From the Cardiovascular Research Group, Departments of Physiology & Biophysics and Biochemistry & Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada
Received for publication, October 10, 2002, and in revised form, November 25, 2002
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ABSTRACT |
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It is well known that the type 3 Ca2+ release channel (ryanodine receptor, RyR3)
exhibits strikingly different pharmacological and functional properties
depending on the tissues in which it resides. To investigate the
molecular basis for this tissue-dependent heterogeneity, we
examined the primary structure of RyR3 from various tissues by reverse
transcription polymerase chain reaction and DNA sequence analysis. As
many as seven alternatively spliced variants of RyR3 were detected.
Ribonuclease protection assays revealed that one of these splice
variants, RyR3 (AS-8a), which lacks a 29-amino acid fragment
(His4406-Lys4434) encompassing a predicted
transmembrane helix, was highly expressed in smooth muscle tissues, but
not in skeletal muscle, the heart, or the brain. Although the RyR3
(AS-8a) splice variant did not form a functional Ca2+
release channel when expressed alone in HEK293 cells, it was able to
form functional heteromeric channels with reduced caffeine sensitivity
when co-expressed with the wild type RyR3. Interestingly, this RyR3
splice variant was also able to form heteromeric channels with and
suppress the activity of the type 2 ryanodine receptor (RyR2).
Tissue-specific expression of RyR3 splice variants is therefore likely
to account for some of the pharmacological and functional
heterogeneities of RyR3. These observations also reveal a novel
mechanism by which a splice variant of one RyR isoform (RyR3) can
suppress the activity of another RyR isoform (RyR2) via a dominant
negative effect.
Ryanodine receptors
(RyRs)1 were initially
described in the sarcoplasmic reticulum of striated muscles. It is now
known that there are three mammalian RyR isoforms (RyR1, RyR2, and
RyR3) and that they are expressed in a variety of cells and tissues (1-3). RyR1 is predominantly expressed in skeletal muscle, whereas RyR2 is mainly expressed in heart, brain, and smooth muscles. The
expression of RyR3 has been detected in a broad range of tissues, including brain, smooth muscles, and skeletal muscle (4). RyR1 and RyR2
function as Ca2+ release channels and play an essential
role in excitation-contraction coupling in striated muscles (5-8).
However, the physiological role and channel properties of RyR3
remain elusive.
Depending on the tissues in which it is expressed, RyR3 exhibits
different functional properties. In skeletal muscle, it functions as a
caffeine- and ryanodine-sensitive Ca2+-induced
Ca2+ release channel similar to RyR1 and RyR2 (9) and is
involved in amplifying the Ca2+ signals generated by RyR1
(10). On the contrary, in smooth muscle cells, RyR3 forms a
ryanodine-sensitive but caffeine-insensitive Ca2+ release
channel (11, 12) and may negatively regulate the activity of RyR2
and/or RyR1 (13). The expression of RyR3 has also been demonstrated in
human Jurkat T-lymphocytes and mink lung epithelial cells.
Interestingly, these cells also exhibit ryanodine-sensitive,
caffeine-insensitive Ca2+ release activity (14, 15). These
observations have led to the notion that RyR3 expressed in smooth
muscles and peripheral tissues possesses unique functional properties,
although the molecular mechanism underlying this tissue-specific
function of RyR3 is unknown.
To account for the pharmacological and functional heterogeneity of
RyR3, it has been proposed that smooth muscle and peripheral tissues
may express a unique isoform of RyR3 as a result of alternative splicing (16, 17). To test this hypothesis, we systematically investigated the existence of alternatively spliced variants of RyR3
expressed in the uterus by amplifying and sequencing the entire
~15-kb coding region of RyR3. Our results show that RyR3 expressed in
smooth muscle tissues is extensively modified by alternative splicing
and that one of the splice variants, RyR3 (AS-8a), is highly and
selectively expressed in smooth muscle tissues. Functional
characterization reveals that this major RyR3 splice variant does not
form a functional channel when expressed alone but is able to form
functional heteromeric channels with the wild type RyR3 and RyR2. Our
data provide the molecular basis for the tissue-dependent
heterogeneity of RyR3 and demonstrate for the first time that a splice
variant of RyR3 is able to interact with and suppress the activity of
another RyR isoform, RyR2, via the formation of heteromeric channel complexes.
Amplification of RyR3 cDNA by RT-PCR--
Total RNA was
isolated from various rabbit tissues as described previously (18).
Briefly, first strand cDNA was prepared from total RNA using the
SuperScript Preamplification System (Invitrogen). The entire 15-kb
coding region of RyR3 was amplified by PCR using Taq DNA
polymerase and eight pairs of primers (CP-1F/CP-1R through CP-8F/CP-8R)
(17). The following PCR primers were used to amplify the splice
regions: AS2-F, 5'-GCATCTCCTTCCGCATC-3'; AS2-R,
5'-TTCAGCCAGCCTGTCTC-3; AS5-F, 5'-TAGCAGCAGTGGGTATG-3'; AS5-R,
5'-CCTTCACCAGCTCTGAG-3'; AS6-F, 5'-ACCAACTCTTCCGCATG-3'; AS6-R,
5'-CCGCTCCTGGTCCTGT-3'; AS7-F, 5'-ATGGAGGCAACGCCGGT-3'; AS7-R,
5'-GTGAACTCATCATTCTGG-3'; AS8a-F, 5'-GGGTTGGAAATCTATCA-3'; AS8a-R,
5'-AGTGCTCTCCTGCAGGA-3'; AS8b-F, 5'-CTGACATGAAGTGTGAC-3'; and AS8b-R,
5'-AGCTGATCTTCATACTGT-3'.
Ribonuclease Protection Assay--
A ribonuclease protection
assay was carried out using the RPA II kit from Ambion according
to the manufacturer's instructions. Briefly, a RyR3 cDNA fragment
(231 bp) encompassing the AS-8a splice region (87 bp), a 114-bp
5'-flanking, and a 30-bp 3'-flanking sequence was generated by PCR and
subcloned into pBluescript. The plasmid was linearized and used as
template to synthesize 32P-labeled RNA probe using the T7
RNA polymerase. The 32P-labeled RNA probe was hybridized
with total RNA isolated from various tissues followed by treatment with
RNases. The protected probes were analyzed by polyacrylamide gel
electrophoresis and autoradiography.
Site-directed Mutagenesis and DNA Transfection--
Construction
of the full-length RyR3 cDNA and insertion of the c-Myc epitope tag
into RyR3 after glutamate 4318 have been described previously (17, 19).
The HA tag (YPYDVPDYA) was inserted into the same position in RyR3 as
the c-Myc tag using the same strategy. A SpeI
(12864)-NotI (vector) fragment containing the AS-8a deletion
was removed from the PCR8 RT-PCR fragment and was used to replace the
corresponding wild type fragment in the full-length RyR3 cDNA. The
SpeI (10659)-SpeI (12864) fragment was ligated
back to form RyR3 (AS-8a). The KpnI (6313)-AflII
(7588) fragment containing the Immunoprecipitation and Immunoblotting Analysis--
Cell
lysates prepared from transfected HEK293 cells as described previously
(21) were incubated with protein G-agarose (30 µl) that was prebound
with 5-10 µg of anti-c-Myc or anti-HA antibodies at 4 °C for
17-19 h. The immunocomplexes bound to the agarose beads were
solubilized by Laemmli's sample buffer (22) and were separated by 6%
SDS-PAGE. The SDS-PAGE resolved proteins were then transferred
to nitrocellulose membranes at 45 mV for 18-20 h at 4 °C in the
presence of 0.01% SDS according to Towbin et al. (23). The
nitrocellulose membrane was blocked for 1 h with a blocking buffer
(phosphate-buffered saline containing 0.5% Tween 20 and 5% skim milk
powder). The blocked membrane was incubated with primary antibodies,
anti-RyR3 (34C), anti-c-Myc, or anti-HA antibodies for 2-4 h and
washed for 15 min three times with the blocking buffer. The membrane
was then incubated with the secondary anti-mouse IgG (H&L) antibodies
conjugated with alkaline phosphatase for 30-40 min. After washing, the
bound antibodies were visualized by the alkaline phosphatase-mediated
color reaction using nitro blue tetrazolium and
5-bromo-4-chloro-3-indolyl phosphate as the substrates.
Ca2+ Release Measurements--
The free cytosolic
Ca2+ concentration in transfected HEK293 cells was measured
using the fluorescence Ca2+ indicator dye fluo-3-AM as
described previously (17), with some modifications. Cells grown for
~18 h after transfection were washed four times with
phosphate-buffered saline (137 mM NaCl, 8 mM
Na2HPO4, 1.5 mM
KH2PO4, 2.7 mM KCl) and incubated
in KRH buffer (125 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 6 mM
glucose, 1.2 mM MgCl2, 2 mM
CaCl2, and 25 mM Hepes, pH 7.4) without
MgCl2 or CaCl2 at room temperature for 45 min
and then at 37 °C for 45 min. After being detached from culture
dishes by pipetting, the cells were collected by centrifugation at
1,000 rpm for 5 min in a Thermo/EC Centra CL2 centrifuge. The cell
pellets were resuspended in Dulbecco's modified Eagle's medium
supplemented with 0.1 mM nonessential amino acids, 4 mM L-glutamine, 100 units of penicillin/ml, 100 mg of streptomycin/ml, 4.5 g of glucose/liter, and 10% fetal calf
serum and were loaded with 10 µM fluo-3-AM at room
temperature for 60 min. The fluo-3-loaded cells were washed with KRH
buffer three times and resuspended in KRH buffer plus 0.1 mg/ml bovine serum albumin and 250 µM sulfinpyrazone. An aliquot of
fluo-3-loaded cells was then added to 2 ml (final volume) of KRH buffer
in a cuvette, and the fluorescence intensity of fluo-3 at 530 nm was measured in an SLM-Aminco series 2 luminescence spectrometer with 480-nm excitation at 25 °C (SLM Instruments, Urbana, IL).
[3H]Ryanodine Binding--
Preparation of cell
lysates from transfected HEK293 cells was carried out as described
previously (21). Equilibrium [3H]ryanodine binding to
cell lysate was also performed as described previously with some
modifications (21). A binding mixture (300 µl) containing 30 µl of
cell lysate (3-5 mg/ml), 500 mM KCl, 25 mM
Tris, 50 mM Hepes, pH 7.4, 5 nM
[3H]ryanodine, 0.4 µM free
Ca2+, and a protease inhibitor mix containing 1 mM benzamidine, 2 µg/ml leupeptin, 2 µg/ml pepstatin A,
2 µg/ml aprotinin, and 0.5 mM phenylmethylsulfonyl
fluoride, was incubated at 37 °C for 2.5 h. The binding mix was
diluted with 5 ml of ice-cold washing buffer containing 25 mM Tris, pH 8.0, and 250 mM KCl and immediately filtered through Whatman GF/B filters presoaked with 1%
polyethylenimine. The filters were washed four times with 5 ml of
ice-cold washing buffer, and the radioactivity associated with the
filters was determined by liquid scintillation counting. Nonspecific
binding was determined by measuring [3H]ryanodine binding
in the presence of 50 µM unlabeled ryanodine. All of the
binding assays were done in duplicate.
Identification of Alternatively Spliced Variants of RyR3--
The
entire coding region of RyR3 from rabbit uterus was amplified by using
RT-PCR and eight pairs of PCR primers (Fig.
1A). The resulting overlapping
cDNA fragments (PCR1 through PCR8) were subcloned into the
pBluescript vector. At least 14 individual clones of each RT-PCR
fragment were isolated and analyzed by restriction endonuclease
digestion. A single digestion pattern was observed when individual
clones of the PCR1, PCR3, and PCR4 fragments were digested with
multiple restriction endonucleases (data not shown). On the other hand,
two or more digestion patterns were detected in clones of PCR2, PCR5,
PCR6, PCR7, and PCR8, indicating that each of these later PCR products
contain heterogeneous DNA species.
The nature of this heterogeneity was further investigated by DNA
sequence analysis. Two or three clones of each PCR fragment representing each unique digestion pattern were sequenced. This analysis revealed that as many as seven regions in RyR3 cDNA were either excluded or included (Fig. 1B and Table
I). Exclusion of regions AS-6, AS-7,
AS-8a, and AS-8b led to deletions of 5, 6, 29, and 51 amino acids,
respectively, without changing the reading frame. Exclusion of region
AS-7 also led to a substitution of Arg for Gly3724. On the
other hand, exclusion of regions AS-2 and AS-5 resulted in a
frameshift. Different from other regions, AS-4 represented an insertion
of a single serine residue after valine 2271 as a result of using an
alternative splicing acceptor site (Table I). A comparison between the
intron/exon boundaries of the human RyR3 gene and the sequences of
these deleted regions (Table I) reveals that each of these regions
corresponds to one or more exons of the human RyR3 gene (Table I and
Fig. 1B). Thus, the exclusion of these regions is most
likely generated by alternative splicing.
Tissue Distribution of Alternatively Spliced Variants of
RyR3--
To examine whether alternative splicing of the RyR3
transcript also occurs in tissues other than uterus, total RNA from
rabbit vas deferens, aorta, stomach, small intestine, heart, brain, and diaphragm were isolated and used for RT-PCR. RyR3-specific primers flanking each splice region identified in uterus (except for AS-4) were
used to amplify cDNA fragments both containing and lacking each
splice region at the same time. RT-PCR products along with those
generated from plasmid clones containing (control 1) or lacking
(control 2) the known splice region were analyzed by polyacrylamide gel
electrophoresis (Fig. 2). The sequences
of RT-PCR fragments obtained from different tissues were confirmed to
be identical to RyR3 by direct sequencing. Both the exclusion and
inclusion of splice regions AS-2, AS-5, AS-7, AS-8a, and AS-8b were
observed in all smooth muscle tissues examined (Fig. 2, a,
b, and d-f, lanes 3-7). However, for
the most part, only the inclusion of these regions was detected in
heart, brain, and diaphragm (Fig. 2, a, b, and
d-f, lanes 8-10). Thus, the deletion of these
regions seems to be smooth muscle-specific. On the other hand, only the exclusion of region AS-6 was detected in smooth muscle tissues, heart,
and diaphragm, whereas both the exclusion and inclusion of this region
were observed in brain. Hence, the inclusion of region AS-6 appears
to be brain-specific.
The AS-8a Splice Variant Is Highly and Selectively Expressed in
Smooth Muscle Tissues--
Of all splice variants detected in smooth
muscle tissues, splice variant AS-8a (containing deletion of the AS-8a
region) appears to be highly expressed (Fig. 2e). To further
quantify the level of the AS-8a splice variant, we performed a
ribonuclease protection assay using total RNA isolated from uterus,
aorta, heart, brain, and diaphragm and an antisense
32P-labeled RNA probe that encompasses the AS-8a region. A
major band with a size of ~114 bases and a minor band of ~231 bases were detected in uterus and aorta (Fig.
3). The 231-base band corresponds to
transcripts that contain the AS-8a region (AS-8a (+)), whereas the
114-base band represents transcripts that lack the AS-8a region (AS-8a
( Formation of RyR3 (AS-8a)/RyR3 (wt) Heteromeric
Complexes--
To investigate the functional properties of the highly
expressed AS-8a splice variant, we constructed a RyR3 cDNA
containing a deletion of the 87-bp AS-8a region and expressed the RyR3
(AS-8a) cDNA in HEK293 cells. Functional characterization revealed
that the AS-8a splice variant did not form a functional
Ca2+ release channel when expressed alone in HEK293 cells.
Because both the alternatively spliced and unspliced forms of AS-8a
transcripts are present in smooth muscle tissues, it is possible that
the AS-8a splice variant and the unspliced RyR3 form heteromeric RyR3
channels. To test this possibility, we inserted the c-Myc antibody
epitope tag into RyR3 (wt) and the HA tag into RyR3 (AS-8a) and
co-expressed them in HEK293 cells. Interactions between RyR3 (wt,
c-Myc) and RyR3 (AS-8a, HA) were examined by immunoprecipitation. Fig.
4a (top panel)
shows that the anti-c-Myc antibody was able to pull down RyR3 (wt,
c-Myc) from lysate of HEK293 cells transfected with RyR3 (wt, c-Myc) or
co-transfected with RyR3 (wt, c-Myc) and RyR3 (AS-8a, HA) (lanes
1 and 3) but not RyR3 (AS-8a, HA) from cells
transfected with RyR3 (AS-8a, HA) alone (lane 2). The same
anti-c-Myc immunoprecipitates were subsequently blotted with the
anti-HA antibody. This antibody recognized a major band in the
anti-c-Myc immunoprecipitate from HEK293 cells co-transfected with RyR3
(wt, c-Myc) and RyR3 (AS-8a, HA) (lane 3, bottom
panel), but not from cells transfected with RyR3 (wt, c-Myc) alone
(lane 1, bottom panel). These data indicate that
the anti-c-Myc and anti-HA antibodies are specific and that RyR3
(AS-8a, HA) when expressed together with RyR3 (wt, c-Myc) can be
co-precipitated with RyR3 (wt, c-Myc) by the anti-c-Myc antibody.
We also performed the reciprocal experiment in which the RyR3 (AS-8a,
HA) was immunoprecipitated by the anti-HA antibody and the presence of
RyR3 (wt, c-Myc) in the anti-HA immunoprecipitates was detected by
Western blotting. As shown in Fig. 4b (top
panel), the anti-HA antibody pulled down the RyR3 (AS-8a, HA)
(lanes 2 and 3) but not the RyR3 (wt, c-Myc)
(lane 1). The anti-c-Myc antibody did not cross-react with
the RyR3 (AS-8a, HA) (lane 2, bottom panel), but
recognized a major band in the anti-HA immunoprecipitate from cells
co-transfected with RyR3 (AS-8a, HA) and RyR3 (wt, c-Myc) (lane
3, bottom panel). Hence, although the anti-HA antibody is unable to pull down RyR3 (wt, c-Myc), it is able to precipitate the
RyR3 (wt, c-Myc) in the presence of RyR3 (AS-8a, HA). Taken together,
these results demonstrate that the RyR3 (AS-8a) splice variant is able
to form heteromeric complexes with the wild type RyR3.
The RyR3 (AS-8a) Splice Variant Is Able to Form Functional
Heteromeric Channels with a RyR3 Mutant--
The ability of the RyR3
(AS-8a) splice variant to form heteromeric complexes was further
assessed by complementation analysis using a RyR3 mutant. The rationale
being that if RyR3 (AS-8a) is able to form heteromeric channels with
other RyR3 variants, co-expression of RyR3 (AS-8a) with a nonfunctional
RyR3 mutant may produce a functional heteromeric channel, because two
nonfunctional mutants may complement each other's defects. During
deletion analysis of RyR3, we generated a caffeine-insensitive RyR3
mutant, RyR3 ( Co-expression of RyR3 (AS-8a) Decreases the Sensitivity of RyR3
(wt) to Caffeine Activation--
The functional aspect of the RyR3
(AS-8a)/RyR3 (wt) heteromeric complexes was further investigated by
examining their caffeine response. HEK293 cells were co-transfected
with RyR3 (wt) and pCDNA3 vector cDNA or with RyR3 (wt) and
RyR3 (AS-8a). The peaks of Ca2+ release from aliquots of
transfected cells induced by different concentrations of caffeine
(0.05-20 mM) were measured. As shown in Fig.
6, Ca2+ release from RyR3
(wt)-transfected cells was activated by caffeine with an
EC50 of 0.87 + 0.09 mM (mean ± S.E.,
n = 3). On the other hand, Ca2+ release
from cells co-transfected with RyR3 (wt) and RyR3 (AS-8a) was activated
by caffeine with an EC50 of 2.4 + 0.02 mM
(n = 3). Therefore, co-expression of RyR3 (AS-8a)
reduces the caffeine sensitivity of RyR3 (wt), suggesting that by
forming heteromeric channels, the RyR3 (AS-8a) splice variant can
influence the activity of RyR3 (wt).
The RyR3 (AS-8a) Splice Variant Is Capable of Interacting with and
Suppressing the Activity of RyR2--
We have recently shown that RyR3
(wt) is capable of forming heteromeric channels with RyR2 (wt) in
HEK293 cells (24). One would expect that the RyR3 (AS-8a) splice
variant would be also able to form heteromeric channels with RyR2 (wt).
To directly test this hypothesis, we co-expressed HA-tagged RyR3
(AS-8a, HA) with c-Myc-tagged RyR2 (wt, c-Myc) in HEK293 cells. The
association of RyR3 (AS-8a, HA) with RyR2 (wt, c-Myc) was examined by
immunoprecipitation followed by immunoblotting. As indicated in Fig.
7A (panel a), in
addition to precipitating RyR2 (wt, c-Myc) from cells transfected with
RyR2 (wt, c-Myc) alone (lane 1, top panel) or
co-transfected with RyR3 (AS-8a, HA) and RyR2 (wt, c-Myc) (lane
3, top panel), the anti-c-Myc antibody was also able to
precipitate RyR3 (AS-8a, HA) from cells co-transfected with RyR3
(AS-8a, HA) and RyR2 (wt, c-Myc) (lane 3, bottom
panel) but not from cells transfected with RyR3 (AS-8a, HA) alone
(lane 2). Similarly, in a reciprocal experiment, the anti-HA
antibody was able to pull down RyR2 (wt, c-Myc) from cells
co-transfected with RyR3 (AS-8a, HA) and RyR2 (wt, c-Myc) (Fig.
7A, panel b, lane 3), but not from
cells transfected with RyR2 (wt, c-Myc) alone (Fig. 7A,
panel b, lane 1). Taken together, these data
indicate that RyR3 (AS-8a, HA) and RyR2 (wt, c-Myc) when expressed
together can be co-precipitated either by anti-HA or anti-c-Myc
antibody, demonstrating that the RyR3 (AS-8a) splice variant, like RyR3
(wt), can form heteromeric complexes with RyR2 (wt).
To examine whether the physical interaction between RyR3 (AS-8a) and
RyR2 (wt) affect the activity of RyR2 (wt), we co-transfected HEK293
cells with RyR2 (wt) and RyR3 (AS-8a) or RyR2 (wt) with vector DNA,
pCDNA3. [3H]Ryanodine binding to lysates of
co-transfected HEK293 cells were determined. As shown in Fig.
7B, no [3H]ryanodine binding was detected in
cells transfected with RyR3 (AS-8a) alone, consistent with the results
of Ca2+ release experiments (Fig. 5a) that
showed that RyR3 (AS-8a) does not form a functional Ca2+
release channel when expressed alone in HEK293 cells. Furthermore, co-expression of RyR3 (AS-8a) reduced [3H]ryanodine
binding to RyR2 (wt) by about 50%. [3H]ryanodine binding
has been widely used as a functional assay for RyR channel activities,
because ryanodine binds to only the open state of the channel. Thus,
these studies indicate that the RyR3 (AS-8a) splice variant is able to
suppress not only the activity of RyR3 (wt) (Fig. 6) but also the
activity of RyR2 (wt) (Fig. 7B) through the formation of
heteromeric channels.
The results of our present study demonstrate that the majority of
the RyR3 transcripts expressed in various smooth muscle tissues contain
a deletion of an 87-base pair region encoding a 29-amino acid fragment
(His4406-Lys4434) encompassing a predicted
transmembrane segment (25, 26) (Fig. 1C), as a result of
alternative splicing. This major RyR3 splice variant, RyR3 (AS-8a),
when expressed by itself in HEK293 cells, does not form a functional
Ca2+ release channel but in combination with the wild type
RyR3 is able to form functional heteromeric channels with reduced
caffeine sensitivity (Figs. 5 and 6). Furthermore, this major RyR3
splice variant is also able to form heteromeric channels with and
suppress the activity of RyR2 (Fig. 7). Together, these observations
reveal a novel mechanism of RyR regulation in which the activity of
RyR3 and RyR2 can be inhibited by a RyR3 splice variant through a
dominant negative effect.
Among the three known RyR isoforms, RyR3 is the most heterogeneous in
both function and regulation (3). One of its major heterogeneities is
its response to caffeine activation. RyR3 expressed in skeletal muscle
and the brain is caffeine- and ryanodine-sensitive (9, 16), whereas
RyR3 expressed in uterine smooth muscle cells is ryanodine-sensitive
but caffeine-insensitive (11, 12). The molecular basis for this tissue-
or cell-dependent RyR3 heterogeneity is unclear. Our
findings that the RyR3 (AS-8a) splice variant is highly expressed in
smooth muscle tissues but not in skeletal muscle and the brain and that
co-expression of this major splice variant with the wild type RyR3
reduces the caffeine sensitivity of RyR3 strongly suggest that
tissue-specific expression of RyR3 splice variants, in particular the
RyR3 (AS-8a) splice variant, may account for the heterogeneous caffeine
response of RyR3 observed in different tissues or cells. In support of
this view, the corresponding AS-8a region was found to be deleted in
RyR3 from mink lung epithelial cells, which also display
ryanodine-sensitive but caffeine-insensitive Ca2+ release
(14). Ryanodine-sensitive but caffeine-insensitive Ca2+
release activity was also observed in human Jurkat T-cells (15), and it
will be interesting to see whether the RyR3 (AS-8a) splice variant is
expressed in these cells.
The physiological function of the AS-8a splice variant is unknown.
Because caffeine activates RyR by sensitizing the channel to
Ca2+ activation, altered caffeine response may reflect
changes in Ca2+ regulation. In this context, it is of
interest to know that RyR3 expressed in nonpregnant myometrial smooth
muscle cells does not respond to activation by Ca2+ and
caffeine under normal sarcoplasmic reticulum Ca2+ loading
but becomes active when sarcoplasmic reticulum Ca2+ loading
is increased (27). This observation suggests that RyR3 from these
smooth muscle cells has a reduced sensitivity to activation by luminal
Ca2+ and that its activity is normally suppressed. It
remains to be explored whether the RyR3 (AS-8a) splice variant is
highly expressed in these smooth muscle cells and whether this splice
variant is involved in luminal Ca2+ regulation.
Another observation that may provide some clues to the physiological
role of the RyR3 (AS-8a) splice variant comes from a study using RyR3
knock-out mice. Cerebral artery smooth muscle cells isolated from these
mice displayed an increased frequency of Ca2+ sparks and
spontaneous transient outward currents and a reduced myogenic tone as
compared with the wild type cells (13). These findings led to the
suggestion that Ca2+ sparks and spontaneous transient
outward currents in arterial vascular smooth muscle cells are
negatively regulated by RyR3. The molecular mechanism by which RyR3
inhibits Ca2+ spark frequency is not clear. It has been
proposed that RyR1 and RyR2, but not RyR3, are responsible for
Ca2+ spark generation and that inhibition of
Ca2+ spark frequency may result from RyR3-mediated
prolonged Ca2+ release, which may then inactivate RyR1 or
RyR2 (13). The findings that RyR3 expressed in myometrial smooth muscle
cells is inactive under normal sarcoplasmic reticulum Ca2+
loading and that the RyR3 (AS-8a) splice variant, the major form of
RyR3 expressed in various smooth muscle tissues, does not form a
functional Ca2+ release channel when expressed alone in
HEK293 cells are inconsistent with the idea of RyR3-mediated prolonged
Ca2+ release. Alternatively, our observation that the RyR3
(AS-8a) splice variant is able to interact physically with and suppress the activity of RyR2 suggests the interesting possibility that RyR3
expressed in smooth muscle cells as a splice variant may suppress
Ca2+ sparks, spontaneous transient outward currents, and
myogenic tone by forming heteromeric channel complexes with RyR2. Thus, it appears that the main functional role of RyR3 in smooth muscle cells
differs from that of RyR3 in skeletal muscle and the brain. Unlike RyR3
expressed in skeletal muscle, where it functions as a Ca2+
release channel, RyR3 expressed in smooth muscles or epithelial cells
in the form of alternatively spliced variants may function largely as a
suppressor of Ca2+ release.
The observation that very low levels of RyR3 transcripts that contain
the AS-8a region were detected in various smooth muscle tissues as
compared with those of the AS-8a splice transcripts is in agreement
with this view (Fig. 2). An excess amount of the AS-8a splice variant
would ensure that all of the wt RyR3 subunits would be oligomerized
with the AS-8a, so that no homomeric wt RyR3 channels or heteromeric wt
RyR3/AS-8a channels with high wt RyR3:AS-8a subunit ratios would be
formed to suppress the activity of the wt RyR3 channels. Because AS-8a
can also form heteromeric channels with wt RyR2, the AS-8a splice
variant may also be involved in oligomerization with wt RyR2 where they
are co-expressed, as shown in vascular smooth muscle. It is important
to note that an excess amount of AS-8a does not completely suppress the
activity of either wt RyR3 or wt RyR2. We were able to detect the
activity of RyR3 or RyR2, although at reduced levels, after
co-expression of AS-8a with wt RyR3 in a ratio of 15:1 (AS-8a:wt RyR3)
or with RyR2 in a 10:1 ratio (AS-8a:wt RyR2) in HEK293 cells (Figs. 6 and 7).
In addition to AS-8a, several other splice regions in RyR3 have also
been detected in various smooth muscle tissues (Fig. 2). Of these
splice regions, AS-2 and AS-5 are of interest. They are located near
the 5'-end and in the middle of the RyR3 cDNA, respectively (Fig.
1). Exclusion of each of these regions leads to a frameshift and is
predicted to result in the synthesis of truncated RyR3 proteins
containing the first NH2-terminal ~800 and ~2900 amino
acid residues, respectively. These truncated RyR3 proteins lack the
COOH-terminal pore-forming region and, if expressed alone, would be
nonfunctional. However, whether they can form heteromeric channel
complexes with and thereby affect the channel activity of the
full-length RyR3 or RyR2 remains to be assessed. We have previously
shown that co-expression of NH2-terminal fragments of RyR2
with overlapping COOH-terminal fragments produces functional Ca2+ release channels in HEK293 cells (28). This
observation indicates that the NH2-terminal region of RyR2
is able to interact functionally with the COOH-terminal region. It will
be of interest to determine whether the NH2-terminal
regions of RyR3, corresponding to the AS-2 and AS-5 splice variants,
are able to interact functionally with the COOH-terminal regions of
RyR3 and RyR2.
The presence of these frameshifted splice variants raises the
possibility that splice variants such as AS-8a, whose splice regions
are located downstream of the frameshifted splice regions, are not made
into proteins in smooth muscles, despite their existence at the RNA
level. Based on the results shown in Fig. 2, it is clear that not all
of the RyR3 transcripts are alternatively spliced in the AS-2 or AS-5
regions, because considerable levels of AS-2 (+) and AS-5 (+)
transcripts were detected in various smooth muscle tissues. Thus, some
RyR3 transcripts without the AS-2 and AS-5 deletions are likely to
exist and be translated. Consistent with this view, RyR3 expressed in
uterine smooth muscle cells has been shown to function as a
Ca2+ release channel with properties different from those
of RyR3 expressed in skeletal muscle and the brain (27). Furthermore, RyR3 expressed in vascular myocytes is capable of binding fluorescent ryanodine, suggesting that vascular RyR3 is also functional (29). These
observations indicate that, although frameshifted alternatively spliced
transcripts are present in smooth muscles, not all of the RyR3 proteins
expressed in smooth muscles were in the truncated form and that some of
the RyR3 transcripts must have been translated all the way to the
3'-end encoding the channel conduction pathway to be functional. The
presence of both the alternatively spliced and nonspliced transcripts
and the detection of functional RyR3 with altered properties suggest
that multiple alternatively spliced variants are co-expressed in smooth
muscles at the protein level. To test this possibility, antibodies that
recognize the NH2-terminal region of RyR3 would be useful
in detecting the expression of the AS-2 and AS-5 proteins. Direct amino
acid determination of the appropriate protease fragments of RyR3
isolated from smooth muscle tissues that encompasses the AS-8a or other
splice regions by the use of mass spectrometry would represent an
alternative approach to definitively demonstrate the existence of AS-8a
or other splice variants at the protein level. Further investigations are required to characterize the functional properties of other potential RyR3 splice variants and to delineate the physiological roles
of RyR3 splice variants in smooth muscles and other cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
E2256-T2429 deletion was generated by the overlap extension method (20) and was used to replace the corresponding wild type fragment in the full-length RyR3 to form RyR3
(
E2256-T2429). HEK293 cells were transfected with RyR cDNAs using Ca2+ phosphate precipitation (17).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic diagrams illustrating the strategy
for identification of RyR3 splice variants and their locations in the
linear sequence of RyR3. A, eight overlapping DNA fragments
(PCR1 through PCR8) that cover the entire ~15-kb coding region of
RyR3 cDNA (depicted by an open rectangle) were amplified
by RT-PCR. B, the transmembrane domain (TM),
three divergent regions (DRI, DRII, and
DRIII), the pore-forming segment (P), the
phosphorylation site (PO4), the calmodulin binding
site (CaM), and the Ca2+ sensor domain
(Ca) are shown. The relative locations of seven potential
alternatively splice regions (AS-2, AS-4, AS-5, AS-6, AS-7, AS-8a, and
AS-8b) and their corresponding exon numbers of the human RyR3 gene are
indicated. C shows the amino acid sequences of the AS-8a
splice region (boxed) and the corresponding regions in RyR2
and RyR1. The AS-8a region encompasses a predicted transmembrane
segment (marked with a bracket).
Locations and sequences of the alternatively spliced junctions
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Fig. 2.
Tissue distribution of RyR3 splice
variants. The presence of alternatively spliced RyR3 variants,
AS-2 (a), AS-5 (b), AS-6 (c), AS-7
(d), AS-8a (e), and AS-8b (f),
expressed in various rabbit tissues was assessed by RT-PCR. PCR
products were electrophoresed in 5% polyacrylamide gels, stained with
ethidium bromide, and visualized under UV light. Plasmid clones
containing RT-PCR fragments (PCR2 through PCR8) isolated from uterus
were used as controls. Control 1 (lane 1) shows PCR products
using a plasmid clone that contains one of the splice regions as the
template, whereas control 2 (lane 2) shows PCR products
using a plasmid clone that lacks one of the splice regions as the
template. Control 1 for AS-6 (c) is not available, because
RyR3 expressed in uterus does not contain the AS-6 region. No signals
were detected in RT-PCR in the absence of reverse transcriptase. The
sizes of PCR fragments that contain (+) or lack ( ) the corresponding
splice region are indicated on the right.
)). The relative level of the 114-base AS-8a (
) band, as
determined by phosphorimaging analysis, was more than 5-fold of that of
the 231-base AS-8a (+) band in uterus and aorta. In contrast, the
relative level of AS-8a (+) band (231 bases) detected in heart, brain,
and diaphragm was about 5-fold of that of the AS-8a (
) band (114 bases). The AS-8a (
) transcript was also found to be the major
transcript expressed in vas deferens and stomach (data not shown).
Hence, consistent with the results of RT-PCR (Fig. 2), these
ribonuclease protection assay results indicate that the AS-8a splice
variant is highly expressed in smooth muscle tissues but not in the
heart, brain, or diaphragm.
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Fig. 3.
Splice variant RyR3 (AS-8a) is highly
expressed in smooth muscle tissues. The relative level of the
AS-8a splice variant expressed in various tissues was examined by
ribonuclease protection assay. An antisense 32P-labeled RNA
probe (307 bases) was hybridized with total RNA isolated from uterus,
aorta, heart, brain, and diaphragm. The probes protected from RNases
digestion were analyzed by polyacrylamide gel electrophoresis and
autoradiography. The 231-base fragment represents RyR3 transcripts
containing the AS-8a region, whereas the 114-base fragment represents
RyR3 transcripts lacking the AS-8a region.
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Fig. 4.
Splice variant RyR3 (AS-8a) is capable of
forming heteromeric channel complexes with RyR3 (wt).
Immunoprecipitation was carried out with anti-c-Myc (a) or
anti-HA (b) antibodies using cell lysates prepared from
HEK293 cells transfected with RyR3 (wt, c-Myc) (12 µg), RyR3 (AS-8a,
HA) (12 µg), or RyR3 (wt, c-Myc) (6 µg) plus RyR3 (AS-8a, HA) (6 µg). The immunoprecipitates (IP) were separated by
SDS-PAGE and transferred to nitrocellulose membranes. The membranes
were probed with the anti-RyR (34C), anti-HA, or anti-c-Myc antibodies
as indicated. WB, Western blot.
E2256-T2429). To test whether RyR3 (AS-8a) can
complement this mutant, HEK293 cells were transfected individually or
in combination with RyR3 (AS-8a) and RyR3 (
E2256-T2429). As
indicated in Fig. 5, HEK293 cells
transfected with RyR3 (AS-8a; Fig. 5a) or RyR3 (
E2256-T2429; Fig. 5b) alone did not respond to caffeine,
whereas co-expression of RyR3 (AS-8a) and RyR3 (
E2256-T2429) led to
a caffeine-sensitive Ca2+ release (Fig. 5,
c-e). These data suggest that the RyR3 (AS-8a) splice
variant is able to form functional heteromeric channels with other RyR3
variants.
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Fig. 5.
RyR3 (AS-8a) is capable of forming functional
heteromeric channels. HEK293 cells were transfected with RyR3
(AS-8a) (16 µg) (a), RyR3 ( E2256-T2429) (16 µg)
(b), RyR3 (AS-8a) (4 µg) plus RyR3 (
E2256-T2429) (12 µg) (c), RyR3 (AS-8a) (8 µg) plus RyR3 (
E2256-T2429)
(8 µg) (d), or RyR3 (AS-8a) (12 µg) plus RyR3
(
E2256-T2429) (4 µg) (e). The transfected cells were
loaded with 10 µM fluo-3-AM, and the fluorescent
intensity was monitored continuously before and after addition of 5 mM caffeine as indicated by the letter C. A
decrease in fluorescence immediately after the addition of caffeine was
the result of fluorescence quenching by caffeine. A transient increase,
although small, was consistently observed in co-transfected cells
(c-e). Similar results were obtained from three separate
experiments.
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Fig. 6.
Co-expression of RyR3 (AS-8a) reduces the
sensitivity of RyR3 (wt) to activation by caffeine. HEK293 cells
were transfected with RyR3 (wt) (1 µg) plus pcDNA3 vector DNA (15 µg) (open circles) or RyR3 (wt) (1 µg) plus RyR3 (AS-8a)
(15 µg) (filled circles). Transfected cells from 20 culture dishes (10 cm in diameter) were pooled together and loaded with
10 µM fluo-3-AM. The fluorescent intensity of an aliquot
of pooled, fluo-3-loaded cells was monitored continuously before and
after the addition of different concentrations of caffeine. The peak
fluorescence induced by each caffeine concentration was measured and
normalized to the peak fluorescence induced by 20 mM
caffeine. The curves shown represent fits using the Hill
equation. The data are shown as the means ± S.E. from three
separate experiments.
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Fig. 7.
RyR3 (AS-8a) is able to form heteromeric
channels with and suppress the activity of RyR2. A,
immunoprecipitation (IP) was carried out with anti-c-Myc
(panel a) or anti-HA (panel b) antibodies using
cell lysates from HEK293 cells transfected with RyR2 (wt, c-Myc) (12 µg), RyR3 (AS-8a, HA) (12 µg), or RyR2 (wt, c-Myc) (6 µg) plus
RyR3 (AS-8a, HA) (6 µg). The immunoprecipitates were separated by
SDS-PAGE and transferred to nitrocellulose membranes. The membranes
were probed with the anti-RyR (34C), anti-HA, or anti-c-Myc antibodies
as indicated. B, [3H]ryanodine binding to cell
lysates prepared from HEK293 cells co-transfected with 2 µg of RyR2
(wt) plus 20 µg of vector pCDNA3 DNA, or with 2 µg of RyR2 (wt)
plus 20 µg RyR3 (AS-8a), or transfected with 20 µg of RyR3 (AS-8a)
alone was determined using 5 nM [3H]ryanodine
and 0.4 µM Ca2+ (pCa = 6.4). The data
shown are the means ± S.E. from 4-6 separate experiments.
WB, Western blot.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Jonathan Lytton for helpful discussions and valuable comments on this manuscript, Dr. Wayne R. Giles and the Ion Channels and Transporters Group for continued support, Dr. Paul Schnetkamp for use of the luminescence spectrometer, Cindy Brown for excellent technical assistance, and Jeff Bolstad for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by research grants from the Canadian Institutes of Health Research.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.
Recipient of an Alex W. Church Graduate Student Award.
§ Alberta Heritage Foundation for Medical Research 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, December 5, 2002, DOI 10.1074/jbc.M210410200
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ABBREVIATIONS |
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The abbreviations used are: RyR, ryanodine receptor; HA, hemagglutinin; RT, reverse transcription; wt, wild type; RPA, ribonuclease protection assay.
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