Ryanodine receptors (RyRs) are
present in the endoplasmic reticulum of virtually every cell type and
serve critical roles, including excitation-contraction (EC) coupling in
muscle cells. In skeletal muscle the primary control of RyR-1 (the
predominant skeletal RyR isoform) occurs via an interaction with
plasmalemmal dihydropyridine receptors (DHPRs), which function as both
voltage sensors for EC coupling and as L-type
Ca2+ channels (Rios, E., and Brum, G. (1987) Nature
325, 717-720). In addition to "receiving" the EC coupling
signal from the DHPR, RyR-1 also "transmits" a retrograde signal
that enhances the Ca2+ channel activity of the DHPR (Nakai,
J., Dirksen, R. T., Nguyen, H. T., Pessah, I. N., Beam,
K. G., and Allen, P. D. (1996) Nature 380, 72-76). A similar kind of retrograde signaling (from RyRs to
L-type Ca2+ channels) has also been reported in
neurons (Chavis, P., Fagni, L., Lansman, J. B., and Bockaert, J. (1996) Nature 382, 719-722). To investigate the molecular
mechanism of reciprocal signaling, we constructed cDNAs encoding
chimeras of RyR-1 and RyR-2 (the predominant cardiac RyR isoform) and
expressed them in dyspedic myotubes, which lack an endogenous RyR-1. We
found that a chimera that contained residues 1,635-2,636 of RyR-1 both
mediated skeletal-type EC coupling and enhanced Ca2+
channel function, whereas a chimera containing adjacent RyR-1 residues
(2,659-3,720) was only able to enhance Ca2+ channel
function. These results demonstrate that two distinct regions are
involved in the reciprocal interactions of RyR-1 with the skeletal
DHPR.
 |
INTRODUCTION |
Dihydropyridine receptors
(DHPRs)1 and ryanodine
receptors (RyRs) are essential for excitation-contraction (EC) coupling
in skeletal muscle (1, 4-6). The DHPRs represent voltage-sensing elements in the plasmalemma (1), and the RyRs function as
Ca2+ release channels in the sarcoplasmic reticulum (7-9).
In response to depolarization, the DHPRs undergo conformational changes
that produce membrane-bound charge movements (10). As one consequence of these conformational changes, a signal is transmitted to the RyRs,
causing them to release Ca2+ from the sarcoplasmic
reticulum. These conformational changes also control a slowly
activating L-type Ca2+ current, which mediates
the entry of extracellular Ca2+ across the plasmalemma.
However, the slow L-type Ca2+ current is not
important for skeletal muscle-type EC coupling because this coupling
persists under conditions that prevent the entry of extracellular
Ca2+ (11). The use of dysgenic myotubes (12), which lack
the endogenous
1 subunit of the skeletal DHPR
(
1S), together with expression of cDNAs encoding
chimeric combinations of
1S and
1C (the
cardiac isoform of the DHPR
1 subunit), has revealed
(13) that the loop linking homology repeats II and III is critical for
transmitting the orthograde, EC coupling signal from the skeletal DHPR
to RyR-1, the predominant skeletal isoform of the RyR.
Recently, cultured myotubes from dyspedic mice (6), which lack a
functional RyR-1 gene, have provided a skeletal muscle system that
makes it possible to express and functionally analyze cDNAs
encoding RyRs. Results with dyspedic myotubes indicate that in addition
to the "orthograde" (EC coupling) signal from DHPRs to RyRs in
skeletal muscle, there also seems to be a "retrograde" signal from
RyRs to DHPRs (2). Specifically, DHPRs appear to be present in the
plasmalemma of dyspedic myotubes and able to undergo the voltage-driven
conformational changes producing charge movement; however, the
magnitude of slow L-type Ca2+ current is
decreased in relationship to that of charge movement, indicating that
the probability of channel opening is reduced and/or the channels open
to less than the full conductance level. Expression in dyspedic
myotubes of cDNA encoding RyR-1 causes the magnitude of the
L-type Ca2+ current to return toward normal and
also restores skeletal-type EC coupling. Expression in dyspedic
myotubes of cDNA encoding RyR-2, the predominant cardiac RyR, fails
to restore either orthograde signaling (skeletal-type EC coupling) or
retrograde signaling (increased density of L-type
Ca2+ current) (14).
To identify regions of RyR-1 important for reciprocal interactions with
the skeletal DHPR, we have expressed cDNAs encoding chimeras of
RyR-1 and RyR-2. We find that two distinct regions of RyR-1 appear to
be important for reciprocal interactions with the DHPR.
 |
EXPERIMENTAL PROCEDURES |
Construction of RyR cDNAs--
Because the RyR-1 plasmid
(pRyR/Hygro) used in our previous experiments (2) lacked convenient
restriction sites at the ends of the cDNA insert, we constructed a
new RyR-1 plasmid (pCIneoRyR-1) as follows. MluI and
XbaI sites were created before the Kozak initiation sequence
(GCCGCC) and after the translation termination codon (15,112-15,114)
of pRyR/Hygro, respectively, by means of polymerase chain reaction
(PCR). The MluI-SalI (PCR-547) fragment amplified
from pRyR/Hygro, the SalI-ClaI (547-14,313)
fragment from pRyR/Hygro, and the ClaI-XbaI
(14,313-PCR) fragment amplified from pRyR/Hygro were ligated to the
MluI/XbaI sites of pCIneo (Promega) to yield
pCIneoRyR-1. Because pRyR/Hygro and pCIneoRyR-1 behaved similarly, data
from both clones are illustrated. The construction of pCIneoRyR-2 was
described previously (14). Chimeric plasmids included the following
sequences (PL designates the polylinker, and an asterisk designates a
restriction site introduced by means of PCR). pCIneoR1:
MluI-NdeI (PL-Sk 11,173) from pCIneoRyR-1 and NdeI-MluI (Ca 11,071-PL) from pCIneoRyR-2;
pCIneoR2: MluI-BamHI (PL-Sk 4,894)
from pCIneoRyR-1 and BamHI-MluI (Ca 4,867-PL)
from pCIneoRyR-2; pCIneoR4: BamHI-NdeI
(Sk 4, 894-11,173) from pCIneoRyR-1 and
NdeI-BamHI (Ca 11,071-4,867) from pCIneoRyR-2;
pCIneoR6: EcoRI-NdeI (Sk 2, 396-11,173) from pCIneoRyR-1 and NdeI-EcoRI (Ca
11,071-2,429*) from pCIneoRyR-2; pCIneoR9:
AflII-NdeI (Sk 7,922*-11, 173) from pCIneoRyR-1
and NdeI-AflII (Ca 11,071-7,820) from
pCIneoRyR-2; pCIneoR10: BamHI-AflII
(Sk 4,894-7,922*) from pCIneoRyR-1 and AflII-BamHI (Ca 7,820-4,867) from pCIneoRyR-2.
PCR was used to create EcoRI (Ca 2,429) and AflII
(Sk 7,922) sites (by the mutations G2430A and C7923T, respectively)
without altering the amino acid code. All fragments amplified by PCR
were sequenced.
Functional Analysis--
The procedures for primary culture of
dyspedic myotubes and for nuclear injection of plasmid DNA were as
described previously (2, 5, 15). RyR cDNAs (0.5 µg/µl except
0.1-0.5 µg/µl for pCIneoRyR-2) were coinjected with CD8 cDNA
(0.1 µg/µl) (16). Cells expressing the injected plasmids were
identified on the basis of contraction and/or binding of CD8
antibody-coated beads (16) and were analyzed 1-4 days after plasmid
injection.
Ca2+ currents were measured with the whole cell patch clamp
technique (2). In some instances, a 5-min exposure of cells to 0.1 mM BAPTA-AM (at room temperature) was used to abolish
spontaneous contractions. The patch pipette contained (mM) 140 cesium
aspartate, 5 MgCl2, 10 Cs2EGTA (20 nM free Ca2+), 5 Na2ATP, and 10 HEPES (pH 7.4 with CsOH) with or without 0.2 or 0.4 pentapotassium
Fluo-3. The bath solution contained (mM) 145 tetraethylammonium+, 165 Cl
, 10 HEPES (pH 7.4 with CsOH), 0.003 tetrodotoxin, and 10 Ca2+. The voltage
clamp command sequence consisted of stepping from the holding potential
(
80 mV) to
30 mV for 1 s, to
50 mV for 25-30 ms, to the
test potential for 200 ms, to
50 mV for 25-30 ms, and then back to
the holding potential.
To analyze intracellular Ca2+ transients in response to
electrical stimulation, myotubes were loaded with Fluo-3 AM, and
fluorescence changes (in arbitrary units) were measured as described
previously (17). The cells were bathed in either normal rodent Ringer
containing (mM) 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES (pH 7.4 with NaOH), or in
Ca2+-free Ringer (made by equimolar substitution of
Mg2+ for Ca2+ in the normal rodent Ringer).
Cells were stimulated with a 10-ms pulse applied via an extracellular
pipette (5, 17). Temperature was 20-22 °C.
 |
RESULTS |
Based on both hydropathy profile (6, 18-22) and comparison of
sequence with the inositol 1,4,5-trisphosphate receptor, the other
intracellular Ca2+ release channel (23), RyRs are predicted
to have two main regions: a cytoplasmic "foot" structure
representing the amino-terminal nine-tenths of the protein and a
channel region comprising the carboxyl-terminal tenth. Additional
support for this general architecture is provided by the recent
observation that even after removal of the majority of the
amino-terminal (~80%), the remaining carboxyl-terminal portion of
RyR-1 is still able to form functional Ca2+ release
channels (24). Because the foot bridges the gap between the
sarcoplasmic reticulum and the sarcolemma (25), it would seem to be the
part of the RyR most likely to participate in reciprocal interactions
with the DHPR. To identify regions of RyR-1 (18, 19) critical for these
reciprocal interactions, we constructed six cDNAs encoding chimeric
RyRs in which a varying portion of the foot region of RyR-2 (20, 21)
was replaced with the corresponding portion of RyR-1 (Fig.
1). The chimeric RyRs were expressed in myotubes obtained from dyspedic mice, which lack an intact RyR-1 gene
(2). This approach is based on previous work with dyspedic myotubes,
which demonstrated that (a) expression of RyR-1 both restored skeletal-type EC coupling (i.e. not requiring entry
of extracellular Ca2+) and enhanced the Ca2+
channel activity of the DHPR (2) and (b) expression of RyR-2 neither restored skeletal-type EC coupling nor enhanced
L-type Ca2+ channel activity (14). The RyR
expression plasmids were co-injected with a plasmid encoding CD8 T-cell
antigen into nuclei of dyspedic myotubes. Myotubes selected for
analysis displayed spontaneous and/or electrically evoked contractions
together with decoration by CD8 antibody-coated beads (16), which had
been added to the bathing medium.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 1.
Schematic representation of the RyR chimeras,
with regions having skeletal (RyR-1) sequence represented by
thick lines and regions having cardiac (RyR-2) sequence
represented by thin lines. The labels D1,
D2, and D3 indicate three regions of RyR-1 highly
divergent from RyR-2 (28). The + and symbols indicate whether
or not the expression of the construct enhanced
ICa2+ or restored skeletal-type EC
coupling. Amino acid composition of the chimeras is given below, with
Sk and Ca indicating RyR-1 and RyR-2,
respectively; when RyR-1 and RyR-2 sequence are identical in the region
flanking a joining site, amino acid numbers are given as if the entire
flanking region were of RyR-1 sequence. R1: Sk(1-3,720),
Ca(3,687-4,968); R2: Sk(1-1,631), Ca(1,623-4,968); R4: Ca(1-1,625),
Sk(1,635-3,720), Ca(3,687-4,968); R6: Ca(1-822), Sk(812-3,720),
Ca(3,687-4,968); R9: Ca(1-2,624), Sk(2,659-3,720), Ca(3,687-4,968);
R10: Ca(1-1,625), Sk(1,635-2,636), Ca(2,603-4,968).
|
|
Fig. 2 illustrates whole cell
Ca2+ currents and Ca2+ transients recorded from
myotubes expressing wild-type or chimeric RyRs. In order to minimize
the amount of exogenous Ca2+ buffering, the
Ca2+ transients were measured in intact myotubes loaded
with Fluo-3 AM. Some of the chimeric RyRs caused spontaneous
oscillatory contractions like those previously described for RyR-2 (14,
26). Therefore, BAPTA-AM was used in some experiments to suppress the
contractions before measurement of Ca2+ currents (see
legend to Fig. 2). Control experiments on normal myotubes demonstrated
that the Ca2+ current density was slightly lower in
BAPTA-AM-treated cells (11.3 ± 2.7 pA/pF, n = 10)
than in nontreated cells (16.8 ± 4.1 pA/pF, n = 15). As reported previously, dyspedic myotubes had a small
Ca2+ current density (2, 27) and lacked EC coupling (2, 6), both of which were restored toward normal after expression of RyR-1
(Fig. 2) (2). The restored EC coupling was skeletal-type because the
depolarization-evoked Ca2+ transient was observed even in
the absence of extracellular Ca2+. Also as reported
previously, expression of RyR-2 restored neither L-type
Ca2+ current (14) nor depolarization-induced
Ca2+ release (even when external Ca2+ was
present) (14, 26). The chimera R1, in which the majority of the
amino-terminal portion of RyR-2 was replaced with RyR-1 sequence (1-3,
720), restored both Ca2+ current density and skeletal-type
EC coupling. Therefore, the foot portion of RyR-1 is important for
reciprocal interactions with the skeletal DHPR.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 2.
Representative Ca2+ currents and
Ca2+ transients of the RyR chimeras expressed in dyspedic
myotubes. Ca2+ currents were recorded from myotubes
depolarized from 80 to +30 mV for 200 ms preceded by a 1-s prepulse
to 30 mV to inactivate T-type Ca2+ current.
Ca2+ transients were measured using Fluo-3 AM. Myotubes
were electrically stimulated (displayed by vertical lines)
with and without extracellular Ca2+. Ca2+
current densities (mean ± S.D. in pA/pF; numbers of cells given
in parentheses): Normal, 11.3 ± 2.7 (10); Dyspedic,
0.98 ± 0.65 (20); RyR-1, 6.2 ± 2.5 (12); RyR-2, 0.17 ± 0.1 (10); R1, 8.9 ± 3.7 (9); R2,0.21 ± 0.27 (10); R4,
5.3 ± 1.9 (8); R6, 7.5 ± 3.6 (14); R9, 5.1 ± 2.2 (10); R10, 3.8 ± 1.7 (9). Data for dyspedic and RyR-1 were from
Ref. 2; data for RyR-2 were from Ref. 14; BAPTA-AM treatment was used
for normal myotubes, R2, and R9.
|
|
To localize more precisely the RyR regions critical for reciprocal
interaction with the skeletal DHPR, we next examined chimeras that
contained segments of RyR-1 sequence smaller than in R1. The chimera
R2, which contained the RyR-1 sequence (1-1,631) corresponding only to
the amino-terminal half of that in R1, restored neither Ca2+ current nor EC coupling. Despite being unable to
interact reciprocally with the DHPR, R2 did encode a functional protein
because myotubes expressing R2 cDNA displayed spontaneous,
oscillatory contractions and could release Ca2+ in response
to application of 0.1 mM caffeine (data not shown). Because
R1 (RyR-1: 1-3, 720) could reciprocally interact with the skeletal
DHPR but R2 (RyR-1: 1-1, 631) could not, we next examined chimeras R6
(RyR-1: 812-3,720), R4 (RyR-1: 1,635-3, 720), and R9 (RyR-1:
2,659-3,720), in which successively longer portions of the RyR-1
sequence at the amino terminus of R1 were replaced with RyR-2 sequence.
The R6 and R4 chimeras were able both to increase Ca2+
current density and to restore skeletal-type EC coupling. Chimera R9
increased Ca2+ current density and restored a
depolarization-induced Ca2+ transient that was present
only when there was extracellular Ca2+. It is
unlikely that Ca2+ entering via the enhanced, slow
L-type Ca2+ current was by itself sufficient to
produce this Ca2+ transient because even a large, rapidly
activating Ca2+ current (resulting from heterologously
expressed L-channels) produces only a small change in
myoplasmic Ca2+ in dyspedic myotubes (14). Thus, R9 appears
capable of supporting Ca2+-induced Ca2+ release
but cannot mediate skeletal-type EC coupling. Because R4 (RyR-1:
1,635-3,720) supported skeletal-type coupling and R9 (RyR-1:
2,659-3,720) did not, we next examined R10 (RyR-1: 1,635-2,636). R10
both supported skeletal-type coupling and increased the density of
L-type Ca2+ current (Fig. 2).
 |
DISCUSSION |
Three regions, which have been designated D1, D2, and D3 (28), are
particularly divergent between RyR-1 (18, 19) and RyR-2 (20, 21).
Yamazawa et al. reported that deletion of D2 (RyR-1:
1,342-1,403) abolishes the ability of RyR-1 to mediate skeletal-type
EC coupling, although EC coupling is preserved when the sequence of the
D2 region is converted to RyR-2 sequence (29). Our data indicate that
neither D2 nor D1 (RyR-1: 4,254-4,631) is important for the difference
between RyR-1 and RyR-2 in mediating skeletal-type EC coupling and
enhanced Ca2+ channel activity of the DHPR (Fig. 1).
However, the D3 region (RyR-1: 1,872-1,923), which contains a cluster
of acidic residues not present in RyR-2, could have specific importance
for the ability of RyR-1 to mediate skeletal-type EC coupling because
the R10 chimera includes the D3 region.
Fig. 3 is a schematic model of
interactions between the DHPR and RyR in skeletal muscle. The skeletal
DHPR is an L-type Ca2+ channel with
voltage-sensing elements that have been adapted to control the gating
of the slow L-type Ca2+ current and also to
initiate EC coupling by triggering the opening of the Ca2+
release channel (i.e. RyR-1). Previous work has shown that
the putative, intracellular loop connecting repeats II and III of the
DHPR is critical for skeletal-type EC coupling (13, 30-32). The
demonstration here that chimera R10 restores skeletal-type EC coupling
suggests the possibility that the II-III loop triggers Ca2+
release by means of contact with RyR-1 in a region delimited by amino
acids 1,635-2,636. Because L-type Ca2+ current
density was increased by both R10 and R9, some of the residues between
1,635 and 2,636, as well as residues between 2,659 and 3,720, may
represent sites of contact for retrograde signaling whereby RyR-1
enhances Ca2+ channel activity of the DHPR (2).
Interestingly, R10 and R9 are contained within two calpain digestion
fragments of RyR-1 (1,401-2,843 and 2,844-4,685), which appear to be
linked by an intrasubunit disulfide bridge (33). No matter what the
actual folding structure of RyRs, the demonstration that R9 enhances L-type Ca2+ channel activity without restoring
skeletal-type EC coupling indicates that the structures of RyR-1
involved in retrograde (channel-enhancing) signaling are not identical
to those involved in orthograde (EC coupling) signaling. Because RyR-1
is expressed in brain (22, 34-36), it will be important to determine
whether the regions we have identified here are also involved in
reciprocal signaling in neurons.

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 3.
A schematic model of reciprocal coupling
between the skeletal DHPR and RyR-1. For simplicity, the
tetrameric structure of the RyR (37) is not represented. In response to
depolarization of the transverse tubules (TT),
voltage-sensing structures (VS) of the DHPR activate RyR-1
to release Ca2+ from the sarcoplasmic reticulum
(SR). This interaction, which may involve a direct, physical
interaction between the DHPR and RyR-1 (30-32), is indicated by the
downward arrow. The DHPR also functions as an
L-type Ca2+ channel. The coupling between the
voltage-sensing structures and opening of the L-type
Ca2+ channel is enhanced by an interaction with RyR-1
(indicated by the upward arrow) (2). The results described
in this paper suggest that region I (amino acids 1,635-2,636) of RyR-1
receives the EC coupling signal, and both region I and region II (amino
acids 2,659-3,720) participate in enhancement of L-type
Ca2+ current.
|
|
We thank Dr. N. Suda for helpful discussions
and K. Lopez for technical help.