From the Cardiovascular Research Group, Departments
of Physiology and Biophysics and of Biochemistry and Molecular
Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada, the
§ Wadsworth Center, New York State Department of Health,
Albany, New York 12201, and the ** Department of Biomedical
Sciences, School of Public Health, State University of New York,
Albany, New York 12201
Received for publication, December 26, 2002, and in revised form, February 5, 2003
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ABSTRACT |
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Of the three divergent regions of
ryanodine receptors (RyRs), divergent region 3 (DR3) is the best
studied and is believed to be involved in excitation-contraction
coupling as well as in channel regulation by Ca2+ and
Mg2+. To gain insight into the structural basis of DR3
function, we have determined the location of DR3 in the
three-dimensional structure of RyR2. We inserted green fluorescent
protein (GFP) into the middle of the DR3 region after Thr-1874 in the
sequence. HEK293 cells expressing this GFP-RyR2 fusion protein,
RyR2T1874-GFP, were readily detected by their green
fluorescence, indicating proper folding of the inserted GFP.
RyR2T1874-GFP was further characterized functionally by
assays of Ca2+ release and [3H]ryanodine
binding. These analyses revealed that RyR2T1874-GFP functions as a caffeine- and ryanodine-sensitive Ca2+
release channel and displays Ca2+ dependence and
[3H]ryanodine binding properties similar to those of the
wild type RyR2. RyR2T1874-GFP was purified from cell
lysates in a single step by affinity chromatography using GST-FKBP12.6
as the affinity ligand. The three-dimensional structure of the purified
RyR2T1874-GFP was then reconstructed using cryoelectron
microscopy and single particle image analysis. Comparison of the
three-dimensional reconstructions of wild type RyR2 and
RyR2T1874-GFP revealed the location of the inserted GFP,
and hence the DR3 region, in one of the characteristic domains of RyR,
domain 9, in the clamp-shaped structure adjacent to the FKBP12 and
FKBP12.6 binding sites. COOH-terminal truncation analysis demonstrated
that a region between 1815 and 1855 near DR3 is essential for
GST-FKBP12.6 binding. These results provide a structural basis for the
role of the DR3 region in excitation-contraction coupling and in
channel regulation.
The ryanodine receptor
(RyR),1 located in the
sarco(endo)plasmic reticulum of a variety of cells, functions as an
intracellular Ca2+ release channel and plays an essential
role in various fundamental cellular processes including muscle
contraction and relaxation, fertilization, and apoptosis (1). Three RyR
isoforms, RyR1, RyR2, and RyR3, have been identified in mammalian
tissues and have been shown to display distinct patterns of expression.
RyR1 is predominantly expressed in skeletal muscle, whereas RyR2 is mainly expressed in cardiac muscle and the brain. RyR3 expression is
widespread but at relatively low levels (2-5). The physiological function and mechanism of activation are also specific to each RyR
isoform. RyR1 is required for excitation-contraction (EC) coupling in
skeletal muscle, in which RyR1 is activated upon depolarization by the
voltage sensor, the L-type Ca2+ channel (dihydropyridine
receptor, DHPR), through a direct physical interaction. RyR2, on the
other hand, mediates EC coupling in cardiac muscle, and it is activated
by Ca2+ influx through the L-type Ca2+ channel
via a mechanism known as Ca2+-induced Ca2+
release. Importantly, neither RyR2 nor RyR3 is capable of substituting for RyR1 in mediating skeletal type EC coupling (2, 6-9). The
physiological role of RyR3 is unclear, but RyR3 has been shown to be
involved in amplification of the Ca2+ signal generated by
RyR1 in skeletal muscle (3, 10) and in regulation of the
Ca2+ release activity of RyR1 or RyR2 in smooth muscle
(11). Some of these functional differences among RyR isoforms are
likely to be attributable to differences in their primary sequences. One of our goals is to determine which differences in the amino acid
sequences of the isoforms are responsible for these functional variations.
The amino acid sequences of the three RyR isoforms share a high degree
of sequence identity (66-70%) with their major variations occurring
in three regions, known as divergent regions 1, 2, and 3 (DR1, DR2, and
DR3) (12). The DR1 region comprises residues 4254-4631 of RyR1 and
residues 4210-4562 of RyR2. The DR2 region includes residues
1342-1403 of RyR1 and residues 1353-1397 of RyR2. Residues 1872-1923
of RyR1 and residues 1852-1890 of RyR2 are denoted as the DR3 region.
These divergent regions have been the focus of a number of structural
and functional studies. For instance, sequence variations in the DR1
region between RyR1 and RyR2 have been shown to account for the
isoforms' different sensitivities to Ca2+ inactivation
(13-15), whereas the DR2 region, which is absent in RyR3, was found to
be critical for skeletal muscle EC coupling, since deletion of DR2 in
RyR1 abolished EC coupling (16).
The DR3 region appears to have multiple roles in RyR channel function.
In RyR1, DR3 consists of a stretch of ~30 negatively charged amino
acid residues that are thought to constitute a low affinity
Ca2+ binding site (17). Consistent with this hypothesis,
deletion of the DR3 region altered the Ca2+- and
Mg2+-dependent regulation and conduction
properties of the RyR1 channel (18-20). A region of RyR1 between
residues 1837 and 2168, encompassing the DR3 region, has also been
shown to interact with the II-III loop of the DHPR and to participate
in EC coupling (21). Interestingly, the interaction between a sequence
in the II-III loop of DHPR and RyR1 was enhanced by the 12-kDa
FK506-binding protein (FKBP12), suggesting that FKBP12 may modulate EC
coupling in skeletal muscle (22). Recently, we have shown that an
NH2-terminal fragment containing the first 1937 residues of
RyR2 is sufficient to enable GST-FKBP12.6 binding, whereas a shorter
NH2-terminal fragment including only the first 1636 residues is unable to bind GST-FKBP12.6, indicating that the region
between residues 1636 and 1937 of RyR2, encompassing the DR3 region, is
required for FKBP12.6 interaction (23). Whether the corresponding
region in RyR1 is essential for FKBP12-RyR1 interaction has yet to be
determined. Taken together, these observations suggest that residues
within or near the DR3 region may be involved in EC coupling and in
channel regulation by Ca2+, Mg2+, or FKBPs. The
molecular mechanisms by which the DR3 region participates in such
seemingly diverse functions are, however, unknown.
One approach to understanding the roles of individual regions or
domains in RyR channel function is to map their three-dimensional locations in the channel structure. The three-dimensional structures of
all three RyR isoforms have been determined to ~30-40 Å by cryoelectron microscopy (cryo-EM) combined with single particle image
processing (24, 25). The three RyR isoforms display virtually identical
three-dimensional structures, as expected based on their high degree of
sequence identity. The three-dimensional structure of RyR is
essentially composed of two major components: a cytoplasmic assembly
and a transmembrane assembly. The cytoplasmic assembly contains at
least 10 domains (26). Large conformational changes, particularly in
the clamp-shaped structure that includes domains 5, 6, 9, and 10, and
is located at the corners of the cytoplasmic assembly, have been
observed when the channel was switched from the closed to the open
state (25, 27). The three-dimensional locations of several ligand
binding sites, including those for calmodulin, FKBP12, FKBP12.6, and
Imperatoxin A, have been identified (24). We have recently mapped the
three-dimensional locations of the NH2-terminal and DR1
regions. The NH2 terminus is located at the corners within
the clamp structure, whereas the DR1 region maps to domain 3, also
known as the "handle" domain, adjacent to the calmodulin binding
site (28, 29).
In the present study, in order to gain insight into the
structural basis and functions of the DR3 region, we have constructed and expressed in HEK293 cells a RyR2 fusion protein in which
green fluorescence protein (GFP) was inserted into the DR3 region after residue Thr-1874 in the sequence (RyR2T1874-GFP). The
RyR2T1874-GFP protein was purified and then subjected to
cryo-EM and single-particle image processing. Three-dimensional
reconstruction of RyR2T1874-GFP revealed the localization
of GFP, and therefore of DR3, to a region in domain 9 in the
clamp-shaped structure, adjacent to the known FKBP12 and FKBP12.6
binding sites. These results suggest a role of the DR3 region in
conformational changes and in EC coupling, and they further demonstrate
the utility of this approach in correlating linear sequence to
three-dimensional structure, thereby allowing an understanding
of the structural basis of RyR function.
Materials--
Restriction endonucleases and DNA-modifying
enzymes were purchased from New England BioLabs Inc. The anti-RyR and
anti-GFP antibodies were obtained from Affinity BioReagents (Golden,
CO). The anti-c-Myc antibody was kindly provided by the Immunology Core
Facility at the Wadsworth Center of the New York State Department of
Health. Soybean phosphatidylcholine was obtained from Avanti Polar
Lipids, Inc. CHAPS and other reagents were purchased from Sigma.
Cell Culture and DNA Transfection--
HEK293 cells were
maintained in Dulbecco's modified Eagle's medium as described
previously (30). HEK293 cells grown on 100-mm tissue culture dishes for
18-20 h after subculture were transfected with 6-12 µg of wild type
(WT) or mutant RyR cDNAs using Ca2+ phosphate
precipitation (31).
Construction of RyR2T1874-GFP--
The cloning and
construction of the 15-kb full-length cDNA encoding the mouse
cardiac RyR2 has been described previously (32). The DNA encoding GFP
flanked by Gly-rich spacers and an AscI site was obtained by
PCR as described previously (29). The AscI site was
introduced into the DR3 region of RyR2 after Thr-1874 in the sequence
by overlap-extension using PCR (33). The "outer" primers used were as follows: forward, 5'-GATCCTCTGCAGTTCATGTCCCTC-3'; reverse,
5'-TCCATCACTGTCTCATGCATCCC-3'. The primers for introducing the
AscI site were as follows: forward,
5'-AGTATAGAAGACGGGCGCGCCGAAGGCGAAGAAGAAGCC-3'; reverse,
5'-TTCTTCGCCTTCGGCGCGCCCGTCTTCTATACTGATCTC-3'. The
Bsu36I (4900)-EcoRV (6439) PCR fragment
containing the AscI site was used to replace the
Bsu36I (4900)-EcoRV (13,873) fragment in the full-length RyR2. The missing EcoRV (6439)-EcoRV
(13,873) fragment was then added back to yield the full-length RyR2
with the inserted AscI site. The
AscI-AscI fragment containing GFP and the spacers was then subcloned into the full-length RyR2 at the introduced AscI site. The sequences of all PCR fragments and the
orientation of the inserted GFP cDNA were verified by DNA
sequencing analysis.
Construction of COOH-terminal Truncation Mutants of
RyR2--
Construction of the 1-1937 and 1-1636 COOH-terminal
truncation mutants was described earlier (23). PCR was used to
introduce a stop codon after residues 1895, 1855, 1815, 1775, and 1735 to generate the COOH-terminal truncation mutants, 1-1895, 1-1855, 1-1815, 1-1775, and 1-1735, respectively. The forward primer used for all truncation mutants was 5'-GATCCTCTGCAGTTCATGTCCCTC-3'. The
reverse primers, each containing a stop codon followed by an
EcoRV site were
5'-ATGATATCTCACATCTGGAGCAAGCCTTCCTT-3' (1895-stop), 5'-ATGATATCTCAAGCTTCCTTAAACACACTGGG-3' (1855-stop),
5'-ATGATATCTCACGTGGTCCCACCCACAGGATC-3' (1815-stop),
5'-GATATCCTAGTCGTTGCTAATGCTCACGA-3' (1775-stop), and
5'-GATATCCTACTTTGTCTCCTCTGTCATAG-3' (1735-stop). The
Bsu36I (4900)-EcoRV (introduced after the stop
codon at various sites) PCR fragments were used to replace the
Bsu36I (4900)-EcoRV (13,873) fragment in the
full-length RyR2 cDNA to yield the various COOH-terminal truncation
mutants. All truncation mutants were confirmed by DNA sequencing.
Cryoelectron Microscopy and Image Processing--
The expression
and purification of RyR2T1874-GFP was carried out as
described previously (29). The purified RyR2T1874-GFP was
diluted 5-10-fold with EM dilution buffer (20 mM Na-PIPES, (pH 7.2), 400 mM KCl, 3 mM EGTA, 0.5% CHAPS, 2 mM dithiothreitol, and 2 mg/ml leupeptin). Grids were
prepared for cryo-EM according to standard methods (34). Micrographs
were recorded using low dose protocols on a Philips EM420, equipped
with low dose kit and a GATAN 626 cryotransfer holder, at a
magnification of 38,600 × (±2%) as verified by a tobacco
mosaic virus standard. Each exposure corresponded to an electron dose
of ~10 e Ca2+ Release and [3H]Ryanodine Binding
Assays--
Measurements of free cytosolic Ca2+
concentrations in the transfected HEK293 cells, using fluorescence
Ca2+ indicator dye fluo-3, and of equilibrium
[3H]ryanodine binding to cell lysate were done as
described previously (38).
GST-FKBP12.6 Pull-down, Immunoprecipitation, and Immunoblotting
Analyses--
GST-FKBP12.6 pull-down, immunoprecipitation, and
immunoblotting were carried out as described previously (23, 29).
Insertion of Green Fluorescent Protein into the DR3 Region of
RyR2--
To understand how the DR3 region participates in channel
function and regulation, we attempted to map the location of the DR3
region onto the three-dimensional structure of RyR2. To this end, we
constructed a RyR2 fusion protein (RyR2T1874-GFP) in which GFP was inserted into the middle of the DR3 region, after residue Thr-1874 in the sequence (Fig. 1). The
strategy was to use GFP, a relatively small protein (238 amino acids,
compared with the ~5000 amino acids of RyR), as a structural marker
(39, 40). Through comparison of the three-dimensional reconstructions
of the WT RyR2 and GFP-inserted RyR2, it would then be feasible to map
the location of GFP and thus the region into which GFP was inserted. To
minimize potential perturbation of the channel folding and function of
RyR2, we added two glycine-rich spacers on either side of GFP (40).
Expression and Functional Characterization of
RyR2T1874-GFP--
Since the fluorescence of GFP
depends on the proper folding of its own structure and the structure of
the inserted region (40, 41), GFP could be used not only as a
structural marker but also as an indicator of proper protein folding.
Fig. 2A shows phase-contrast
and fluorescent microscopic images of the HEK293 cells transfected with
RyR2T1874-GFP. Green fluorescent HEK293 cells were readily
detected after transfection (Fig. 2A, a), whereas no
fluorescence was observed in HEK293 cells transfected with the WT RyR2
(data not shown). The green fluorescence indicates that the inserted
GFP and its neighboring region are likely to have folded properly. A
reticular pattern of distribution of RyR2T1874-GFP was
observed in most of the fluorescent cells, as expected based on its
function as an intracellular Ca2+ release channel (Fig.
2B). In some cells, however, the fluorescence appeared as an
amorphous mass due to heavy expression of the RyR2T1874-GFP protein.
To further demonstrate that RyR2T1874-GFP retains proper
folding, we assessed its functional properties. We measured
Ca2+ release from transfected HEK293 cells using the
fluorescent Ca2+ indicator dye fluo-3. As shown in Fig.
2B, HEK293 cells transfected with RyR2T1874-GFP
cDNA displayed multiple Ca2+ release events in response
to repeated stimulation by caffeine (Fig. 2B, a),
an activator of RyRs. Transfected cells pretreated with ryanodine, on
the other hand, responded only to the first and not the second or third
caffeine stimulation (Fig. 2B, b), suggesting
that RyR2T1874-GFP is sensitive to ryanodine modification (42). The immediate drop in fluorescence after the second and third
caffeine stimulations was caused by fluorescence quenching by caffeine
(42). Similar Ca2+ release responses to caffeine and
ryanodine were observed in HEK293 cells transfected with WT RyR2 (42).
No caffeine- or ryanodine-induced Ca2+ release was detected
in nontransfected HEK293 cells or in cells transfected with pCDNA3
vector DNA (data not shown). Thus, RyR2T1874-GFP is able to
form a caffeine- and ryanodine-sensitive Ca2+ release
channel in HEK293 cells.
The functional properties of RyR2T1874-GFP were further
characterized by [3H]ryanodine binding studies, which
have been widely used as a functional assay for RyR channel activity,
since ryanodine has access to its binding site only when the channel is
open (43). Fig. 3A shows
[3H]ryanodine binding to a cell lysate prepared from
HEK293 cells transfected with RyR2T1874-GFP in the presence
of a wide range of Ca2+ concentrations. Analysis of the
Ca2+ dependence of [3H]ryanodine binding by
the Hill equation yielded an EC50 of 0.20 ± 0.02 µM (mean ± S.E., n = 3). This
EC50 value is similar to that reported for the WT RyR2
(29). We also performed binding in the presence of various
concentrations of [3H]ryanodine (Fig. 3B).
Scatchard analysis of these binding data revealed that
RyR2T1874-GFP exhibited high affinity
[3H]ryanodine binding with a Kd of
2.75 ± 0.43 nM (n = 3), which is also
similar to the behavior of [3H]ryanodine binding to WT
RyR2 reported previously (38). No ryanodine binding activity was
detected in lysates from HEK293 cells transfected with either
pCDNA3 vector DNA or no DNA. Taken together, these results suggest
that the insertion of GFP after Thr-1874 does not significantly alter
the structure or function of RyR2.
Purification of Recombinant RyR2T1874-GFP Protein by
GST-FKBP12.6 Affinity Chromatography--
RyR2T1874-GFP
retains the ability to interact with GST-FKBP12.6. Taking advantage of
this property, we were able to purify the RyR2T1874-GFP
protein from transfected HEK293 cell lysate by affinity chromatography
in a single step using GST-FKBP12.6 as the affinity ligand. The
purified protein was analyzed by SDS-PAGE and immunoblotting. A single
high molecular weight band, which migrated at a slightly slower rate
than the WT RyR2 (as expected due to the addition of GFP), was detected
in the purified sample of RyR2T1874-GFP (Fig.
4A). This band was recognized
by both the anti-RyR and anti-GFP antibodies and hence corresponds to
RyR2T1874-GFP. On the other hand, the purified WT RyR2
protein was recognized only by the anti-RyR antibody and not by the
anti-GFP antibody (Fig. 4, B and C).
RyR2T1874-GFP appears to be expressed in an intact form; no
degradation products were detected.
Cryo-EM and Three-dimensional Reconstruction of
RyR2T1874-GFP--
The purified RyR2T1874-GFP
proteins were preserved in a thin layer of vitreous ice and imaged by
cryo-EM. Fig. 5 shows a typical electron
micrograph of frozen-hydrated RyR2T1874-GFP in which individual RyR2T1874-GFP particles are readily visualized.
The particles displayed an apparently intact structure with multiple orientations similar to those observed in the WT RyR2 (29).
Single particle image processing was then applied to obtain a more
detailed structure of RyR2T1874-GFP. Fig.
6A shows a surface representation of the three-dimensional reconstruction of
RyR2T1874-GFP in three orientations (top, bottom, and side
views). The overall form of the reconstructed RyR2T1874-GFP
structure resembles a mushroom in shape and consists of two major
components: a large squarelike cytoplasmic assembly (290 × 290 × 130 Å) encompassing 10 distinct domains (labeled by
numerals in Fig. 6) and a smaller transmembrane assembly
(120 × 120 × 70 Å, labeled TA).
Overall, the three-dimensional reconstruction of
RyR2T1874-GFP is very similar to that of the
recombinant WT RyR2 obtained previously (29), but close examination
reveals some small differences. The most noticeable difference was
found on domain 9, one of the domains located within the "clamp"
structures that form each of the corners of the RyR2 square-shaped
cytoplasmic assembly (25, 27). Specifically, the volume of domain 9 of
RyR2T1874-GFP appears to be larger than the volume in the
corresponding domain in WT RyR2. These differences could result either
directly from the GFP insertion or from conformational changes caused
by GFP insertion. To more precisely determine the differences, we
generated a three-dimensional difference map by subtracting the
three-dimensional volume of WT RyR2 from that of
RyR2T1844-GFP. The difference regions were displayed in
green and superimposed on the three-dimensional reconstruction of WT
RyR2 (shown in blue) using nearly the same threshold as was
used in the display of RyR2T1874-GFP and WT RyR2 (Fig.
6B). This difference map clearly indicates four significant
differences located in each domain 9 in the cytoplasmic assembly. This
significant difference, repeated four times in the difference map, is
not only due to the 4-fold symmetry enforcement, since RyR2 is a
homotetramer composed of four identical monomers. Because GFP was
inserted into each RyR2 monomer, the difference as a result of GFP
insertion would be expected to repeat four times in the
three-dimensional difference map.
We believe that these differences are directly attributable
to the excess mass contributed by the GFP insertion in
RyR2T1874-GFP, because they are the
only significant differences that appear when the three-dimensional
difference map is displayed at a density threshold nearly the same as
used to image the RyR2T1874-GFP and WT RyR2 structures.
Furthermore, the calculated volume of each of the four difference
features in Fig. 6B corresponds to a molecular mass of 28 kDa, assuming a protein density of 1.37 g/cm3 (26), and
this volume agrees well with the molecular mass of GFP. Other minor
differences are small and insignificant, both in volume and density,
and are unlikely to correspond to the inserted GFP. Hence, we conclude
that the DR3 region, as indicated by the inserted GFP, is located in
domain 9, one of the characteristic domains that form the
cytoplasmically located clamp structures of RyR2.
The FKBP12 binding site was previously mapped to a region at or near
the junction of domains 3 and 9 in the three-dimensional structure of
RyR1 (indicated by an asterisk in Fig. 6B) (44, 45). Recently, the FKBP12.6 binding site has also been mapped to a
similar region in the three-dimensional structure of RyR2.2
Interestingly, the three-dimensional location of the DR3 region, as
indicated by the localization of the inserted GFP, is in close proximity to the FKBP12 and FKBP12.6 binding
sites.2 The distances between
the DR3 location and the binding sites for FKBP12 and FKBP12.6 are ~3
and 2.5 nm, respectively. The close localization of the DR3 region and
FKBP binding sites in the three-dimensional structure of RyR suggests
that residues near the DR3 region may be involved in FKBP12/12.6 interaction.
A Region Upstream of DR3 Is Essential for FKBP12.6 Binding--
We
have previously shown that the first 1937 NH2-terminal
amino acid residues of RyR2, a span that includes the DR3 region, are
sufficient for FKBP12.6 binding (23). To examine the role of the DR3
region in FKBP12.6 interaction, we constructed a series of c-Myc-tagged
COOH-terminal truncation mutants by inserting a stop codon between
residues 1636 and 1937 (Fig.
7A). These COOH-terminal truncation mutants were then expressed in HEK293 cells and their expression was ascertained by immunoprecipitation using the anti-c-Myc antibody, whereas their ability to interact with FKBP12.6 was assessed
by a GST-FKBP12.6 pull-down assay. As shown in Fig. 7B, NH2-terminal fragments containing the first 1815 or fewer
residues are unable to bind GST-FKBP12.6, whereas
NH2-terminal fragments including the first 1855 or more
residues are sufficient for FKBP12.6 binding. These data indicate that
the DR3 region (1852-1890) is unlikely to be critical for FKBP12.6
binding, whereas a region between residues 1815 and 1855 upstream of
DR3 is essential for FKBP12.6 interaction. The GST-FKBP12.6
binding-deficient NH2-terminal fragment, 1-1815, is able
to restore the function of an NH2-terminal truncation
mutant, D1-1636 (Fig. 8A).
HEK293 cells transfected with the 1-1815 NH2-terminal
fragment and the D1-1636 NH2-terminal truncation mutant
alone exhibited no caffeine-sensitive Ca2+ release
activity, whereas caffeine-induced Ca2+ release was readily
observed in cells co-transfected with both the 1-1815 and D1-1636
fragments (Fig. 8B). These observations indicate that the
1-1815 NH2-terminal fragment is functional when co-expressed with an overlapping COOH-terminal fragment and that the
lack of GST-FKBP12.6 binding to this fragment is unlikely to result
from abnormal expression or gross structural alterations.
By applying cryo-EM and three-dimensional reconstruction to
recombinant RyR2 containing a GFP insertion at position 1874, near the
center of the divergent region, DR3, we have mapped DR3 to a subregion
within domain 9 in the cytoplasmic assembly of the receptor. Also, new
evidence has been presented to further implicate a region of the RyR2
sequence that is just upstream of the DR3 region as being required for
the binding of the accessory protein, FKBP12.6. Consistent with this
interpretation, previous mapping studies by cryo-EM of FKBP12-RyR1 and
FKBP12.6-RyR2 complexes have placed the FKBP12/FKBP12.6 binding
sites adjacent to domain 9 in both receptor isoforms. Although our
mapping of the DR3 region was obtained on RyR2, we are confident that
essentially the same result would have been obtained for RyR1 and RyR3,
because the high degree of sequence identity (~70%) among the
isoforms means that their tertiary structures should also be very
similar. Indeed, the three-dimensional reconstructions that have been
reported for the three RyR isoforms show strict conservation of the
domain architecture of the cytoplasmic region and few significant
differences at the 2-4-nm resolution level (24). Thus, we conclude
that the DR3 region maps to domain 9 in all three RyR isoforms. Much of
the following discussion focuses on the skeletal muscle receptor, RyR1,
which has been more intensively characterized than RyR2, with respect
to the function of DR3.
Implications for Excitation-Contraction Coupling in Skeletal
Muscle--
Comparison of the three-dimensional reconstructions of the
closed and open states of RyR1 reveals that several domains, including 5, 6, 9, and 10, in the clamp-shaped structures at the corners of the
cytoplasmic assembly, undergo large conformational changes and/or
movements (25, 27). Based on these findings and on the observation that
the spacing between the receptor's clamp structures is similar to that
of a group of four DHPRs (also known as the tetrad) that abut on RyR1
in situ at triad junctions, it has been proposed that the
clamp structures undergo direct physical interaction with DHPRs (25,
27, 46). The amino acid sequences that make up domains 5, 6, 9, and 10 are, however, unknown. Several regions in the linear sequence of RyR1,
including the DR2 and DR3 regions (16, 21), and regions between
residues 1076 and 1112 (47) and between residues 2659 and 3720 (48)
have been implicated as being important for EC coupling or for
interactions between RyR1 and DHPR. Until now, it was not known with a
high level of confidence which, if any, of these sequences is located in the clamp-shaped structure. This work reveals, for the first time,
that the DR3 region forms part of domain 9, one of the domains comprising the clamp-shaped structure, and raises the possibility that
DR3 is involved in the conformational changes in RyRs that are
associated with channel gating. This interpretation is supported by
comparative studies of RyR1 and RyR3 in closed and open states, which
reveal structural differences between the two states in the vicinity of
domain 9 (25, 27, 49). Thus, our observations further support the
notion that domain 9 is important for EC coupling.
DR3 Is Located Near the FKBP12/FKBP12.6 Binding Sites on
RyRs--
Our results are also suggestive of a structural link between
EC coupling and the binding of FKBP12, which is tightly associated with
RyR1 (50). FKBP12 has been suggested to be an important regulator of EC
coupling, in part because FK506, an immunosuppressant drug that
dissociates FKBP12 from RyR1, has been shown to impair EC coupling in
skeletal muscle (51). Moreover, the interaction between RyR1 and the
II-III loop of DHPR is apparently potentiated by FKBP12 (22). The
molecular mechanism by which FKBP12 enhances DHPR-RyR1 interaction and
modulates EC coupling is, however, unclear. We found that a region
including residues 1815-1855, upstream of the DR3 region, is essential
for GST-FKBP12.6 binding and that the three-dimensional location of the
DR3 region is very close to the FKBP12 and FKBP12.6 binding sites. It
is thus possible that FKBP12, by binding to a site near the DR3 region
may either stabilize a conformation required for DR3-DHPR interaction,
as suggested previously (22), or may stabilize the DR3-DHPR interaction directly.
DR3 and Regulation of RyRs by Ca2+ and
Mg2+--
The DR3 region encompasses a highly negatively
charged sequence that is thought to form the low affinity
Ca2+ binding site. Deletion of the DR3 region affected both
Ca2+-dependent activation and inactivation and
Mg2+ inhibition of the RyR1 channel (17-20).
Mg2+ is an essential regulator of EC coupling in skeletal
muscle. It has been proposed that the initial activation of RyR1 by the voltage sensor during EC coupling involves the removal of the inhibitory effect of cytoplasmic Mg2+ (52). However, the
molecular mechanisms underlying Mg2+ inhibition of RyR
channel activity and EC coupling are unknown. Localization of the DR3
region, and consequently the putative Mg2+ inhibitory site,
to domain 9 within the clamp structure suggests certain hypotheses as
to the role of Mg2+ in conformational changes and in EC
coupling. For instance, it is possible that Mg2+ binds to
domain 9, stabilizing RyR1 in an inactive conformation, and that the
II-III loop of DHPR may bind to the same domain, relieving
Mg2+ inhibition and resulting in channel openings. Binding
of Mg2+ to domain 9 may also affect the binding of FKBP,
which binds adjacent to domain 9 (45). This may explain the observation that Mg2+ influences the interaction between FKBP and RyR
(53, 54). It will be of great interest to determine whether mutations
in the DR3 region, particularly among the negatively charged residues, alter Mg2+ inhibition and skeletal muscle EC coupling.
DR3 and Channel Conduction--
The DR3 region appears to be
involved not only in channel gating but also in channel conduction,
since deletion of the DR3 region has been shown to alter the conduction
properties of RyR1 (20). The DR3 region is located along the edge of
the cytoplasmic assembly, far away from the channel pore-forming region
located in the transmembrane assembly, so how this region influences
channel conduction is unclear. It is unlikely that the DR3 region is
directly involved in the formation of the ion conduction pathway.
It is, however, possible that conformational changes in the DR3 region may have long range effects on both gating and conduction.
In summary, domain 9, which is involved in the structural changes
that occur upon channel activation and is also in close contact with
bound FKBPs, appears to encompass, within its DR3-containing region, a
binding site for the II-III loop of DHPR and a low affinity cation
binding site for Ca2+ and Mg2+. Accordingly, we
propose that domain 9 constitutes an essential part of the
macromolecular apparatus that carries out EC coupling. It is probable
that multiple regions or domains are involved in EC coupling. The most
likely additional candidates for EC coupling are the DR2 region and the
regions between residues 1076 and 1112 and between residues 2659 and
3720 or domains 5, 6, and 10, located within the clamp-shaped structure
(16, 25, 27, 46-48). The DR2 region is completely missing in RyR3, and
comparison of the three-dimensional reconstructions of RyR1 and RyR3
revealed a significant difference in domain 6, suggesting that domain 6 may include the DR2 region (49). The three-dimensional locations of
residues 1076-1112 and residues 2659-3720 and the sequence identity
of domains 5 and 10 are unknown. A systematic and comprehensive study
by cryo-EM and single particle image processing of modified RyR
proteins containing GFP insertions into various regions in RyR will
allow us to further define the structural basis of EC coupling and
other functions of the RyR channel.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/Å2. Micrographs were
checked for drift, astigmatism, and presence of Thon rings by optical
diffraction. Selected electron micrographs were digitized on a
Zeiss/Imaging scanner (Z/I Imaging Corp., Huntsville, AL) with a step
size of 14 µm. Images were processed using the SPIDER/WEB software
package (35), and three-dimensional reconstructions were obtained
through use of the projection matching procedure (29, 36). The final
three-dimensional reconstructions of WT RyR2 and
RyR2T1874-GFP were computed, respectively, from 4365 and
4374 particles. 4-Fold symmetry was enforced in both three-dimensional
reconstructions. The final resolutions of both reconstructions were
estimated to be 34 Å by Fourier shell correlation using a cut-off
value of 0.5 (37). The difference map was calculated by subtracting the
three-dimensional volume of the WT RyR2 from the
RyR2T1844-GFP volume.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Insertion of GFP into RyR2 after residue
Thr-1874 in the sequence. The amino acid sequences of the three
RyR isoforms differ primarily in three regions known as the divergent
regions (DR1, DR2, and DR3) shown by the shaded
areas. GFP flanked by two Gly-rich spacers was inserted into
the DR3 region after Thr-1874, as indicated by a filled
box. The linear sequence of RyR2 is denoted by an
open rectangle. The hatched
area depicts the transmembrane domain (TM). The
phosphorylation site (S2808), the calmodulin binding site
(CaM; 3614-3643), the proposed Ca2+ sensor
(E3987), and the proposed pore-forming segment
(4820-4829) are also shown.
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Fig. 2.
Expression and functional characterization of
RyR2T1874-GFP. HEK293 cells grown on glass coverslips
were transfected with RyR2T1874-GFP cDNA. Transfected
cells were fixed with 1% formaldehyde 24 h after transfection.
Fluorescence (a) and phase-contrast images (b)
were recorded under the fluorescence microscope at ×40 magnification
(A). B, HEK293 cells were transfected with 12 µg of RyR2T1874-GFP cDNA. Fluorescence intensity of
the fluo-3-loaded transfected cells was monitored continuously before
and after the addition of 2.5 mM caffeine (C) or
50 µM of ryanodine (R). The sharp decreases in
fluorescence intensity immediately after the second and third additions
of caffeine were due to fluorescence quenching by caffeine. Similar
results were obtained from three separate experiments.
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Fig. 3.
Ca2+ dependence and
[3H]ryanodine binding properties of
RyR2T1874-GFP. A,
[3H]ryanodine binding to cell lysate prepared from HEK293
cells transfected with RyR2T1874-GFP was carried out at
various concentrations of Ca2+ in the presence of 5 nM [3H]ryanodine. B,
[3H]ryanodine binding to RyR2T1874-GFP
transfected cell lysate was performed at various concentrations of
[3H]ryanodine in the presence of 100 µM
Ca2+. Data shown are from representative experiments, each
repeated three times. The inset in B shows a
Scatchard plot. The binding affinity (Kd) is
2.75 ± 0.43 (mean ± S.E., n = 3). The
binding density (Bmax) is 0.25 ± 0.06 pmol/mg protein (mean ± S.E., n = 3).
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Fig. 4.
Purification of RyR2T1874-GFP by
GST-FKBP12.6 affinity chromatography. The
RyR2T1874-GFP protein was purified from cell lysate by
affinity chromatography using GST-FKBP12.6 as the affinity ligand. The
purified RyR2T1874-GFP and WT RyR2 proteins were
solubilized and separated in SDS-PAGE. The gel was then stained with
Coomassie Brilliant Blue (CBB) (A). A similar
SDS-PAGE gel was transferred to nitrocellulose membrane. The membrane
was probed either with the anti-RyR antibody (B) or the
anti-GFP antibody (C) for Western blotting. Note that the
RyR2T1874-GFP protein migrated at a slightly slower rate in
SDS-PAGE than did the WT RyR2, due to the addition of GFP. Also note
that the anti-GFP antibody reacted with RyR2T1874-GFP but
did not recognize the WT RyR2, indicating that the antibody is
specific.
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Fig. 5.
Cryoelectron microscopy of
RyR2T1874-GFP. A portion of a cryo-EM micrograph of
the purified RyR2T1874-GFP proteins embedded in a thin
layer of vitreous ice is shown. The tetrameric structure of
RyR2T1874-GFP is well preserved. Several individual
RyR2T1874-GFP particles are marked with white
circles. Scale bar, 500 Å.
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Fig. 6.
Three-dimensional surface representation of
RyR2T1874-GFP and difference map. A, the
three-dimensional reconstruction of RyR2T1874-GFP is shown
in green. B, difference map
(RyR2T1874GFP WT RyR2) shown in green is
superimposed on the three-dimensional reconstruction of the WT RyR2 (in
blue). The asterisk indicates the equivalent
location of the FKBP12 binding site. The three-dimensional
reconstructions are shown in three views. Left, top views
from the cytoplasmic surface, which in situ would face the
transverse-tubule; middle, views toward the bottom of the
receptor (i.e. as it would appear if viewed from the lumen
of the sarcoplasmic reticulum; right, side views. The
numerals on the cytoplasm assembly indicate the
distinguishable domains.
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Fig. 7.
GST-FKBP12.6 binding to COOH-terminal
truncation mutants of RyR2. A, COOH-terminal truncation
mutants are depicted by rectangles (constructs 1-7). The
remaining NH2-terminal residues of each COOH-terminal
truncation mutant are shown on the left of the corresponding
rectangle. All truncation mutants were tagged with the c-Myc
antibody epitope near the NH2 terminus, as indicated by
small open boxes. B, HEK293
cells were transfected with truncation mutants as indicated. The
c-Myc-tagged mutant proteins were precipitated from cell lysates by the
anti-c-Myc antibody and by GST-FKBP12.6 glutathione-Sepharose. The
precipitates were solubilized and separated in SDS-PAGE and stained
with Coomassie Brilliant Blue. The expression of and GST-FKBP12.6
binding to mutants 1-1937 and 1-1636 have been shown previously
(23).
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Fig. 8.
Co-expression of overlapping NH2-
and COOH-terminal fragments leads to the synthesis of functional RyR2
channels in HEK293 cells. The NH2-terminal fragment
containing residues 1-1815 and the COOH-terminal fragment missing the
first 1636 NH2-terminal residues are shown by
filled boxes in A. B,
HEK293 cells were transfected with NH2-terminal fragment
1-1815 and the COOH-terminal fragment D1636 either individually (12 µg each) (Ba and Bb) or in combination (8 µg
each) (Bc). Fluorescence intensity of the fluo-3-loaded
transfected cells was monitored continuously before and after the
addition of 2.5 mM caffeine, indicated by C. A
transient increase in fluorescence was detected only in cells
co-transfected with mutants 1-1815 and D1636, indicating
caffeine-induced Ca2+ release from intracellular stores.
The drops in fluorescence signals immediately after the addition of
caffeine in Ba and Bb were due to fluorescence
quenching by caffeine. Similar results were obtained from three
separate experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank the Wadsworth Center's molecular genetics, immunology, and electron microscopy core facilities and the Resource for Visualization of Biological Complexity, the Immunology Core Facility at the Wadsworth Center of the New York State Department of Health for providing the anti-c-Myc antibody, Dr. Wayne R. Giles for continued support, Cindy Brown for excellent technical assistance, and Jeff Bolstad for a critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by research grants from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Alberta, N.W.T. and Nunavut (to S. R. W. C.) and by the Muscular Dystrophy Association and National Institutes of Health Grant AR40615 (to T. W.). The Resource for Visualization of Biological Complexity was supported by National Institutes of Health Biotechnological Resource Grant RR01219.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by the Uehara Memorial Foundation (Japan) and Postdoctoral Fellowships from the Heart and Stroke Foundation of Canada and the Alberta Heritage Foundation for Medical Research (AHFMR).
Recipient of the Alex W. Church Graduate Student Award.
AHFMR Senior Scholar. To whom correspondence should be
addressed. Tel.: 403-220-4235; Fax: 403-283-4841; E-mail:
swchen@ ucalgary.ca.
Published, JBC Papers in Press, February 7, 2003, DOI 10.1074/jbc.M213164200
2 M. Sharma, L. Jeyakumar, S. Fleischer, and T. Wagenknecht, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: RyR, ryanodine receptor; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate; EC, excitation-contraction; cryo-EM, cryoelectron microscopy; GFP, green fluorescent protein; PIPES, 1,4-piperazinediethanesulfonic acid.
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