(Received for publication, September 28, 1995; and in revised form, December 27, 1995)
From the
Although the RNA for the third isoform (Ryr3) of ryanodine
receptor (RyR), a Ca release channel, is detected in
specific regions of mammalian brain, little is known about the protein.
We investigated Ryr3 in rabbit brain, using an antibody raised against
the synthetic peptide corresponding to amino acid sequence
4375-4387 of rabbit Ryr3, the homologue of bullfrog
-RyR.
The antibody which reacted with bullfrog
-RyR, but not with the
other isoforms, Ryr1 or Ryr2, specifically precipitated a single
polypeptide from rabbit brain microsomes having a size similar to
-RyR. Sucrose gradient ultracentrifugation revealed that Ryr3
forms a homotetramer, as true of the other isoforms. Being consistent
with the distribution of its RNA, Ryr3 was abundantly expressed in
hippocampus, corpus striatum, and diencephalon. Ryr3 demonstrated
Ca
-dependent [
H]ryanodine
binding, and caffeine increased its Ca
sensitivity.
The Ca
sensitivity of Ryr3 was also enhanced in a
medium containing 1 M NaCl, as observed with
-RyR.
[
H]Ryanodine binding gave an estimate of Ryr3
which would be only 2% or less of total RyR in rabbit brain. These
results confirm the expression of functional Ryr3 in mammalian brain
which is similar to nonmammalian
-RyR.
Increase in cytoplasmic Ca in neurons plays an
important role in the regulation of neuronal functions(1) .
Ca
can be released from endoplasmic reticulum via two
different types of release channel: inositol 1,4,5-trisphosphate
receptors (IP
Rs) (
)(2, 3) and
ryanodine receptors
(RyRs)(4, 5, 6, 7, 8) . In
brain, IP
R is more abundant than RyR. However, the
contribution of IP
R and RyR may be nearly equipotent in the
regulation of cytoplasmic Ca
in neurons because the
unit conductance of RyR is about 4 times as large as that of
IP
R(2) . The two receptors also differed in their
distribution in the brain.
Cloning and sequencing of cDNAs for RyR revealed that there are three genes coding RyRs in mammals(9, 10, 11, 12, 13, 14) . The ryr1 gene encodes RyR in skeletal muscle (Ryr1)(9, 10) , and the RyR in cardiac ventricles (Ryr2) is the gene product of ryr2(11, 12) . More recently, the third gene ryr3 has been identified in rabbit brain(13) , mink lung epithelial cells(14) , and human Jurkat T-cell (15) .
The major isoform of RyR isolated from brain is very similar to Ryr2 (16, 17, 18, 19) , although all three ryr genes have been found in the central nervous system by Northern blot (13) or in situ hybridization(20, 21) . It has been shown that Ryr1 is exclusively localized in cerebellar Purkinje cells (17, 20-26; see also (8) ). mRNA of the ryr3 gene has been detected in specific regions of brain, e.g. hippocampus, thalamus, and corpus striatum(13, 20, 21) , suggesting that this isoform, i.e. Ryr3, may have an important role in specific brain functions. The properties of Ryr3, however, are still unclear.
We purified two isoforms of RyR (- and
-RyR) from bullfrog skeletal muscle which coexist in non-mammalian
vertebrate skeletal muscle(27) . The sequencing of their cDNAs
revealed that these two isoforms are most homologous to Ryr1 and Ryr3,
respectively(28) . Taking full advantage of this similarity
between mammalian Ryr3 and bullfrog
-RyR, we here studied the
properties of the former in rabbit brain using the polyclonal antibody
specific for Ryr3.
Figure 1:
Amino acid sequence for antigen peptide
in rabbit Ryr3 and its alignment with counterparts for the other RyR
isoforms of rabbit and bullfrog. The amino acid sequence of rabbit Ryr3
used to immunize rabbits is shown at the top (Rabbit
Ryr3), and corresponding sequences of rabbit Ryr1 and Ryr2, and
bullfrog - and
-RyRs are aligned below. Number of amino acid
residues of each sequence is given at either end. Dots represent residues identical with those of rabbit
Ryr3.
Figure 2: Specificity of anti-Ryr3 antibody. A, microsomal proteins (30 µg) prepared from bullfrog skeletal muscle (lane 1), rabbit skeletal muscle (lane 2), and rabbit cardiac muscle (lane 3) were separated on a 2-12% linear gradient SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue. Arrowheads indicate the band of RyR in each specimen. B, similar gel was transferred onto a PVDF membrane and probed with anti-Ryr3 antibody. The bands in lane 3 (asterisk) are due to the direct reaction of the secondary antibody.
Figure 3: Detection of Ryr3 in rabbit brain microsomes. A, Western blot analysis by monoclonal antibody RY-3 and anti-Ryr3 antibody. 100 µg of rabbit whole brain microsomes was separated on a 2-12% SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue (lane 1). Similar gels were transferred onto PVDF membranes and probed with monoclonal antibody RY-3 (lane 2) or anti-Ryr3 antibody (lane 3). While monoclonal antibody RY-3 showed a strongly reacted band of Ryr2 (arrowhead), anti-Ryr3 antibody recognized no specific bands. B, immunoprecipitation of Ryr3 from rabbit brain microsomes by anti-Ryr3 antibody. 10 mg of solubilized rabbit whole brain microsomes was precipitated with anti-Ryr3 antibody as described under ``Experimental Procedures'' in the absence (lane 2) and presence (lane 3) of 30 µM antigen peptide. The immunoprecipitated products were resolved on 2-12% SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue (left panel). A similar gel was transferred onto a PVDF membrane and probed with anti-Ryr3 antibody (right panel). The bullfrog skeletal muscle microsomes were used as markers for mobilities of RyR isoforms (lane 1). The band which selectively disappeared in the presence of the antigen peptide is indicated by an asterisk.
It is well known that RyR forms a tetramer showing a large sedimentation coefficient (about 30 S)(31, 32) . Fig. 4shows the pattern of sucrose density gradient ultracentrifugation of microsomal proteins from rabbit brain. Using monoclonal antibody RY-3, we observed Ryr2 in brain which sedimented to high density fractions as reported previously (16, 18) (Fig. 4A). Immunoprecipitation of the identical gradient fractions showed that Ryr3 also sedimented to the same fractions as Ryr2 (Fig. 4B). Monoclonal antibody RY-3 did not react with the product immunoprecipitated by anti-Ryr3 antibody (data not shown), nor did anti-Ryr3 antibody react with the product immunoprecipitated by monoclonal antibody RY-3 (data not shown). Thus it appears that Ryr3 as well as Ryr2 forms a homotetramer in brain as is true of Ryr1 in skeletal muscles and Ryr2 in cardiac muscle.
Figure 4: Sedimentation of RyR isoforms expressed in rabbit brain through sucrose gradient ultracentrifugation. 20 mg of solubilized rabbit whole brain microsomes was sedimented through 5-20% sucrose gradient by ultracentrifugation and divided into 8 fractions (5 ml each, numbered from the top of the gradient). A, 30 µl of each fraction was resolved on 2-12% SDS-polyacrylamide gel, transferred onto a PVDF membrane, and probed with monoclonal antibody RY-3. B, the immunoprecipitated products from 4 ml of each gradient fraction with anti-Ryr3 antibody were separated, transferred, and probed with anti-Ryr3 antibody as described under ``Experimental Procedures.''
The distribution of Ryr3 in rabbit brain was determined by immunoprecipitation assay of solubilized microsomal proteins prepared from each region of the brain (Fig. 5). The isoform was abundantly expressed in hippocampus, diencephalon, and corpus striatum and slightly in cerebral cortex and mesencephalon. In cerebellum, no bands could be detected. This distribution well corresponded to the previous results of Northern blots (13) and in situ hybridization (20) in rabbit brain.
Figure 5: Distribution of Ryr3 in rabbit brain. Immunoprecipitation and detection of Ryr3 was carried out as in Fig. 3B using 4 mg (corpus striatum) or 10 mg (other regions) of the microsomes prepared from each region of rabbit brain.
We determined the cross-reactivity of anti-Ryr3 antibody with Ryr3 in mammalian brains other than rabbit. A single band showing mobility similar to rabbit Ryr3 was detected in the immunoprecipitated products of mouse, rat, and guinea pig whole brain microsomes. The intensities of the reacted bands appeared to be similar among these animals (data not shown). These results indicate that Ryr3 exists in similar quantities in the brains of these animals.
As shown in Fig. 6, the immunoprecipitated radioactivity due to
[H]ryanodine bound in 2.5 mg of solubilized brain
microsomes increased with the added amounts of antibody-agarose beads
and reached a plateau around 350 cpm at 50 µl of beads or more.
Radioactivity was no longer immunoprecipitated from the supernatant
that had been incubated with 100 µl of the beads (data not shown).
Thus, 100 µl of the antibody-agarose beads immunoprecipitated
almost all Ryr3 in rabbit brain microsomes. This allowed us to estimate
the relative amount of Ryr3 to total RyRs in rabbit brain. As shown in Fig. 7A, total [
H]ryanodine
binding determined by filtration assay increased linearly with the
amount of microsomes, which gave the specific binding activity of 8200
cpm/mg of microsomal protein. The immunoprecipitated activities were
also linearly proportional to the amount of solubilized microsomes, the
specific activity of which was 160 cpm/mg of protein (Fig. 7B). Ratio of the immunoprecipitated ryanodine
binding activity to total activity was calculated to be about 0.02. The
fraction of 1.6% was obtained with another lot of microsomal
preparation (
in Fig. 7). Assuming that all RyR isoforms
have almost identical properties of ryanodine binding, the fraction of
Ryr3 would be only 1.6-2% of total RyRs in rabbit whole brain.
Figure 6:
Immunoprecipitation of
[H]ryanodine binding activity by anti-Ryr3
antibody. [
H]Ryanodine binding to 2.5 mg of
solubilized rabbit whole brain microsomes was carried out as in Table 1in the presence of 10 mM Ca-EGTA (pCa
4). Immunoprecipitation was performed using antibody-agarose beads in
the amount indicated on the abscissa. The data are the means
of duplicate determinations.
Figure 7:
Estimation of the fractional amount of
Ryr3 in rabbit brain based on [H]ryanodine
binding activity. [
H]Ryanodine binding to
solubilized rabbit whole brain microsomes was carried out as in Table 1in the presence of 10 mM Ca-EGTA (pCa
4). A, total [
H]ryanodine binding
determined by filtering samples through polyethyleneimine-treated
Whatman GF/B glass filters (27) . B, the
[
H]ryanodine binding immunoprecipitated by 100
µl of anti-Ryr3 agarose beads. The specific activities for total
and immunoprecipitated [
H]ryanodine binding (circles) were calculated from each slope to be 8200 and 160
cpm/mg of microsomes, respectively. This indicates that the
immunoprecipitable fraction, i.e. that of Ryr3, is 2.0% of
total. Similar determinations were carried out using a different
microsomal preparation at a fixed amount of microsomes (squares), giving an estimate of 1.6%. Error bars indicate half-range of deviations of duplicate determinations (circles) or S.E. of four determinations (squares).
No error bars were shown in cases where the deviation was
smaller than the diameter of a circle.
Figure 8:
Effect of Ca and
caffeine on [
H]ryanodine binding to Ryr3 in media
containing 0.17 M and 1 M NaCl. Determinations were
carried out as in Table 1under four different Ca
concentrations (pCa 6, 5.4, 3.9, and 2.5) in medium
containing 0.17 M (A) or 1 M (B)
NaCl with (hatched columns) or without (filled
columns) 10 mM caffeine. The optimum Ca
was around pCa 3.9 in either concentration of NaCl.
Averages of 2-4 determinations with the S.E. are
shown.
We examined the effect
of caffeine on [H]ryanodine binding to Ryr3,
since several groups have claimed that the isoform may be insensitive
to this substance(14, 15) . As shown in Fig. 8B, 10 mM caffeine markedly increased the
[
H]ryanodine binding to near maximum at pCa 6 in 1 M NaCl media. Thus, caffeine did increase
Ca
sensitivity for Ryr3; this effect was also
observed in the 0.17 M NaCl medium (data not shown). The
results in Fig. 8B are consistent with the finding that
the effects of caffeine and 1 M NaCl were additive with
respect to the increase in Ca
sensitivity(35) .
The results of this study have revealed several new findings
about mammalian Ryr3 in the brain. First, mammalian Ryr3 is expressed
in specific regions (hippocampus, diencephalon, and corpus striatum) of
rabbit brain; this distribution is consistent with that of mRNA.
Second, Ryr3 was estimated to be only 2% or less of the total RyR in
the rabbit brain, while the major isoform was Ryr2. Third, Ryr3 forms a
homotetramer in spite of being a minor fraction as do the other RyR
isoforms and may constitute a functional Ca release
channel. Finally, but no less important, mammalian Ryr3 shares the
functional characteristics of Ca
-induced
Ca
release channel with bullfrog
-RyR:
enhancement of Ca
sensitivity in the presence of 1 M NaCl and response to caffeine action. This functional
coincidence is consistent with the homologous primary structures of
both.
The sequence of the antigen peptide was very similar to the
corresponding portion of bullfrog -RyR, but was not homologous to
those of rabbit Ryr1 or Ryr2 nor to bullfrog
-RyR (Fig. 1).
Furthermore, this portion is highly conserved in the deduced amino acid
sequence of Ryr3 of mink lung epithelial cell and human Jurkat T cell;
in mink, the sequence is identical (14) and in human only one
amino acid is replaced(15) . The antibody recognized Ryr3s not
only of rabbit but also of mouse, rat, and guinea pig. Chicken skeletal
muscle
-RyR, which is homologous to bullfrog
-RyR(36, 37) , also reacted positively with the
antibody (data not shown). Thus, this sequence may be highly conserved
among Ryr3 of various species.
The anti-Ryr3 antibody was able to
precipitate the cognate RyR while retaining its
Ca-dependent [
H]ryanodine
binding activity from solubilized microsomes that had been incubated
with [
H]ryanodine. Similar results of ryanodine
binding were obtained when immunoprecipitation preceded the
[
H]ryanodine binding assay (data not shown).
Thus, the binding of the antibody may not affect the ryanodine binding
activity of RyR. We could not exclude the possibility that the antibody
might alter the properties of RyR channels as previously
reported(38, 39) , because we could not obtain Ryr3
without complex formation with the antibody. However, well-consistent
properties of ryanodine binding with those of bullfrog
-RyR (Fig. 8) suggest that the antibody may not significantly affect
the RyR channel properties.
The response to caffeine of mammalian
Ryr3 has, to date, been controversial. Giannini et al.(14) first reported that caffeine did not release
Ca from intracellular stores in transforming growth
factor
-treated mink lung epithelial cells which express Ryr3.
Recently, Hakamata et al.(15) demonstrated that human
Jurkat T-cell expresses only Ryr3 among the three isoforms, and that an
increase of intracellular Ca
was evoked by ryanodine
but not by caffeine. In contrast, skinned skeletal muscle fibers of
``dispedic mouse'' which lack Ryr1 have been shown by
Takeshima et al.(40) to release Ca
from sarcoplasmic reticulum by caffeine. They concluded on the
basis of mRNA analysis that the Ca
release was
probably mediated by Ryr3. The [
H]ryanodine
binding of this study (Fig. 8) clearly indicated that caffeine
markedly enhanced the Ca
sensitivity of Ryr3 as well
as other isoforms reported to date. The lack of caffeine response,
therefore, may be due to the low levels of expression of the RyR in
those cells, as suggested by Giannini et al.(21) .
Alternatively, it is possible that Ryr3 expressed in peripheral tissues
may not be identical because the transcription product may be subject
to alternative splicing(13) .
It has been widely accepted that the major isoform of RyR in mammalian brain is Ryr2 (11, 12, 18, 19, 41) and that the other isoforms are expressed in specific regions(13, 20, 21) . Very recently, Giannini et al.(21) reported low levels of Ryr3 in the mouse brain, and we have estimated that this fraction is about 2% or less of total RyRs expressed in rabbit brain (Fig. 7). This is also the case with other mammalian brains. This limited amount can clearly explain the failure to detect the isoform by Western blot (Fig. 2) (see also (37) ), as well as earlier observations that RyR purified from rabbit brain was very similar to Ryr2(19) . This is also consistent with the results of Furuichi et al.(20) , who reported exposure time of 5 days for ryr2 and 18 and 14 days for ryr1 and ryr3, respectively, for radioautography of in situ hybridization.
Study of the distribution of mRNA indicated that Ryr3 should be expressed in specific regions (hippocampus, thalamus, and corpus striatum) of rabbit brain(13, 20) . The enriched expression of Ryr3 in hippocampus, corpus striatum, and diencephalon was confirmed as demonstrated by immunoprecipitation (Fig. 5). More recently, expression of Ryr3 in specific regions of mouse brain has been demonstrated by Western blot analysis using isoform-specific antibody(21) . The results between rabbit and mouse are similar in some points: abundant expression in hippocampus and corpus striatum, suggesting their important functions in these regions. Several differences are also noted, however. In cerebral cortex, marked expression was observed in mouse, while only a small amount was expressed in rabbit. Expression in diencephalon (or thalamus) seems more abundant in rabbit than in mouse. These might be due to the difference of the animal species examined.
In the central nervous
system, the dominant Ca release channel protein is
IP
R which also has at least three isoforms and forms a
tetrameric complex of these subunits. The possibility of heterotetramer
as well as homotetramer has recently been suggested, resulting in
potential diversity of the function as the Ca
release
channels, although this theory is yet to be proved(42) . In
contrast, our evidence shows that RyR isoforms, other Ca
release channel proteins, make only homotetramers even in a
fraction as small as 2% or less of total RyR. The
- and
-isoforms (Ryr1 and Ryr3, respectively) of bullfrog skeletal
muscle were found to have very similar properties of CICR channel in
the myoplasm, although they differed in the 1 M NaCl medium (33) (concerning RyR isoforms of chicken and fish, see (43, 44, 45) , respectively). It might be
generalized that all three isoforms of RyR have similar properties of
CICR channels. Then, what is the biological reason that the three
isoforms occur and coexist in some places? Why is the Ryr3 isoform
localized in three specific and very interesting regions of the brain,
hippocampus, thalamus, and corpus striatum? The key to the answers to
these questions may be found in the mechanism(s) of signal transduction
like that between the voltage sensor in the T-tubule (dihydropyridine
receptor) and RyR in the sarcoplasmic reticulum in the skeletal muscle.
Further investigations will clarify the functional role of the Ryr3 in
specific neuronal cells.