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Journal of Cell Science
N-terminus oligomerization regulates the function of
cardiac ryanodine receptors
Spyros Zissimopoulos
1,
*, Cedric Viero
1
, Monika Seidel
1
, Bevan Cumbes
1
, Judith White
1
, Iris Cheung
1
,
Richard Stewart
1
, Loice H. Jeyakumar
2
, Sidney Fleischer
2
, Saptarshi Mukherjee
1
, N. Lowri Thomas
1
,
Alan J. Williams
1
and F. Anthony Lai
1
1
Wales Heart Research Institute, Cardiff University School of Medicine, Cardiff CF14 4XN, UK
2
Departments of Biological Sciences and Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
*Author for correspondence (zissimopouloss@cf.ac.uk)
Accepted 30 July 2013
Journal of Cell Science 126, 5042–5051
ß2013. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.133538
Summary
The ryanodine receptor (RyR) is an ion channel composed of four identical subunits mediating calcium efflux from the endo/
sarcoplasmic reticulum of excitable and non-excitable cells. We present several lines of evidence indicating that the RyR2 N-terminus is
capable of self-association. A combination of yeast two-hybrid screens, co-immunoprecipitation analysis, chemical crosslinking and gel
filtration assays collectively demonstrate that a RyR2 N-terminal fragment possesses the intrinsic ability to oligomerize, enabling
apparent tetramer formation. Interestingly, N-terminus tetramerization mediated by endogenous disulfide bond formation occurs in
native RyR2, but notably not in RyR1. Disruption of N-terminal inter-subunit interactions within RyR2 results in dysregulation of
channel activation at diastolic Ca
2+
concentrations from ryanodine binding and single channel measurements. Our findings suggest that
the N-terminus interactions mediating tetramer assembly are involved in RyR channel closure, identifying a crucial role for this
structural association in the dynamic regulation of intracellular Ca
2+
release.
Key words: Cardiac ryanodine receptor, N-terminus, Calcium release channel, Oligomerization
Introduction
The integral membrane ryanodine receptor (RyR) governs the
sarcoplasmic reticulum (SR) Ca
2+
release that is essential for
initiating skeletal muscle contraction and also for triggering the
activation of each heartbeat (Zissimopoulos and Lai, 2007). In
mammals, RyR1 is the predominant isoform in skeletal muscle,
whereas the heart primarily expresses RyR2. The vital
importance of RyRs is highlighted in pathological conditions
where inherited or acquired defective channel regulation results
in abnormal Ca
2+
handling and leads to neuromuscular disorders,
cardiac arrhythmias and heart failure (Blayney and Lai, 2009;
George and Lai, 2007). RyRs comprise four identical subunits of
,560 kDa that combine to form high-conductance, cation-
permeable channels (Fleischer, 2008). The RyR C-terminus
(,10% of the protein) contains the membrane-spanning domains
and is known to tetramerize and form a constitutively open Ca
2+
-
conducting pore (Bhat et al., 1997a; Bhat et al., 1997b; George
et al., 2004; Wang et al., 1996). The large N-terminal
cytoplasmic portion is associated with the transmembrane
assembly, as indicated by partial proteolysis studies of native
RyR1 (Chen et al., 1993) and by protein complementation assays
between overlapping N- and C-terminal RyR2 fragments (George
et al., 2004; Masumiya et al., 2003). Intra- and inter-subunit
interactions are believed to regulate the opening and closing of
the channel pore (Zissimopoulos and Lai, 2007). Importantly,
defective inter-domain interactions have been implicated in RyR
pathophysiology. For example, Ikemoto, Yano, Matsuzaki and
colleagues have proposed that mutation-induced disruption of the
association between N-terminal and central domains results in
abnormal RyR Ca
2+
release and is involved in the pathogenesis of
malignant hyperthermia (MH) and catecholaminergic
polymorphic ventricular tachycardia (CPVT) (Ikemoto and
Yamamoto, 2002; Yano et al., 2009).
The N-terminus of RyR1 and RyR2 is the location of one of the
three major disease clusters each containing numerous genetic
mutations associated with MH and CPVT respectively, implying
that it is a functionally important module of the channel. Initial
reports using cryo-electron microscopy (EM) of GFP-RyR or
GST-RyR protein fusions, or homology modeling and docking
analysis, have placed the N-terminus within the clamp region at the
corners of the RyR three-dimensional architecture (Baker et al.,
2002; Liu et al., 2001; Serysheva et al., 2008; Wang et al., 2007).
By contrast, a very different N-terminal topology that specifically
located it surrounding the central fourfold axis was recently
proposed, based on docking the crystal structure of an RyR1 N-
terminal fragment (amino acids 1–559) within the native RyR1
cryo-EM density map (Tung et al., 2010).
Here, we report on the identification of a novel inter-subunit
interaction occurring between the N-terminal domains within the
RyR2 tetrameric channel and present functional evidence implicating
RyR2 N-terminus tetramerizationinstabilizationoftheclosed
conformation of the channel. Part of these results has previously been
presented in abstract form (Zissimopoulos et al., 2005).
Results
RyR2 N-terminus assembles into tetramers
In order to identify whether any of the RyR2 domain(s)
associates with the N-terminal fragment of RyR2 (AD4L;
5042 Research Article
Journal of Cell Science
residues 1–906 of human RyR2 fused with GAL4 AD; see
Fig. 1), we used the yeast two-hybrid (Y2H) system to screen for
the potential protein interaction of AD4L with overlapping RyR2
domain expression constructs spanning the entire protein (Fig. 1).
Notably, we found that the human RyR2 N-terminus (AD4L)
associated most potently with itself (BT4L; residues 1–906 fused
with GAL4 DNA-BD), and weakly interacted with the RyR2 C-
terminal tail (BT8) (Fig. 1, inset table). Quantitative b-
galactosidase assays indicated a very strong interaction for the
BT4L–AD4L association (i.e. N-terminus–N-terminus; Fig. 1,
inset histogram), whereas the BT8–AD4L (C-terminus–N-
terminus) interaction was considerably weaker (,10%). Central
domain constructs (BT5, BT6 and their overlapping BT2
fragment) showed negligible binding to AD4L. These data
suggest that the N-terminal portion can specifically confer cogent
self-association, thus facilitating RyR2 subunit oligomerization.
To extend the Y2H observations, we co-expressed in
mammalian HEK293 cells the RyR2 N-terminus (1–906),
differentially tagged with either the Myc or HA peptide epitope
(BT4L and AD4L, respectively), and we then performed co-
immunoprecipitation (co-IP) assays. HA-AD4L from CHAPS-
solubilized HEK293 cell lysate was immunoprecipitated with
anti-HA antibodies (Ab
HA
) and the presence of co-precipitated
Myc-BT4L was analyzed by immunoblot using antibodies
against Myc (Ab
Myc
). Initial observations revealed the presence
of an oligomeric species and so further experiments were
specifically carried out in either reducing (10 mM DTT) or
oxidizing (1 mM H
2
O
2
) conditions. Fig. 2 shows that Myc-
tagged BT4L (,100 kDa) was recovered only in the Ab
HA
IP,
and not in the control IP lacking antibody. Without DTT
(ambient), an additional Ab
Myc
-immunoreactive high M
r
(,400610
3
) band was detected in the Ab
HA
IP, indicating that
a mixed oligomer had formed comprising BT4L and AD4L. This
oligomeric species was abolished by DTT addition, suggesting the
existence of disulfide bonding between the protomers. However,
the oxidizing reagent H
2
O
2
did not substantially enhance
conversion of the monomeric into the oligomeric form. Possibly,
the same cysteines participating in disulfide bond formation in
some subunits are S-glutathionylated or S-nitrosylated in other
subunits, thus preventing their oxidation to disulfides. Indeed, it
has been reported that several RyR1 cysteines, including cysteines
within the N-terminus, displayed all three types of modification;
oxidation to RyR1 intra- or inter-subunit disulfide bonds, S-
glutathionylation and S-nitrosylation (Aracena-Parks et al., 2006).
As the BT4L–AD4L domain is ,100 kDa, formation of the
,400 kDa protein strongly suggests that the RyR2 N-terminus
may be self-associating into DTT-sensitive tetramers when
expressed in mammalian cells.
Notably, BT4L–AD4L self-interaction occurred in the
presence of the reducing agent DTT (Fig. 2), indicating that
disulfide bonds are dispensable and RyR2 N-terminal self-
association is primarily mediated by non-covalent protein-
protein interactions. This was further tested with the use of
the linear zwitterionic detergent zwittergent 3-14, which was
previously shown to cause dissociation of native RyR1, as well
as RyR2 C-terminal tetramers into monomers (Lai et al., 1989;
Stewart et al., 2003). We found that zwittergent 3-14 prevented
the interaction between BT4L and AD4L in co-
immunoprecipitation assays, whereas robust BT4L–AD4L self-
interaction was retained in the presence of CHAPS
(supplementary material Fig. S1), which suggests that
zwittergent 3-14 disrupts non-covalent, RyR2 N-terminal inter-
subunit interactions. An alternative explanation is that
zwittergent 3-14, as relatively powerful detergent, causes
protein conformational changes and/or partial unfolding of the
RyR2 N-terminus, thereby preventing its self-association.
Fig. 1. The RyR2 N-terminus interacts with itself in yeast cells. Schematic diagram depicting the series of human RyR2 overlapping protein fragments
tested in the Y2H system for interaction with the RyR2 N-terminal AD4L construct. Quantitative liquid b-galactosidase assays are shown in the inset (pVA3
encodes GAL4 DNA-BD fusion with p53 protein; pTD1 encodes GAL4 AD fusion with SV40 large T antigen). Results are means 6s.e.m.
RyR2 N-terminus tetramerization 5043
Journal of Cell Science
To examine the precise stoichiometry of the RyR2 N-terminus
oligomer, we used glutaraldehyde to crosslink HEK293 cell
homogenates expressing the ,100 kDa BT4L protein. We
observed time-dependent formation of a ,400 kDa band
(Fig. 3A,B) indicating existence of a tetrameric assembly of
,100 kDa BT4L protomers, irrespective of DTT pre-treatment.
Cumulative data (n54) following densitometry analysis are
presented in Fig. 3C. The predominant protein band observed
corresponds to a tetramer, with minimal dimer and no trimer
bands detected. Although it is a minor component, the tetrameric
form is evident in ambient conditions even before glutaraldehyde
addition (i.e. at the 0 minute time point), but is abolished by DTT
pre-treatment (0 minute time point, 10 mM DTT) (Fig. 3B). As
with the co-IP assays, these experiments indicate that a small
proportion of the N-terminus already exists as a disulfide-linked
tetramer.
In order to accurately determine the apparent molecular mass
of the BT4L oligomer, we used 4–15% gradient SDS-PAGE gels
and protein standards with a range of 30–460 kDa (Fig. 3D).
From the resulting standard curve, we calculated the oligomer to
be 358615 kDa (n54), consistent with a tetramer arranged in a
closed circular fashion, as expected from the arrangement of the
four subunits within the native RyR2 channel. A closed circular
tetrameric protein species should experience less gel retardation
and would run with greater relative mobility during SDS-PAGE
than the equivalent linear form, and therefore it could have an Rf
that corresponds to a slightly smaller size than the expected
400 kDa. To address whether disulfide bonds are formed by air
oxidation during the experiment, we included 5 mM NEM (a
thiol-reactive, alkylating reagent) during cell homogenization to
covalently modify free sulfhydryls. Tetramers were still obtained
after NEM treatment (Fig. 3D), suggesting that sulfhydryl
oxidation occurs endogenously within the cells.
During the course of our experiments, the crystal structure of a
RyR1 N-terminal fragment (residues 1–559) was determined and
topological docking onto the native RyR1 cryo-EM electron
density map suggested a tetrameric arrangement (Tung et al.,
2010). However, size exclusion chromatography indicated that
the purified, bacterially-expressed RyR1 fragment remained a
monomer in solution. We therefore used gel filtration to assess
oligomerization of the human RyR2(1–906) (GST-BT4L) protein
recombinantly expressed in, and purified from, bacteria
(supplementary material Fig. S2). Thus, GST-BT4L (,130 kDa)
was separated by size exclusion under reducing conditions
(10 mM DTT) and then the eluted fractions, analyzed by
immunoblot using Ab
GST
, were compared with gel-filtrated
protein standards in the range of 29–669 kDa (Fig. 4). GST-
BT4L was detected in two distinct areas of the elution profile, early
fractions (46–52 ml) and later elution fractions (64–68 ml)
corresponding to the tetramer and monomer, respectively. We
note that gel filtration is a technique that separates proteins
according to their hydrodynamic size under non-denaturing
conditions, and therefore protein oligomers held together by
non-covalent protein–protein interactions will be preserved.
However, as a result of SDS-PAGE denaturing conditions,
which disrupt protein–protein interactions, gel-filtrated GST-
BT4L tetramers will dissociate into monomers and a protein
band at only ,130 kDa will be detected (Fig. 4A,B). Tetramer
formation by purified GST-BT4L was also demonstrated by
chemical crosslinking, with no appreciable dimer or trimer bands
observed (supplementary material Fig. S3).
Native RyR2 N-terminus assembles into disulfide-linked
tetramers
The cumulative evidence from the Y2H, co-IP, chemical
crosslinking and gel-filtration data strongly suggest that the
discrete RyR2 N-terminus region can tetramerize. However, this
oligomerization might not be present in the full-length RyR2,
where potentially disparate protein folding of adjacent RyR2
domains and/or accessory regulatory proteins might not favor this
interaction. We therefore investigated the oligomeric state of the
N-terminus in the native RyR2 from pig cardiac SR, under
reducing (10 mM DTT) or oxidizing conditions (1 mM H
2
O
2
).
Native RyR2 was incubated with calpain protease to generate the
characteristic ,150 kDa N-terminal and ,400 kDa C-terminal
proteolytic fragments. Untreated and calpain-cleaved RyR2
fragments were monitored by immunoblot using two different
N-terminal-specific antibodies (Ab
D2
and Ab
H300
) and with an
antibody against the C-terminus (Ab
1093
).
On the basis of the results obtained (Fig. 5A,B), we can
distinguish between the different possibilities, as follows. Lanes
1, 3 and 5, in the absence of exogenous calpain digestion, show
that the full-length RyR2 (550 kDa) subunit is largely intact;
however, some degradation by endogenous protease that
generates the ,150 kDa N-terminal (and ,400 kDa C-
terminal) fragment is also evident. If the RyR2 and its N-
terminus exist in monomeric form, only the 550 kDa and
,150 kDa bands should be observed upon immunoblot
analysis of the N-terminus. If the RyR2 and its N-terminus
exist as tetramers but are not linked by endogenous disulfide
bonds, only the 550 kDa and ,150 kDa bands should be
observed (tetramers are dissociated as a result of SDS-PAGE
denaturing conditions). This is what we observed in Fig. 5A,B
lane 3, where samples were treated with the reducing agent, DTT.
If the RyR2 and its N-terminus form into tetramers that are
covalently linked by endogenous disulfide bonds, then two bands
in addition to 550 kDa and ,150 kDa would be expected; the
two additional bands would be the full-length tetramer at
2200 kDa and the N-terminus tetramer at ,600 kDa. We
Fig. 2. Self-association of RyR2 N-terminal fragment in mammalian
cells. Co-immunoprecipitation assays from HEK293 cell lysate co-expressing
Myc-tagged (BT4L) and HA-tagged (AD4L) RyR2 residues 1–906, pre-
treated with or without 10 mM DTT or 1 mM H
2
O
2
as indicated. AD4L was
immunoprecipitated with Ab
HA
from CHAPS-solubilized HEK293 lysate
and the presence of associated BT4L was analyzed by SDS-PAGE (6% gel)
and immunoblotting using Ab
Myc
. Cell lysate, 1/50th of the volume
processed in IP samples, was also included to serve as molecular mass
standard. Monomer and mixed tetramers are shown with the arrows.
Journal of Cell Science 126 (21)5044
Journal of Cell Science
observed the ,600 kDa, but not the 2200 kDa band in Fig. 5A,B
lanes 1 and 5 (ambient and H
2
O
2
, respectively). The reason why
the 2200 kDa band is not visible is most probably due to low
abundance and/or poor electrophoretic transfer of such a large
protein. Fig. 5A,B lanes 2, 4 and 6, following exogenous calpain
digestion, show that the full-length 550 kDa subunit is abolished
to yield the ,150 kDa N-terminal fragment (and ,400 kDa C-
terminus). If the RyR2 and its N-terminus were monomeric, only
the ,150 kDa band should be observed. If the RyR2 and its N-
terminus form tetramers that are not linked by endogenous
disulfide bonds, only the ,150 kDa band should be observed.
This is what we observed in Fig. 5A,B lane 4, where samples
were treated with the reducing agent, DTT. If the RyR2 and its N-
terminus exist as tetramers that are covalently linked by
endogenous disulfide bonds, the N-terminus tetramer at
,600 kDa in addition to the ,150 kDa monomer band would
be expected. This is what we observed in Fig. 5A,B lanes 2 and 6
(ambient and H
2
O
2
, respectively).
Thus, two different antibodies, Ab
D2
and Ab
H300
, which
recognize different epitopes within the N-terminus, detected a
,600 kDa oligomeric protein (Fig. 5A,B). The band at ,600 kDa
persisted following calpain cleavage, but it was abolished by DTT,
consistent with disulfide-linked tetramerization of the ,150 kDa
N-terminal fragment. Addition of H
2
O
2
did not substantially
enhance the ,600 kDa species. Densitometry analysis indicated
that following exogenous calpain cleavage, the disulfide-linked N-
terminus tetramer to monomer relative abundance is 10.864.0%
and 13.064.7% for ambient and H
2
O
2
conditions, respectively
(n54). By contrast, Ab
1093
(specific for RyR2 C-terminus) failed
to detect the ,600 kDa band, confirming that it does not contain
the RyR2 C-terminus (Fig. 5C). Instead, Ab
1093
detected a band at
,1600 kDa, which resisted calpain cleavage but was abolished
by DTT, which would be consistent with disulfide-linked
tetramerization of the ,400 kDa C-terminal fragment. These
results suggest that the native pig RyR2 N-terminus region
associates with itself and, after calpain cleavage, remains
tetrameric through DTT-sensitive disulfide bonds. Note that both
the ,600 kDa N-terminus and ,1600 kDa C-terminus tetramers
did not appear to substantially increase following exogenous
calpain digestion. Interestingly, it seems that native RyR2 subunits
that are disulfide-linked are already cleaved by endogenous
calpain. This could imply that endogenous calpain digestion
primes RyR2 for inter-subunit disulfide linkage. Alternatively,
disulfide-linked RyR2 might be specifically targeted for calpain
digestion.
Similar calpain cleavage experiments were carried out for
native RyR1 from rabbit skeletal muscle SR, using antibodies
specific for the N- and C-terminal RyR1 fragments (Ab
2142
or
Fig. 3. Tetramer is the predominant oligomeric form of RyR2 N-terminal fragment. (A,B) Chemical crosslinking assays from HEK293 cell homogenate
expressing BT4L, pre-treated without (A) or with (B) the reducing agent, dithiothreitol (10 mM DTT). Cell homogenate was incubated with glutaraldehyde for
the indicated time points and analyzed by SDS-PAGE (6% gels) and immunoblotting using Ab
Myc
. Oligomeric forms are indicated by the arrows.
(C) Densitometry analysis (n54) was carried out on the bands corresponding to tetramer and monomer and used to express tetramer to monomer ratio. Data
are given as mean value 6s.e.m. (D) HEK293 cells expressing BT4L were homogenized in the presence of 10 mM DTT, 10 mM b-mercaptoethanol or 5 mM
N-ethylmaleimide. Proteins were separated through 4–15% gradient SDS-PAGE gel together with high molecular mass markers (HiMark, Invitrogen). A
calibration standard curve was subsequently prepared by plotting Rf values versus log M
r
and fitting the data using a polynomial curve (not shown).
RyR2 N-terminus tetramerization 5045
Journal of Cell Science
Ab
H300
and Ab
2149
, respectively). Unlike RyR2, the RyR1
,150 kDa N-terminal fragment was detected only as a
monomer, irrespective of the presence of DTT or H
2
O
2
(Fig. 5D,E). The RyR1 ,400 kDa C-terminal fragment was
found as a monomer (ambient, DTT-treated), but H
2
O
2
induced
appearance of a ,1600 kDa band, which is consistent with a
tetramer (Fig. 5F).
RyR2 N-terminus dissociation enhances channel activity
N-terminus tetramerization confers a structural role, but could
also potentially contribute to functional regulation of the RyR2
channel. To assess its putative functional significance, we
investigated the effects that the BT4L fragment exerts on the
activity of native and recombinant RyR2 channels. Our reasoning
was that the exogenous BT4L fragment could compete for N-
terminal binding sites presumed to exist within the RyR2
oligomer, thereby disrupting endogenous N-terminal inter-
subunit interactions and altering channel function. DTT was
included in the assays to reduce the covalent disulfide bonds,
leaving only the non-covalent protein–protein interactions within
the RyR2 N-terminus, thus facilitating the interaction of
exogenous BT4L with the endogenous N-terminal region
within the native RyR2 channel. Using GST pull-down assays,
we observed that the purified GST-BT4L polypeptide interacts
with native RyR2 and the 150 kDa calpain-cleaved N-terminal
fragment (supplementary material Fig. S4). Initial [
3
H]ryanodine
binding assays of pig cardiac SR indicated that 10 nM GST-
BT4L caused a ,twofold binding increase at low Ca
2+
concentrations (supplementary material Table S1), although the
results did not reach statistical significance. This could be due to
the low concentration of GST-BT4L used (dictated by the low
protein expression and purification yield from bacteria) and/or
insufficient binding assay incubation time.
To overcome this problem of the low yield of isolated protein,
we co-expressed BT4L with full-length RyR2 in HEK293 cells,
enabling an enhanced concentration of BT4L protein to
potentially interact with and disrupt the N-terminus self-
association within RyR2 channels upon recombinant
expression. Sub-cellular fractionation and immunoblot analysis
indicated that the recombinant BT4L specifically translocates
from the cytosol to a predominantly microsomal localization only
upon co-expression with the full-length RyR2 (supplementary
material Fig. S5). [
3
H]Ryanodine binding assays performed
under reducing conditions (2 mM DTT) on HEK293 microsomes
that contained either co-expressed RyR2+BT4L, or RyR2 alone,
revealed a maximum binding value (in 100 mM free Ca
2+
and
10 mM caffeine) of 37.964.3 and 41.063.7 fmol/mg,
respectively, indicating equivalent RyR2 protein expression,
which was also verified by immunoblotting. The presence of co-
expressed BT4L did not affect ryanodine binding to RyR2 at
Ca
2+
§1mM (Fig. 6). By contrast, at low Ca
2+
concentrations
(#250 nM), the presence of BT4L induced a statistically
significant ,twofold increase (P,0.05) in ryanodine binding,
suggesting that RyR2 channel activation by the recombinant
BT4L occurs in low Ca
2+
conditions.
The functional effects of BT4L on RyR2 activity were further
investigated in single-channel recordings. RyR2 from CHAPS-
solubilized HEK293 cells isolated by sucrose density gradient
centrifugation was incorporated into lipid bilayers and cation
channel activity was monitored at 100 nM free cis Ca
2+
,
approximating the diastolic Ca
2+
concentration in cardiac
myocytes. Upon recombinant BT4L polypeptide (+10 mM
DTT) addition to the cis chamber, the open probability of a
third of RyR2 channels increased, following a variable delay. The
unresponsiveness and/or variable delay of some channels could
be due to the relatively low (10 nM) BT4L protein concentrations
applied, and the kinetic mechanism of BT4L action potentially
involving interaction of BT4L with the full-length RyR2 to
promote N-terminus dissociation within the oligomeric channel.
As shown in Fig. 7A, RyR2 channel activity was extremely low
at 100 nM cytosolic Ca
2+
(P
o
50.004). Addition of 10 nM GST-
BT4L to the cis chamber resulted in a significant increase of open
probability (P
o
50.064). Under these conditions, the average
increase in P
o
by GST-BT4L was 9.864.4 fold (n55 channels).
Fig. 4. Gel filtration of purified RyR2 N-terminal fragment.
(A,B) Western blot analysis using Ab
GST
of eluted fractions following gel
filtration of purified GST-BT4L (RyR2 residues 1–906). (C) Gel-filtration
calibration curve (elution volumes in ml plotted against log M
r
) was generated
using thyroglobulin (669 kDa), apoferritin (443 kDa), b-amylase (200 kDa),
alcohol dehydrogenase (150 kDa), BSA (66 kDa) and carbonic anhydrase
(29 kDa).
Journal of Cell Science 126 (21)5046
Journal of Cell Science
To control for the GST moiety, we carried out experiments where
GST alone and GST-BT4L were sequentially added to the cis
chamber. Fig. 7B shows a channel where 100 nM GST alone had
a minimal effect (P
o
50.019 for GST versus P
o
50.014 for
control), but the subsequent addition of 10 nM GST-BT4L
resulted in a substantially increased open probability (P
o
50.084).
On average, GST-BT4L increased P
o
by 7.462.8 fold relative to
GST alone (n55 channels). The two datasets pooled together
indicate that GST-BT4L specifically induced a statistically
significant (P,0.05) increase in channel P
o
by 8.662.5 fold
(n510 channels). By contrast, GST alone resulted in a 1.760.4-
fold increase in P
o
(n57 channels).
Discussion
The aim of our study was to identify the RyR2 domain(s) that
potentially interact with the N-terminus to further elucidate its
precise structural and functional role. Our Y2H screening of a
series of overlapping fragments spanning the entire RyR2 coding
sequence revealed that the N-terminus region, BT4L (residues 1–
906), interacts strongly with itself (Fig. 1). This self-interaction
was verified in HEK293 mammalian cells by co-IP assays using
disparate fusion tags (Fig. 2), whereas chemical crosslinking
experiments indicated tetramer formation by the N-terminus
Fig. 5. Native RyR2 N-terminus assembles into disulfide-linked tetramers. Calpain cleavage of pig cardiac SR (A–C) and rabbit skeletal muscle SR
(D–F), that were pre-treated with or without 10 mM DTT or 1 mM H
2
O
2
, as indicated. Samples were analyzed by SDS-PAGE (4% gels) and immunoblotting
using Ab
D2
against RyR2 N-terminus (A), Ab
H300
against RyR2 N-terminus (B), Ab
1093
against RyR2 C-terminus (C), Ab
2142
against RyR1 N-terminus
(D), Ab
H300
against RyR1 N-terminus (E) and Ab
2149
against RyR1 C-terminus (F). Full-length (FL) RyR subunit, calpain-cleaved ,150 kDa N-terminal and
,400 kDa C-terminal fragments as well as oligomeric forms are indicated by arrows.
Fig. 6. RyR2 displays increased [
3
H]ryanodine binding in the presence of
BT4L. [
3
H]ryanodine binding assays of HEK293 microsomes expressing
RyR2 alone or RyR2 with BT4L, over a range of free Ca
2+
concentrations.
Summary of three separate experiments each performed at least in duplicate.
Data are normalized against maximum binding (obtained in the presence of
100 mM free Ca
2+
and 10 mM caffeine) and expressed as mean value 6s.e.m.
RyR2 N-terminus tetramerization 5047
Journal of Cell Science
domain (Fig. 3). Consistent with this observation, a tetrameric
species was also identified by gel filtration of the purified N-
terminal fragment (Fig. 4). Interestingly, in the native pig heart
RyR2, the N-terminus was found to form stable tetramers involving
intrinsic disulfide bonds (Fig. 5). However, disulfide bonds appear
not to be essential for N-terminal oligomerization, because the
discrete RyR2 N-terminus domain was still able to form tetramers
in the presence of DTT (Fig. 3B; Fig. 4). These findings suggest
that the RyR2 N-terminus domain can intrinsically assemble into
tetramers through non-covalent protein–protein interactions, and
that N-terminal tetramerization could be further stabilized by
covalent bonds between cysteine residues in the native protein. The
observed tetramerization favors a model where the N-terminal
regions of the four subunits within a single RyR2 channel are
associated with each other in a closed circular fashion (Fig. 8). If
the N-termini were involved in inter-oligomeric interaction
between adjacent channels within the two-dimensional membrane
lattice (Yin et al., 2005; Yin and Lai, 2000), the formation of dimers
rather than tetramers would be expected. This is because tetramer
assembly (or any oligomer .2) should in theory be mediated by at
least two interacting domains per monomer, whereas a single site of
interaction would only lead to dimers.
Our model for RyR2 N-terminus tetramerization based on
empirical evidence derived from the current biochemical analysis
Fig. 7. BT4L enhances RyR2 channel open probability. Single-channel recordings of RyR2 using K
+
as the charge carrier at 260 mV in the presence of cis
100 nM free Ca
2+
. (A) Channel activity before and after addition of 10 nM GST-BT4L to the cis chamber. (B) Channel activity before and after sequential
addition to the same channel of 100 nM GST alone, followed by 10 nM GST-BT4L, in the cis chamber. Please note the difference in scale bars between A and B.
Fig. 8. Proposed model of RyR2 N-terminus tetramerization. Drawing on the left is a schematic representation of the RyR2 N-terminus interactions that
form a tetramer surrounding the central fourfold axis. A simplified model of proposed RyR2 N-terminus dissociation induced by exogenous BT4L peptide
resulting in a ‘leaky’ channel is shown on the right.
Journal of Cell Science 126 (21)5048
Journal of Cell Science
(Fig. 8), appears incompatible with the proposed ‘clamp’ location
for the N-terminus at the periphery of the RyR initially suggested
by cryo-EM studies of GFP-RyR or GST-RyR fusions and/or
homology modeling and docking (Baker et al., 2002; Liu et al.,
2001; Serysheva et al., 2008; Wang et al., 2007). By contrast, our
N-terminal tetramerization model is entirely consistent with, and
supports the recently proposed N-terminus location surrounding
the central fourfold axis of RyR1 (Tung et al., 2010). Although
the N-terminal fragment of RyR1 (amino acids 1–559) remained
a monomer in the crystal, a precise docking of its tertiary
structure within the native RyR1 EM density map placed the four
N-terminal domains immediately adjacent to each other at the
center of the molecule (Tung et al., 2010). In the previous studies,
GST or GFP was fused at the extreme N-terminus of RyR3 or at
Ser437 within RyR2, respectively, and observed differences in
the resultant cryo-EM density maps relative to wild-type RyR2 or
RyR3, were taken as evidence for the precise location of the GST
or GFP insertion (Liu et al., 2001; Wang et al., 2007). However
the insertion of GST or GFP, both discrete proteins of ,27 kDa,
potentially could have perturbed the intrinsic RyR structure
locally and might also induce long-range allosteric effects.
Hence, distally-altered RyR conformational changes (e.g. at the
RyR periphery) might result from N-terminal GST or GFP
insertions (e.g. at the RyR centre) and are therefore attributed
incorrectly as the actual location of the N-terminus. Additionally,
cryo-EM sample processing involving freezing of solubilized
RyR might potentially cause conformational changes in GST-
RyR and GFP-RyR fusion proteins that do not occur in the native
or wild-type recombinant RyR. Furthermore, the use of long
flexible linkers on either side of the insertion, together with the
size of a GST or GFP protein, could span a sphere of ,8nm
radius, as already highlighted by Tung and colleagues (Tung
et al., 2010).
In other studies, computational methods were used to generate
structural models for the RyR1 N-terminus, which were
subsequently docked into the clamp region of the full-length
RyR1 architecture (Baker et al., 2002; Serysheva et al., 2008).
Although the model generated based on the prediction that the
RyR1 N-terminus forms an oxidoreductase-like domain (Baker
et al., 2002) is not consistent with the structure empirically
determined by X-ray crystallography (Tung et al., 2010), the
model that was based on the crystal structures of inositol
trisphosphate binding core and suppressor domains (Serysheva
et al., 2008) is quite similar. However, as previously raised by
Tung and colleagues, docking of the latter model at the RyR1
clamps might have been an artifact due to the very electron-dense
nature of the clamp region (Tung et al., 2010). Thus, it appears
that the initial studies (Baker et al., 2002; Liu et al., 2001;
Serysheva et al., 2008; Wang et al., 2007) placing the N-terminus
within the clamp region at the corners of the RyR architecture are
not compatible with the empirically determined N-terminal
location at the centre of the RyR.
Unlike RyR2, we did not detect disulfide-linked N-terminal
tetramers for the native RyR1 (Fig. 5D,E) in agreement with a
previous report (Wu et al., 1997). However, this does not exclude
the presence of RyR1 N-terminal inter-subunit interactions, but
simply argues against the requirement for disulfide bonds.
Potentially, the RyR2 N-terminal cysteines involved in
disulfide bonds are not conserved in RyR1 or else they
might be post-translationally modified (e.g. glutathionylation,
nitrosylation) in RyR1. Alternatively, these cysteines could be
occluded in RyR1 because local conformational differences
prevent their oxidation into disulfides. Interestingly, the disparate
local folding between skeletal and cardiac RyR N-termini has
previously been proposed to explain the differential effects of
dantrolene. Despite its binding site being 100% conserved (RyR1
residues 590–609 is identical to RyR2 residues 601–620) and
observation of dantrolene binding to RyR1, the wild-type RyR2
does not appear to bind this drug (Paul-Pletzer et al., 2002; Paul-
Pletzer et al., 2005). However, dantrolene is capable of inhibiting
the failing or mutant (R2474S) RyR2 and can restore normal
cardiac function (Kobayashi et al., 2009; Kobayashi et al., 2010;
Uchinoumi et al., 2010), suggesting that its interaction site on
RyR2 is conformation sensitive, becoming accessible only in
disease states. Notably, disulfide-linked N-terminal oligomers
were induced in solubilized, purified RyR1 possibly because
altered folding exposes previously buried cysteine(s) residues
(Wu et al., 1997). Given the high degree of N-terminus sequence
homology between the three mammalian RyRs (,85% similarity
for residues 1–900 of RyR1, RyR2 and RyR3), the determinants
of N-terminus tetramerization could therefore be conserved in all
three isoforms. However, although both RyR2 and RyR1 might
be capable of N-terminus self-association, cysteine oxidation to
form inter-subunit disulfide bonds might occur in an isoform- or
tissue-dependent manner.
An intimate association between the RyR N-terminal and
central domains has been suggested previously with well-
documented functional effects of central-domain-derived, short
synthetic peptides (DP4; RyR1 amino acids 2442–2477 and
DPc10; RyR2 amino acids 2460–2495) (Ikemoto and Yamamoto,
2002; Yano et al., 2009). However, our Y2H screen notably
failed to detect any interaction between the AD4L N-terminal
fragment and any of the RyR2 central domain constructs. It is
possible that the RyR2 central domain fragments that we used in
the present study have an altered, non-native protein
conformation that precludes access of the N-terminus to the
DPc10 sequence, or prevents the reporter gene transcription
necessary for the Y2H protein interaction assay. An alternative
explanation is that the robust BT4L self-interaction detracts from
the detection of significantly weaker interactions with other
RyR2 domains. The use of a series of small N-terminus truncated
constructs could possibly help in accurately dissecting the
specific sequences involved in mediating N-terminal self-
interaction versus central domain binding.
It is apparent that RyR2 N-terminus tetramerization may have
more than just a structural role. We observed that RyR2 co-
expressed with the N-terminus fragment, BT4L, displayed a
,twofold increase in [
3
H]ryanodine binding at low Ca
2+
concentrations (#250 nM) (Fig. 6 and supplementary material
Table S1). At 100 nM Ca
2+
, exogenous BT4L increased the
channel open probability by ,eightfold (Fig. 7). These results
indicate that BT4L enhances activity at low Ca
2+
concentrations,
with both native and recombinant purified RyR2 channels,
suggesting that its effect on RyR2 is direct, not requiring any
other cytosolic proteins. BT4L might be exerting its functional
effects by competing with the corresponding RyR2 subunit N-
terminal sequence for its local binding partner, thereby disrupting
endogenous inter-domain associations of the oligomeric N-
terminus in the native RyR2. Further, exogenous BT4L was
found to interact with RyR2 and in particular with its N-terminus
(supplementary material Fig. 4). Given the wide array of
biochemical data consistent with N-terminus self-association, it
RyR2 N-terminus tetramerization 5049
Journal of Cell Science
seems reasonable to predict that when exogenous BT4L interacts
with full-length RyR2, the primary site of its interaction is with
the N-terminus of one of the four subunits. In doing so, BT4L
displaces the N-terminal domain of this subunit from its
tetrameric association with the other three N-termini, thus
resulting in disruption or destabilization of the native N-
terminal inter-subunit interactions (Fig. 8). We note that rather
than being direct evidence, this is the most likely interpretation
for the mechanism of action of exogenous BT4L in enhancing
RyR2 channel activity.
Thus, stable N-terminus oligomerization is likely to play a
functional role in maintaining the RyR2 channel closed at low
Ca
2+
. Accordingly, conditions that weaken or disrupt these
oligomeric interactions within the RyR2 N-terminus could lead to
SR Ca
2+
leak during the diastolic phase in cardiac myocytes.
RyR2 bearing CPVT mutations are known to result in ‘hyper-
sensitive’ channels and diastolic Ca
2+
leak, often leading to
delayed after-depolarizations and fatal arrhythmias (Blayney and
Lai, 2009; Thireau et al., 2011). In light of the present findings,
the potential involvement of RyR2 N-terminus tetramerization in
the pathogenesis of CPVT is very plausible and this prospect
should be further investigated. Indeed, a recent study suggested
that the N-terminal region is involved in Ca
2+
release
termination, which could be defective in RyR2-associated
cardiomyopathies (Tang et al., 2012).
In summary, we have identified the N-terminus as an important
RyR2 structural locus that plays a direct role in promoting
channel closure. First, we find that the N-termini of the four
RyR2 subunits within a channel assemble into stable tetramers.
Second, we present evidence for RyR2-specific, inter-subunit
disulfide bonds located specifically within the N-terminal region.
Third, disruption of inter-subunit N-terminal interactions
enhances channel activity at diastolic Ca
2+
concentrations
consistent with a ‘leaky’ phenotype. We therefore suggest that
defective N-terminal tetramerization has an important role in
RyR2 pathophysiology.
Materials and Methods
Materials
Cell culture reagents were obtained from Invitrogen (Life Technologies), high
molecular weight markers (HiMark) from Invitrogen (Life Technologies), all other
electrophoresis reagents from Bio-Rad, protease inhibitor cocktail (Complete) from
Roche, CHAPS and calpain-2 from Calbiochem (Merck), [
3
H]ryanodine from
Perkin-Elmer, synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine
from Avanti Polar Lipids; all other reagents from Sigma.
Chemical crosslinking assays
HEK293 cells were homogenized on ice (in 0.3 M sucrose, 5 mM HEPES, pH 7.4
and protease inhibitors) by 20 passages through a needle (23 G, 0.6625 mm) and
dispersing the cell suspension through a half volume of glass beads (425–600 mm).
Cell nuclei and glass beads were removed by centrifugation at 1500 gfor
10 minutes at 4 ˚
C, and the supernatant was retained. Cell homogenate (20 mg) was
incubated with or without 10 mM DTT for 30 minutes and then incubated with
0.0025% glutaraldehyde. Reaction was stopped with 2% hydrazine and SDS-
PAGE loading buffer (60 mM Tris-HCl, 2% SDS, 10% glycerol, 5 mM EDTA,
0.01% Bromophenol Blue, pH 6.8) and samples were then analyzed by
immunoblotting using Ab
Myc
.
Calpain cleavage of native RyR1/2
Rabbit skeletal muscle SR (10 mg) or pig cardiac SR (100 mg) was treated with
redox reagent (10 mM DTT or 1 mM H
2
O
2
) for 30 minutes (in 10 mM Na
2
PIPES,
120 mM KCl, pH 7.4). SR vesicles were recovered at 20,000 gfor 10 minutes at
4˚
C, resuspended in fresh buffer supplemented with 2 mM CaCl
2
and incubated
with 4 units of calpain-2 for 2 minutes at room temperature. Proteolysis was
stopped with SDS-PAGE loading buffer and analyzed by immunoblotting using
RyR antibodies.
Protein expression and purification, and gel filtration of GST-BT4L
GST-BT4L with C-terminal 66His tag was created by PCR in pGEX6P1 (GE
Healthcare). Protein expression in bacteria (E.coli Rosetta, Novagen) was induced
for 18 hours at 12˚
C with 0.1 mM isopropyl b-D-thiogalactoside at OD
600
50.8.
Bacteria were permeabilized in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM
Na
2
HPO
4
, 1.8 mM KH
2
PO
4
, pH 7.4, 10 mM DTT and protease inhibitors) by
three freeze-thaw and sonication cycles, and the insoluble material was removed at
10,000 gfor 10 minutes at 4˚
C. The supernatant was incubated with glutathione-
Sepharose (GE Healthcare) for 2 hours at 4˚
C and the captured GST-BT4L was
eluted with 10 mM glutathione. Eluate was dialyzed (SnakeSkin 10,000 M
r
cut-off, Pierce) against buffer (50 mM NaH
2
PO
4
, 300 mM NaCl, 5 mM b-
mercaptoethanol) and incubated with Ni-NTA agarose (Qiagen) for 2 hours at 4˚C.
Captured GST-BT4L was eluted with 250 mM imidazole and dialyzed against
PBS (+10 mM DTT).
A Hiload 16/60 Superdex 200 PG column (GE Healthcare), pre-equilibrated in
buffer (10 mM HEPES, 250 mM KCl, 10 mM DTT, pH 7.4), was calibrated with
thyroglobulin, apoferritin, b-amylase, alcohol dehydrogenase, BSA and carbonic
anhydrase (Sigma) using a 1 ml sample loop on an A
¨KTA FPLC (GE Healthcare)
at 4˚
C with a flow rate of 0.5 ml/minute. GST-BT4L (10 mg), purified in the same
buffer, was separated under identical conditions, 2 ml elution fractions were
collected and analyzed by immunoblotting using Ab
GST
.
[
3
H]Ryanodine binding assays
Ryanodine binding was performed using 8 nM [
3
H]ryanodine and 200 mgof
HEK293 microsomes for 2 hours at 37 ˚
C (in 25 mM PIPES, 1 M KCl, 2 mM
DTT, pH 7.4). Free Ca
2+
concentration was buffered using a combination of CaCl
2
and 1 mM EGTA, HEDTA and NTA (calculated using MaxChelator software,
www.stanford.edu/,cpatton/downloads.htm). Bound [
3
H]ryanodine was separated
from unbound by vacuum filtration through glass fiber filters (Whatman GF/F).
Radioactivity was quantified by liquid scintillation counting. Specific binding was
calculated from total by subtracting non-specific binding (in 10 mM unlabelled
ryanodine) from three separate experiments, each performed at least in duplicate.
Purification of recombinant RyR2
HEK293 cells transfected with human RyR2 cDNA expression plasmid were
homogenized and microsomes pelleted at ,100,000 g(28,000 rpm, Beckman
50.2Ti rotor) for 1 hour at 4˚
C. The pellet was solubilised for 1 hour at 4 ˚
Cin
buffer (25 mM Na
2
PIPES, 1 M NaCl, 0.6% CHAPS, 0.6% phosphatidylcholine,
0.15 mM CaCl
2
, 0.1 mM EGTA, 2 mM DTT, pH 7.4, and protease inhibitors) and
the insoluble material was removed at 14,000 gfor 30 minutes at 4 ˚
C. The
supernatant was layered on top of a continuous (5–40%) sucrose gradient prepared
in buffer (25 mM Tris-HCl, 50 mM HEPES, 300 mM NaCl, 0.3% CHAPS, 0.3%
phosphatidylcholine, 0.1 mM CaCl
2
, 0.3 mM EGTA, 2 mM DTT, pH 7.0). The
gradient was spun at ,100,000 g(28,000 rpm, Beckman 32Ti rotor) for 18 hours
at 4˚
C, and fractions were collected. Typically, the ,26–28% sucrose fraction
contained functional RyR2 channels.
Single-channel recordings
Planar phospholipid bilayers using phosphatidylethanolamine were formed across
a 200-mm-diameter hole in a partition that separated the cis and trans chambers
containing 20 mM HEPES, 210 mM KCl, pH 7.2. Purified RyR2 was added to the
cis chamber and channel incorporation was induced by the addition of aliquots of
3 M KCl. After fusion, the cis chamber was perfused to re-establish symmetrical
210 mM K
+
and the presence of active single RyR2 channels was verified by
recording at 260 mV in contaminating Ca
2+
.Ca
2+
dependence was tested by cis
addition of 1 mM EGTA, HEDTA and NTA that should cease channel activity; cis
free Ca
2+
concentration was then adjusted to 100 nM. Single-channel current
fluctuations were low-pass filtered at 5 kHz, digitized at 20 kHz and displayed
with Acquire 5.0.1 software (Bruxton Corporation). To characterize the effect of
GST-BT4L (+10 mM DTT) on RyR2, at least 120 seconds of recording data were
analyzed for each channel using QuB software (SUNY, Buffalo, www.qub.buffalo.
edu). Currents were idealized using the hidden-Markov modeling algorithm of the
QuB 1.5.0.39 suite using a simple two state scheme (closed /
?open).
Other methods
The yeast two-hybrid system, HEK293 cell culture and transfection, SR
preparation, co-immunoprecipitation, GST pull-down assays and immunoblotting
were carried out as described previously (Zissimopoulos and Lai, 2005;
Zissimopoulos et al., 2006). RyR1-specific Ab
2142
was raised against residues
830–845, RyR consensus Ab
2149
was raised against residues 4933–4948 of RyR2,
RyR2-specific Ab
1093
was raised against residues 4454–4474 (Fitzsimmons et al.,
2000; Mackrill et al., 1997; Zissimopoulos et al., 2007); RyR2-specific Ab
D2
(raised against residues 1344–1365) has been described previously (Jeyakumar
et al., 2001); RyR consensus Ab
H300
(raised against N-terminal 300 residues) was
obtained from Santa Cruz Biotechnology. Densitometry analysis was performed
using a GS-700 scanner (Bio-Rad) and Quantity One software (Bio-Rad).
Journal of Cell Science 126 (21)5050
Journal of Cell Science
Statistical analysis was performed using unpaired Student’s t-test. Data are
expressed as mean values 6s.e.m.
Author contributions
S.Z. conceived the study, designed the experiments and wrote the
paper; F.A.L. and A.J.W. contributed to study design; S.Z., C.V.,
M.S., B.C., J.W., I.C., R.S., S.M., N.L.T. performed and analyzed
the experimental data; L.H.C. and S.F. contributed reagents and
materials; S.Z. and F.A.L. edited the manuscript.
Funding
This work was supported by a British Heart Foundation Fellowship
[grant number FS/08/063 to S.Z.]; and Wales Heart Research
Institute Training Placement Scholarships to J.W. and I.C.
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.133538/-/DC1
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