1 Department of Molecular and Cellular Neurobiology, Neurobiology Institute,
Campus UNAM-Juriquilla, Universidad Nacional Autónoma de México,
Querétaro 76230, Mexico
2 Department of Developmental Neurobiology and Neurophysiology, Neurobiology
Institute, Campus UNAM-Juriquilla, Universidad Nacional Autónoma de
México, Querétaro 76230, Mexico
* Authors for correspondence (e-mail: juan{at}zool.unizh.ch; mdiaz{at}calli.inb.unam.mx)
Accepted 5 March 2003
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Summary |
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Despite evolutionary distances, our functional results demonstrate that Drosophila ryanodine and inositol triphosphate receptors and Ca2+-ATPase are reasonably similar to vertebrate counterparts. Our protein expression data are consistent with the known functions of these proteins in the Drosophila digestive tract and nervous system. Overall, results show Drosophila as a valuable tool for intracellular Ca2+ dynamics studies in eukaryotes.
Key words: Calcium release channel, Intracellular calcium, Sequence analysis, Confocal microscopy, Drosophila
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Introduction |
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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 Drosophila RyR, 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.
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Materials and Methods |
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Cellular fractions
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]-IP3
binding 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 and [3H]-IP3-binding
assays
[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.
Measurement of Ca2+ and Mg2+ ATPases
activities
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 1 µm 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).
Confocal microscopy
Embryos
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).
Adults
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.
Database searches
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.
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Multiple-sequence alignment and phylogeny
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
).
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Results |
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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+.
[3H]-IP3-binding assays
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 Kd 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.
|
Mg2+ and Ca2+-ATPases activities
thapsigargin sensitivity
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
Mg2+ and Ca2+-ATPase activities. Then, we determined the
`basal' or `background' ATPase activity; that is, ATPase activity under
conditions where Ca2+-ATPase is inhibited. The difference between
`total' and `basal' is SERCA activity. Fig.
3A shows that both WT and YW stocks of Drosophila
presented 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).
Confocal microscopy studies
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).
Sequence analysis and phylogenetic classification
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. 5B shows 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).
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Discussion |
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Biochemical properties of Drosophila RyR, IP3R and
SERCA
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, Kd 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 Drosophila
RyR.
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
Kd 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
).
Drosophila RyR, IP3R and SERCA anatomical
localization
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.
Molecular evolution of Drosophila RyR, IP3R and SERCA
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. elegans
RyRs 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.
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