Cloning and characterization of fiber
type-specific ryanodine receptor isoforms in skeletal muscles of
fish
Jens P. C.
Franck,
Jeffery
Morrissette,
John E.
Keen,
Richard L.
Londraville,
Mark
Beamsley, and
Barbara A.
Block
Stanford University, Hopkins Marine Station, Pacific Grove,
California 93950
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ABSTRACT |
We have cloned a
group of cDNAs that encodes the skeletal ryanodine receptor isoform
(RyR1) of fish from a blue marlin extraocular muscle library. The cDNAs
encode a protein of 5,081 amino acids with a calculated molecular mass
of 576,302 Da. The deduced amino acid sequence shows strong sequence
identity to previously characterized RyR1 isoforms. An RNA probe
derived from a clone of the full-length marlin RyR1 isoform hybridizes
to RNA preparations from extraocular muscle and slow-twitch skeletal
muscle but not to RNA preparations from fast-twitch skeletal or cardiac
muscle. We have also isolated a partial RyR clone from marlin and
toadfish fast-twitch muscles that shares 80% sequence identity with
the corresponding region of the full-length RyR1 isoform, and a RNA
probe derived from this clone hybridizes to RNA preparations from
fast-twitch muscle but not to slow-twitch muscle preparations. Western
blot analysis of slow-twitch muscles in fish indicates the presence of
only a single high-molecular-mass RyR protein
corresponding to RyR1. [3H]ryanodine binding
assays revealed the fish slow-twitch muscle RyR1 had a greater
sensitivity for Ca2+ than the
fast-twitch muscle RyR1. The results indicate that, in fish muscle,
fiber type-specific RyR1 isoforms are expressed and the two proteins
are physiologically distinct.
fiber types
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INTRODUCTION |
THE PROCESS OF excitation-contraction (EC) coupling in
muscles involves the release of
Ca2+ from sarcoplasmic reticulum
(SR) stores. This release is mediated by the SR
Ca2+ release channel, a large
tetrameric protein complex of ~2.2 × 106 Da. The affinity of the SR
Ca2+ release channel for the plant
alkaloid ryanodine has provided a tool for the purification and
biochemical characterization of this protein commonly referred to as
the ryanodine receptor (RyR) (22, 23). Vertebrate RyRs identified to
date are all similar-sized homotetrameric proteins composed of four
polypeptide subunits with molecular masses of 500-600 kDa (37).
mRNAs for the three known isoforms of the RyR gene family have been
cloned and sequenced. The primary sequences for these isoforms were
first characterized from mammalian tissues, including RyR1 from rabbit
and human skeletal muscle (39, 42), RyR2 from rabbit cardiac muscle
(29), and RyR3 from rabbit brain (20). Giannini et al. (19) extensively surveyed the expression of the three RyR isoforms in various murine tissues. With the use of both RNA antisense probes and isoform-specific antisera, a much wider tissue distribution of the mammalian isoforms of
the RyRs emerged.
Three homologous isoforms of the RyR have also been identified in
nonmammalian tissues based on molecular, biochemical, immunologic, and
physiological results (1, 25, 28, 30, 31). In contrast to adult mammals
that predominantly express only the RyR1 isoform in skeletal muscle,
nonmammalian skeletal muscle expresses two isoforms, originally
described as
- and
-RyRs, due to unknown homologies to mammalian
isoforms (1, 25, 28). The cloning and characterization of the two
skeletal RyR isoforms from bullfrog have revealed the
- and
-RyRs
are homologous to the mammalian RyR1 and RyR3 gene products,
respectively (30, 31). Partial sequence data indicate that in lower
vertebrates the homolog of the RyR2 gene family is also present in
cardiac muscle (J. Keen and B. A. Block, unpublished data). In Western
blot analysis of nonmammalian skeletal muscles, RyR1 and RyR3 occur as
discrete high-molecular-weight bands, with RyR3 having a slightly
higher mobility. Exceptions to this expression pattern exist in certain nonmammalian muscles. In specialized muscle fibers of nonmammals, such
as the super-fast-contracting swim bladder muscle of toadfish, immunoblotting experiments have determined that RyR1 is the sole isoform expressed, making the muscle a pure source of the nonmammalian RyR1 isoform (3, 25). The ryanodine binding characteristics and
single-channel conductance of the fish RyR1 channel indicate it is the
functional homolog of the mammalian RyR1 protein (9, 32, 38). A second
exception to the two RyR isoform expression pattern of nonmammalian
skeletal muscles is the slow-twitch muscle fibers in fish. In a
preliminary report of the results in this paper, we demonstrated that
fish slow-twitch muscles do not express RyR3 (17).
Recent physiological studies of fish skeletal muscle fiber
Ca2+ release kinetics indicate
distinct Ca2+ transients are
present in the slow- and fast-twitch muscles of fish (34). Studies in
mammalian skeletal muscles also indicate that the mechanism of
Ca2+ release differs between
muscle fiber types (12, 13, 18, 36). The most prominent difference
between fiber types is the time course of intracellular
Ca2+ transients. In addition,
different sensitivities of the RyR
Ca2+ release mechanism (in fiber
and SR vesicle preparations) to known modulators of the RyR channel,
such as Ca2+,
Mg2+, ATP, caffeine, doxorubicin,
and ruthenium red, indicate distinct differences between slow- and
fast-twitch vesicle preparations (24, 36). The molecular basis for the
distinct Ca2+ transients has also
been attributed to the presence of two isoforms of
Ca2+-ATPase in skeletal muscles of
tetrapods (5) as well as differences in the troponin off-rate of
Ca2+ (34). In mammals and fish,
slow fibers have longer Ca2+
transients and more sensitive force-pCa relationships. In this paper,
we present evidence for two distinct fiber type-specific SR
Ca2+ release channels in the
skeletal muscles of fish that may also be contributing to the
physiological differences between fiber types (34).
The discrete anatomic separation of fast- and slow-twitch muscle fibers
in fish provides an unparalleled system for studying the biochemical
and molecular components of different muscle fiber types. One of the
richest sources of slow-twitch muscle is found in the large open ocean
fishes (marlin and tunas) that use this muscle type to power endurance
swimming across ocean basins as well as for thermogenic purposes (2).
The slow-twitch (red) muscles of tuna and marlin are composed of 100%
slow-twitch fibers, and the fast or white muscles are 99% pure sources
of fast-twitch fibers (40). In this study, we constructed a cDNA
library from the superior rectus muscle to characterize the message for
the fish RyR1 isoform. Our interest in the superior rectus muscle stems
from studies on the thermogenic potential of eye muscles in marlin (3).
We report the cloning and characterization of a unique vertebrate RyR1
message from blue marlin (Makaira
nigricans) eye muscle (a mixed fiber type muscle)
that is also expressed in slow-twitch muscles of fish. We have also
cloned and sequenced partial RyR1 messages from cDNA libraries
derived from super-fast-contracting toadfish swim bladder muscle
and fast-twitch muscle of marlin. Hybridization of RNA probes from
these three sources reveals a fiber type-specific distribution of the
fish RyR1 isoforms, indicating the presence of both slow and fast RyR1
messages. Ryanodine binding assays with heavy SR vesicles isolated from
slow- and fast-twitch muscle fibers reveal distinct sensitivities of
the Ca2+ dependence of the
ryanodine receptor proteins.
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MATERIALS AND METHODS |
cDNA cloning and sequencing.
Total RNA was extracted from blue marlin (M. nigricans) tissues by either the guanidium
isothiocyanate/cesium chloride method of Chirgwin et al. (8) or by
using Tri-reagent (Molecular Research Center, Cincinnati, OH).
First-strand cDNA was synthesized from 10 µg total RNA using an
oligo(dT) primer. Reaction conditions for PCR included 200 ng template,
1 µM of each primer, 200 µM dNTPs, and 0.5 U Taq DNA polymerase
(Promega Biotech, Madison, WI). First-strand cDNA synthesized from
superior rectus muscle of blue marlin was used to amplify an ~750-bp
PCR product with primers RyR24
(5'-AAGGCATCAATGATCAGACCC-3') and RyR25
(5'-CTGTACATCACAGAGCAGCC-3'). The PCR product
corresponds to nucleotides 14056-14819 in the rabbit RyR1 open
reading frame (ORF) (42). This PCR product was used to screen a
commercially prepared oligo(dT)/random-primed cDNA library derived from
the superior rectus eye muscle of blue marlin (Stratagene, La Jolla,
CA). For the initial screening and all subsequent library screenings,
probes were labeled by random priming with
[
-32P]dCTP
according to Feinberg and Vogelstein (15) or with the Ready-to-Go
random-priming kit (Pharmacia Biotech). The initial screening yielded
clone
BMRR1 (ORF 14,044-15,332; Fig.
1).

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Fig. 1.
Schematic diagram of clones used to compile complete fish ryanodine
receptor (RyR1) cDNA message. Central line indicates full-length cDNA
in kilobases. Protein coding region of full-length cDNA is indicated by
an open box, and 5'- and 3'-untranslated regions are
indicated by solid lines. Location of restriction endonuclease
recognition sites is indicated as annotations above schematic. cDNA
clones used to compile complete sequence are illustrated at bottom.
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The cDNA library was also screened using a monoclonal antibody, Ab34C
(28), which yielded clone
BMRR2 (ORF 5133-8127). For the
immunoscreening procedure, the library was plated at a density of
50,000 pfu/plate and incubated for 3.5 h at 42°C. The recombinant
clones were induced to express by placing nylon membranes impregnated
with 10 mM
isopropyl-
-D-thiogalacto-pyranoside
(Hybond-N, Amersham, Arlington Heights, IL) on the plates and
continuing incubation at 37°C for an additional 4 h. The membranes
were preblocked in 2% dried milk and 1× Tris-buffered saline and
0.2% Tween (TBST) for 1 h followed by a wash for 10 min in 1×
TBST. The primary antibody was diluted 1:1,000 in TBST and incubated
with the membranes for 2 h. After the primary antibody incubation, the
membranes were washed three times for 10 min each in 1× TBST. The
membranes were subsequently incubated with the secondary
antibody/alkaline phospatase conjugate diluted 1:1,000 (goat
anti-mouse) for 1 h. All incubations were performed at room
temperature.
After the secondary antibody incubation, the membranes were washed
three times for 10 min each in TBST. Positive clones were detected
colorimetrically with the substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma Chemical, St. Louis, MO). The
human-specific RyR primer pair RR61FX
(5'-ATCTCTAGATCAAAAGCTGGGGAGCAGGAGGAG-3') and RR20XR
(5'-ACTTCTAGATTCAGAGCCCACAATGTCCTTGAG-3') amplified an approximate 1,200-bp PCR product from blue marlin first-strand cDNA
derived from the superior rectus muscle. This PCR product was
subsequently used to screen the random/oligo(dT)-primed superior rectus
muscle library, yielding two clones
BMRR3 and
BMRR4 (ORF 10,003-11,685). Screening with this PCR product also yielded
clones
BMRR5 (ORF 11,700-13,685) and
BMRR6 (ORF
11,375-11,725). The gap between clones
BMRR4 and
BMRR6 was
closed by PCR amplification from first-strand cDNA with primers
specific to the two clones. The PCR product pBMRR1 was cloned into the
vector pGEM and sequenced, which determined its identity to clones
BMRR4 and
BMRR7 in overlapping regions. An approximate 800-bp PCR
product amplified from the 3'-terminus of clone
BMRR2 was used
to screen the random/oligo(dT)-primed library, resulting in the
isolation of the clone
BMRR7 (ORF 6800-8647). Clone
BMRR8
(ORF 2879-6000) was isolated from the library after screening with
an ~800-bp PCR product derived from the 5'-end of clone
BMRR2. Two of the clones used in the assembly of the final RyR1
sequence were isolated from a primer extension library. An 18-mer
primer complementary to nucleotide residues 3030-3047 in the final
fish RyR1 ORF was used to prime first-strand synthesis from 2.5 µg
poly(A)+ RNA isolated with a
Poly(A) Quik mRNA isolation kit from the superior rectus muscle of
M. nigricans (Stratagene). After
second-strand synthesis was completed, the cDNAs were blunt ended by
adding 2 U Pfu DNA polymerase
according to the manufacturer's protocol (Stratagene).
EcoR I adaptors were blunt-end ligated
to the cDNAs and kinased using 10 U T4 polynucleotide kinase. Removal
of excess adaptors and size fractionation of the cDNAs were performed
by centrifugation through a S-500 Sephacryl column according to the manufacturer's instructions. The entire aliquot of the
size-fractionated cDNA was ligated to the
ZAPII vector arms and
packaged using the Gigapack III Gold packaging extract. The primer
extension library was screened with a PCR product derived from the
5'-terminus of the
BMRR8 cDNA clone. This screening yielded
clone
BMRR9, which corresponds to nucleotides 985-3,191 in the
final RyR1 ORF. Clone
BMRR9 was partially restriction mapped, and a
400-bp BamH I/Hind III restriction fragment from
the 5'-end of the clone was used to rescreen the primer extension
library. This yielded clone
BMRR10, which corresponds to nucleotides
869-3,190 in the final ORF. The clone that codes for the
NH2 terminus of the fish RyR1 sequence was isolated from the original oligo(dT)/random-primed cDNA
library using an ~350-bp probe amplified from the 5'-end of
clone
BMRR10. This screening yielded clone
BMRR11, which contains
65 bp of 5'-untranslated sequence and extends to base 1,100 in
the final ORF. PCR-amplified regions of the 3'- and 5'-ends of clones
BMRR7 and
BMRR4 were radiolabeled and used to screen the oligo(dT)/random-primed library, resulting in the isolation of
clone
BMRR12 corresponding to nucleotides 8,948-10,374 in the
final ORF. Rescreening of the library with a PCR-amplified region from
the 5'-end of clone
BMRR12 yielded clone
BMRR13, which
corresponds to nucleotides 6,941-8,690. The missing sequence between clones
BMRR12 and
BMRR13 was amplified from first-strand cDNA using a primer derived from the clones. The PCR clone was identical in sequence to the overlapping region of clone
BMRR12 and
BMRR13. The insert from clone pBMRR2 was radiolabeled and used to
rescreen the oligo(dT)/random-primed library, which yielded two clones,
BMRR14 and
BMRR15, which correspond to nucleotides 8,736-9,732 and 8,715-9,450, respectively, in the final ORF.
cDNA libraries were constructed from RNA isolated from marlin white
muscle RNA and toadfish swim bladder RNA using the
ZAP kit of
Stratagene. Both libraries were screened with a radiolabeled probe
derived from the
BMRR1 clone using the primers RyR24 and RyR25 (see
above). This screening yielded four clones from the toadfish swim
bladder (TFSB) library, named
TFSB1 through
TFSB4, and one clone
from the blue marlin white muscle (BMWM) library, named
BMWM1. The TFSB clones encompassed sequence corresponding to
nucleotides 13,695-14,950 of the blue marlin ORF, whereas the BMWM
clone contained sequence corrresponding to nucleotides
13,750-15,150.
Ribonuclease protection assays.
The RyR1-specific antisense probe was synthesized from a subcloned
region amplified from clone
BMRR8 using U-strand primer RyR1Eco
(5'-TATGAATTCCTCAAGAAGTCTGCT-3') and L-strand primer RyRXho (5'-GATCTCGAGGTCGTCCCTGTCGTC-3'). The amplified
product was digested with the restriction enzymes
EcoR I and
Xho I and unidirectionally cloned into
Bluescript SK+ digested with EcoR I
and Xho I. The subcloned region
corresponds to nucleotides 4,075-4,315 in the blue marlin RyR1
ORF. The antisense probe was synthesized from the
EcoR I linearized clone with T7 RNA
polymerase according to the Ambion Maxiscript T7/T3 in vitro
transcription kit protocol (Ambion, Austin, TX). An antisense probe was
synthesized from a 375-bp region of clone
TFSB1 using U-strand
primer (5'-AGGATTGAATTCATGAACTACTTG-3') and L-strand primer
(5'-AGGGAGCTCGAGGCAGTTGTATTC-3'). The PCR product was
digested with the restriction enzymes
EcoR I and
Xho I and unidirectionally cloned into
Bluescript SK+ digested with EcoR I
and Xho I. The subcloned region
corresponds to nucleotides 13,737-14,130 in the blue marlin RyR1
ORF. The antisense probe was synthesized from the
EcoR I linearized clone with T7 RNA
polymerase. Total RNA for the ribonuclease protection assays (RPA) was
prepared using Trisol reagent. The assay was performed according the
protocol of the Ambion Direct Protect RPA kit except that total RNA (20 µg) was used instead of tissue homogenates. All hybridizations were
performed at 37°C. Samples were separated on a 6% sequencing gel
that was dried and exposed to X-ray film for 24-72 h at
70°C with intensifying screens.
Heavy SR protein preparation.
Approximately 10-25 g of blue marlin and tuna fast-twitch muscle,
slow-twitch muscle, or toadfish swim bladder muscle were homogenized in
10 vol of homogenization buffer containing 300 mM sucrose, 5.0 mM
Na2EGTA, 10.0 mM
Na2EDTA, 20 mM K-PIPES, pH 7.3, 1.1 µM diisopropyl fluorophosphate, and various protease inhibitors using a Tekmar tissue homogenizer. Sodium pyrophosphate (25 mM) and 100 mM KCl were added to the homogenization, and the slurry was
stirred on ice for 45 min to separate myofibrillar proteins from
triads. The homogenate was centrifuged for 50 min at 100,000 g in a Ti50.2 Beckman rotor. The
supernatant was discarded, and the pellet was resuspended in
homogenization buffer and centrifuged at 2,000 g for 20 min in a Sorval SS34 rotor.
The supernatant was passed through two layers of cheese cloth, and the
crude microsomes were pelleted by centrifugation at 100,000 g for 50 min. The pellets were
resuspended in 300 mM sucrose and 5 mM K-PIPES, pH 7.0. This material
was layered onto discontinuous sucrose gradients (6 ml 20%, 8 ml 30%,
8 ml 36%, and 4 ml 45%) containing 0.4 M KCl, 0.1 mM
Na2EGTA, 0.1 mM
CaCl2, and 5.0 mM K-PIPES, pH 6.8, and centrifuged 16 h at 25,000 rpm in Beckman SW25 rotor. Heavy SR
membrane fractions were collected by aspiration from the 36%-45%
interface, diluted with ice-cold water, and pelleted at 100,000 g in a Beckman Ti50.2 rotor. Pelleted
membranes were resuspended in 300 mM sucrose and 5 mM K-PIPES, pH 7.0, and frozen in liquid nitrogen until use.
[3H]ryanodine binding
assays.
Heavy SR vesicles were incubated for 2 h at 30°C in binding medium
containing 10 nM
[3H]ryanodine, 0.2 M
KCl, 1.0 mM 
-methyleneadenosine 5'-triphosphate, 1.0 mM
Na2EGTA, and 20 mM
K-PIPES, pH 7.1. The free
Ca2+ concentration of the medium
was adjusted to 0.1-1,000 µM by the addition of
CaCl2 according to the affinity
constants of Fabiato (14). After incubation, the samples were filtered
onto Whatman GF/B glass fiber filters and washed twice with 5 ml
ice-cold distilled water. Nonspecific binding was measured in the
presence of 10 µM unlabeled ryanodine and was subtracted
from each sample.
Sequence and phylogenetic analysis.
For the determination of putative transmembrane regions, the protein
structure was predicted using the PredictProtein server (35). Sequences
were submitted using the PredictProtein interactive Web site
(http://www.embl-heidelberg.de/predictprotein). Transmembrane predictions were made using a neural network method with a >95% expected accuracy per residue.
The deduced amino acid sequences for the fish RyR1 sequences were
aligned to all published RyR amino acid sequences using the program
CLUSTAL W (21). A phylogenetic tree based on parsimony was generated
using the ProtPars algorithm of PHYLIP (16). Confidence values on the
major nodes of the tree were calculated by generating 500 replicate
data sets with replacement using the SeqBoot program of PHYLIP. The
multiple data sets were used as the input for the ProtPars program.
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RESULTS |
Sequence determination of a full-length RyR1 isoform in fish.
Screening of an oligo(dT)/random-primed library and a specific primer
extension library with DNA and antibody probes resulted in the
identification of a series of overlapping clones (Fig. 1). Compilation
of the cDNA clones resulted in a 16,313-bp contiguous sequence. The
contiguous sequence codes for an ORF of 15,243 bp with the initiation
methionine at position 66 and the termination codon (TAG) at position
15,309. A termination codon TAA is found 15 bp upstream of the
initiator methionine. The 3'-untranslated region is 1,002 nucleotides long. Polyadenylation signals with the motif AATAAA were
located at 961, 892, and 247 bases upstream from the 3'-terminus.
The complete fish RyR cDNA sequence encodes a protein of 5,081 amino
acids with a deduced molecular mass of 576,302 Da, including the
initiator methionine. A multiple alignment of the deduced amino acid
sequence to RyR1 sequences of frog, rabbit, and human illustrates that
the four sequences share large regions of sequence identity especially
toward the highly conserved COOH terminus of the molecule (Fig.
2.
The fish sequence is longer than the other three vertebrate RyR1
sequences, which is largely accounted for by two insertions, one of 30 amino acids from amino acid 1,851-1,880 and one of 25 amino acids
from amino acids 1,926-1,950. The overall sequence identities
between the fish RyR1 sequence and published sequences are given in
Table 1. The deduced amino acid sequence
from fish shares the greatest sequence identity with the frog RyR1
sequence (77%) and is 72 and 73% identical in pairwise comparisons
with human and rabbit RyR1 amino acid sequences, respectively.

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Fig. 2.
Multiple alignment of fish RyR1 amino acid sequence to published
RyR1 isoforms. Amino acid sequence deduced fish RyR1 amino acid
sequence was aligned to RyR1 isoform sequences from frog skeletal
muscle (31), rabbit skeletal muscle (42), and human skeletal muscle
(39). Amino acid positions that are identical to fish residue are
indicated by black shading. Gaps have been introduced to permit
alignment. Amino acid residue numbers are indicated at
right.
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The fish RyR1 amino acid sequence was analyzed for conserved motifs for
putative regulatory domains (Table 2). The
RyR protein is known to be phosphorylated by kinases. With the use of
the consensus sequence RXXS/T for calmodulin-dependent protein kinase, a computer block search of the deduced amino acid sequence revealed 16 sites. Six of the sites were conserved in all RyR1 sequences, and five
were unique to the fish RyR1 sequence. The activity of the RyR is also
known to be modulated by adenine nucleotides. With the use of the
nucleotide binding site consensus sequence, GXGXXG, seven sites were
identified in the fish amino acid sequence. Five of the sites were
completely conserved in all RyR1 sequences, and two were unique to the
fish sequence. Takeshima et al. (39) in their description of the first
RyR isoform characterized from human skeletal muscle (RyR1) identified
a putative Ca2+ binding domain
between residues 4,253 and 4,499. Site-directed antibodies raised
against specific fusion proteins further resolved that the
Ca2+ binding domain lies between
residues 4,478 and 4,512 (6, 7). Within this region is a
PE amino acid repeat motif that is reiterated six times in
the mammalian RyR1 isoforms. It was hypothesized that this region or
region(s) adjacent to it may be the sites of
Ca2+ binding. The multiple
alignment of the fish RyR1 sequence to other RyR1 isoforms reveals that
the PE repeat motif is not conserved (residues 4,519-4,532). The
multiple alignment does, however, reveal a stretch of six amino acids
(EPEKAD) adjacent to the PE repeat that is completely conserved between
all RyR1 isoforms.
The fish RyR1 sequence was analyzed for potential transmembrane regions
using the Predict Protein algorithm (35). Predictions improve with the
addition of more evolutionary divergent vertebrate taxa. Transmembrane
regions using this algorithm are predicted at a >95% expected
accuracy per residue for membrane proteins. Four transmembrane regions
were predicted (4,596-4,615, 4,686-4,712, 4,885-4,900,
and 4,910-4,931) that correspond closely to the four regions M1-M4
originally predicted by Takeshima et al. (39) for the mammalian RyR1
isoform.
To determine the evolutionary relationship of the RyR isoform
characterized from the superior rectus muscle library, the deduced amino acid sequence of the fish RyR isoform was aligned to the published mammalian and amphibian RyR sequences using the
CLUSTALW multiple alignment program. The
Drosophila RyR sequence was included in the analysis to serve as the designated outgroup. The multiple alignment was analyzed using the protein parsimony algorithm ProtPars of PHYLIP (16). The full-length RyR1 isoform clustered with the RyR1
isoforms of frog, rabbit, and human (Fig.
3). The tree also clustered the RyR3
isoforms of rabbit, frog, and chicken in a separate clade. The RyR2
isoform sequence of rabbit is found on a separate branch and appears to
be the most primitive of the three isoforms.

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Fig. 3.
Phylogenetic tree of RyR isoforms. A multiple alignment of fish RyR1
sequence to all known RyR isoforms was performed and used as input for
phylogenetic analysis. Multiple alignment was analyzed using protein
parsimony program ProtPars of PHYLIP (16). Confidence values on major
nodes of tree were calculated by boot-strapping input data set 500 times with replacement and using multiple data set option of ProtPars.
Confidence values are given on tree as a percentage of time that this
node was identified in 500 replicate analyses. Tree was rooted using
deduced amino acid sequence for
Drosophila RyR gene.
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An RPA was performed to qualitatively determine the distribution of the
RyR1 message cloned from the marlin eye muscle cDNA library. The
message was detected in RNA isolated from marlin eye muscle, marlin
slow-twitch muscle, and tuna slow-twitch muscle. The message could not
be detected by hybridization in RNA isolated from marlin or tuna
fast-twitch muscle (Fig. 4). Longer
exposures revealed a very low level of expression in the fast-twitch
muscle RNA preparations from marlin and tuna (data not shown).

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Fig. 4.
Tissue distribution of RyR1 isoform cloned from extraocular eye muscle
of blue marlin. Ribonuclease protection assays were used to
qualitatively determine expression of RyR1 isoform. A
32P-labeled synthetic RNA probe
was generated from a linearized region of complete RyR1 message (open
reading frame 4,075-4,315) and hybridized to 20 µg of total RNA
from blue marlin fast-twitch white muscle (BMWM), blue marlin
slow-twitch red muscle (BMRM), blue marlin superior rectus eye muscle
(BMSR), tuna fast-twitch white muscle (Tuna WM), and tuna slow-twitch
red muscle (Tuna RM). Probe was also hybridized against 20 µg of
yeast RNA as a negative control.
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Identification of fast-twitch muscle specific RyR1 isoforms.
To determine if fish express fiber type-specific RyR1 isoforms, we
screened cDNA libraries derived from toadfish swim bladder (RyR1 only
muscle) and blue marlin fast-twitch muscles (RyR1 and RyR3) (25). Both
libraries were screened with a radiolabeled probe amplified from the
BMRR1 clone. This screening yielded four clones from the TFSB
library named
TFSB1 through
TFSB4 and one clone from the BMWM
library named
BMWM1. The TFSB clones generated a contiguous sequence
corresponding to nucleotides 13,695-14,950 of the blue marlin eye
muscle RyR ORF, whereas the BMWM clone contained sequence corresponding
to nucleotides 13,750-15,150.
The derived amino acid sequences from these two contiguous sequences
were aligned to the previously described RyR1 sequence from marlin eye
muscle and the RyR2 and RyR3 sequences from rabbit and frog (Fig.
5). The alignment reveals several
fast-twitch muscle-specific amino acid residues (conserved in toadfish
swim bladder and marlin white muscle but not present in the RyR1
sequence from extraocular muscle). Phylogenetic comparison of the
toadfish swim bladder and marlin fast-twitch muscle sequences to the
marlin eye muscle sequence and other RyR sequences clustered the two
fast-twitch sequences together to the exclusion of the marlin eye
muscle sequence and in a larger cluster with all the RyR1 sequences
(Fig. 6).

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Fig. 5.
Multiple alignment of marlin white muscle and toadfish swim bladder
partial RyR sequence. Partial amino acid sequences from marlin white
muscle (BM fast) and toadfish swim bladder muscle (TFSB) were aligned
to corresponding regions of rabbit RyR2 isoform (RabRyR2; Ref. 29),
frog RyR3 isoform (FrogRyR3; Ref. 31), rabbit RyR3 isoform (RabRyR3;
Ref. 20), and RyR isoform characterized from eye muscle (BM slow).
Highlighted residues indicate fast-twitch specific (BM fast and TFSB)
and slow-twitch specific (BM slow) amino acids. The amino acid
alignment is from 4618 to 4955 in the fish RyR1 (Fig. 2).
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Fig. 6.
Phylogenetic analysis of partial RyR sequences. A multiple alignment of
blue marlin white muscle RyR1 cDNA and toadfish swim bladder RyR1 cDNA
sequences to marlin slow-twitch RyR1 isoform and previously
characterized RyR sequences was used as input for phylogenetic
analysis. Analysis was performed using protein parsimony algorithm
ProtPars of PHYLIP package (16). Confidence values on major nodes of
tree were calculated by boot-strapping multiple alignment using SeqBoot
algorithm of PHYLIP and using multiple data set option of ProtPars.
Boot-strap confidence values are given as percentage of 500 replicates
that node was identified in analysis. Tree was rooted using deduced
amino acid sequence for Drosophila RyR
gene.
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|
Tissue distribution of the fast-twitch RyR1 message was determined
using RPAs with a probe synthesized from the toadfish swim bladder
clone
TFSB1 (Fig. 7). The RNA probe
hybridized to RNA preparations from toadfish swim bladder, toadfish
fast-twitch muscles, marlin fast-twitch muscle, and tuna fast-twitch
muscle. Importantly, the message could not be detected by
hybridization, even after long exposures, in marlin or tuna slow-twitch
muscle. An RNA probe constructed from the blue marlin fast-twitch
muscle clone also hybridized to RNA isolated from the fast-twitch
muscles but not the slow-twitch muscles (data not shown).

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Fig. 7.
Tissue distribution of RyR message coded for by toadfish swim bladder
RyR1 cDNA. A 32P-labeled antisense
RNA probe derived from toadfish swim bladder RyR sequence was
hybridized to 20 µg of total RNA from toadfish swim bladder (TFSB),
toadfish fast-twitch white muscle (TFWM), blue marlin slow-twitch red
muscle (BMRM), blue marlin fast-twitch white muscle (BMWM), tuna
slow-twitch red muscle (Tuna RM), and tuna fast-twitch white muscle
(Tuna WM). Probe was also hybridized against 20 µg of yeast RNA as a
negative control.
|
|
Ryanodine binding and Western blot analysis.
The previous results suggest that two distinct RyR1 isoforms are
expressed in fish skeletal muscle in a fiber-type specific manner.
[3H]ryanodine binding
was performed to characterize the properties of the RyR isoforms in
fish fast- and slow-twitch muscles.
[3H]ryanodine binding
to RyR1 in mammalian skeletal muscle SR vesicles displays a classic
bell-shaped Ca2+ dependency, with
activation occurring at micromolar
Ca2+ concentrations and
inactivation occurring at millimolar
Ca2+ concentrations.
[3H]ryanodine binding
to SR preparations from marlin fast-twitch muscle exhibited this
bell-shaped Ca2+ dependency with a
peak at pCa 4 (Fig.
8A). The
SR preparation from marlin slow-twitch muscle also exhibited a
bell-shaped Ca2+ dependency but
with the peak shifted to a lower
[Ca2+] of pCa 5 (Fig.
8B). Additional ryanodine binding
assays with SR preparations from tuna fast-twitch muscle and tuna
slow-twitch muscle confirmed the marlin results (Fig. 8,
D and
E). The data from the binding curves
in Fig. 8 were normalized to show the amount of
[3H]ryanodine binding
relative to the peak for each curve (Fig. 9). The left shift in the
Ca2+ sensitivity for the
slow-twitch preparations is clearly evident. Western blot analysis
using the antibody C010, which reacts against an epitope common to all
RyRs (25), detected two high-molecular-weight bands in the fast-twitch
(white) muscle preparations previously identified as RyR1 and RyR3
(25). Suprisingly, only one band, with a mobility similar to the RyR1
protein of fast-twitch muscles, was found in the slow-twitch muscle SR
preparations (Fig. 10). Thus the
properties examined in the
[3H]ryanodine binding
assays can be attributed to this one RyR protein expressed in the
slow-twitch preparations.

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Fig. 8.
Ca2+ dependence of
[3H]ryanodine binding
to sarcoplasmic reticulum (SR) preparations from blue marlin and tuna
fast- and slow-twitch muscle and toadfish swim bladder muscle. SR
preparations were incubated with 10 nM
[3H]ryanodine under
various free Ca2+ concentrations
as described in MATERIALS AND METHODS.
Blue marlin fast-twitch (white, A) muscle and marlin
slow-twitch (red, B) muscle both exhibit biphasic responses
to Ca2+ concentration with peak
binding at pCa 5 for white muscle and pCa 4 for red muscle. Tuna white
muscle SR (C) also exhibits a biphasic response to
Ca2+ concentration with a peak
binding at pCa 4 (n = 9), whereas tuna
red muscle SR (D) demonstrates a bell-shaped dependence on
Ca2+ concentration with peak
binding occurring at pCa 5 (n = 6).
Toadfish swim bladder muscle SR (E), which solely expresses
a RyR1 isoform, also shows a bell-shaped dependence on
Ca2+ concentration with a peak
binding at pCa 4 similar to fast-twitch muscle fibers
(n = 4).
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Fig. 9.
Normalized Ca2+ sensitivity curves
for [3H]ryanodine
binding to fast- and slow-twitch muscle. Binding data from Fig. 8 were
normalized to show amount of
[3H]ryanodine binding
relative to peak for each curve. Left shift in slow-twitch muscle
preparations is clearly evident. , Tuna slow-twich red muscle; ,
blue marlin slow-twitch red muscle; , tuna fast-twitch white muscle;
, blue marlin fast-twitch white muscle; , toadfish swim bladder
muscle.
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Fig. 10.
Western blot analysis with C010 antisera. C010 antisera were reacted
against SR protein preparations from toadfish swim bladder (TFSB), blue
marlin superior rectus eye muscle (BMSR), blue marlin fast-twitch
muscle (BMWM), and blue marlin slow-twitch muscle (BMRM). Antisera
recognize a single RyR1 isoform in toadfish swim bladder, blue marlin
eye muscle, and blue marlin slow-twitch muscle and react against 2 polypeptides (RyR1 and RyR3) in SR preparation of blue marlin
fast-twitch muscle.
|
|
To establish that the differences in
[3H]ryanodine binding
between slow- and fast-twitch muscle SR preparations were not because of the influence of RyR3 in the fast-twitch muscle preparations of
marlin, we performed binding with an SR preparation from the super-fast
toadfish swim bladder muscle, which does not express RyR3 (25). The
binding characteristics for the toadfish swim bladder SR preparation
were similar to the fast-twitch muscle preparations with a peak of
binding at pCa 4 (Fig. 8E). This
indicated that the differences observed in the
Ca2+ sensitivity of fast- and
slow-twitch muscle were because of the presence of the distinct RyR1
isoforms and not because of the presence or absence of RyR3 in the
preparation.
 |
DISCUSSION |
The results of this study suggest that fast- and slow-twitch muscles of
fish express specific RyR isoforms. The full-length RyR1 sequence
presented here represents a novel vertebrate isoform of the RyR1 gene
family. The cDNA isolated from the superior rectus muscle library
encodes a deduced amino acid sequence that is 77% identical in
pairwise comparison to the frog RyR1 isoform. The calculated molecular
mass of 576 kDa is also similar to the RyR1 isoforms of mammals and
amphibians (31, 39, 42). Molecular phylogenetic analyses also confirm
that the cDNA codes for a RyR isoform that is most closely related to
the RyR1 isoforms (Fig. 3).
Hybridization of an antisense RNA probe was performed to resolve the
tissue distribution of the fish eye muscle RyR1 isoform. The message
for the fish RyR1 isoform is preferentially expressed in the
slow-twitch (red) muscle of fish (Fig. 4). Surprisingly, this RyR1
message is not expressed in fast-twitch muscles of three fish species
examined (marlin, tuna, and toadfish). The message can only be detected
in minor abundance if exposure times are significantly increased, and
this most likely corresponds to the presence of a small number of
slow-twitch fibers in these muscles. The message is detected in the
mixed fiber type superior rectus muscle (40) from where the library was
originally constructed. The full-length cDNA we have cloned and
sequenced is designated RyR1 slow. The expression results prompted us
to determine if a second, fast-twitch muscle RyR1 isoform may also be
expressed in fish muscles. We constructed and screened cDNA libraries
derived from marlin fast-twitch muscle and toadfish swim bladder muscle and were able to obtain partial RyR sequences from both tissues. Previous expression studies based on the presence of isoforms of the
Ca2+-ATPase (SERCA1 and SERCA2)
have shown the toadfish swim bladder muscle is composed of only
fast-twitch fibers (40). The toadfish swim bladder muscle and marlin
fast-twitch muscle sequences share high identity with the RyR1 gene
family and are similar but not identical to the RyR sequence derived
from the extraocular eye muscle library. Phylogenetic analyses
determined that these partial sequences are closely related to the fish
slow-twitch isoforms but significantly distinct. Importantly, the
marlin fast-twitch muscle and toadfish swim bladder muscle sequences
are more closely related to each other than either is to the marlin
slow-twitch sequence (Fig. 6). A probe derived from the toadfish swim
bladder muscle RyR1 clone hybridizes to messages in swim bladder muscle and fast-twitch muscle fibers of marlin, toadfish, and tuna, but not to
slow-twitch muscle from marlin and tuna (Fig. 7). These results indicate that fish express fiber type-specific RyR1 isoforms.
Although several splice variants of the mammalian RyR1 message have
been described (33, 43), the current study represents the first
evidence of unique RyR1 gene products in fast- and slow-twitch muscle
fibers. The two partial sequences isolated from the fast-twitch fish
muscles share significant amino acid substitutions (shared derived
characters) when compared with the slow-twitch isoform. This indicates
that within the channel region for which the limited comparative
sequence data exist, there are significant differences between the fast
and slow isoforms that represent evolutionary divergences in the RyR1
isoforms. The fact that fiber type-specific RyR1 isoforms have not been
characterized in mammals or amphibians may be associated with a loss of
one of the isoforms in higher vertebrates, but it could also be
attributed to the bias of selecting fast-twitch muscles for the
construction of cDNA libraries, since these muscles contain a high
content of SR (26, 31, 39, 42). Londraville et al. (unpublished data)
have recently shown that SERCA1b, a neonatal form of the
Ca2+-ATPase in mammals, is
expressed in adult extraocular muscles of fish and birds but not in
extraocular muscles of adult mammals. Thus distinct expression patterns
in the SR proteins of lower vertebrates may be a common finding once
investigated in further detail.
The anatomic arrangement of fish muscles provides the tool for
separating the pure slow-twitch from fast-twitch muscle, making the
expression and binding studies possible. To determine whether the
expression of unique RyR1 isoforms in fast- and slow-twitch muscles of
fish results in functional differences, we assayed SR fractions from
the different muscle fiber types for their affinity for
[3H]ryanodine.
Ryanodine binding in skeletal muscle SR preparations is typically
activated by submicromolar Ca2+
concentrations and inhibited by millimolar
Ca2+ concentrations.
[3H]ryanodine binding
to both fast- and slow-twitch SR preparations from fish exhibited the
classic bell-shaped dependence on
Ca2+ concentration that is
characteristic of skeletal RyR isoforms. The pCa for peak binding was,
however, different for the two muscle fiber types. The fast-twitch
muscle showed peak binding at pCa 4, whereas the slow-twitch muscle
fibers exhibited peak binding at pCa 5 (Figs. 8 and 9). This indicates
that the RyR isoforms of slow-twitch muscle fibers have a significantly
lower threshold for Ca2+
activation.
Nonmammalian skeletal muscles typically coexpress both the RyR1 and
RyR3 isoforms (25). Immunoblot analysis of SR preparations using an
antisera that recognizes an epitope common to all RyR isforms revealed
that although two isoforms can be detected in marlin fast-twitch muscle
(RyR1 and RyR3), the slow-twitch muscle SR preparation only expresses a
single RyR isoform (Fig. 9). The mobility of the single band on the
protein gels and recognition by the C010 antibody along with the RNase
hybridization results with the probe generated from the full-length
cDNA for RyR1 slow isoform indicate this is the RyR1 slow protein. In
addition, we have generated an RyR1-specific antibody that recognizes
only the RyR1 protein in fast- and slow-twitch muscles of marlin (17). Because of this result, the differences observed between the
fast-twitch and slow-twitch SR preparations for ryanodine binding could
be ascribed to the influence of the coexpression of the RyR1 and RyR3
isoforms. However, the toadfish swim bladder muscle is known to be
composed of a homogeneous fiber type that only expresses the RyR1
isoform (25). A SR preparation derived from toadfish swim bladder
muscle also exhibits the bell-shaped dependency on Ca2+ concentration with a peak
binding at pCa 4, similar to the fast-twitch white muscle preparations
of marlin and tuna (Fig. 8E).
Therefore, the shift in peak binding for ryanodine binding for the
slow-twitch muscle preparation cannot be attributed to the absence of
the RyR3 isoform in the preparation. These results were demonstrated for two fish species from which significant quantities of slow-twitch muscle can be isolated (tuna and marlin). Toadfish have extremely small
amounts of slow-twitch muscle fibers, which limits the ability to do
protein analyses.
The absence of the RyR3 protein as revealed by Western blots raises
interesting questions. It may be due to a reduced need to amplify the
RyR1 signal as has been proposed in the two-component model for
Ca2+ release (27) or a property of
the RyR1 slow isoform that necessitates building triads with only this
protein. It is possible that the slow-twitch muscle fibers in fish
operate in vivo at lower thresholds of
Ca2+ than fast-twitch fibers,
possibly necessitating the construction of the triad with RyR isoforms
that share similar properties (low threshold for activation via
Ca2+). If triads were built with
a mixture of RyR isoforms that had different sensitivities to
Ca2+ for opening and closing,
Ca2+ release may not be as
efficient.
Previous studies have described physiological differences in the
mechanism of EC coupling between fast- and slow-twitch muscles. Salviati and Volpe (36) compared the kinetics of
Ca2+ release from rabbit skinned
fast and slow-twitch fibers, determining that the
Ca2+ release channels of both
tissue types respond to known modulatory agents but show different
sensitivities. The SR preparations of slow-twitch muscle fibers had a
lower threshold for caffeine, whereas the fast-twitch SR preparations
were found to be more sensitive to ryanodine. Lee et al. (24) further
showed with planar lipid bilayer recordings that rat fast- and
slow-twitch muscle Ca2+ release
channels have different rates of initial
Ca2+ release and mean channel
closed times. The Ca2+ released
from the slow-twitch vesicles was 28% less than from fast-twitch SR
vesicles. Recently, Delbono and Meissner (11) compared the
intracellular transients in rat fast- and slow-twitch fibers using the
low-affinity Ca2+ indicator
Mag-fura 2 and demonstrated that rat fast-twitch muscles removed
myoplasmic Ca2+ faster than
slow-twitch muscle. They also determined with binding experiments that
slow-twitch muscles have a lower ratio of dihydropyridine receptors to
RyRs, concluding that a lower number of the RyRs in slow-twitch muscle
are directly controlled by the dihydropyridine receptor. Fish muscle
fibers also demonstrate markedly different Ca2+ transients (34). In fish, the
observed differences in Ca2+
transients and force generation by specific muscle fibers are most
likely because of several molecular and biochemical modifications of SR
and myofibrillar proteins, including the expression of fiber type-specific skeletal RyR isoforms.
 |
ACKNOWLEDGEMENTS |
We thank Dr. D. MacLennan for contribution of several primers.
 |
FOOTNOTES |
The research was supported by National Institute of Arthritis and
Musculoskeletal and Skin Diseases Grant AR-40246 and National Science
Foundation Grant IBN-9507499 to B. Block. J. Franck was supported by a
National Sciences and Engineering Research Council Postdoctoral
Fellowship, J. Morrissette by National Institute of Arthritis and
Musculoskeletal and Skin Diseases Grant AR-08425, and J. Keen by the
British Columbia Heart and Stroke Foundation.
The blue marlin RyR1 slow accession number in GenBank is U97329.
Present addresses: J. P. C. Franck, Dept. of Biology, Occidental
College, 1600 Campus Rd., Los Angeles, CA 90041; R. L. Londraville, Dept. of Biology, Univ. of Akron, Akron, OH 44325-3908.
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. §1734 solely to indicate this fact.
Address for reprint requests: B. A. Block, Stanford University, Hopkins
Marine Station, Oceanview Boulevard, Pacific Grove, CA 93950.
Received 16 March 1998; accepted in final form 23 April 1998.
 |
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