Further Characterization of the Type 3 Ryanodine Receptor (RyR3)
Purified from Rabbit Diaphragm*
Takashi
Murayama
§,
Toshiharu
Oba§¶,
Eisaku
Katayama§
,
Hideto
Oyamada**,
Katsuji
Oguchi**,
Masakazu
Kobayashi
,
Kazuyuki
Otsuka
, and
Yasuo
Ogawa
§§
From the
Department of Pharmacology, Juntendo
University School of Medicine, Tokyo 113-8421, the ¶ Department
of Physiology, Nagoya City University Medical School, Nagoya 467-8601, the
Department of Fine Morphology, Institute of Medical Science,
The University of Tokyo, Tokyo 108-8639, ** Department of Pharmacology,
School of Medicine, Showa University, Tokyo 142-8555, and

Fujisawa Pharmaceutical Co. Ltd.,
Osaka 541-8541, Japan
 |
ABSTRACT |
We characterized type 3 ryanodine receptor (RyR3)
purified from rabbit diaphragm by immunoaffinity chromatography using a specific antibody. The purified receptor was free from 12-kDa FK506-binding protein, although it retained the ability to bind 12-kDa
FK506-binding protein. Negatively stained images of RyR3 show a
characteristic rectangular structure that was indistinguishable from
RyR1. The location of the D2 segment, which exists uniquely in the RyR1
isoform, was determined as the region around domain 9 close to the
corner of the square-shaped assembly, with use of D2-directed antibody
as a probe. The RyR3 homotetramer had a single class of high affinity
[3H]ryanodine-binding sites with a stoichiometry of
1 mol/mol. In planar lipid bilayers, RyR3 displayed cation channel
activity that was modulated by several ligands including
Ca2+, Mg2+, caffeine, and ATP, which is
consistent with [3H]ryanodine binding activity. RyR3
showed a slightly larger unit conductance and a longer mean open time
than RyR1. Whereas RyR1 showed two classes of channel activity with
distinct open probabilities (Po), RyR3
displayed a homogeneous and steeply
Ca2+-dependent activity with
Po ~1. RyR3 was more steeply affected in the
channel activity by sulfhydryl-oxidizing and -reducing reagents than
RyR1, suggesting that the channel activity of RyR3 may be transformed
more precipitously by the redox state. This is also a likely
explanation for the difference in the Ca2+ dependence of
RyR3 between [3H]ryanodine binding and channel activity.
 |
INTRODUCTION |
Intracellular Ca2+ stores play a critical role in
regulation of the cytosolic Ca2+ concentration in various
cells. Ryanodine receptors
(RyRs)1 belong to a family of
Ca2+ release channels of the Ca2+ stores
(1-7). To date, three isoforms of RyR that are encoded by distinct
genes have been identified in mammalian tissues. Type 1 RyR (RyR1) is
primarily expressed in skeletal muscles and plays a crucial role in
excitation-contraction (E-C) coupling, a process whereby depolarization
in the sarcolemma triggers Ca2+ release from sarcoplasmic
reticulum (SR) resulting in muscle contraction (6, 8, 9). Type 2 RyR
(RyR2) is the primary isoform in heart and is involved in the E-C
coupling in cardiac muscle. cDNA and mRNA for type 3 RyR (RyR3)
was first identified in mink lung epithelial cells and in specific
regions (hippocampus, thalamus, and corpus striatum) of rabbit brain
(3, 4). Recent studies revealed that mRNAs for all
these isoforms can be widely detected in various tissues, suggesting
potential coexpression of multiple isoforms (5).
RyR1 and RyR2 have been purified from skeletal and cardiac muscles,
respectively, and are well characterized (1-3, 5, 7). A homotetramer
of the ~560-kDa subunit is a functional unit and shows a
characteristic structure with 4-fold symmetry that is identical to feet
which span the gap between the transverse tubule and terminal cisterna
of SR (6, 7). A homotetramer can specifically bind one
[3H]ryanodine molecule with a KD of
nanomolar order, after which it is named RyR (1-3). When
incorporated into planar lipid bilayers, the receptor demonstrates
cation channel activity with a large conductance showing properties
of Ca2+-induced Ca2+ release (CICR) (2, 3).
Knowledge about RyR3, in contrast, is much less than that about RyR1 or
RyR2 because of its minuscule amount. An excess amount of other
coexisting isoforms of similar biochemical characteristics has
prevented us from isolating RyR3 by conventional purification procedures. To overcome this difficulty, we utilized an antibody specific to RyR3 (anti-RyR3) as a tool for isolation of the protein. Frog skeletal muscle expresses two isoforms of RyRs (
- and
-RyR) in almost equal amounts (10). Cloning and sequencing of their cDNAs
revealed that
- and
-RyRs are homologues of RyR1 and RyR3, respectively (11). Taking advantage of the similarity between RyR3 and
-RyR, we have successfully raised and purified anti-RyR3 against a
peptide of amino acid sequence 4375-4387 of rabbit RyR3 (12).
Selective immunoprecipitation with the antibody revealed properties of
RyR3 in rabbit brain (12) and diaphragm (13). RyR3 forms a homotetramer
of ~550-kDa subunit, which is slightly smaller than those of RyR1 and
RyR2, as is the case for
-RyR. It shows high affinity
[3H]ryanodine binding which is activated by micromolar
Ca2+, adenine nucleotides, and caffeine, and inhibited by
millimolar concentrations of Ca2+ and Mg2+,
procaine, and ruthenium red, indicating that it may function as a CICR
channel. The contents of RyR3 in rabbit diaphragm and brain were
estimated to be less than 1 and 0.06%, respectively, of RyR1 in
skeletal muscle (12, 13).
In this paper, we describe the structural and functional
characterization of RyR3 that was successfully purified from rabbit diaphragm by immunoaffinity chromatography. [3H]Ryanodine
binding and single channel recordings of the molecule revealed several
unique properties of RyR3. We also defined the location of the
differential segment in the assembly of the RyR1 molecule, which could
be one of the origins for the difference in function between the two
isoforms. During the course of publication of our results, Jeyakumar
et al. (14) independently reported the purification of RyR3.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Anti-RyR3 antibody against a peptide
corresponding to the amino acid sequence 4375-4387 of rabbit RyR3
(RyR3-peptide) was produced in rabbits and purified by affinity
chromatography with frog
-RyR-bound polyvinylidene difluoride (PVDF)
membranes (12, 13). Monoclonal antibody 3F4 against a 12-kDa
FK506-binding protein (FKBP12) and human recombinant FKBP12 (hFKBP12)
were produced as described previously (15, 16). Monoclonal antibody XA7 which reacts with RyR1 (17) and 34C which recognizes all the three
mammalian RyR isoforms (18) were purchased from Upstate Biotechnology,
Inc., and Affinity Bioreagents, Inc., respectively. [3H]Ryanodine (60-90 Ci/mmol) was purchased from NEN
Life Science Products. Goat anti-rabbit IgG-agarose and
glutathione-agarose were obtained from Sigma. Egg lecithin (egg total
phosphatide extract) was from Avanti Polar-Lipids. All other reagents
were of analytical grade.
Isolation of Sarcoplasmic Reticulum (SR) Vesicles--
Heavy
fraction of SR vesicles was prepared from rabbit diaphragm according to
Murayama and Ogawa (13) in the presence of a mixture of protease
inhibitors (2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 µg/ml
antipain, 2 µg/ml pepstatin A, and 2 µg/ml chymostatin). The
isolated membrane vesicles were quickly frozen in liquid N2 and stored at
80 °C until used.
Purification of RyR3--
RyR3 was purified by a combination of
sucrose density gradient ultracentrifugation and immunoprecipitation.
Anti-RyR3 beads were prepared by mixing anti-rabbit IgG-agarose beads
with the purified anti-RyR3 antibody, followed by washing with a buffer containing 0.15 M NaCl, 20 mM sodium phosphate,
pH 7.2. These anti-RyR3 beads can be stored at 4 °C for 3 months
without a significant decrease in the RyR3-binding activity. Rabbit
diaphragm SR vesicles (10 mg) were solubilized in 4% CHAPS and 2% egg
lecithin in 2.5 ml of 0.5 M NaCl, 20 mM
Tris-HCl, pH 7.4, and 2 mM dithiothreitol (DTT) including
the above protease inhibitors (buffer A) to isolate tetrameric RyRs by
ultracentrifugation on 5-20% linear gradients of sucrose (10).
Fractions around ~15% sucrose containing tetrameric RyRs were
incubated overnight at 4 °C with 100 µl of the anti-RyR3 beads.
The beads were washed five times with buffer A containing 1% CHAPS and
0.5% egg lecithin, and then incubated overnight at 4 °C with 30 µM RyR3- peptide in the same buffer to dissociate RyR3
from the antibody. Finally, the supernatant containing RyR3 was applied
to a small gel filtration column (Centri-sep, Applied Biosystems Inc.)
to remove a large amount of the RyR3-peptide.
RyR1 was purified from rabbit back muscle in which virtually no RyR3
protein was detected (13) by sucrose gradients and Mono-Q anion
exchange column chromatography as described (10).
Protein Assay--
Protein concentrations of the purified
RyR1 were determined by the method of Kaplan and Pedersen (19) with
Amido Black 10B using bovine serum albumin as a standard. The amount of
RyR3 was determined by densitometry of its band on Coomassie Brilliant Blue-stained SDS-polyacrylamide gel with a MasterScan densitometer. The
purified RyR1 or RyR2 was used as a standard in determinations where it
showed a linear relationship up to 100 ng (data not shown).
SDS-Polyacrylamide Gel Electrophoresis (PAGE) and Western
Blotting--
SDS-PAGE was performed on 2-12% linear gradient gel
with standards of 205 (in kDa) (myosin heavy chain), 116 (
-galactosidase), 97.4 (phosphorylase b), 66 (bovine
serum albumin), 45 (ovalbumin), and 29 (carbonic anhydrase) (10, 13).
Gels were stained with Coomassie Brilliant Blue. The separated proteins
were electrophoretically transferred overnight onto PVDF membranes at
40 V in the presence of 0.02% SDS (10, 13). Immunodetection was
carried out with an ECL system (Amersham Pharmacia Biotech) using
peroxidase-conjugated secondary antibodies. Primary antibodies were
diluted as follows: 1:1,000 for anti-RyR3, 1:5,000 for XA7, 1:5,000 for
34C, and 1:1,000 for 3F4. When probed by these antibodies, the same
transferred PVDF membrane was repeatedly used.
FKBP12 Binding Assay--
Binding of FKBP12 to RyR3 was
determined using glutathione S-transferase (GST)-hFKBP12
fusion protein (GST-FKBP) (20). The plasmid construction of GST-FKBP
was essentially the same as previously reported (21). A plasmid
pFKBP333 for hFKBP12 (15) was subjected to PCR to place
BamHI and EcoRI restriction sites at the 5'- and 3'-end of hFKBP12, respectively. The set of primers was as
follows: 5'-CGGGATCCAGATGGGAGTGCAGGTGGAAAC-3' (hFKBP12-F) and
5'-CGGAATTCTCATTCCAGTTTTAGAAGCTCC-3'(hFKBP12-R). The PCR product
was digested with BamHI and EcoRI restriction enzymes and ligated into the BamHI/EcoRI sites of
pGEX-3X vector (Amersham Pharmacia Biotech). Expression and
purification of the fusion protein was carried out according to the
manufacturer's instructions. The purified GST-FKBP was recognized by
the anti-FKBP12 antibody (data not shown).
25 µl of glutathione-agarose beads was incubated for 2 h at room
temperature with 10 µg of control GST or GST-FKBP in 0.17 M NaCl, 20 mM Tris-HCl, pH 7.4, 0.1% CHAPS, 2 mM DTT, and 0.3 M sucrose (buffer B) with
gentle mixing. After washing twice with buffer B, the beads were
incubated for 1 h at 37 °C with 1 µg of the purified RyR1 or
RyR3 in buffer B. The beads were washed three times with buffer B and
proteins bound to the beads were subjected to SDS-PAGE.
[3H]Ryanodine Binding Assay--
To determine with
high sensitivity the [3H]ryanodine binding activity with
a small amount of RyR3 protein, we separated protein-bound ryanodine
from the free ligand using a small scale gel filtration column
(Centri-sep) in a centrifuge. The purified RyR3 was incubated with 8.5 nM [3H]ryanodine for 5 h at 25 °C in
30-50 µl of a binding buffer containing 0.17 M NaCl, 10 mM MOPSO/NaOH, pH 6.8, 2 mM DTT, 1% CHAPS,
0.5% egg lecithin, 4 mM AMP and various concentrations of
Ca2+ buffered with 10 mM EGTA. Free
Ca2+ concentrations were calculated using the value of
8.79 × 105 M
1 as the
apparent binding constant for Ca2+ of EGTA (22). Then, an
aliquot of the sample (10-20 µl) was applied to the Centri-sep
column that had been equilibrated with 1 M NaCl, 10 mM MOPSO/NaOH, pH 6.8, 1% CHAPS, 0.5% egg lecithin, 2 mM DTT, and 0.1 mM CaCl2. The
radioactivity of the eluate was determined by a liquid scintillation
counter. Nonspecific radioactivity was determined in the presence of 50 µM nonradioactive ryanodine. By using this assay system,
we were able to determine the ryanodine binding activity with only 20 ng of the purified RyR protein, which is one-tenth the minimum amount
for the conventional filtration assay. [3H]Ryanodine
binding to the purified RyR1 was determined under the identical
condition to the filtration assay with polyethyleneimine-treated glass
filters as described (10).
Electron Microscopy--
The structure of RyR molecules was
examined by electron microscopy after negative staining. It was
necessary to completely eliminate phospholipid and to reduce detergent
from the preparation, since their presence hampered the visualization
of structural details of the proteinaceous receptors. To meet such
requirements, the materials for the structural study were treated as
follows. The RyR3 immunoprecipitated by anti-RyR3 beads was washed with a buffer containing 0.1% CHAPS, instead of 1% CHAPS and 0.5% egg lecithin, and eluted by the RyR3-peptide. Then, the peptide was removed
by passing the solution through a Centri-sep column pre-equilibrated with a buffer containing 0.5 M NaCl, 5 mM
sodium phosphate (pH 7.2), 0.1 M sucrose, 0.1% CHAPS, and
2 mM DTT (buffer C). For RyR1, the initial buffer was
exchanged with buffer C by gel filtration on a Superose 6 column
(Amersham Pharmacia Biotech). Those procedures did not deteriorate the
[3H]ryanodine binding activity of RyRs unless the
preparations were left standing for a long period. For examination by
electron microscopy, 2-3 µl of the sample was applied to fresh thin
carbon film over a 200-mesh copper grid. After 3 min, the residual
solution was thoroughly rinsed off with the above buffer without CHAPS,
and the sample was stained negatively with 1% uranyl acetate
containing bacitracin (23). Specimens were set in a side entry
goniometer for the JEOL 2000EX electron microscope with the carbon side
facing upward. Stereo-paired micrographs were taken by tilting ±10°
at 80 kV acceleration voltage (23).
Location of the D2 segment in the RyR1 architecture was determined
similarly by negative staining but with use of a specific anti-D2-region polyclonal antibody as a probe. Since the dimension of
the IgG molecule is quite small and thin (~150 kDa) as compared with
the gigantic RyR molecule (~2,200 kDa) standing upright on the
carbon-support, it was anticipated that the IgG probe bound to the
receptor molecule might be hard to identify by itself, in the thick
stain layer embedding the total receptor assembly. Thus, we used a
two-step procedure (24, 25) to search for the epitope site of the
antibody, tentatively with IgG conjugated with a probe large enough to
be easily found, and later with the antibody by itself, to finally
pinpoint the precise site. The antibody against the D2 region (amino
acid sequence 1358-1413) of RyR1 was produced with rabbits using
GST-fusion protein as an antigen. A cDNA fragment (residues
4073-4239) was obtained from a cassette pBS-RyR1cs4 (26) and cloned
into the pGEX-3X vector (Amersham Pharmacia Biotech). Expression and
purification of the GST-fusion protein was carried out according to the
manufacturer's instructions. The IgG was affinity-purified by the
RyR1-bound PVDF membranes (10). This antibody stained a single band of RyR1 on Western blot analysis of SR vesicles which, in turn, were specifically immunoprecipitated by the same antibody (data not shown).
In order to produce a probe with a large marker, the antibody was
biotinylated and conjugated with oligomeric avidin, a linear rod-like
assembly. Biotinylation of the antibody was carried out by incubation
of the antiserum with sulfo-NHS-LC-biotin (Pierce). An oligomeric
avidin was prepared by addition of a divalent biotin (generous gift
from Dr. Kazuo Sutoh to E. K.) to neutravidin (Pierce) at a molar
ratio of 2:1 (divalent biotin/neutravidin) (27) and was mixed with the
biotinylated antibody before use. For negative staining, purified RyR1
receptor was first applied onto a carbon film and left standing for 3 min. The grid was briefly washed and the solution containing the
antibody with or without avidin conjugate was added. After 3-5 min
incubation period, it was exhaustively washed with the buffer and
stained negatively with uranyl acetate, as described above.
Planar Lipid Bilayer Experiments and Single Channel Data
Acquisition--
Single channel recordings were carried out as
described previously (28). Briefly, lipid bilayer consisting of a
mixture of L-
-phosphatidylethanolamine,
L-
-phosphatidyl-L-serine, and L-
-phosphatidylcholine (5:3:2 by weight) in decane
(40 mg/ml) was formed across a hole (~200 µm in diameter) in a
polystyrene partition separating two chambers referred to as
cis (volume 1 ml) and trans (volume 1.5 ml). The
trans chamber was held at virtual ground potential, and the
cis chamber was voltage-clamped at
40 mV relative to
the ground, unless noted otherwise. Incorporation of the purified RyR
channel was performed in asymmetrical KCl solutions containing 500:50
mM (cis/ trans) KCl, 20 mM
HEPES/Tris, pH 7.4, and 0.1 mM CaCl2. The
protein was added to the cis chamber. After confirming the
channel incorporation by the occurrence of flickering currents, further
incorporation of the protein was prevented by supplement of an aliquot
of 3 M KCl dissolved in 20 mM HEPES/Tris to the
trans chamber. Single channel currents were recorded in
symmetrical solutions containing 500 mM KCl, 20 mM HEPES/Tris, pH 7.4, and various concentrations of free
Ca2+ buffered with 1 mM EGTA. Free
Ca2+ was calculated using the apparent binding constant of
EGTA for Ca2+ by Harafuji and Ogawa (22) and was confirmed
by potentiometry with a handmade ETH1001-based Ca2+
electrode. Only bilayers containing a single channel were used in this
study. Experiments were carried out at room temperature (18-22 °C).
We found that the purified RyRs could be incorporated into the bilayers
in either orientation. The sidedness of the single channel was
determined by response to Ca2+, because the RyR channel
sensitively responded to cytoplasmic Ca2+. About 90%
of the RyR channels was blocked by lowering the cis free
Ca2+ to 10-100 nM with EGTA, indicating that
the cytoplasmic side of most channels faced the cis chamber.
Single channel currents amplified by an Axopatch 1D patch clamp
amplifier (Axon Instrument, CA) were displayed on an oscilloscope, filtered at 1 kHz using an eight-pole low-pass Bessel filter, and
digitized at 5 kHz for analysis. Data were saved on the hard disk of an
IBM personal computer. Po and the lifetime of
open and closed events from records of duration >2 min were calculated by 50% threshold analysis using pClamp (version 6.0.4) software. The
results were presented as means ± S.E.
 |
RESULTS |
Purification of RyR3 from Rabbit Diaphragm SR--
RyR3 was
purified from rabbit diaphragm using immunoprecipitation with an
antibody specific to RyR3 (anti-RyR3) (12, 13) which was raised against
a synthetic peptide corresponding to residues 4375-4387 of rabbit RyR3
(RyR3-peptide) (see "Experimental Procedures"). The diaphragm was
found to express about 10-fold more abundant RyR3 than brain in rabbits
(13). As shown in Fig. 1A, SR
protein prepared from rabbit diaphragm contained a large amount of RyR1
that was easily seen on Coomassie Brilliant Blue-stained gel and
positively detectable on Western blot with anti-RyR1 monoclonal antibody XA7 and a low level of RyR3 that was identified on anti-RyR3 blot. After solubilization with CHAPS, SR proteins were incubated with
anti-RyR3-agarose beads to precipitate RyR3 (12, 13). The band for RyR3
was clearly detected in the precipitate on both protein staining and
anti-RyR3 blot (Fig. 1B, left lanes). Addition of 30 µM RyR3-peptide during incubation failed to precipitate RyR3 (Fig. 1B, right lanes), indicating specific interaction
of RyR3 with the anti-RyR3. RyR1 band was detected neither on protein staining (Fig. 1B, left panel) nor on anti-RyR1 blot (data
not shown, but see Fig. 2B).
RyR3 was thus effectively concentrated and separated from the other SR
proteins. The immunoprecipitated RyR3 was then dissociated from the
antibody by incubation with excess RyR3-peptide. Preliminary attempts
of treatment with 0.1 M glycine HCl (pH 2.8) or 5 M potassium thiocyanate, which is conventionally used to
release antigen from antibody, caused total loss of
[3H]ryanodine binding activity, indicating irreversible
degeneration of RyR3 (data not shown). As shown in Fig. 1C,
RyR3 (arrowhead) was detected in supernatant after
incubation with 30 µM RyR3-peptide, whereas it did not
come out from the beads without the peptide. Finally, the pure RyR3 was
eluted from a small gel filtration column that held therein the
RyR3-peptide. By this procedure, 0.5-0.7 µg of RyR3 was purified
from 10 mg of SR vesicles.

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Fig. 1.
Selective immunoprecipitation by the
anti-RyR3 antibody and recovery of RyR3 by the RyR3-peptide.
A, Western blotting of rabbit diaphragm SR vesicles. 30 µg
of the SR vesicles was subjected to SDS-PAGE on a 2-12% linear
gradient gel and stained with Coomassie Brilliant Blue
(CBB). Similar gel was transferred onto a PVDF membrane and
probed with anti-RyR1 monoclonal antibody XA7 (XA7) or with
anti-RyR3 (Anti-RyR3). B, selective
immunoprecipitation of RyR3. Five mg of the solubilized rabbit
diaphragm SR was incubated with anti-RyR3 beads in the absence
(Control) and presence (RyR3-pep.) of 30 µM RyR3-peptide. The resultant beads were subjected to
SDS-PAGE and stained with Coomassie Brilliant Blue (panel
CBB) or probed with the anti-RyR3 antibody (panel
Anti-RyR3). C, recovery of RyR3 by the addition
of the RyR3-peptide. RyR3 immunoprecipitated with anti-RyR3 beads was
incubated with (panel RyR3-pep.) or without
(panel Control) 30 µM RyR3-peptide.
Proteins in the supernatant (Sup.) and the precipitated
beads (Ppt.) were subjected to SDS-PAGE and stained with
Coomassie Brilliant Blue. Arrowheads indicate the position
of RyR3. The RyR3-peptide is detected as a heavily stained band near
dye front (asterisk).
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Fig. 2.
SDS-PAGE and Western blotting of the purified
RyR3. A, 15 µg of rabbit diaphragm SR vesicles
(SR), 0.2 µg of the purified RyR1 (RyR1), and
0.1 µg of the purified RyR3 (RyR3) were subjected to
SDS-PAGE on a 2-12% linear gradient gel and stained with Coomassie
Brilliant Blue. B, 0.2 µg of the purified RyR1
(RyR1) and 0.1 µg of the purified RyR3 (RyR3)
were subjected to SDS-PAGE and transferred onto a PVDF membrane. The
identical PVDF membrane was probed with 34C (34C), XA7
(XA7), and anti-RyR3 antibody (Anti-RyR3). The
positive bands were developed by ECL system.
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A single band of purified RyR3 moved slightly ahead of RyR1 on a
Coomassie Brilliant Blue-stained SDS-polyacrylamide gel (Fig. 2A). Western blots by isoform-specific antibodies are shown
in Fig. 2B. Monoclonal antibody 34C, which can recognize all
three mammalian RyR isoforms, clearly detected both RyR1 and RyR3.
Anti-RyR1 antibody XA7 positively reacted with the purified RyR1 but
did not detect any bands in the RyR3 preparation, indicating no
contaminating RyR1 in the purified RyR3. Anti-RyR3 reacted with RyR3
but not with the purified RyR1. These findings confirm our previous
results of homotetrameric formation of each isoform (13).
FKBP12 Binding of RyR3--
It has been demonstrated that FKBP12
is tightly associated with RyR1 with a stoichiometry of 1 mol/mol of
the monomer and modulates channel activity (24, 29, 30). To know
whether RyR3 associated with FKBP12 as well, we carried out Western
blotting using an antibody against FKBP12. As shown in Fig.
3A, rabbit diaphragm SR
vesicles contained a band recognized with anti-FKBP12 monoclonal
antibody 3F4 whose mobility was almost identical with human recombinant
FKBP12 (hFKBP12) (lower panel). This corresponds to previous
results that RyR1 associates with FKBP12 in skeletal muscles (29, 31).
In contrast, no band was detectable in either RyR1 or RyR3 preparation.
Because the charged amount of the RyR in these experiments was
comparable to that in the SR vesicles on the basis of the density of
RyR bands (upper panel), negative immunoreactivity is not
due to a small amount of charged proteins. These results indicate that
FKBP12 may be dissociated from RyR1 or RyR3 during purification
procedures. This is consistent with the results of Wagenknecht et
al. (24) who reported a substantial loss of FKBP12 from the RyR1
preparation on addition of CHAPS.

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Fig. 3.
FKBP12 binding of the purified RyR3.
A, detection of FKBP12 in the purified RyR preparations. 0.2 µg of hFKBP12 (hFKBP12), 30 µg of rabbit diaphragm SR
vesicles (SR), and 0.5 µg of the purified RyR1
(RyR1) and RyR3 (RyR3) each were subjected to
SDS-PAGE with a 5-15% linear gradient gel, transferred onto a PVDF
membrane, and probed with anti-RyR antibody 34C (panel
Anti-RyR) and with anti-FKBP12 antibody 3F4
(panel Anti-FKBP12). The positive bands were
developed by ECL system. B, 1 µg of the purified RyR1
(panel RyR1) and RyR3 (panel RyR3)
each were incubated for 1 h at 37 °C with control GST beads
(lane 1) or GST-FKBP beads (lanes 2 and
3) in a buffer containing 0.17 M NaCl, 20 mM Tris-HCl, pH 7.4, 0.1% CHAPS, 0.3 M
sucrose, and 2 mM DTT. In lane 3, 10 µM FK506 were supplemented. The beads were washed three
times with the buffer, and proteins bound to the beads were subjected
to SDS-PAGE and stained with Coomassie Brilliant Blue.
Arrowheads indicate the position of RyR.
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To address whether RyR3 possesses an ability to bind FKBP12, we carried
out FKBP12 binding assays using glutathione S-transferase (GST)-hFKBP12 fusion protein (GST-FKBP) (Fig. 3B). RyR1
(left panel) and RyR3 (right panel) were
incubated with glutathione-agarose beads that had been incubated with
GST-FKBP or control GST in a buffer containing 0.17 M NaCl
and 0.1% CHAPS. After washing the beads, proteins bound to the beads
were detected by SDS-PAGE. When incubated with GST-FKBP beads, RyR1 and
RyR3 were bound to the beads (lane 2). When incubated with
control GST beads (lane 1), in contrast, bound RyRs were
hardly detectable. In the presence of 10 µM FK506, which
was known to dissociate FKBP12 from RyR1, RyRs were also severely
prevented from binding the GST-FKBP beads (lane 3). These
results strongly indicate that RyR3 can specifically bind FKBP12, as is
true of RyR1.
Electron Microscopy of Purified RyR3--
Fig.
4 exhibits negatively stained images of
RyR3 particles, in comparison with those of RyR1. As shown in the
general view (Fig. 4A), RyR3 preparation showed homogeneous
distribution of the particles of rectangular shape similar to the
reported image of RyR1. A gallery of the selected particles shows that
both isoforms are indistinguishable in shape or in overall dimension
(about 25 nm on each side) from each other (compare Fig. 4,
B with C). They showed 4-fold symmetry reflecting
their tetrameric nature, and the substructures were well defined under
higher magnification. The central core with a crossed groove and outer
regions with several cavities or pockets were the features commonly
found in both isoforms, although closer examination revealed delicate
differences in appearance from particle to particle, probably owing to
uncontrollable differences in stain depth. It is also notable that both
isoforms shared a pinwheel-like appearance with the same rotational
handedness.

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Fig. 4.
Negatively stained images of purified
receptor molecules. All the images are printed so that the
particles are seen from their top surface, i.e. cytoplasmic
side. A, general view of RyR3 preparation indicating the
homogeneity of the purified molecules in their size (25 nm on each
side) and overall square shape. Scale bar represents 50 nm.
B and C, galleries of selected images of RyR3 and
RyR1 molecules, respectively. Both receptor isoforms share a
pinwheel-like appearance with the same rotational handedness. A crossed
groove in the central core and the stain-filling pockets in the outer
regions were also commonly observed and indistinguishable from each
other. D, four pairs (alternate columns in each row) of
stereo images of the RyR3 molecules including some oblique views. These
particles seem to show the details of polypeptide folding probably
because of less contribution of lipid and/or detergent in the
polypeptide images. One might trace a complexed organization of the
skeleton-like polypeptide chain similar to that visualized in the
reconstructed three-dimensional model (32). Scale bar
indicates 20 nm for B-D.
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We also obtained several images of RyR3 particles with less
contribution of phospholipid and/or detergent. Four pairs of
stereo-micrographs for RyR3 (alternate columns in each row of Fig.
4D) show the tubular network-like structure forming several
stain-filling pockets, which appears very similar to the published
solid models of RyR1 (32, 33). Despite careful comparison of the two
isoforms, we could not find any significant difference between them
which exceeds the image variance due to staining conditions. Thus, we concluded that the actual difference in the molecular architecture of
the isoforms might be subtle, even if it exists.
We then switched to another approach to find the structural difference
in a more positive way, using a structural probe specific to the
segments unique to each isoform. One of such candidates is naturally
the antibody used to isolate RyR3 in the present study. However, since
the epitope of this antibody in the amino acid sequence of RyR is close
to the membrane-spanning region, it might not be an easy task to
identify the epitope site in the geometry of the total RyR assembly
that is attached to the carbon support through the membrane-spanning
region. So, we decided to use the other antibody probe which is not
only easier to visualize but also specific to some functionally
important region.
The fact that RyR3 lacks a stretch of about 100 amino acid residues
corresponding to 1305-1405 in RyR1 is one of the most substantial
differences between them (34). The region including this stretch is
generally termed D2 because of its high divergency among RyR1-3 (35).
As an appropriate candidate to search for the subtle difference between
RyR1 and RyR3 in the local architecture, we made an attempt to define
the location of the D2 region in the organized structure of the RyR1
assembly by negative staining. We utilized an antibody specific to the
D2 region of RyR1 (anti-D2 antibody) that was produced against the
amino acid sequence 1358-1413 of rabbit RyR1 (see "Experimental
Procedures"). Because this antibody could immunoprecipitate native
RyR molecules in solution, it is clear that the antibody can attach to
RyR without significantly affecting its organized architecture. In
order to visualize unambiguously the bound antibody in negatively
stained images, we initially used the antibody conjugated with an
oligomeric avidin which might form a characteristic rod-like shape and
would be easily distinguished from the other structures (27). Fig.
5A exhibits a general view of
such specimen. Most of the receptor molecules in the field were
accentuated with strongly stain-excluding dots and many of them with
short rod-like structures (indicated by black arrows), both
close to the corners of the square-shaped assembly. This indicates that
the anti-D2 antibody should be associated with the RyR1 assembly at the
sites close to the corner of the square. This probe, however, was too
large to pin-point the precise epitope site on the assembly. With the
knowledge that the D2-specific antibody might bind close to the corners
of the RyR1, we then carried out similar experiments with the
unconjugated antibody. Fig. 5B shows the images obtained in
such way. After incubation with antibody solution and exhaustive
washing, many receptor molecules appeared as square-shaped particles
with small but strongly stain-excluding spots close to the corners. A
substantial fraction of them also had the protrusions from the same
position close to the corner of the square assembly. These spots often
consisted of a triangular or tripodal structure that would represent
the anti-D2 IgG molecule (free IgG molecules are shown in the
insets as reference). They were reproducibly found at
several nanometers apart counterclockwise from the exact corners of the
rectangular receptor molecules. More convincing evidence for the
location of the epitope site was the presence of cross-linked particles
through fine Y- or V-shaped pieces, the dimension of which is similar
to that of free IgG, connected to the sites equivalent to those
suggested above as the epitope site. It is notable that the disposition of the two adjacent receptors seen here was opposite to the model motif
II for self-association of the purified RyR which was suggested by
Wagenknecht et al. (25). Hence, it seems unlikely that these connected receptors were due to self-association without the antibody. These lines of evidence strongly suggest that the epitope site of
anti-D2 antibody, i.e. the D2 region, exists at the position several nanometers apart counterclockwise from the corner of the tetrameric receptor particles. It has been shown that RyR adheres almost exclusively with its transmembrane side toward the carbon film
(36). Because the images in this paper are printed so that the receptor
molecules are observed from their cytoplasmic side, the location of the
D2 region may be safely interpreted to be at or close to "domain 9"
in the three-dimensional architecture of RyR1 molecule (32), close to
the top of the cytoplasmic assembly (see also the side view in Fig.
5B).

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Fig. 5.
Location of the D2 segment in the molecular
architecture of RyR1 probed with anti-D2 antibody. A, a
general view of the purified RyR1 molecules mixed with the anti-D2
antibody which was conjugated to oligomeric avidin. Antibody probes are
observed typically as short stain-excluding rods and sometimes merely
as spots, both attached near the corners of the quatrefoil structure
(receptor particles with rods are indicated by black
arrows). Scale bar exhibits 40 nm. B,
gallery of the negatively stained images of the purified RyR1 complexed
with the unconjugated anti-D2 antibody. Note that small stain-excluding
particles (arrowheads) are consistently bound to the edge of
the receptor at the identical site close to the corner. These particles
look similar in shape and size to those of the free IgG molecules
(two small insets). The images indicated in the panels of
the right column exhibit RyR molecules that are
cross-connected by small V- or Y-shaped pieces corresponding to IgG.
The IgG probe is also seen bound near the other corner of the
molecules. It should be noted that the cross-linked sites are
equivalent to those decorated by the spots shown in the panels of the
left column. The bottom panel of the left
column of Fig. 5B represents the cross-linked receptors
whose side and oblique views are seen. Antibody binding occurs at the
same site as above. Scale bar represents 20 nm.
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[3H]Ryanodine Binding to the Purified
RyR3--
[3H]Ryanodine binding to the
purified RyR3 was determined by gel filtration method (see
"Experimental Procedures"). The preliminary results indicate that
properties of [3H]ryanodine binding to the purified RyR3
are consistent with those already reported with the immunoprecipitated
receptors (12, 13); it was activated by micromolar Ca2+,
adenine nucleotides, and caffeine, inhibited by millimolar
concentrations of Ca2+ and Mg2+, procaine, and
ruthenium red, and affected by salt concentration of the medium (data
not shown). In this study, we focused on some important characteristics
that so far remained to be determined or were controversial.
Fig. 6 shows dose-dependent
[3H]ryanodine binding to the purified RyR3. The amounts
of the bound [3H]ryanodine in the medium containing 1 M NaCl, 1 mM AMP-PCP, and 0.1 mM
Ca2+ increased with increases in
[3H]ryanodine concentrations and approached the
asymptotic value. The Scatchard plot (inset) gave a straight
line within the range of 0.5-21 nM
[3H]ryanodine, indicating a single class of high affinity
ryanodine-binding sites with an apparent KD of 1.5 nM, which is similar to that of the immunoprecipitated RyR3
(1.6 nM) (13). In the previous study (13), we were unable
to estimate the stoichiometry of ryanodine bound to RyR3, because we
could not determine the amount of the receptor. The
Bmax of the purified RyR3 (522 pmol/mg protein)
shown here revealed that it binds [3H]ryanodine with a
stoichiometry of 1 mol of ryanodine per 1 mol of the homotetramer, as
is true of the other isoforms (3). Based on the binding sites of
[3H]ryanodine binding to RyR3 in rabbit diaphragm SR
(Bmax = 0.065 pmol/mg) (13), the amount of RyR3
in 10 mg of the SR is calculated to be 1.2 µg (10 × 0.065
522 × 1,000). We obtained 0.5-0.7 µg of the purified RyR3
from 10 mg of the SR vesicles. These results show that the yield of
RyR3 was 42-58% and that the extent of purification was more than
8,000-fold.

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Fig. 6.
Dose-dependent
[3H]ryanodine binding to the purified RyR3. 20.2 ng
of the purified RyR3 was incubated with 0.5-21 nM
[3H]ryanodine for 4 h at 25 °C in 1 M
NaCl, 20 mM MOPSO/NaOH, pH 6.8, 1% CHAPS, 0.5% egg
lecithin, 2 mM DTT, 1 mM AMP-PCP, and 0.1 mM free Ca2+. Bound ryanodine was separated by
small-scale gel filtration columns as described under "Experimental
Procedures." The data were mean ± half-range of deviation of
duplicate determinations. Linear Scatchard plot (inset)
indicates that RyR3 had a single class of binding sites. The
KD and Bmax values were 1.5 nM and 522 pmol/mg protein, respectively.
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We previously reported that RyR3 in rabbit diaphragm showed unique
characteristics with regard to sensitivity to Ca2+ and
Mg2+; it was less sensitive to activating Ca2+
than RyR1 and resistant to inhibition by Mg2+ (13). Since
these conclusions were obtained from the results of
[3H]ryanodine binding with the immunoprecipitated
receptors, one might assume that binding of anti-RyR3 would modify the
cation sensitivity of RyR3. We therefore determined Ca2+
and Mg2+ sensitivities of RyR3 with the purified receptors.
Fig. 7A shows the
Ca2+ dependence of the [3H]ryanodine binding
to the purified RyR1 and RyR3 in an isotonic medium containing 0.17 M NaCl. The Ca2+ sensitivity of RyR3 for
activation (open circles) (EC50 = 14 µM) was 4-fold lower than that of RyR1 (filled
circles) (EC50 = 3.5 µM). On the other
hand, it was apparent that both receptors were effectively inactivated
by millimolar Ca2+; IC50 values were calculated
to be 1.9 and 2.9 mM for RyR1 and RyR3, respectively. These
results confirm the previous conclusion (13) that RyR3 shows a lower
sensitivity to activating Ca2+ than RyR1, whereas they are
similar with regard to the Ca2+ sensitivity in
inactivation.

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Fig. 7.
Ca2+ and Mg2+
dependences of [3H]ryanodine binding to the purified
RyR3. A, Ca2+ dependence; B,
Mg2+ dependence. 20.2-24.6 ng of the purified RyR3
(open circles) and 3.3 µg of the purified RyR1
(filled circles) were incubated with 8.5 nM
[3H]ryanodine for 5 h at 25 °C in 0.17 M NaCl, 20 mM MOPSO/NaOH, pH 6.8, 1% CHAPS,
0.5% egg lecithin, 2 mM DTT, and 4 mM AMP with
various free Ca2+ buffered with 10 mM EGTA
(A) or with 0.1 mM free Ca2+ plus
various Mg2+ (B). For experiments in
B, the ionic strength of the assay medium was kept constant
by reducing the NaCl concentration as Mg2+ increased.
Assays were carried out as under "Experimental Procedures." Data
were mean ± S.E. of 3-6 determinations. 100% represents 132 and
156 pmol/mg protein for RyR1 and RyR3, respectively.
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The effect of Mg2+ on [3H]ryanodine binding
of the purified RyR3 is shown in Fig. 7B. The amount of
binding to RyR3 that had been activated by 100 µM
Ca2+ was dose-dependently reduced by
Mg2+ with IC50 of 2.1 mM, which was
similar to that of RyR1 (2.5 mM). The IC50
value for Mg2+ was consistent with that for inactivating
Ca2+ (see Fig. 7A). Because
dose-dependent inhibition by Mg2+ was also
observed on single channel recordings (Fig. 9C), it is
concluded that RyR3 is as effectively inhibited by Mg2+ as
RyR1. This is in contrast to our previous results with the immunoprecipitated receptors (13). It is unlikely that binding of
anti-RyR3 antibody may alter the sensitivity to Mg2+ of
RyR3, because the inhibition by Mg2+ of the purified RyR3
was unchanged, irrespective of the presence or absence of the antibody
(data not shown). The reason for the discrepancy remains to be solved.
Single Channel Recordings of the Purified RyR3 Channel--
Single
channel currents through the purified RyR3 were recorded in a solution
containing symmetrical 500 mM KCl, 20 mM
HEPES/Tris, pH 7.4. As shown in the right column in Fig.
8A, the RyR3 channel was
activated by exposure to cis pCa 6.7 (Po = 0.236). The Po remained unchanged at voltages between
60 and +60 mV, indicating no
voltage dependence of the open probability. The counterparts for RyR1
with a similar Po (0.259) in the presence of 10 µM cis Ca2+ were drawn in the
left column of Fig. 8A. It should be noted that
the subconductance opening was not as common as claimed to be
characteristic of FKBP12-free RyR, although the frequency of subconductance opening was only slightly higher with the purified RyR1
than that with the receptor in SR vesicles. The current-voltage plot
for RyR3 (open circles in Fig. 8B) gave a linear
relationship between
60 and +60 mV, as is true of RyR1 (filled
circles in Fig. 8B). This indicates the non-rectifying
property of either channel. The conductance of the RyR3 channel ranged
from 719 to 794 pS with an average of 743 ± 11 pS (mean ± S.E., n = 6). This value was significantly larger than
630 ± 9 pS (n = 8) for the RyR1 channel (ranging
from 582 to 669 pS), when K+ was used as current
carrier.

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Fig. 8.
Conductance and gating kinetics of purified
RyR1 and RyR3 channels. The purified RyR1 and RyR3 were
incorporated into lipid bilayers as described under "Experimental
Procedures." A, single channel currents of the RyR1
channel activated by 10 µM Ca2+ (left
column) and the RyR3 channel activated by 0.2 µM
Ca2+ (right column) were recorded in a
symmetrical recording solution containing 500 mM KCl and 20 mM HEPES/Tris, pH 7.4, at indicated holding potentials. The
levels of the base lines are indicated by a short line to the
right of each current trace. B, current-voltage
relationships of the purified RyR1 (filled circles) and the
purified RyR3 (open circles) channels. Data were mean ± S.E. of eight and six independent experiments for RyR1 and RyR3,
respectively. C, histograms of the dwell time for the RyR1
(left) and RyR3 (right) channels. The histograms
were constructed from channels with similar open probabilities of RyR1
(Po = 0.259) and RyR3 (Po = 0.236). Open time constants and their relative areas (in parentheses)
were calculated as follows: o1 = 0.58 ms (76%),
o2 = 1.97 ms (24%) for RyR1 channel; o1 = 0.59 ms (57%), o2 = 2.51 ms (43%) for RyR3 channel.
Calculated closed time constants and their relative areas (in
parentheses) are as follows: c1 = 0.57 ms (36%),
c2 = 2.53 ms (46%), c3 = 8.85 ms (18%)
for RyR1 channel; c1 = 0.31 ms (27%), c2 = 4.48 ms (40%), c1 = 17.04 ms (33%) for RyR3
channel.
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The gating kinetics for RyR1 and RyR3 channels were determined by open
and closed time histograms. Typical results are shown in Fig.
8C where RyR1 and RyR3 showed similar
Po values of 0.2-0.25. Mean open time for the
RyR3 channel (4.85 ± 1.16 ms, n = 7) was significantly (p < 0.05) longer than that for the RyR1
channel (2.58 ± 0.35 ms, n = 10). The open
lifetime distributions were best fit by two exponentials for both
channels (dashed lines). The fast (
o1) and
the slow (
o2) open time constants and their relative
areas (in parentheses) which were averaged from independent experiments
were 0.50 ± 0.04 ms (80.2 ± 2.3%) and 2.59 ± 0.25 ms
(19.8 ± 2.3%) for the RyR1 channel (n = 10), and
0.53 ± 0.05 ms (48.0 ± 12.8%) and 5.61 ± 1.75 ms
(52.0 ± 12.8%) for the RyR3 channel (n = 7),
respectively. Thus, the longer mean open time for RyR3 is due both to a
larger fraction of the
o2 component and to its longer
dwell time. In contrast, mean closed time for the RyR3 channel
(8.93 ± 1.39 ms) was similar to that for the RyR1 channel
(8.68 ± 2.36 ms). Closed time constants in both channels were
best fit by three similar exponentials (dashed lines). The fast (
c1), medium (
c2), and slow
(
c3) components of closed time constants and their
relative areas (in parentheses) were 0.52 ± 0.06 ms
(34.0 ± 3.2%), 2.91 ± 0.34 ms (43.3 ± 2.9%), and 11.94 ± 1.46 ms (22.7 ± 3.3%) for the RyR1 channel and
0.55 ± 0.10 ms (32.4 ± 9.7%), 3.01 ± 0.59 ms
(35.6 ± 5.1%), and 12.31 ± 1.26 ms (32.0 ± 7.9%)
for the RyR3 channel, respectively.
Modulation of the RyR3 channels by Ca2+, caffeine, ATP,
Mg2+, ryanodine, and ruthenium red is demonstrated in Fig.
9. At 0.1 µM Ca2+ (control), the RyR3 channel showed no open events
(Fig. 9A). The channel was partially activated by 0.2 µM cis Ca2+. Addition of 1 mM caffeine increased Po from 0.007 to 0.101, being consistent with the result of
[3H]ryanodine binding (12, 13). ATP also enhanced the
channel activity; Po increased from 0.007 to
0.498 by 1 mM ATP in the presence of 0.25 µM
free Ca2+ (Fig. 9B). Mean open time and numbers
of open events were 0.81 ms and 5.1/s in the control, 1.08 ms and
104.4/s in 0.2 mM ATP, and 1.44 ms and 323.1/s in 1 mM ATP, respectively. Thus, the effect of ATP appears to be
more marked in increasing the frequency of the opening than in
prolonging the open time duration. Mg2+ over the range of
1-10 mM dose-dependently inhibited the RyR3 channel which had been activated almost fully by 10 µM
Ca2+ (Po = 0.991) (Fig.
9C). However, an increase in Mg2+ concentration
to 20 mM could not completely inhibit the channel activity
(data not shown). All the channels examined were modified by 10 µM ryanodine showing typically long lasting
subconductance open state and completely blocked by subsequent
application of 5 µM ruthenium red (Fig.
9C).

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Fig. 9.
Ligand gating properties of the purified RyR3
channel. Mean open probability (Po) is
indicated at the right of each trace. A, effects
of Ca2+ and caffeine. The channel was first inhibited by
1.63 mM EGTA (0.1 µM free Ca2+)
and then activated partially by 0.2 µM Ca2+.
The concentrations of ligands shown in the figure stand for their final
concentrations. Subsequent application of 1 mM caffeine
increased the Po of the channel. B,
effect of ATP on the purified RyR3 channel. 1 mM ATP was
subsequently added to the cis chamber in the presence of
0.25 µM cis-Ca2+. C,
effects of Mg2+, ryanodine, and ruthenium red.
MgCl2 (1 and 10 mM)
dose-dependently reduced the Po of
the RyR3 channel that had been activated by 10 µM
Ca2+. Subsequent addition of 10 µM ryanodine
shifted the channel to a long lasting open state with about half of
normal conductance. The channel was completely blocked by the addition
of 5 µM ruthenium red.
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Fig. 10 depicts Ca2+
dependence of single channel activities of purified RyR1 and RyR3
channels. The channels were activated by Ca2+ alone without
any addition of other ligands such as ATP or caffeine. The RyR3 channel
displayed no open event at pCa 7, and partial open events
(Po = 0.172) were seen at pCa 6.7 (Fig. 10A). The channel was further activated to a great
extent (Po = 0.817) at pCa 6.3 and
showed almost full opening (Po ~1.0) over the
wide range of pCa 6-3. Around 3 mM
Ca2+, on the contrary, the channel activity markedly
declined (Po = 0.191). Po
values that were obtained from 4 to 7 separate experiments are plotted
against varied cis Ca2+ concentrations in Fig.
10C. At 0.1 µM Ca2+, five of six
determinations showed no open events, whereas one experiment showed
Po = 0.181. Thus, the threshold of
Ca2+ concentration for the RyR3 channel opening seemed to
be around 0.1 µM, and the channel was already fully
activated at 1 µM or more Ca2+. The
pCa-Po relationship was very steep
and appeared to be in almost all-or-none fashion. This Ca2+
dependence is similar to the results reported with RyR2 (1, 2) as well
as those with RyR3 which were determined by lipid bilayer experiments
(14, 37). The RyR3 channel was inactivated by Ca2+
concentrations above 1 mM. The dose dependence of
inactivation by high Ca2+ appeared similar to that of
inhibition by Mg2+ (see Fig. 9).

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Fig. 10.
Ca2+ dependence of the purified
RyR3 channel. A, typical current traces of the purified
RyR3 channel. The RyR3 channel was activated by 0.2 µM or
more Ca2+ and inhibited by 3 mM
Ca2+. Note that the RyR3 channel was nearly fully activated
(Po ~1) over the wide range of pCa
6-3. B, typical current traces of the RyR1 channel with low
open probability (low Po, left
column) and high open probability (high Po,
right column). C,
pCa-Po relationship of each type of
channel. Data were mean ± S.E. of 4-7, 9-12, and 4-7 separate
experiments for RyR3 (open circles), low
Po RyR1 (open triangles), and high
Po RyR1 (filled circles) channels,
respectively. There was no spontaneous transition between low and high
Po channels.
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We determined Ca2+ dependence of the RyR1 channel activity
to compare with that of the RyR3 channel (Fig. 10B). RyR1
channels demonstrated the following two distinct populations of single channel current fluctuations: high and low open probability (high and
low Po channel, respectively), just as recently
revealed with terminal cisterna of rabbit skeletal muscle SR (38, 39).
The high Po channel (7 of 19 determinations) was
activated by cis Ca2+ concentrations up to 10 µM and inhibited at higher than 100 µM Ca2+ (right column in Fig. 10B). In
contrast, the low Po channel (12 of 19 determinations) showed only a few open events even at the optimum
Ca2+, although it showed biphasic Ca2+
dependences (left column in Fig. 10B).
Po values against pCa values for high
Po (n = 4-7) and low
Po (n = 9-12) channel are also
plotted in Fig. 10C. Their Ca2+ dependences
appeared to be similar, although the relationship for low
Po channel was less clear. Whereas the RyR3
channel showed homogeneous channel activity with nearly full opening
(Po ~1), RyR1 displayed heterogeneous
populations of the channel activity with high Po
(~0.3) and low Po (<0.05), respectively, at
the optimum Ca2+. Ca2+ concentrations that
would give half the maximum activation were estimated to be ~1 and
~0.3 µM for RyR1 and RyR3 channels, respectively. In
the presence of 0.1 µM Ca2+, interestingly,
the high Po RyR1 showed a larger
Po than the RyR3 channel.
It has been demonstrated that the RyR channel is modulated by the redox
state of RyR molecules that could be attained by sulfhydryl-oxidizing and -reducing reagents (39-43). We therefore examined effects of sulfhydryl reagents on single channel activity of RyR1 and RyR3 channels. Typical data are depicted in Fig.
11. Addition of 50 µM DTT
to the cis side of the high Po RyR1
channel led to a remarkable reduction in Po
(from Po = 0.217 ± 0.061 in controls to
Po = 0.071 ± 0.046, n = 7;
see Fig. 11A for a typical result). Increase in DTT to 500 µM induced a further decrease in
Po (Po = 0.035 ± 0.002, n = 7). All DTT-treated channels were modified
by 10 µM ryanodine and blocked by 5 µM
ruthenium red (data not shown). The channel activity inhibited by
application of 100-500 µM DTT (from
Po = 0.104 ± 0.051 in controls to
Po = 0.002 ± 0.001, n = 3)
was overshot to Po = 0.313 ± 0.006 by
subsequent cis addition of 100-500 µM p-chloromercuriphenylsulfonic acid (pCMPS), a specific
organic sulfhydryl reagent (bottom traces in Fig.
11A). On the other hand, the low Po
RyR1 channel was activated by the addition of 10-50 µM
cis pCMPS (from Po = 0.004 ± 0.001 in controls to Po = 0.116 ± 0.034, n = 9), which was similar to the high
Po state (Fig. 11B). Subsequent
exposure of such pCMPS-activated channel to 25-50 µM DTT
reversed the Po to a low level (0.019 ± 0.008, n = 4). In 3 of 14 experiments,
cis-pCMPS elicited irreversible closure after transient
increase in Po, which could not be recovered by any ligands including a large amount of DTT (data not shown), suggesting the existence of at least two different sulfhydryls associated with activation and inactivation of the channel in the RyR1
molecule. Similar results on sulfhydryl reagents were observed with RyR
channels in frog skeletal muscle SR (43). The conversion between low
Po and high Po channels
occurred immediately after addition of these reagents. During channel
activation or inactivation by these sulfhydryl-modifying reagents, the
channel conductance remained unchanged. These findings suggest that the two populations of the RyR1 channel showing different
Po against pCa values might reflect
the extent of their redox state as follows: the high
Po channel at a more oxidized state and the low
Po channel at a more reduced state.

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Fig. 11.
Effects of sulfhydryl-oxidizing and
-reducing reagents on the purified RyRs. A, the high
Po RyR1 channel (Po = 0.197) at 0.1 µM Ca2+ (control)
was markedly reduced in Po to 0.049 by 50 µM DTT (+50 µM DTT). Subsequent
application of 500 µM DTT further decreased
Po to 0.009 (+500 µM
DTT). The Po diminished by DTT
rebounded remarkably (Po = 0.315) by addition of
500 µM pCMPS (+500 µM pCMPS).
B, the low Po RyR1 channel
(Po = 0.001) at 0.1 µM
Ca2+ (control) was strongly activated by
addition of 10 µM pCMPS to Po = 0.062 (+10 µM pCMPS). This activation
disappeared (Po = 0.007) by subsequent addition
of 25 µM DTT (+25 µM DTT).
C, the RyR3 channel was activated at 0.3 µM
Ca2+ (Po = 0.729)
(control). The four rows of current traces in the
middle were recorded 118 s after addition of 250 µM DTT. Current fluctuations of the channel remained
unchanged for the first 2 min and then suddenly ceased. The closure of
the channel with no detectable open events lasted for about 10 min
(+250 µM DTT). The channel activity was
overshot (Po = 0.904) immediately after addition
of 100 µM pCMPS (+100 µM pCMPS).
D, the RyR3 channel activity (Po = 0.020) at 0.15 µM Ca2+ (control)
was markedly increased by 10 µM pCMPS to
Po = 0.650 (+10 µM
pCMPS).
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Fig. 11, C and D, exhibits the effects of
sulfhydryl-oxidizing and -reducing reagents on the RyR3 channels. When
250 µM DTT was added to the cis side, four of
five RyR3 channels that had been activated by 0.15-0.3
µM cis-Ca2+
(Po = 0.466 ± 0.175, n = 5) suddenly switched to the closed state after a latent period (2-10
min) (Fig. 11C). In some channels, the addition of DTT
resulted in alternation between the burst of opening and the closed
state in the duration of several tens of seconds to minutes (data not
shown). One channel in five, however, did not respond to DTT even at 1 mM (data not shown). Subsequent addition of 100 µM pCMPS to channels that had been inhibited by DTT
treatment recovered usual current fluctuations with high
Po (0.543 ± 0.161, n = 3).
The RyR3 channel showing low Po (0.054 ± 0.026, n = 4) at 0.13-0.3 µM
Ca2+, on the other hand, was markedly activated by the
addition of 10 µM pCMPS to the cis side to
high Po (0.599 ± 0.192) (Fig.
11D). Thus, the effect of sulfhydryl reagents on the RyR3
channel was similar to that on the RyR1 channel; a sulfhydryl-oxidizing
reagent activates the channel activity, whereas a sulfhydryl-reducing reagent inhibits it. The channel activity of RyR3, however, appears to
depend steeply on the redox state showing almost all-or-none fashion,
whereas the dependence is more gradual with RyR1. This may be reflected
in the observation that the RyR3 channel showed no opening in the
closed state after addition of DTT, whereas some opening was still
detected with RyR1 even at Po < 0.001.
 |
DISCUSSION |
In this study, we purified homotetrameric RyR3 from rabbit
diaphragm by immunoprecipitation with highly specific antibody to RyR3
and by dissociation under gentle conditions using an epitope peptide.
The purified RyR3 demonstrated the characteristic structure of a
quatrefoil under electron microscopy. It also showed
Ca2+-dependent high affinity
[3H]ryanodine binding and cation channel activity. Thus,
RyR3 purified by this procedure retained structural and functional
integrity, which enabled us to characterize the protein.
Our recent studies with the immunoprecipitated RyR3 indicate that RyR3
forms a homotetramer of a single polypeptide with a mobility slightly
larger than that of RyR1 on SDS-PAGE (12, 13). It showed
Ca2+-dependent high affinity
[3H]ryanodine binding which was modulated by several
ligands, indicating that it constitutes a Ca2+ release
channel (12, 13). The results shown here with the purified receptor
confirmed most of these previous results. In the previous results,
[3H]ryanodine binding was partially inhibited by
Mg2+ (13). As shown in Fig. 7B in this report,
however, it was fully inhibited by Mg2+, and its
IC50 was similar to that for Ca2+. There is no
satisfactory explanation for this discrepancy at the present time.
Furthermore, several new findings were added. RyR3 was free of FKBP12,
although it retained the ability to bind FKBP12. This is also the case
with RyR1. Observations with negative staining electron microscopy
revealed that RyR3 forms a characteristic structure with 4-fold
symmetry, which is morphologically very similar to RyR1. RyR3 had a
single class of [3H]ryanodine-binding sites with a
stoichiometry of 1 mol of [3H]ryanodine per 1 mol of a
homotetramer. When incorporated into planar lipid bilayers, RyR3
displayed a cation channel current with a large conductance, which is
activated by micromolar Ca2+, caffeine, and ATP, inhibited
by millimolar concentrations of Ca2+, and Mg2+,
and ruthenium red, and modified by ryanodine. Thus, RyR3 constitutes a
homotetrameric Ca2+ release channel that shares several
basic properties with RyR1.
In addition to the overall similarities as mentioned above, some
functional differences between RyR1 and RyR3 were also noted. First,
RyR3 displays a significantly larger monovalent cation conductance
(743 ± 11 pS in 500 mM KCl) than RyR1 (630 ± 9 pS) (Fig. 8). Second, the purified RyR3 channel differs in gating kinetics from the RyR1 channel. The RyR3 channel showed longer durations of channel opening than the RyR1 channel under similar Po values as reflected in a larger fraction of
the component of the slow open time constant (
o2) (Fig.
8).
Single channel recordings of RyR3 have recently been reported with
recombinant rabbit uterine RyR3 that was expressed in HEK293 cells
(37). The properties of the purified RyR3 channel shown here well
correspond to those of the recombinant RyR3 channel in several respects
as follows: (i) a large monovalent cation conductance, (ii) modulation
by ligands, (iii) gating kinetics showing longer duration of open time,
(iv) a high open probability, and (v) biphasic Ca2+
dependence for channel activation and inactivation. Our results are
also consistent with more recent reports by Jeyakumar et al. (14) using RyR3 channels isolated from bovine diaphragm.
It was shown that FKBP12 tightly bound to RyR1 with a stoichiometry of
1 mol/mol of the RyR1 monomer and affected the function (24, 29, 30,
44). We found that FKBP12 was associated with SR vesicles isolated from
rabbit diaphragm but was not detected either in RyR1 or RyR3
preparations (Fig. 3). Wagenknecht et al. (24) recently
reported that FKBP12 was considerably dissociated from
CHAPS-solubilized RyR1 preparation. Considering the abundance of RyR1
in the SR vesicles (>99%), these results indicate that FKBP12 may be
dissociated from RyR1 during solubilization and purification
procedures. A binding study with GST-FKBP12 fusion protein revealed
that RyR3 can bind FKBP12, as is true of RyR1 (Fig. 3). Because FKBP12
is widely and abundantly expressed in mammalian tissues including
skeletal muscles (31), this suggests that RyR3 may bind FKBP12 in
situ. It should also be noted that functional properties of RyR1
and RyR3 shown here were determined with receptors lacking FKBP12. It
has been reported that removal of FKBP12 from RyR1 makes the channel
unstable, resulting in increase in the number of openings to
subconductance levels (30). Although we observed channels showing
subconductance states in both RyR1 and RyR3 preparations, populations
of such channels were minor (see Figs. 8-11). We are currently
investigating the effect of FKBP12 on the RyR3 channel activity.
With the hope of correlating the functional differences of RyR1 and
RyR3 to the specific structural features, we examined the negatively
stained images of the two isoforms. Although we could not recognize any
significant differences in their submolecular architecture, we found
that a site-directed antibody against the D2 region of RyR1 which is
absent in RyR3 binds to domain 9 or its vicinity (Fig. 5). Considering
the three-dimensional architecture of the receptor molecule, the
epitope site may correspond to the "clamp-shaped" domain according
to the terminology of Orlova et al. (45), in which they
found the occurrence of certain conformational change accompanied with
opening and closing events of the channel. Several attempts were
recently made to determine the interaction sites of the receptor
molecules with some specific proteinaceous ligands, e.g.
calmodulin (46) and FKBP12 (24). Although such studies have
successfully pin-pointed the binding sites of those proteins in the
three-dimensional architecture of the receptor molecule, none of them
has determined the position of those sites in the primary amino acid
sequence. Our result thus provides the first case to link the
three-dimensional architecture of RyR with its primary structure.
Since the distance between two adjacent voltage sensors of a tetrad is
almost equal to the dimension of a "foot" structure, it has been
postulated that the corners of a quatrefoil including domain 9 or
clamp-shaped domain might interact directly with dihydropyridine receptor of the T-tubules (45, 47, 48). On the other hand, the crucial
role of the D2 region in the E-C coupling of skeletal muscle was
claimed based on the fact that the deletion of the D2 region causes the
functional loss of Ca2+ release activity triggered by the
electrical stimulation while preserving caffeine-induced
Ca2+ release (49). Our assignment of the D2 region to
domain 9 or its vicinity may reasonably link those two issues and could
suggest the importance of the D2 region in the interaction of RyR1 with dihydropyridine receptor. Recently, two additional regions of RyR1
were also shown to interact with dihydropyridine receptor (50, 51). It
is also of interest where these stretches are localized in the
three-dimensional structure of the RyR molecule since these regions are
relatively close to the D2 region in the primary structure of the receptor.
It has been demonstrated that a number of sulfhydryl-oxidizing reagents
activate RyR channels (40, 41, 43). Sulfhydryl-reducing reagents such
as glutathione and DTT have been shown to reduce the RyR channel
activity (39, 42). On single channel recordings, we found that a
sulfhydryl-oxidizing reagent pCMPS activated both RyR1 and RyR3
channels, whereas a sulfhydryl-reducing reagent DTT inhibited them
(Fig. 11). The channel activity stimulated by the oxidizing reagent was
reversed by the subsequent addition of the reducing reagent, and vice
versa. Transition between the two states is reversible. We observed two
populations of channel activity (high Po and low
Po channel) in RyR1 preparation (Fig. 10).
Multiple populations of RyR1 channel activity were also reported with
SR vesicles prepared from skeletal muscles (38, 39). Because the
channel activity of RyR is dependent on the redox state of the receptor
molecule (Fig. 11), distinct populations of RyR1 channels may be
attributed to the heterogeneity of the redox state of the receptor in
the lipid bilayer, as recently suggested by Marengo et al.
(39). In contrast, only a single population of current fluctuations
with Po ~1 was detected in RyR3 preparation
(Fig. 10). Consistently, no heterogeneity of RyR3 channels was so far
reported (14, 37). Homogeneous channel activity was also shown in
cardiac RyR (RyR2) (38). These results indicate that RyR3 may be much
more steeply dependent on the redox state in the channel activity than
RyR1, showing almost all-or-none fashion. This may be reflected in
homogeneous channel activity of RyR3 in contrast to heterogeneous
population of RyR1.
The results of the Ca2+ sensitivity of RyR3 for activation
and inactivation were so far controversial (13, 37, 52). We determined
the Ca2+ dependence of the purified RyR3 both in
[3H]ryanodine binding and in single channel recordings,
and surprisingly we found that they were entirely different.
EC50 of RyR3 for Ca2+ in
[3H]ryanodine binding was about 14 µM which
was about 4-fold larger than that of RyR1 (Fig. 7A),
confirming the previous results with the immunoprecipitated RyR3 (13).
This is consistent with the CICR activity of RyR1-knock-out (dyspedic)
mice skeletal muscle which is probably attributed to RyR3 (52). In
contrast, the RyR3 channel in single channel recordings was steeply
activated to Po ~1 between 0.1 and 1 µM Ca2+ in almost all-or-none fashion (Fig.
10C). A very similar relationship was obtained with the
recombinant rabbit uterine RyR3 channel (37) and the RyR3 purified from
bovine diaphragm (14). Although a higher salt concentration in the
channel recordings (500 mM KCl) than in the ryanodine
binding (170 mM NaCl) might be a possible cause, it is
unlikely because similar results of the single channel experiments with
the recombinant RyR3 were obtained in relatively low salt concentration
(250 mM KCl) (37). These findings indicate that the
difference in Ca2+ dependence is simply attributed to the
method adopted. RyR3 channel showed almost full opening
(Po ~1) at 10 µM
Ca2+ in the absence of adenine nucleotide or caffeine (Fig.
10) (37). [3H]Ryanodine binding activity at the same
Ca2+ concentration, in contrast, was further stimulated by
adenine nucleotides or caffeine (13). It was so low in the presence of
Ca2+ alone that a supplement of 4 mM AMP was
required to obtain the precise results (Fig. 7A).
Dithiothreitol (2 mM), on the other hand, was included in
the medium for ryanodine binding but not for channel recordings. The
low Po activity of RyR1 appears to correspond
well to [3H]ryanodine binding in the presence of
Ca2+ alone. Marengo et al. (39) reported that
the Ca2+-dependent channel activity of RyR was
sensitively dependent on the redox state. We observed that the channel
activity of RyR3 was likely to be varied in almost all-or-none fashion
by the redox state, whereas that of RyR1 was gradually dependent. The
steep increase in the channel activity of RyR3 between 0.1 and 1 µM Ca2+ (Fig. 10C) may indicate
that some Ca2+-dependent process(es) could also
be involved in the abrupt transition of the channel activity in RyR3.
This steep dependence may be a probable explanation for the difference
in the Ca2+ dependence.
 |
ACKNOWLEDGEMENTS |
E. K. is indebted to Dr. Kazuo Sutoh,
the University of Tokyo, for the generous gift of the divalent-biotin.
We thank Kazunao Wakatsuki, Showa University, for technical assistance
in preparing the GST fusion protein of the D2 region.
 |
FOOTNOTES |
*
This work was supported in part by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Science, Sports and
Culture, by the Uehara Memorial Foundation (to T. M.), and by the
Naito Foundation (to H. O.).
§
These authors equally contributed to this work.
§§
To whom reprint requests and correspondence should be addressed:
Dept. of Pharmacology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-3-5802-1034; Fax:
81-3-5802-0419; E-mail: ysogawa{at}med.juntendo.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
RyR, ryanodine
receptor;
CHAPS, 3-[(3-cholamidopropyl)
dimethylammonio]-1-propanesulfonic acid;
CICR, Ca2+-induced Ca2+ release;
DTT, dithiothreitol;
FKBP12, 12-kDa FK506-binding protein;
GST, glutathione
S-transferase;
MOPSO, 3-(N-morpholino)-2-hydroxypropanesulfonic acid;
PAGE, polyacrylamide gel electrophoresis;
SR, sarcoplasmic reticulum;
PVDF, polyvinylidene difluoride;
AMP-PCP, adenosine
5'-(
,
-methylenetriphosphate);
pCMPS, p-chloromercuriphenylsulfonic acid.
 |
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