Published online before print May 24, 2001, doi: 10.1161/hh1101.091268
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© 2001 American Heart Association, Inc.
Molecular Medicine |
Coupled Gating Between Cardiac Calcium Release Channels (Ryanodine Receptors)
From the Center for Molecular Cardiology, Department of Medicine, and Department of Pharmacology, Columbia University College of Physicians and Surgeons, New York, NY, and the Institute of Molecular Physiology and Genetics (K.O.), Slovak Academy of Sciences, Bratislava, Slovak Republic.
Correspondence to Andrew R. Marks, MD, Center for Molecular Cardiology, Box 65, Columbia University College of Physicians and Surgeons, Room 9-401, 630 W 168th St, New York, NY 10032. E-mail arm42{at}columbia.edu
Abstract
Abstract—Excitation-contraction coupling in heart muscle requires the activation of Ca2+-release channels/type 2 ryanodine receptors (RyR2s) by Ca2+ influx. RyR2s are arranged on the sarcoplasmic reticular membrane in closely packed arrays such that their large cytoplasmic domains contact one another. We now show that multiple RyR2s can be isolated under conditions such that they remain physically coupled to one another. When these coupled channels are examined in planar lipid bilayers, multiple channels exhibit simultaneous gating, termed "coupled gating." Removal of the regulatory subunit, the FK506 binding protein (FKBP12.6), functionally but not physically uncouples multiple RyR2 channels. Coupled gating between RyR2 channels may be an important regulatory mechanism in excitation-contraction coupling as well as in other signaling pathways involving intracellular Ca2+ release.
Key Words: ryanodine receptors • Ca2+channels • coupled gating • FKBP12.6 • excitation-contraction coupling
Ryanodine receptors (RyRs) are intracellular ion channels that provide a pathway for the release of Ca2+ from the sarcoplasmic reticulum (SR)/endoplasmic reticulum into the cytosol. The release of intracellular stores of Ca2+ occurs in virtually all types of cells as a means of amplifying external signals that modulate intracellular signaling events. In cardiac myocytes, type 2 RyRs (RyR2s) are activated during excitation-contraction (E-C) coupling by Ca2+-induced Ca2+ release (CICR) triggered by Ca2+ influx across the sarcolemma.
If RyR2s act independently, then the activation and inactivation of individual channels should be stochastic, based on the probability of an individual channel being open or closed. Although the process is complicated by cellular geometry, the distribution of RyR2s, and local activation of CICR in cardiac myocytes,1 2 the probability that any single RyR2 opens should still be determined in part by CICR.3 4 5 6 However, we now present data showing that RyR2 channels are physically and functionally coupled such that the activity of an individual channel is coordinated with that of its neighbors in a manner analogous to that in skeletal muscle.7
Ca2+-release channels in cardiac muscle comprise four 565-kDa RyR2 subunits and four FK506 binding proteins (FKBP12.6). FKBP12, originally identified as the peptide KC 7 that copurifies with the type 1 RyR (RyR1),8 and FKBP12.6 (found in cardiac muscle9 ) are members of the immunophilin family of cis-trans peptidyl-prolyl isomerases that serve as cytosolic receptors for the immunosuppressant drugs rapamycin and FK506.10 11 In the absence of FKBP12/12.6, a homotetrameric RyR1 or RyR2 channel can be examined in planar lipid bilayers but reveals subconductance states, consistent with a defect in coordinated activity of the four subunits that form the RyR channel.12 13 The addition of FKBP12 to recombinant RyR1 stabilizes the channel complex, resulting in channels with full conductance.12 These stabilizing effects are reversed by treating the channels with rapamycin or FK506 to remove FKBP12/12.6 from the RyR channels.12 13 From these findings, we hypothesize that the FKBPs serve to stabilize interactions among RyR subunits.12
Skeletal muscle Ca2+-release channels (RyR1s) exhibit a phenomenon termed "coupled gating."7 Coupled gating enables RyR1 Ca2+-release channels in a given SR/T-tubule junction to open and close in a coordinated manner. The spatial organization of RyR1s on the SR junctional membrane into clustered arrays of channels is such that each channel physically contacts four of its neighbors.14 Clustering of channels is critical if coupled gating is to be physiologically important. In the heart, the spatial organizations of RyR2s and the sarcolemmal Ca2+ channels required to activate CICR have no clear relation to one another, as opposed to skeletal muscle, in which every other RyR1 is closely apposed to four Ca2+ channels in the T tubule.15 However, the RyR2s are clustered into arrays on the junctional SR such that each channel physically contacts four of its neighbors.16 17 The assembly of multiple RyR2s into functional Ca2+-release units mediated by coupled gating in cardiomyocytes provides a means of regulating the Ca2+ signal required for E-C coupling in the heart.
Materials and Methods
Isolation of RyR2s From Cardiac SR
Canine cardiac muscle heavy SR was isolated as
described,13 incubated with
[3H]ryanodine, solubilized with CHAPS, and
centrifuged on a 10% to 32% linear sucrose gradient at
26 000 rpm and 2°C in a Sorvall AH-629
rotor.7 12 In some
experiments, cardiac SR was preincubated with rapamycin (2 µmol/L) to
remove FKBP12.6 from RyR2s.
Immunoblots
Immunoblots were performed as
described18 by use of the
following: anti-FKBP12 (1:1000, recognizes both FKBP12 and FKBP12.6)and
anti-RyR (5029,
1:3000).19 After they were
washed, the membranes were incubated with peroxidase-conjugated goat
anti-rabbit IgG antiserum (1:3000, Boehringer-Mannheim) for 60
minutes at room temperature, washed 3 times with Tris-buffered saline
and 0.1% Tween 20, and developed by use of enhanced chemiluminescence
(ECL, Amersham).
Single-Channel Recordings
Cardiac SR microsomes or purified RyR2s (dialyzed
into lipid liposomes) were fused to planar lipid bilayers as
described.7 12
Single-channel experiments were conducted under voltage-clamp
conditions. Cardiac SR vesicles or liposomes were added to the
cis chamber near the planar
lipid bilayer composed of 3:1 phosphatidyl ethanolamine/phosphatidyl
serine (Avanti Polar Lipids). The bilayer cup was polystyrene with a
0.15-mm aperture. Fusion of vesicles/liposomes was promoted by KCl
added to the cis chamber
(
500 mmol/L). After incorporation of a
Ca2+ channel, the KCl gradient was
eliminated by perfusion of cis
chamber with cis solution (10
mL). Solutions were as follows:
trans, 250 mmol/L HEPES,
53 mmol/L Ca(OH)2 or
Ba(OH)2, and 50 mmol/L KCl, pH 7.35;
cis, 250 mmol/L HEPES,
125 mmol/L Tris, 1 mmol/L EGTA, 0.5 mmol/L
CaCl2, and 50 mmol/L KCl, pH 7.35. Free
Ca2+ concentration
(cis) was calculated by using
CHELATOR software.20 The
trans chamber was connected to
the head-stage input of an Axopatch 200 amplifier (Axon Instruments)
with Ag/AgCl electrodes and an agar/KCl bridge. The
cis chamber was held at ground
with a similar electrode. Single-channel currents were filtered at 1
kHz with an 8-pole Bessel filter (Warner Instruments) and digitized at
4 kHz. Data were collected on a Pentium computer with the use of
AxoScope1 (Axon Instruments) and a Digidata 1200 (Axon Instruments)
interface. pClamp 6.0.1 (Axon Instruments) was used for
analyses. Open probability (Po) was
identified by using >2 minutes of continual recording. Time
constants and proportions (area under the curve for each exponential
distribution) were calculated by using SigmaPlot 4.00 (time constant
exponentials were fitted by using the Levenberg-Marquardt least-squares
method). Open-time distributions were generated by pooling data for
individual channels (n=29), coupled channels with
Ca2+ as the charge carrier (n=16), and
coupled channels with Ba2+ as the charge
carrier (n=2). Po for individual channels in
experiments in which there was more than one channel in the membrane
(eg, one set of coupled channels and one or more single channels) was
analyzed by manually identifying each opening and by using
pClamp to determine its duration. Current-amplitude histograms are
all-points histograms. The numbers of channel openings
analyzed for each histogram are as follows: Figure 2B
, 76;
Figure 2D, 84; Figure 2F, 24; Figure 5B, 223; Figure 5G, 216; Figure 6B, 47; Figure 6G, 133; and Figure 6I, 35). Results are mean±SD.
Significance of differences were analyzed by the Student
t test and are regarded as
statistically significant at
P<0.05.
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Results
RyR2 channels were isolated from myocardium
by sucrose density gradient centrifugation. The 30S
complex represents single tetrameric RyR2 channels, and the
60S complex represents two physically associated RyR2
channels
(Figure 1A). Immunoblot analyses showed
that RyR2 protein was in both the 30S and 60S complexes as well as in
denser complexes, consistent with multiple channels (
2) being
physically associated
(Figure 1B, top). Pretreatment of SR microsomes with
rapamycin (2 µmol/L) caused dissociation of FKBP12.6 from RyR2s
(Figure 1B, bottom) but did not alter sedimentation of RyR2
complexes
(Figure 1B, second panel; densitometric analyses of
the immunoblots showed no significant differences in the
amount of immunoreactive RyR2 protein in each fraction of the sucrose
gradient for control versus rapamycin-treated samples). In the
rapamycin-treated samples, most of the FKBP12.6 was in upper fractions
of the gradient (
14%
sucrose).
In the samples not treated with rapamycin, most of the FKBP12.6 was in
fractions containing RyR2s (18% sucrose,
Figure 1B). Removal of FKBP12.6 by rapamycin is such that we
cannot detect any FKBP12.6 cosedimenting with RyR2s, nor is there any
FKBP12.6 detectable in RyR2 immunoprecipitates (data not shown). As
noted above, there is no significant reduction in RyR2 protein in the
FKBP12.6-stripped RyR2 samples compared with control nonstripped RyR2s
on the basis of immunoblotting and
[3H]ryanodine binding
(Figures 1A and 1B). Thus, removal of FKBP12.6 does not
influence the physically associated coupling of RyR2
channels.
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Cardiac microsomes containing RyR2 channels, as well as RyR2
channels from the 30S complex isolated by sucrose density gradient
centrifugation, were incorporated into planar lipid
bilayers and revealed stable openings of 4 pA (eg,
Figure 2A
). A current-amplitude histogram
(Figure 2B) revealed two discrete populations of events,
closed channels (0 pA) and openings to the full amplitude of a single
channel (4 pA). Channels similar to these have been well described
in the past and have been shown to be due to the
Ca2+-activated RyR2s from the heart.
However, some channels from cardiac microsomes as well as RyR2 channels
from the 60S complex opened to 8 pA, twice the normal current
amplitude and conductance (eg,
Figure 2C), a finding consistent with the
simultaneous opening of two RyR2s. Double-amplitude
"coupled" channels opening to 8 pA were observed in 19 of 205
consecutive experiments (9.3%) using canine cardiac microsomes, and
triple-amplitude coupled channels opening to 12 pA were observed in
3 of 205 consecutive experiments (1.5%). A current-amplitude histogram
(Figure 2D) revealed closed channels (0 pA) and openings to
8 pA. The RyR2 has a conductance for Ca2+
of 100 pS.4 The
conductances were 100±12 pS for the single-amplitude openings compared
with 200±17 pS for the double-amplitude openings. Double-amplitude
coupled channels opening to 8 pA were also observed by using
Ba2+ (n=2) as the charge carrier, indicating
that Ca 2+ fluxing through the channel is
not required for coupled gating.
Ultrastructural studies suggest that RyR2s in
cardiomyocytes are present in dense arrays on the SR,
with the four corners of each channel appearing to contact corners of
each of four neighboring
channels.17 We estimate that
10% of these RyR2 channels remain physically connected in native
cardiac SR preparations. This is based, in part, on electron
micrographs of isolated cardiac SR showing that 10% of the channels
remain physically contacting one another after isolation of the
vesicles with the use of methods similar to the ones used in the
present study.21 With
use of slightly modified conditions for cardiac SR vesicle fusion to
the bilayer (see Materials and Methods), coupled RyR2s were observed in
11% of our experiments (n=22 of 205).
RyR2s are organized in large arrays (up to 300); thus, we
expect to find interacting RyR2 homotetrameric units with a
multiplicity >2. This is consistent with the presence of RyR2
complexes sedimenting below the 30S complex on the sucrose gradient
(Figure 1B, top two panels) and our finding (in planar lipid
bilayer experiments) of channels that open and close with current
amplitudes equivalent to three RyR2 homotetrameric channels (n=3,
Figures 2E and 2F). The probability of obtaining still larger
arrays of interacting and functional RyR2 channels is reduced as the
number increases because of the likely instability of these arrays when
purified biochemically.
Ryanodine (5 µmol/L) induced the characteristic 50%
subconductance state when it was added to coupled channels
(Figure 3A), resulting in a current amplitude of 4 pA
(equivalent to two coupled channels, each contributing 50% of
the normal single-channel current amplitude of 4 pA). The effect of
ryanodine further supports the concept that the 8-pA openings
represent two coupled RyR2 channels. In this experiment
(Figure 3A), ryanodine partially blocks the pore of one
channel (resulting in a reduction from 8 pA to 6 pA,
representing coupled gating of one half-conducting channel
and one fully open channel). Subsequently, ryanodine blocks the pore of
the second channel, resulting in a final current amplitude of 4 pA,
representing two half-conducting channels,
consistent with the known effect of ryanodine on the RyR2
channel. Ruthenium red (20 µmol/L), an RyR2 inhibitor,
blocked coupled RyR2 channels
(Figure 3B). In this experiment, two coupled RyR2 channels
were first activated with caffeine (5 mmol/L) and then
blocked with 20 µmol/L ruthenium red
(Figure 3B). In contrast to ryanodine, which sequentially
modified each of the two coupled RyR2 channels
(Figure 3A), ruthenium red simultaneously blocked
both coupled channels
(Figure 3B). Coupled RyR2 channels also responded to other
known modulators of RyR2 channel function in the same manner as single
RyR2 channels. For example, coupled RyR2 channels were
activated by 5 mmol/L caffeine
(Figures 4A, middle tracing, and 4B), and 1 mmol/L
MgCl2 decreased the Po
and current amplitude
(Figure 4A, bottom tracing). Taken together, these data show
that coupled RyR2 channels respond to modulators in the same manner as
single RyR2 channels.
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One possible explanation for coupled gating between RyR2s is
that Ca2+ fluxing through one channel
activates a neighboring channel via CICR. To exclude this
possibility, we also recorded coupled RyR2 channels by using
Ba2+ as the charge carrier (n=2; eg, see
Figures 5A and 5B). We also observed coupled gating between
RyR2 channels with K+ used as the charge
carrier (n=3; with use of 250 mmol/L KCl
trans/50 mmol/L KCl
cis, data not shown).
Dissociation of FKBP12.6 from RyR2s with rapamycin (2 µmol/L) first
caused the 8-pA openings to be halved to 4 pA (two uncoupled
channels) and then induced subconductance states in each of the
uncoupled channels, as shown in
(Figures 5A through 5H). Twenty-five minutes after addition of
rapamycin (2 µmol/L), the two coupled channels first uncoupled
(Figure 5D); 27 minutes after adding rapamycin, the two RyR2
channels were clearly uncoupled
(Figures 5F and 5G); 32 minutes after the addition of
rapamycin, the uncoupled RyR2 channels exhibited subconductance states
(Figure 5H). In this experiment
(Figure 5E), the Po of the coupled
channels decreases to zero after the complete uncoupling that is due to
the removal of FKBP12.6 is achieved. We conclude that removal of
FKBP12.6 causes RyR2s to gate independently despite the fact that they
remain physically associated (see
Figure 1B). Interestingly, in some experiments, the open
states (but not the closed states or baseline) of the coupled channels
were noisier than those of single channels. This was particularly
evident during the removal of FKBP12.6 (ie, during the transition from
coupled to uncoupled channels, as shown in
Figures 5C, 5D, and 6C), consistent with the concept
that FKBP12 and FKBP12.6 "stabilize" RyR1s and RyR2s,
respectively.12 Longer
treatment with rapamycin (2 µmol/L) leads to the appearance of
subconductance states of the individual uncoupled RyR2 homotetrameric
channels
(Figure 5H), similar to those previously reported when single
RyR2 channels are treated with
rapamycin.13 Thus, we
conclude that FKBP12.6 facilitates the coordination of single RyR2
subunits with the other members of the homotetramer and also permits
homotetrameric RyR2s to exhibit coupled gating with each
other.
An important question is whether dissociation of FKBP12.6
from RyR2s results in functional uncoupling of the channels followed by
the induction of subconductance states in each channel versus the
induction of subconductance states in each individual channel without
functionally uncoupling them. In either case, the result would be the
appearance of subconductance states, as shown in
Figure 5H. Examination of the transition from two coupled
channels to two uncoupled channels after dissociation of FKBP12.6 by
rapamycin (2 µmol/L) shows that the two channels first uncouple
(25 minutes after the addition of rapamycin) and gate independently
and that they subsequently (>27 minutes after rapamycin addition)
develop subconductance states
(Figures 5A through 5H). Because each RyR2 channel binds 4
FKBP12.6 (one per subunit), one possible explanation for this sequence
of events is that dissociation of one or two FKBP12.6 molecules is
sufficient to uncouple the channels and that subconductance states
appear after dissociation of the remaining FKBP12.6.
The role of FKBP12.6 in coupled gating was further supported
by experiments showing that two physically coupled channels that are
functionally uncoupled because of the removal of FKBP12.6 with
rapamycin become functionally coupled with the readdition of FKBP12.6
(Figure 6). In these experiments, two coupled channels were
uncoupled by the addition of rapamycin
(Figures 6A through 6G). Rapamycin was washed out, and then 30
minutes after addition of FKBP12.6, coupled gating of the two channels
was restored
(Figures 6H through 6I).
Measured under identical conditions
(cis
Ca2+=150 nmol/L), the
Po of coupled channels was significantly
increased compared with uncoupled channels (two coupled channels
[Po=0.43±0.37, n=16] versus single channels
[Po=0.03±0.03, n=8],
P<0.01;
Figures 7A and 7B). In some experiments (n=9), two coupled
channels and an uncoupled channel were active in the same bilayer
membrane
(Figures 7A and 7B). These experiments show the increased
Po of two coupled channels compared with single
channels under the same conditions, ie, when the
cis and
trans solutions are the same
for both the coupled and uncoupled channels (eg,
cis
Ca2+=150 nmol/L). The distributions of open
times for individual versus coupled channels are shown in
Figure 7C. Fitting with three exponentials yielded mean open
times of 
1=0.48±0.06 ms,
2=1.88±0.24 ms, and
3=13.60±0.91 ms for the individual channels
and 1=75.8±2.8 ms,
2=285.7±32.7 ms, and
3=2500±625 ms for the coupled channels. For
each of these mean open times, there is a statistically significant
difference between individual and coupled channels
(P<0.01). The open-time
distributions for coupled channels recorded with
Ba2+ used as the charge carrier were fit
with two exponentials, yielding mean open times of
1=86.06±34.81 ms and
2=176.90±50.07 ms. As with the coupled
channels recorded with Ca2+ used as the
charge carrier, the mean open times for coupled channels recorded
with Ba2+ used as the charge carrier were
longer than those observed for individual RyR2 channels with
Ba2+ used as the charge carrier.
Interestingly, the longest time constant observed for coupled RyR2
channels with Ca2+ used as the charge
carrier (3=2500 ms,
Figure 7C) was not observed for coupled channels with
Ba2+ used as the charge carrier. These
longest openings of coupled channels may reflect the effect of CICR in
which Ca2+ fluxing through one open channel
could help keep the second channel open. In 8 of 9 such experiments
(when two coupled RyR2s and a single RyR2 channel were recorded in
the same bilayer), the coupled RyR2 channels exhibited a higher
Po than the single RyR2 channels
(Figure 7B). This may be due to the fact that two coupled
RyR2 channels have twice as many
Ca2+-activating sites as single RyR2
channels. Compared with single RyR2 channels, coupled channels also
exhibited increased caffeine sensitivity (data not shown). This finding
further supports the concept that two coupled RyR2 channels have twice
as many Ca2+-activating sites as a single
RyR2 channel, inasmuch as caffeine is known to activate RyR2s
by increasing the Ca2+ sensitivity of the
channel. Moreover, the ability to record coupled and single RyR2
channels in the same bilayer excludes the possibility that
recording of double-amplitude RyR2 channels could be due to
artifacts or other technical explanations (eg, drift in amplifier
gain).
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) RyR2 channels from 9 experiments in which both coupled and single channels were observed in the same bilayer membrane. C, Open-time distributions for individual vs coupled RyR2 channels. Curves were fit with three exponentials to derive Discussion
Could observations that suggest functionally important
coupled gating of RyR2s in the heart be due to some other channel? This
is unlikely for several reasons: (1) The pharmacological profile of
coupled channels is the same as that of individual RyR2s. The
characteristic 50% conductance state was observed in response to
appropriate concentrations of ryanodine but scaled by the number of
coupled RyR2s
(Figure 3A). Additionally, caffeine activates coupled
RyR2 channels, MgCl2 inhibits them, and
ruthenium red blocks the coupled channels just as they block the single
RyR2 channels
(Figure 4). (2) Uncoupling of the coupled channels leads to
RyR2 channels with half the current of two coupled RyR2 channels (eg,
Figure 5). (3) The conversion of coupled RyR2s into
uncoupled RyR2s depends on the presence or absence of a specific
protein, FKBP12.6
(Figure 5), and the addition of FKBP12.6 can restore coupled
gating between channels
(Figure 6). (4) When highly purified RyR2 protein containing
FKBP12.6 is fused to planar lipid bilayers, we observe coupled
channels.
The present study of coupled gating between cardiac RyR2 channels supports the following conclusions: (1) Cardiac RyR2s can exhibit coupled gating. (2) Physical coupling between RyR2s does not require FKBP12.6, whereas functional coupling between RyR2s does require FKBP12.6. (3) Removal of FKBP12.6 from RyR2s with rapamycin first uncouples two coupled channels, and then each individual channel exhibits subconductance states. (4) Two coupled RyR2 channels exhibit an increased Po compared with a single channel.
Taken together, the data of the present study show that
two or more physically connected RyR2 channels can gate
simultaneously, a phenomenon termed coupled
gating.7 Coupled gating
between individual RyR2s, which requires FKBP12.6, provides a mechanism
for the coordinated activation and inactivation of
RyR2/Ca2+-release channels during cardiac
muscle E-C coupling. Coupled gating may explain how the coordinated
termination of cardiac SR Ca2+ release
occurs
(Figure 8). Coupled gating provides a mechanism for
simultaneously closing all RyR2s in a T-tubule/SR junction,
thereby reducing the probability that individual RyR2 channels will be
reactivated stochastically by Ca2+
fluxing through their neighbors
(Figure 8).
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It has been proposed that the
Ca2+ efflux via the RyR2s leads to an
elevated [Ca2+]i
that produces RyR2
inactivation.22 However,
experiments suggest that inactivation processes dependent on cytosolic
[Ca2+]i are too
slow to account for the termination of Ca2+
release.23 24 25
A second mechanism proposed for closing RyR2s during cardiac E-C
coupling relies on the stochastic closing of RyR2s. However, modeling
of the stochastic closing of RyR2s suggests that this mechanism may
also be too slow.26 A third
possibility is that a reduction in SR Ca2+
(that is due to Ca2+ release) signals the
RyR2s to close because the RyR2 Po depends (in
part) on SR luminal
Ca2+.5
SR Ca2+ depletion combined with coupled
gating could account for RyR2 closure and termination of
Ca2+ release. Under this proposed model
(Figure 8), SR Ca2+ depletion
reduces the Po of RyR2s in a T-tubule/SR
junction. When the first channel closes because of decreased
Po that is signaled by falling SR
Ca2+ levels, all RyR2s in the junction close
because of coupled gating.
Treatment of cardiomyocytes with FK506, which can dissociate FKBP12.6 from RyR2s, alters Ca2+ sparks.27 28 FK506 prolonged Ca2+ sparks,28 a finding that is consistent with a model in which uncoupling RyR2 channels causes a defect in RyR2 closure. However, another study found that FK506 did not significantly change spark duration, although spark frequency was increased.27 The fact that Ca2+ sparks are observed despite dissociation of FKBP12.6 from RyR2s indicates that coupled gating is not required for the generation of sparks. There is clear evidence that in vivo FKBP12.6 is physically associated with RyR2s.9 18 Defects in cardiomyocyte Ca2+ signaling were recently reported in an FKBP12.6 knockout mouse model.29 These defects included an increase in the amplitude and duration of Ca2+ sparks in cardiomyocytes from FKBP12.6-deficient mice.29 Thus, although the physiological impact of coupled gating has not been proven, currently available data involving FKBP12.6 knockout mice support the concept that it plays a role in modulating E-C coupling in cardiac muscle. The present findings may have important implications for understanding the mechanisms underlying human disease states, such as heart failure30 and sudden cardiac death. For example, we have recently shown that protein kinase A hyperphosphorylation of RyR2s occurs in failing hearts.30 Protein kinase A hyperphosphorylation dissociates FKBP12.6 from the RyR2 macromolecular complex.30 These data suggest that coupled gating of RyR2s would be reduced in failing hearts because of the dissociation of FKBP12.6. Reduced coupled gating between RyR2s might result in defective closure of RyR2 channels, which could deplete SR Ca2+, reduce E-C coupling gain, and/or contribute to diastolic depolarizations that can initiate fatal cardiac arrhythmias. However, these speculations as to the in vivo consequences of the inhibition of coupled gating will have to be tested with the use of physiological animal models.>
Acknowledgments
This study was supported by the National Institutes of Health (Dr Marks), the American Heart Association (Drs Marks and Marx), and Vedecka Grantova Agentura 7019 (Dr Ondrias). Dr Marks is a Doris Duke Distinguished Clinical Scientist. We thank R. Kass and J. Lederer for helpful discussions.
Footnotes
Original received January 10, 2001; revision received April 10, 2001; accepted April 10, 2001.
1 These authors contributed equally to this study. 
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