filtered through Whatman GF/F glass fiber filters using a multifilter harvester (Brandel, Gaithersburg, MD). The filters were then washed with five 5 ml aliquots of a buffer containing 25 mM Tris-HCl, pH 8.0, 5 mM NaHCO₃ and 1 mM EDTA, and counted in a liquid scintillation counter, after the addition of 5 ml of Tritosol(Fricke, 1975). In both assays, Scatchard plots were analyzed by linear regression.

ATPase activities were measured following Saborido et al.(Saborido et al., 1999), by using the coupled enzymatic assay of Chu et al.(Chu et al., 1988), where the rate of ATP hydrolysis is calculated from the spectrophotometric data of NADH oxidation at 340 nm.

To measure the combined Ca²⁺ and Mg²⁺ ATPases activities, the reaction mixture (1 ml final volume) contained 1 μg of protein of microsomal membranes, 19.5 mM MOPS, pH 7.0, 0.78 mM EGTA, 11.7 mM MgCl₂, 156 mM KCl, 10 mM phosphoenol pyruvate, 4 μM of the Ca²⁺ ionophore A23187, 9.1 units of pyruvate kinase, 5.7 units of lactate dehydrogenase, 0.3 mM NADH and 0.78 mM CaCl₂. To eliminate the contribution of the Ca²⁺ ATPase to the reaction, 16.4 mM CaCl₂ was added to the reaction mixture in parallel assays. The assays started with the addition of 4 mM ATP and the ATPases activities were followed by a decrease in optical density at 340 nm for the next 3-5 minutes. Additionally, we repeated this assay including two ionophores (nigericin 1μm and valinomycine 10 μm) besides the calcium ionophore A23187, to control for possible iontophoretic effects of thapsigargin at the concentrations used. Results were not significantly different from assays without the ionophores (n=5).

The sensitivity of the Ca²⁺ ATPase to thapsigargin was measured in the same conditions described above, but in the presence of 20, 50, 80,100, 120 and 150 μM of thapsigargin (Calbiochem, San Diego, CA).

Adults were allowed to lay eggs on fruit juice agar plates seeded with yeast paste at 25°C for 3 hours. The embryos were collected with a paintbrush onto a mesh, rinsed with distilled water, and dechorinated in 50%chlorox, rinsed with water, and transferred to microfuge tubes. Next, they were permeabilized in heptane for 30 seconds, followed by the addition of fixing solution (37% formaldehyde in phosphate buffered saline solution (PBS)and 50 mM EGTA, pH 7.5) and incubated with gentle agitation for 1 minute. The viteline membrane was removed by shaking the embryos vigorously for 1 minute after adding 1 ml of 100% methanol to the tubes and having removed the fixing solution. Finally, embryos were rehydrated in PBS and incubated with 1 μM BODIPY TR-X Ryanodine, or 5 μM BODIPY FL-Thapsigargin, or 2 μM FL-Heparin (Molecular Probes, Eugene, Oregon), or combinations of the above for 2 hours. To perform the experiments with FL-Heparin, it was necessary to treat the embryos with 0.3% Triton-X 100 for 2 hours before the addition of the fluorescent compound. To estimate non-specific binding, control embryos were incubated with 100 μM ryanodine, thapsigargin, and heparin, and the correspondent fluorescent derivative, respectively. After incubation, embryos were washed twice with PBS, placed on microscope slides and observed on a NIKON PCM2000 confocal microscope(Cifuentes et al., 2001).

Organisms under CO₂ anesthesia were immersed on Tissue-Tek and frozen (Leica, Nussloch, Germany). 8-μm-thick cryostat sections were cut(Leica, Nussloch, Germany), dried for 30 minutes at 60°C, fixed in 3%glutaraldehide for 30 minutes at 37°C, and incubated with the fluorescent compounds using the same conditions as embryos(Thompson et al., 1997). Controls for non-specific binding were done as mentioned above.

Complete amino acid sequences corresponding to 15 RyRs (10 species), 13 IP₃Rs (8 species), and 21 SERCAs (14 species) were obtained from the Swiss-Prot and NCBI sequence banks. Drosophila, other invertebrate, and different vertebrate isoforms were used for sequence examination and construction of phylogenetic trees. On average, the number of total amino acids corresponding to each protein was: RyR, 5200;IP₃Rs, 3250; SERCAs, 300. Proteins and species used in the study are listed in Table 1.

Full-length proteins were initially aligned using the program CLUSTALX,version 1.8.1 (Thompson et al.,1997). We employed the Program BLAST-2 SEQ 2.2.2 to assess sequence relatedness using the whole set of amino acids of each protein and a PAM distance (number of accepted point mutations per 100 residues separating two sequences) below 250 (Tatusova and Madden, 1999). Using PHYLO_WIN(Galtier et al., 1996),phylogenetic trees were constructed by maximum parsimony and distance methods. A distance matrix of pairwise comparisons of the proportion of different amino acids per site was constructed using PROTDIST of PHYLIP, version 3.572c(Hillis, 1991). This program was used to derive a neighbor-joining tree whereas maximum-parsimony analysis was done using PHYLIP-PROTPARS (Hillis,1991). To assess support at each node, Bootstrap resampling analysis was performed (Galtier et al.,1996). However, only a limited number of replicates were done with Bootstrap analysis, since the capacity of the program was reached due to the large size of the protein sequences studied. The hierarchical structure of the trees was confirmed by the g1 statistic test(Felsenstein, 1996). Phylogenetic trees were displayed with the program TREEVIEW 1.6.6(Page, 1996).

Fig. 1A shows a saturation curve with increasing concentrations of [³H]-ryanodine in TM fractions of adult Drosophila. The saturation curve was hyperbolic but linear with Scatchard analysis (Fig. 1A, inset). The parameters obtained were: Bmax=0.42±0.06 pmol/mg of protein, with a K_d of 8.1±1.2 nM and a Hill coefficient of 1.0±0.2 (n=5). Similar binding constants were found when Oregon and Yellow-White stocks of Drosophila were used as sources of subcellular fractions (data not shown). Comparable results were attained when the microsomal fraction was used (data not shown).

Fig. 1B illustrates the pharmacological profile of Drosophila RyR evaluated by[³H]-ryanodine-binding assays(Chu et al., 1990; Antaramián et al.,2001). In agreement with results from vertebrate type 1, 2 and 3 RyRs (Antaramián et al.,2001; Zarka and Shashan-Barmatz, 1993; Manunta et al., 2000; Holmberg and Williams, 1990), AMP-PCP (1 mM) promoted a significant increment in [³H]-ryanodine binding (8.6 times over control). Caffeine (10 mM) enhanced [³H]-ryanodine binding in low (10 nM) free Ca²⁺, consistent with vertebrate results(Fig. 1B). We also tested the activator xanthine in the nM range. Some of us have characterized xanthine(5-10 nM) as an excellent activator of the rabbit RyR type 1(Butanda-Ochoa et al., 2003). Xanthine also promoted an important activation of the Drosophila RyR(5.3 times more than control; Fig. 1B). MgCl₂ (1 mM), Ruthenium Red (10 μM) and Dantrolene (50 μM) drastically inhibited (90-98%) [³H]-ryanodine binding.

Fig. 1C depicts the effect of increasing free Ca²⁺ concentrations on [³H]-ryanodine binding with Drosophila TM fractions. Like vertebrate RyRs, Drosophila RyR shows a gaussian profile of [³H]-ryanodine binding as a function of Ca²⁺ concentration, with a maximum at approximately 100 μM. The Ca²⁺-promoted activation presented an EC₅₀ between 1 and 10 μM, whereas the IC₅₀ occurred between 100 μM and 1 mM of free Ca²⁺. [³H]-ryanodine binding was always detectable in these assays, even with 10 mM free Ca²⁺.

Fig. 2 shows a representative [³H]-IP₃ saturation curve, the corresponding Scatchard analysis (Fig. 2A), and the effects of heparin 10 μg/ml, 2-aminoethoxydiphenyl borate (2-APB) 75 μM, and xestospongin C 5 μM on the[³H]-IP₃ binding(Fig. 2B). Only one high affinity binding site was detected for [³H]-IP₃ in the TM fraction. The Scatchard analysis indicated a Bmax=6.1±0.8 pmol/mg protein, a K_d of 7.3±0.9 nM and a Hill coefficient of 1.0±0.1. Similar binding constants were found when WT and YW stocks of Drosophila were used (data not shown).[³H]-IP₃ binding to Drosophila TM fractions was inhibited 75% by 1 mg/ml heparin, 85% by 75 μM 2-APB, and 90% by 5 μM xestospongin C.

We measured Mg²⁺ and Ca²⁺ ATPases activities in Drosophila microsomal fractions to characterize the thapsigargin-sensitive Ca²⁺ ATPase(Fig. 3). We employed the procedure reported by Saborido et al.(Saborido et al., 1999). First, so-called `total' ATPase activity was determined: Mainly Mg<sup>2+</sup> and Ca<sup>2+</sup>-ATPase activities. Then, we determined the`basal' or `background' ATPase activity; that is, ATPase activity under conditions where Ca<sup>2+</sup>-ATPase is inhibited. The difference between`total' and `basal' is SERCA activity. Fig. 3A shows that both WT and YW stocks of Drosophilapresented similar total activities. However, WT activities were ≈70% of YW,with ≈4 μmols NADH/mg protein/minute in WT, and ≈6.2 μmols NADH/mg protein/minute in YW.

Drosophila SERCA was sensitive to thapsigargin(Fig. 3B). This is shown by inhibition of SERCA activity in both WT and YW microsomal membranes in the presence of different amounts of thapsigargin. The IC₅₀ for this sesquiterpene lactone was approximately 80 μM. In this assay,Ca²⁺-ATPase activity was also inhibited by the addition of EGTA (1 mM) or by high Ca²⁺ concentrations (≈20 mM) (data not shown).

To characterize RyR, IP₃R and SERCA protein distribution in fly tissues, we used fluorescent compounds specific for RyR(TX-R-BODIPY-ryanodine), IP₃R (FL-Heparin), and thapsigargin-sensitive SERCA (FL-BODIPY-thapsigargin) (Figs 4, 5). Fig. 4A shows the signal associated with fluorescent ryanodine present in practically all cells of early embryos. Fig. 4D shows the label localized mainly to the cytoplasm of cells. The fluorescent ryanodine signal observed in older embryos (stages 15-17) clearly shows RyR at higher concentrations in the digestive tract(Fig. 4A'). The label associated with Drosophila SERCA(Fig. 4C) and IP₃R(Fig. 4B) in early embryos is also present in practically all cells. In late embryos[Fig. 4C' (SERCA); B'(IP3R)], label is present in nearly all tissues and is distributed more homogeneously than ryanodine signals. Labeling for all three fluorescent compounds is seen in tissues derived from all germinal layers:ectoderm (epidermis), mesoderm (muscle), and endoderm (digestive tract). As seen for the ryanodine receptor, higher magnification views of cells labelled with thapsigargin and heparin also show cytoplasmic staining(Fig. 4F,E, respectively)Co-localization of these compounds with fluorescent ryanodine illustrates that SERCA and RyR are highly coexpressed in the digestive tract(Fig. 4H), whereas coexpression of IP₃R and RyR is evenly distributed(Fig. 4G). Label observed in these experiments is specific, since coincubation with excess ryanodine,heparin or thapsigargin abolished labeling(Fig. 4A” for ryanodine, Fig. 4B” for heparin, and Fig. 4C” for thapsigargin).

Adult tissues were stained with BODIPY TR-X Ryanodine, BODIPY FL-Thapsigargin and FL-Heparin. A generalized RyR expression was observed,with higher levels in the digestive tract(Fig. 5A,E), muscle(Fig. 5A,D,D'), and adult optic lobe and retina (Fig. A,C). Label is cytoplasmic(Fig. 5D,D',E), as in embryos. Staining is seen in tissues of ectodermal origin (nervous system, Fig. 5A,C), mesodermal origin(indirect flight and leg muscles, Fig. 5A,D,D'), and endodermal origin (digestive tract, Fig. 5A,E). Staining for heparin was seen also in practically all adult tissues(Fig. 5B), and more homogeneous in levels than RyR. Most tissues show extensive colocalization of both labels(compare Fig. 5A and B). Colocalization of fluorescent ryanodine with fluorescent thapsigargin was coincidental (Fig. 5C).

We performed sequence analysis of RyR, IP₃R and SERCA as another way to address similarities to and differences from their vertebrate counterparts and within themselves. Computer-generated alignments of 15 RyRs,13 IP₃Rs and 21 SERCAs were analyzed. Table 1 shows the percentage identities between the Drosophila RyR, IP₃R and SERCA compared with corresponding proteins from other species. The extent of identity between Drosophila RyR and other RyRs considered in this study was the lowest and ranged from 37% (with Homo sapiens RyR type 1) to 45% (with Caenorhabditis elegans unique RyR isoform). It was thus not possible by this means to recognize an accentuated identity among Drosophila RyR and any of the three vertebrate isoforms in Table 1.

The identity detected between Drosophila IP₃R and other IP₃Rs was intermediate and ranged from 36% (with Caenorhabditis elegans unique IP₃R isoform) to 57% (with Panulirus argus unique IP₃R isoform). In contrast with RyR isoforms,vertebrate IP₃R type 1 isoform showed a slightly higher percentage of identity with the fruit fly receptor (56%), than IP₃R type 2(53%) and type 3 (50%). SERCA enzymes had the highest percentage of identity within themselves. The range went from 67% (with both Rattus norvergicus and Homo sapiens SERCA type 3) to 81% (with the Procambarus clarkii unique SERCA isoform). Drosophila SERCA had a slightly higher identity with vertebrate type 1 and 2 SERCAs (71-73%)than with type 3 (67-69%).

Equal-weight (`unrooted') Parsimony and Neighbor Joining analyses of the sequences were performed for RyRs, IP₃Rs(Fig. 6) and SERCAs(Fig. 7). Both programs yielded virtually identical topologies suggesting, as expected, that the three Drosophila proteins grouped together with all other invertebrate genes. Fig. 6A shows a phylogram where full-sequences of RyRs and IP₃Rs were analyzed together. Both types of calcium release channels were separated in the tree very clearly. Drosophila RyR was sister to C. elegans RyR,and both were in a different node from vertebrate RyRs. Type 2 and 3 RyRs were co-segregated in one group and separated from type 1 RyRs.

Drosophila IP₃R was sister to crustacean P. argus IP₃R, whereas the receptor of C. elegans split from all other IP₃Rs. Vertebrate type 1 and 2 IP₃Rs shared a common node. Fig. 5Bshows the unrooted cladogram following Bootstrap analysis to determine support at each node. Topology is very similar using the PROTDIST algorithm(Fig. 6A). The cladogram shows vertebrate and invertebrate RyRs and IP₃Rs forming distinct clades within each type of calcium release channel.

Fig. 7A shows a phylogram and a cladogram (Fig. 7B) based on the analysis of amino acid sequences of SERCAs considered in this study. Drosophila SERCA grouped with other invertebrates and was closer to vertebrate type 1 SERCAs. Vertebrate type 3 SERCAs were the more distal proteins compared with invertebrate SERCAs(Fig. 7A,B).

Intracellular calcium dynamics is governed mainly by the evolutionarily conserved Ca²⁺-mobilizing elements RyR, IP₃R and SERCA. Insights into the mechanisms of intracellular calcium signaling using Drosophila is facilitated by virtue of the existence of single RyR,IP₃R and SERCA molecules in this model. In addition, Drosophila melanogaster presents a unique possibility of studying cellular signaling by multidisciplinary approaches with well established molecular, genetic,cellular, physiological, and biochemical tools. This allows better opportunities of identification and characterization of essential properties and physiological significance of these proteins in cellular extracts. Once these basic parameters are defined, the relationship of the RyR,IP₃R and SERCA from Drosophila with their vertebrate counterparts can be worked out. The data obtained in this study lead in this direction.

Functional studies of the Drosophila intracellular calcium regulation proteins are scarce. The published information does not include studies done with native membranes. For example, a report indicating the biochemical characteristics of the Drosophila IP₃R used S2 cells, a cellular line derived from late embryonic states(Swatton et al., 2001).

The [³H]-ryanodine-binding assay is a specific, conformationally sensitive probe for the RyRs of skeletal and cardiac muscles(Takeshima et al., 1994; Pessah et al., 1987). Using this assay, we estimated the Bmax, K_d and activation state of this calcium release channel. [³H]-ryanodine bound a single class of sites in the Drosophila microsomal preparation. The Bmax obtained for Drosophila RyR (0.42±0.06 pmol/mg of protein) is lower than the value reported for rabbit sarcoplasmic reticulum heavy fraction, but is in the range of values reported for total membrane fractions of skeletal and cardiac muscles of several species of rodents(Martinez-Merlos et al.,1997). In general, the number of [³H]-ryanodine-binding sites in Drosophila microsomal membranes is higher than the Bmax described for cerebral fractions obtained with different membrane preparations(Martinez-Merlos et al., 1997; McPherson and Campbell, 1990). The affinity of [³H]-ryanodine for Drosophila RyR (8.1 nM)is in the range reported for almost all known vertebrate and invertebrates RyRs (Martinez-Merlos et al.,1997; Pozzan et al.,1994).

In general, the pharmacological profile of[³H]-ryanodine-binding assays was similar to previously reported results for vertebrate RyRs (Chu et al.,1990; Antaramián et al.,2001). Type 1 and 3 RyRs are more prone to respond to AMP-PCP than type 2 RyRs (Antaramián et al.,2001; Zarka and Shashan-Barmatz, 1993; Manunta et al., 2000; Holmberg and Williams, 1990). Thus, the large Drosophila RyR activation promoted by 1 mM AMP-PCP (Fig. 3B) is closer to responses elicited by type 1 and 3 RyRs. Xanthine(at the μM range) is an oxidized purine that is a good activator of the type 1 RyR from rabbit skeletal muscle(Butanda-Ochoa et al., 2003).[³H]-ryanodine binding in Drosophila RyR was enhanced 4-5 times by xanthine (Fig. 3B). This result is similar for both the Drosophila RyR and the rabbit type 1 RyR. [³H]-ryanodine binding to Drosophila RyR was inhibited by Mg²⁺ (2 mM) and ruthenium red (10 μM), in the same way as vertebrate RyR isoforms. However, inhibition by dantrolene(Fig. 3B) makes the Drosophila RyR more similar to type 1 and 3 RyRs, since Zhao et al.(Zhao et al., 2001) reported that RyR type 2 is not a target for dantrolene inhibition.

The calcium sensitivity of the [³H]-ryanodine-binding assay is one of the most important factors discriminating among different RyR isoforms. Whereas the type 1 and 3 RyRs show an unambiguous bell-shaped calcium dependence curve with increased sensitivity at low calcium concentrations for the RyR type 1 (Murayama et al.,1999), the RyR type 2 is not sensitive to inactivation by high calcium concentrations up to pCa 2 (Du et al., 1998). The result obtained with the calcium dependence curve of Drosophila RyR (Fig. 3C), indicates a pronounced similarity with RyR type 1, since the[³H]-ryanodine binding was visibly present at 1 μM of free Ca²⁺ in the assay, a condition where the activity of type 3 RyRs is not observed (Murayama et al.,1999). A distinctive characteristic of Drosophila RyR was its capacity to bind [³H]-ryanodine even at Ca²⁺concentrations in the millimolar range(Fig. 3C). This ability is in some way similar to the low Ca²⁺ dependence of inactivation of RyR type 2. Thus, Drosophila RyR shares features of both RyR type 1 and 2, but is closer to RyR type 1.

Drosophila RyR, as well as vertebrate RyR isoforms, are activated by 3-methyl xanthine and caffeine (2 mM). Zhang et al.(Zhang et al., 1999), reported that caffeine activation of lobster RyR is insensitive to Ca²⁺concentrations, which is different from caffeine activation of vertebrate RyRs. Further experiments are needed to confirm whether this caffeine-binding site reported for lobster skeletal RyR is also present in DrosophilaRyR.

In binding experiments using [³H]-IP₃ in Drosophila microsomal membranes, the ligand was bound to a single class of sites (Hill coefficient=1.0). The Bmax value indicates that, in Drosophila, the IP₃R is 15 times more abundant than the RyR in microsomes, a proportion that is similar to that reported for cerebral tissue in several species (McPherson and Campbell, 1990; Diaz-Muñoz et al.,1999). The affinity for [³H]-IP₃ found in Drosophila microsomal membranes (7.3 nM) was similar to the K_d reported for S2 cells(Swatton et al., 2001) and for the mammalian IP₃Rs subtypes including type 1 from rat cerebellum and type 2 from rat liver (Correa et al.,2001).

[³H]-IP₃ binding to microsomal membranes of Drosophila was inhibited by the competitive antagonist heparin(Fig. 2B) to a similar extent to that of vertebrate IP₃Rs(Mikoshiba et al., 1994). Unexpectedly, the noncompetitive IP₃R inhibitors, 2-APB and xestospongin C, reduced notably the [³H]-IP₃ binding to Drosophila microsomal membranes. These results are in contrast with reports showing that 2-APB and xestospongin C abolished ion transport through IP₃R without affecting the ability of[³H]-IP₃ to bind to microsomes from CHO cells and cerebellum, respectively (Kukkonen et al.,2001; Gafni et al.,1997). Further experiments exploring the inhibitory mechanism(s)of these drugs are needed to clarify the discrepancy between the pharmacological properties of Drosophila and vertebrate IP₃Rs.

Another difference in the pharmacological profile between Drosophila IP₃R and its vertebrate counterparts, is its increased sensitivity to the agonist adenophostin A(Swatton et al., 2001). This means that the recognition site for adenophostin could have different properties in the Drosophila IP₃R.

Given the high homology (≈70%) between Drosophila SERCA and the rest of the SERCA enzymes included in Table 1, it is very likely that most of the structure/function relationships and the mechanism of SERCA Ca²⁺ transport in vertebrate fast twitch skeletal muscle(MacLennan et al., 1997), are also present in this insect ATPase. The SERCA activity measured in Drosophila microsomal membranes was in the same range as those reported elsewhere for vertebrate isoforms(Saborido et al., 1999; Chu et al., 1988). Thapsigargin is a specific inhibitor of SERCAs, and has been used as a pharmacological probe to detect this family of enzymes. The inhibitory mechanism of this sesquiterpene lactone is to bind the enzyme during the Ca²⁺-deprived intermediate state, being usually effective at sub-nM concentrations (Mintz and Guillan,1997). High micromolar concentrations of thapsigargin (like the ones we used) can act as a Ca²⁺ ionophore(Favero and Abramson, 1994),but since our SERCA activity assay is done in the presence of the well known Ca²⁺ ionophore A23187, an iontophoretic activity of thapsigargin is not critical for interpretation of our results. The thapsigargin sensitivity that we observed in Drosophila microsomes was in the micromolar range(Fig. 3B). This clear discrepancy with previous reports, and also with the observation made in saponine-permeabilized S2 cells, could involve changes in the properties of the enzyme due to microsomal membrane preparation. Further experiments are also needed to clarify this point. Also, the fact that the YW stock had a higher activity than the WT stock might be related to the amount of pigment present within the cells.

The Mg²⁺-ATPase activity measured according to Saborido et al.(Saborido et al., 1999), is designed to evaluate the activity of E-type ATPases, and so it could be that the activity we measured in the Drosophila microsomal fraction is of E-type. Additional experiments are necessary to characterize the sensitivity of this enzymatic activity to specific inhibitors and substrates, to compare SERCA with other E-type Mg²⁺-ATPases(Plesner, 1995).

Fluorescently tagged ryanodine labeled nearly all cells in early and late embryos (Fig. 4). Label is cytoplasmic, consistent with labeling of endoplasmic reticulum(Fig. 4D). This generalized labeling is maintained throughout embryogenesis(Fig. 4A'), but some areas,like the digestive tract, accumulate higher amounts of label. Staining of adult fly cryostat sections showed similar results. Notably, the nervous system showed staining, albeit not particularly high, in agreement with behavioral and electrophysiological studies that have yet to show nervous system functional deficits in RyR and IP₃R mutants(Acharya et al., 1997) and a report showing adaptation deficits to odorant stimuli in antennal electrophysiological recordings in IP₃R receptor mutants(Deshpande et al., 2000). Results with both fluorescently tagged heparin and thapsigargin show overall similarities: early embryos show promiscuous staining in nearly all cells, and late embryos show staining in nearly all tissues with the digestive tract showing higher levels of staining. This higher level of digestive tract staining is more prominent with heparin. In both cases staining appears to be cytoplasmic, consistent with labeling of the endoplasmic reticulum(Fig. 4). Double labeling experiments show that, as expected, the digestive tract has generalized overlapping staining, with a preponderance of ryanodine staining(Fig. 4G,H).

These RyR results are in contrast with in situ hybridization experiments,where expression of RyR is seen at stage 9 at the earliest(Sullivan et al., 2000), and not as widespread as here. We interpret this as maternal contribution:ryanodine is well known as a very specific pharmacological binding agent for the RyRs (Chu et al., 1990; Pessah et al., 1987). Since we were able to outcompete the staining with non-fluorescent ryanodine, we conclude that RyR protein is present from the very early stages, with a subcellular localization consistent with its purported role in intracellular Ca²⁺ homeostasis.

The difference between our results and those obtained through in situ hybridization experiments could be due to the presence of maternally deposited RyR protein, and also, to low levels of RyR expression, levels not readily demonstrable by in situ hybridization. Our data complements mRNA expression data, and thus, offer a more comprehensive view of localization and function of RyR. It would be of interest to stain developing oocytes and females ovaries to further these results.

The localization of IP₃R also shows similar differences with respect to in situ hybridization data: published in situ data offer a very restricted expression pattern, with higher levels in late embryos in prospective antenno-maxillary complex (a complex comprising the dorsal and terminal organs) and the labial organ(Hasan and Rosbash, 1992; Raghu and Hasan, 1995)[(Campos-Ortega and Hartenstein,1997) for sensory organ nomenclature]. In contrast, our data reveal widespread expression of the IP₃R protein; consistent with mutant defects, expression of the protein occurs at all stages and tissues. There is also high expression in the digestive tract, again consistent with the requirement for intracellular Ca²⁺ dynamics in visceral muscle function and consistent with immunocytochemistry data(Raghu and Hasan, 1995). Staining has less marked regional differences than RyR staining. Our data support the idea that IP₃R protein, like RyR protein, is also contributed maternally and/or is expressed at levels not readily detectable by in situ hybridization at all stages and tissues. This underscores the value of examining both transcript and protein expression data, although some caution should be exercised as heparin may label other proteins besides IP₃R protein.

Finally, Drosophila SERCA expression has not been reported before. We find widespread embryonic expression, with little or no regional differences (Fig. 4C,C'). Thapsigargin also shows widespread staining in adult tissues(Fig. 5C). There is ample colocalization of RyR and SERCA protein expression in embryonic and adult tissues, evidenced by the general overlap between RyR and SERCA (thapsigargin)staining. The embryonic digestive tract stands out in double labeling experiments because the amount of RyR is so strong that it quenches the SERCA staining present in the digestive tract (compare Fig. 4C',H). Elsewhere, levels are less disparate (Fig. 5C). This generalized co-localization is consistent with the largely complementary function of both proteins in intracellular Ca²⁺ dynamics. Overall,our data support the involvement of these proteins in intracellular Ca²⁺ dynamics and muscle function, but point to a more general role in all cells and tissues.

The homology between the Drosophila RyR and other RyRs, like the C. elegans gene and the three vertebrate ones, was in all cases around 40% (Table 1). Thus, it is not possible to deduce relationships between the invertebrate receptors and their vertebrate counterparts from sequence data. However, inspection of the phylogram and cladogram in Fig. 6 indicates that the Drosophila and C. elegansRyRs form a separate group from the vertebrate isoforms. It might seems premature to assign the Drosophila and C. elegans RyRs as type 1 based only in their conspicuous muscular localization(Takeshima et al., 1994; Maryon et al., 1996), but biochemical data support such a tenet. Taken together, evidence points to a closer relationship between vertebrate RyR type 1 and invertebrate RyR.

IP₃Rs are different: the homology between the Drosophila IP₃R and the IP₃R from C. elegans was smaller (36%) than with the vertebrate isoforms 2 (53%), 3(50%) and 1 (56%), or the lobster IP₃R (57%)(Table 1). The data may suggest that the Drosophila IP₃R is closer to the vertebrate IP₃R type 1 than the other 2 isoforms. One can speculate that this state of affairs is due to evolutionary divergence since the last common ancestor between nematodes and arthropods/vertebrates happened earlier in time than the split between arthropods and vertebrates. Once the vertebrate lineage split from the arthropods, several duplication events in the vertebrate lineage gave rise to the current three isoforms. It can be added that similarities in homology values among all RyRs considered could be explained by somewhat different structural constraints in RyRs compared to IP₃Rs.

RyRs and IP₃Rs are homologous proteins sharing 30-35% homology at the amino acid level. However, there are three regions where the homology is higher: (1) the first 600 amino acids (numbering based on RyRs sequences),(2) the central region between amino acids 1500 and 2600, and (3) the C-terminal domain starting from residue 3900, containing the transmembrane domains (Sorrentino et al.,2000). The phylogenetic tree of the intracellular calcium release channel family presented in Fig. 6 is an extension of previous studies(Takeshima et al., 1994; Franck et al., 1998). Both the phylogram and the cladogram in Fig. 6 show that the RyRs and IP₃Rs from invertebrates are grouped separately from vertebrate isoforms. Vertebrate isoforms could have diverged because they specialized to fulfill physiological requirements of determined tissues; for example, RyR type 1 allows the excitation-contraction coupling of skeletal muscle, whereas RyR type 2 does the same in cardiac muscle. What is the strategy used in invertebrates? There are three possibilities: first, that in Drosophila and other invertebrates the specialized roles of each receptor can be accomplished by alternatively spliced forms of the RyR and IP₃R genes; second, that the intrinsic molecular properties of each receptor enable them to carry out all the different functions encompassed by their vertebrate counterparts; or third,that owing to the different nature of tissues in vertebrates and invertebrates, such specialized roles are not required.

The Ca²⁺ ATPases of intracellular stores clearly derived from their plasma membrane counterparts early in the evolution of eukaryotes(Carafoli and Klee, 1999). Table 1 shows that, overall,SERCAs from Drosophila and other invertebrates present somewhat higher levels of identity with vertebrate SERCA1 and SERCA2 (71-73%), and somewhat less with SERCA type 3 (67-69%). From the phylogram and cladogram in Fig. 7 it seems that invertebrate SERCAs are perhaps closer to vertebrate type 1 SERCAs. Vertebrate type 1 SERCAs are characteristic of fast-twitch skeletal muscle in mammals(Brandl et al., 1987). Loss of its function in humans causes Brody disease, a debilitating but not lethal human disorder (Ordermatt et al., 1996). Interestingly, SERCA type 1 of Makaira nigricans (blue marlin) is in a different node from the rest of the other vertebrates, in a position closer to invertebrates. It may be that this pelagic fish has retained more of characteristics of the common SERCA ancestor due to its demand for high speed travel. It would be of interest to examine SERCAs from other fast swimming fish, such as Tuna, to see whether this is indeed a possibility.

We thank Fernando López-Barrera for help with figures. The assistance of Rafael Favila-Humara and Leopoldo González-Santos in the use of the PHYLO_WIN program is appreciated, and of Luis Vaca, Arturo Hernández-Cruz and Luis Oropeza for use of the confocal microscope at the Instituto de Fisiología Celular. This work was supported by CONACyT(projects 28055-N to M.D.-M., J27954-N and 36040-N to J.R.R.-E.) and DGAPA(projects IN 200500 to M.D.-M. and IN 209798 to J.R.R.-E.).