Allen and José R. Lopez
J. Biol. Chem.
Tadeusz F. Molinski, Isaac N. Pessah, Paul D.
José M. Eltit, Tianzhong Yang, Hongli Li,
in Skeletal Myotubes
Entry Determine Resting Intracellular Ca
Leak and Ca
doi: 10.1074/jbc.M110.107300 originally published online March 5, 2010
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RyR1-mediated Ca2?Leak and Ca2?Entry Determine Resting
Intracellular Ca2?in Skeletal Myotubes*
Jose ´ M. Eltit‡§, Tianzhong Yang‡, Hongli Li‡, Tadeusz F. Molinski¶, Isaac N. Pessah?, Paul D. Allen‡1,
and Jose ´ R. Lopez‡
?DepartmentofMolecularBiosciences,SchoolofVeterinaryMedicine,UniversityofCalifornia,Davis, California 95616
The control of resting free Ca2?in skeletal muscle is thought
to be a balance of channels, pumps, and exchangers in both the
sarcolemma and sarcoplasmic reticulum. We explored these
mechanisms using pharmacologic and molecular perturbations
of genetically engineered (dyspedic) muscle cells that constitu-
tively lack expression of the skeletal muscle sarcoplasmic retic-
ulum Ca2?release channels, RyR1 and RyR3. We demonstrate
total resting Ca2?concentration ([Ca2?]rest) measured in wild
a result of active gating of the RyR1 channel but instead is
formation. In addition, we demonstrate that basal sarcolemmal
in the regulation of [Ca2?]restin skeletal myotubes.
In skeletal muscle active Ca2?efflux from the sarcoplasmic
reticulum (SR)2occurs fundamentally through RyR1 via an
orthograde signal from DHPR. In the absence of stimuli the
open probability of RyR1 is very low, and [Ca2?]restis main-
tained near 100 nM in frog (1), mammalian (2), and human
skeletal muscle (3) and in mammalian skeletal myotubes (4).
This stems from the fact that in the absence of depolarization,
as evidenced higher RyR1 activity in mdg myotubes that lack
expression of the ?1s-DHPR (7, 8).
In addition to the “classical” release pathway mediated by
of a second less defined SR Ca2?efflux pathway that has been
referred to as ryanodine (Ry)-insensitive “Ca2?leak” (4, 9, 10).
This Ca2?leak can be broadly defined as a passive efflux of
not all of the Ry-insensitive Ca2?leak pathway has been pro-
posed to represent a conformation of RyR1 with a low conduc-
tance that is constitutively open (PO?1) and represents a dis-
tinct conformation from that of actively gated RyR1 channels
involved in excitation contraction coupling (4, 10). It has been
shown that Ry-insensitive Ca2?leaks may contribute signifi-
cantly to SR Ca2?loading capacity and that they may have a
significant contribution to regulation of [Ca2?]restin skeletal
muscle. If this is correct, then RyR1 leak may have relevance in
physiological and pathological regulation of muscle Ca2?
thella basta are novel modulators of RyR1. Bastadin 5 (B5) has
constants of single RyR1 channels reconstituted in bilayer lipid
membranes without changing their unitary conductance or
overall open probability (11). Importantly, under conditions
where RyR1 channels are pharmacologically blocked (with
micromolar ryanodine or ruthenium red) both B5 and its
related congener bastadin 10 have been shown to increase sig-
nificantly the Ca2?loading capacity in SR vesicles and increase
the capacity of SR membranes to bind [3H]Ry ?4-fold (Bmax)
We hypothesized that expression of RyR1 in RyR-null
(NullRyR) myotubes would increase [Ca2?]restand that this
increase would be secondary to passive Ca2?efflux from SR
stores mediated by Ry-insensitive Ca2?leak. As expected,
dine did not modify [Ca2?]restlevels. When B5 was added to
examine the contribution of RyR1 leaks toward the [Ca2?]rest,
we found that Ry?B5 in combination reduced resting
[Ca2?]restto essentially dyspedic levels in RyR1-expressing
cells, but had no effect inNullRyR cells. [Ca2?]restwas further
reduced when Ry?B5-pretreated
Similar results were obtained in primary myotubes generated
These results show that a fraction of RyR1 within the SR mem-
brane exists in a Ry-insensitive conformation that mediates
Ca2?leak that determines [Ca2?]restlevels in skeletal muscle.
In addition, RyR1 expression also regulates basal sarcolemmal
Ca2?influx, which also contributes to [Ca2?]restin skeletal
NullRyR and wild type
Grants 2R01AR43140 and 5P01 AR052354 (to P. D. A. and I. N. P.).
1To whom correspondence should be addressed: Dept. of Anesthesiology
cis St., Boston, MA 02115.
dine receptor; Ry, ryanodine; [Ca2?]rest, resting Ca2?concentration; B5,
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 18, pp. 13781–13787, April 30, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
APRIL 30, 2010•VOLUME 285•NUMBER 18JOURNAL OF BIOLOGICAL CHEMISTRY 13781
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MATERIALS AND METHODS
Isolation of B5—B5 was extracted from lyophilized Ianthella
basta sponge collected from Guam using methods described
Cell Culture and Infection with RyR1 Herpes Simplex Virus
Virions—1B5 cells that lack expression of RyR-1, RyR-2, and
RyR-3 (NullRyR) were cultured on Matrigel- (BD Bioscience)
coated 10-cm dishes as described previously (4, 12, 13) and
allowed to differentiate for 5 days. Grid plates containing dif-
ferentiated myotubes were infected with helper-free herpes
simplex virus type 1 virion particles containingWtRyR1 cDNA
(10, 14). Transduced cells were identified after making mea-
surements of resting [Ca2?] using immunofluorescence with
Primary myoblast cell lines were generated from the hind-
limb and forelimb muscles of E18 RyR1/RyR3 double-null dys-
pedic mice and their Wt littermates (15, 16). Myoblasts were
described previously (14).
Ca2?-selective Microelectrodes—Single- and Double-bar-
reled Ca2?-selective microelectrodes were prepared using
thin walled borosilicate glass capillaries (WPI 2B150F-4, and
WPI PB-150F-4 Sarasota, FL) as described previously (4). They
were back-filled first with the neutral carrier ETH 129 (Fluka,
Ronkontioma, NY) and then with pCa7 solution. Each Ca2?-
selective microelectrode was individually calibrated as de-
scribed previously (1), and only those with a linear relationship
between pCa3 and pCa7(Nernstian response, 29.5 mV/pCa
unit) and at least 25 mV between pCa7 and pCa 8 were used
To better mimic intracellular ionic conditions, all calibra-
tion solutions were supplemented with 1 mM Mg2?. After
making measurements of resting [Ca2?], all electrodes were
then recalibrated, and if the two calibration curves did not
agree within 3 mV, the data from that microelectrode were
discarded. Before starting the studies, we determined by
direct calibration that the calcium sensitivity of the Ca2?
microelectrodes was not affected by any of the drugs used in
the present study.
viously (1, 4). The potential from the 3 M KCl microelectrode
(Vm) was subtracted electronically from potential of the Ca2?
tial (VCa) that represents the [Ca2?]rest. Vmand VCa were fil-
tered (30–50 KHz) to improve the signal-to-noise ratio and
stored in a computer for further analysis.
Mn2?Quench—PrimaryNullRyR andWtRyR myotubes
were loaded with 5 ?M fura-2/AM to measure the rate of dye
quench by Mn2?entry (Molecular Probes, Eugene, OR) at
36 °C for 20 min in imaging buffer, pH 7.4. The myotubes
were then washed three times with imaging buffer and trans-
ferred to the stage of a Nikon TE2000 inverted microscope
and illuminated at the isosbestic wavelength for fura-2 (360
nm). Fluorescence emission was captured from regions of
interest within each myotube from 3–10 individual cells at 5
frames/s using an Olympus 40 ? oil 1.3 NA objective. Mn2?
influx into myotubes was measured as described previously
with minor modification (17, 18). Final concentrations of
500 ?M MnCl2and 1.2 mM Mg2?were added to a nominally
Ca2?-free (?7 ?M free Ca2?).
SR Ca2?Loading Content Determination—Relative SR Ca2?
content levels of primaryNullRyR andWtRyR myotubes were
at 37 °C. Cells were incubated in Ca2?-free solution to avoid
Ca2?entry from the extracellular medium. Total SR calcium
content was expressed as the area under the curve of the Ca2?
Membrane Vesicle Preparation and Immunoblotting—
Microsomal vesicles were prepared from cultured myotubes.
mm imidazole, pH 7.4, 300 mm sucrose supplemented with
protease inhibitor (CompleteTM; Roche Applied Science) and
collected as described previously (19). Proteins were separated
ride membranes. Expression of specific proteins was assessed
bodies against; RyR1 (34C; Sigma-Aldrich), SR Ca2?-ATPase 1
(SERCA-1) (ABR-Affinity BioReagents, Rockford, IL), Na?-
Ca2?exchange 3 (NCX3) (a gift from Dr. Kenneth Philipson
and 95209 Swant, Bellinzona, Switzerland), plasma membrane
technology), and glyceraldehyde-3-phosphate dehydrogenase
(FL-335, Santa Cruz Biotechnology).
Solutions—Ionic composition of the mammalian Ringer
solution was: 125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 25 mM
HEPES, 6 mM glucose, 2 mM CaCl2. Ryanodine (500 ?M) and
B5 (20 ?M) solutions were prepared by adding these com-
pounds to the desired concentration in normal mammalian
Ringer solution. The low Ca2?solution was prepared using
the same protocol as the corresponding regular Ringer solu-
tion, but Ca2?was omitted, and Mg2?(2 mM) was added.
Ca2?-free solution was prepared by omitting Ca2?and add-
ing Mg2?(2 mM) and EGTA (1 mM). All solutions were
adjusted to pH 7.4. Solution exchange was realized by gentle
aspiration of the media and application of the new media
with a transfer pipette. Solution replacement was repeated
several times to assure the complete exchange of media.
Experiments were performed at 23 °C.
Drug Treatments—In the drug treatment studies, all cells
B5 was added to the cells for 10 min prior to [Ca2?]restmea-
surements. For studying the effect of Ry and B5 together, the
myotubes were first incubated with Ry for 45 min followed by
an additional 10-min incubation with both Ry and B5 before
[Ca2?]restmeasurements were made. To explore the effect of
low Ca2?solution in cells treated with Ry and B5 together, the
myotubes were first incubated with Ry 45 min followed by an
additional 10-min incubation with both Ry and B5 and then
were incubated in low Ca2?in the presence of Ry and B5 for 2
min before [Ca2?]restmeasurements were made. We avoided
13782 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 18•APRIL 30, 2010
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making [Ca2?]restmeasurements after long incubations in low
Ca2?solution (more than 10 min) because despite the fact that
2 mM Mg2?was added to the low Ca2?solution all myotubes
began to show a significant depolarization after this interval
Statistics—All values are expressed as mean ? S.D. Paired
and unpaired t tests were used to compare the [Ca2?]restin
single myotubes and groups of myotubes before and after drug
treatment(s); p ? 0.05 was considered significant.
RyR1 Expression Significantly Increases [Ca2?]restinNullRyR
Myotubes—Resting membrane potentials and [Ca2?]restwere
measured in differentiatedNullRyR 1B5 myotubes and those
transduced withWtRyR1 virions. The [Ca2?]restobserved in
observed inNullRyR myotubes (123 ? 4.7 nM, n ? 83 versus
65 ? 4 nM, n ? 84, p ? 0.0001) (Fig. 1). There were no differ-
ences in membrane potential of
compared withNullRyR myotubes (63 ? 2.1 mV, n ? 84 versus
63 ? 1.8 mV, n ? 83, p ? 0.05) (Fig. 1).
Ry Does Not Lower [Ca2?]rest—It is well established that low
concentrations Ry can enhance the open probability of RyR1
and that high micromolar concentrations (such as the condi-
tions used in the present study) fully block channel conduc-
tance (21–23) and prevent Ca2?release induced by direct RyR
agonists (e.g. caffeine, 4 CmC). Incubation of myotubes
expressingWtRyR1 with 500 ?M Ry completely abolished Ca2?
responses to the first and any subsequent exposure to caffeine
(20 mM) (data not shown). Incubation ofWtRyR1-expressing
myotubes andNullRyR myotubes in 500 ?M Ry for 45 min did
not modify [Ca2?]restin either group of cells. InNullRyR
nM (n ? 30) (p ? 0.05) after Ry treatment, and inWtRyR1-
expressing myotubes, [Ca2?]iwas 123 ? 3.6 nM (n ? 24)
before and 122 ? 3.2 nM (n ? 24) (p ? 0.05) after Ry incu-
bation (Fig. 2A). There was no change in VminNullRyR and
WtRyR1myotubes after the treatment with Ry.
tion ofNullRyR myotubes for 10 min with B5 did not affect the
levels of [Ca2?]rest(65 ? 4 nM, n ? 22 before versus 63 ? 4 nM,
n ? 22, p ? 0.05) (Fig. 2B). Interestingly, B5 alone diminished
FIGURE 1. Resting membrane potentials (left) and resting intracellular
electrodes in RyR-null and Wt RyR1-expressing 1B5 myotubes. Data are
expressed as mean ? S.D., n ? 20 cells/group.
FIGURE 2. Resting intracellular free Ca2?concentrations measured in
RyR-null and Wt RyR1-expressing 1B5 myotubes. A, after treatment with
500 ?M ryanodine. B, after treatment with 20 ?M B5. C, after treatment with
500 ?M ryanodine and 20 ?M B5. Data are expressed as mean ? S.D., n ? 20
cells/group.***, p ? 0.0001.
APRIL 30, 2010•VOLUME 285•NUMBER 18JOURNAL OF BIOLOGICAL CHEMISTRY 13783
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0.0001 (Fig. 2B). There was no change in VminNullRyR and
WtRyR1 myotubes during the treatment with B5 from pretreat-
ment values (data not shown).
els in RyR1-expressing Cells—Incubation ofNullRyR myotubes
with Ry?B5 had no effect on [Ca2?]rest(64 ? 3 nM (n ? 22)
before and 65 ? 4.2 nM (n ? 22), p ? 0.05 after). However in
WtRyR1-expressing myotubes, incubation with Ry?B5 signifi-
cantly decreased [Ca2?]restin myotubes to a level not signifi-
cantly different from that measured inNullRyR myotubes, from
123 ? 4.8 nM (n ? 36) to 70 ? 4.7 nM (n ? 44, p ? 0.0001) (Fig.
bation with Ry?B5 (data not shown).
Effect of Ry?B5 in low [Ca2?]e—To explore the contribution
andWtRyR myotubes were incubated in Ry?B5 (see under
“Materials and Methods”), and then the bath solution was then
substituted with one containing low Ca2?in the presence of
Ry?B5. InNullRyR myotubes, [Ca2?]restin normal Ringer was
67 ? 2.9 nM (n ? 11), and after Ry?B5 incubation [Ca2?]rest
was 65 ? 2.6 nM (n ? 11, p ? 0.05) After substitution with the
Ca2?solution [Ca2?]restdeclined to 42 ? 5.6 nM (n ? 12, p ?
0.0001). Following the same experimental protocol inWtRyR-
expressing myotubes resulted in [Ca2?]restvalues that shifted
from 123 ? 3.4 nM (n ? 13) in normal Ringer to 70 ? 4.2 nM
presence of Ry?B5 and low [Ca2?]eresulted in [Ca2?]restin
WtRyR andNullRyR myotubes that were nearly identical (45 ?
5.8 nM versus 42 ? 5.6 nM, respectively) (Fig. 3).
[Ca2?]restinNullRyR andWtRyR1 Primary Myotubes—Rest-
ing membrane potentials and [Ca2?]restwere measured in dif-
ferentiated dyspedic and wild type primary myotubes. Similar
to the data for null and RyR1-transduced 1B5 myotubes,
[Ca2?]restobserved inWtRyR1 primary myotubes was signifi-
cantly higher than that observed in dyspedic myotubes (122 ?
3.6 nM, n ? 17 versus 66 ? 5.2 nM, n ? 17, p ? 0.001) (Fig. 4)
FIGURE 4. Resting membrane potentials (left) and resting intracellular
electrodes in Wt and RyR-null primary myotubes. Data are expressed as
mean ? S.D., n ? 20 cells/group.
FIGURE 5. Measurements of resting cation entry using Mn2?quench in
RyR-null and Wt primary myotubes. Upper, fura-2 fluorescence raw traces
the absence of Ca2?, and Mn2?in the absence of Ca2?after the addition of
extracellular Cd2?and La3?. Lower, comparison of the rate of Mn2?quench
n ? 15 cells/group. **, p ? 0.0001.
FIGURE 3. Effects of removal of extracellular Ca2?on intracellular free
expressed as mean ? S.D., n ? 20 cells/group.
13784 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 18•APRIL 30, 2010
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with no difference in the resting membrane potential (64 ? 1.6
mV, n ? 17 versus 64 ? 1.5 mV, n ? 17, p ? 0.05) between the
from the values seen in corresponding 1B5 cells.
Ca2?Entry inNullRyR andWtRyR1 Primary Myotubes—Us-
ing the Mn2?quench technique, the quenching of fura-2 fluo-
rescence was measured at its isosbestic point in primary
NullRyR andWtRyR myotubes at rest in a nominally Ca2?-free
external solution containing 500 ?M Mn2?. Under these assay
conditions, the rate of Mn2?quench of fura-2 signal can be
attributed to Ca2?entry (Fig. 5, upper). The rate of Mn2?
quench at rest was ?2-fold greater inWtRyR than inNullRyR
myotubes (1.26 versus 0.67 fluorescence (arbitrary units)?s
the first time that resting Ca2?entry is greater inWtRyR1 com-
pared withNullRyR myotubes.
SR Ca2?Content—To quantify
the level of SR Ca2?content, we
exposedNullRyR andWtRyR1 pri-
mary myotubes loaded with Fluo-
5N/AM and exposed to 5 ?M iono-
mycin in Ca2?free solution, to
avoid Ca2?entry from the extracel-
lular medium. Fig. 6 shows that
under these conditions the there is
no significant difference in ampli-
tude of the fluorescence signal (Fig.
6A) or the total Ca2?released (area
under the curve, Fig. 6B) from
NullRyR myotubes compared with
Ca2?Handling Protein Expres-
sion—Western blot analysis for
expression of RyR1, PMCA, NCX3,
and SERCA performed on mem-
branes isolated fromNullRyR and
WtRyR1 primary myotubes is shown
in Fig. 7 (representative blot, Fig. 7,
accompanied with increased expression of PMCA and a
decreased expression of NCX3 and SERCA.
The purpose of this study was to examine whether the
expression of RyR1 has any effect on [Ca2?]rest, the resting
Ca2?entry, and SR Ca2?loading in skeletal muscle. Our study
demonstrates that expression ofWtRyR1 is associated with a
significant increase in [Ca2?]rest, in resting Ca2?entry, with no
significant change in SR Ca2?loading compared withNullRyR
myotubes. The fact that we observed the same results with
tubes were transduced with virion particles containing wild
type RyR1 cDNA. A significant part of this elevation in
[Ca2?]restappears to be related to the presence of RyR1 leaks
those observed inNullRyR myotubes. In addition, our results
show clearly that extracellular Ca2?also plays an important
role in maintaining [Ca2?]restat physiological levels.
From these results it is clear that in addition to the control
of well known intracellular Ca2?regulatory mechanisms
(PMCA, NCX, SERCA) on steady-state [Ca2?]restin skeletal
Ca2?release, which appears to be the result of a fraction of
WtRyR1s within SR that are in a Ry-insensitive Ca2?leak con-
formation and by the Ca2?influx via the sarcolemma that is
of expression of RyR1 elevated the [Ca2?]restby ?2-fold. The
precise mechanism underlying how RyR1 leaks and the expres-
sion of the RyR1 lead to a chronic elevation [Ca2?]restneeds
further study. If RyR1 leak exceeds Ca2?uptake by the SR and
rium of Ca2?mobilization must be established that allows the
FIGURE 6. Fluo-5N fluorescence signals after the addition of 5 ?M inomycin to Wt and RyR-null primary
myotubes in the presence of nominal free extracellular Ca2?buffer. A, representative curve of Wt and
mean ? S.D., n ? 20 cells in each group. p ? 0.05.
FIGURE 7. Expression of Ca2?-handling proteins in Wt and RyR-null pri-
mary myotubes. Left, representative Western blots using antibodies
differentiation state), and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) as a loading control. Right, expression of RyR1 associated with a
significant decrease in the expression of SERCA and NCX3 and a significant
n ? 5 Western blots/group.
APRIL 30, 2010•VOLUME 285•NUMBER 18JOURNAL OF BIOLOGICAL CHEMISTRY 13785
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higher [Ca2?]rest. If the currently proposed mechanism that
Ca2?extrusion processes of the plasma membrane (PMCA,
by elevations in resting [Ca2?]i, and in the steady state, such as
those defined by our experimental conditions, these transport
mechanisms should be sufficient to compensate for the
increased Ca2?leak/sarcolemmal Ca2?entry, resulting in a
Because this does not happen, then the changes in [Ca2?]rest
observed with the expression of RyR1 must involve a modifica-
tion of the set points of the activity of both SERCA and these
sarcolemmal Ca2?transport mechanisms and/or the amount
In fact, it was found that expression of RyR1 was accompanied
by an increase in the expression of PMCA and a decreased
expression of NCX3 and SERCA, all of them linked to the reg-
ulation of intracellular [Ca2?]. As modeled in Fig. 8, the
decreased expression of SERCA, an increased expression of
PMCA, elevated resting Ca2?entry, and an elevated cytoplas-
stores at levels equal to that found in dyspedic cells. One expla-
the level of SERCA expression was maintained in the face of a
sizable Ca2?leak, futile cycling of Ca2?between the SR lumen
and the extracellular space would come at a great energy cost.
Conversely, dyspedic cells express higher levels of SERCA
because without the RyR1 Ca2?leak there is a reduced energy
cost. One intriguing discovery in the present study is that RyR1
expression appears to confer significant regulation of the den-
sity of SERCA protein found in SR membranes. These results
are consistent with previous findings that indicated up-regula-
tion of SERCA levels in skeletal muscle membranes isolated
from dyspedic mice compared with those isolated from wild
type (Fig. 6 in Ref. 24).
Second, the higher PMCA expression could help offset
decreased rates of Ca2?transient recovery (relaxation) in light
of lower SERCA capacity by removing a larger fraction of
NCX protein is down-regulated and how it contributes to
maintenance of resting Ca2?are unclear.
B5, through its modulatory actions on the FKBP12?RyR1
complex, has been previously shown to increase SR Ca2?load-
ing capacity and concomitantly attenuate RyR1 Ca2?leak.
This property of the bastadins is the result of their ability to
convert Ry-insensitive leak states (RyR1 leak) into ryanod-
ine-sensitive channels (RyR1 Ca2?channels) (10) and is dem-
Bmaxof Ry binding/mg of protein
B5 was used to examine the rela-
tionship between Ry-sensitive and
that coexist in the SR ofWtRyR1-ex-
pressing myotubes. We found that
B5 in combination with blocking
concentrations of Ry decreased
sis that bastadins can promote the conversion of RyR1 in the Ry-
insensitive Ca2?leak conformation into Ry-sensitive RyR1 chan-
nels. B5 alone, also reduced [Ca2?]restinWtRyR1-expressing
myotubes but to a lesser degree (25%) compared with its effect in
verted into gating channels do not have the same degree of nega-
Another interesting result is that the resting Ca2?entry is
nitude of this entry is modulated by the presence of RyR1. The
physiological role of this resting Ca2?entry is poorly understood,
but appears to be independent of resting membrane potential
(myotubes polarized based on the Nernst equation for 23°C)
and/or the degree of SR depletion as we showed in Fig. 6, as has
The existence of a RyR1-mediated Ca2?leak pathway in
the SR may have some implications for the pathophysiology
of two well characterized disorders of skeletal muscle, malig-
nant hyperthermia and central core disease. In muscle cells
from the majority of patients with either disorder, there is a
treating the muscle cells with B5 in combination with blocking
concentrations of Ry (4). In summary, our results demonstrate
that that expression ofWtRyR1 is associated with an increase in
[Ca2?]restand that in addition to traditionally proposed mech-
anisms involving SERCA, NCX, and PMCA, [Ca2?]restin skel-
etal muscle is determined in part by passive Ca2?leak through
WtRyR1 and increased basal sarcolemmal Ca2?entry.
Acknowledgments—We thank Dr. Kenneth Philipson for antibodies
against NCX3 and Dr. Peter Schupp (University of Guam, Marine Lab-
oratory) for I. basta. Monoclonal antibody 34C was developed by J. A.
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