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INTRODUCTION |
In cardiac as well as skeletal muscles, the
SR1 plays a central role in
excitation and contraction coupling (1). Important molecular components
for excitation-contraction coupling include the ryanodine receptor
(RyR), Ca2+ ATPase, ion channels including K+
channels (2), and several kinds of Cl
channels (3-8). SR
Cl channels have been reported to have conductances of 55 pS (cardiac
SR in 260 mM Cl
) (4), 200 pS (skeletal SR in
100 mM Cl
) and 130 pS (cardiac SR in 250 mM Cl
) (8). This latter channel is also
voltage-sensitive. We have characterized a protein kinase A-activated
Cl
channel in the cardiac SR with a conductance of 116 pS
(500 mM Cl
in the cis, 50 mM Cl
in the trans solution) (5,
6). Channel opening is regulated by PKA-dependent
phosphorylation and by Ca2+-calmodulin, but insensitive to
voltage or the levels of cytosolic Ca2+. It has been
suggested that Cl
channels may function to maintain
charge neutrality across the SR membrane generated by Ca2+
movement during excitation-contraction coupling, but their functional characteristics are not well elucidated.
Recently, it has been suggested that certain types of anion channels in
the plasma membrane conduct adenine nucleotides such as ATP (9-11),
but this suggestion has been contested (12-14). It has also been
suggested that the mitochondrial voltage-dependent anion
channel (VDAC) (15) may conduct adenine nucleotides. It is not known,
however, whether the 116-pS Cl
channel in cardiac SR
conducts ATP or not. In the present study, we examined the anion
permeability and whether ATP might permeate through this
Cl
channel or not. We found that this channel could
conduct adenine nucleotides, ATP, ADP, and AMP. A preliminary report of
this work was presented in abstract form (16).
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EXPERIMENTAL PROCEDURES |
Preparation--
Porcine cardiac heavy SR was isolated by
discontinuous sucrose gradient centrifugation as reported previously
(5, 6).
Solutions and Drugs--
The cis bath solution
contained (mM) CsCl, 500; EGTA, 1; HEPES, 10; MgATP, 2; and
CaCl2, 5. The free Ca2+ concentration was 1 µM calculated with the method proposed by Fabiato (18).
The trans solution contained (mM) CsCl, 50;
EGTA, 1; HEPES, 10; and 1 µM free Ca2+. The
pH of these solutions was adjusted to 7.3 by adding CsOH. In the
experiments to determine anion permeability, CsCl was replaced by 500 mM CsBr, CsF, CsNO3, or CsI. The 200 mM ATP solutions were made by replacement of 500 mM CsCl with 198 mM Tris ATP,
Na2ATP, K2ATP, or Cs2ATP and 2 mM MgATP. In some experiments, 500 mM CsCl was
replaced with 200 mM MgATP. The 100 mM ATP
solutions were made by replacing 100 mM CsCl with 98 mM Tris ATP or Na2ATP and 2 mM
MgATP. 10 µM ryanodine (Wako Chemical Co., Osaka, Japan) was added to the cis solution to block the RyR channels. A
protein kinase inhibitor (PKI) specific to PKA (Sigma P3294) was
purchased from Sigma Chemical Co. Enzymes were dialyzed against the
cis solution at 0 °C for 1 h before use. DIDS
(Sigma) was dissolved in dimethyl sulfoxide (Me2SO).
Concentrations of Me2SO in final solutions were less than
0.1%.
Electrophysiological Methods and Data Analysis--
The planar
bilayer was composed of brain phosphatidylethanolamine and brain
phosphatidylserine (Avanti Polar Lipids, Alabaster, AL) at a ratio of
1:1, dissolved in decane (20 mg/ml). Purified cardiac heavy SR vesicles
were added to the cis chamber and fused into the lipid
bilayer formed in the hole (0.25 mm in diameter) in a Lexan
polycarbonate partition. In the present experiments, the cis
chamber was defined as the side to which SR vesicles were added, and
the opposite side was referred to as the trans chamber. The
cis side was equivalent to the cytoplasmic side of the
incorporated channel, and the trans side was equivalent to
the lumen of the SR as determined previously (5, 6). Currents flowing
through the ion channels were measured by using the voltage-clamp
technique. Applied voltages were defined with respect to the
trans chamber held at ground. Channel activities were
recorded at room temperature (22 ± 1 °C), amplified by a
patch-clamp amplifier (Axopatch 1C, Axon Instruments, Inc, Foster City,
CA), and stored on a videocassette tape recorder through a PCM
converter system (RP-880, NF Instruments, Yokohama, Japan) digitized at
10 kHz. Data were reproduced and low pass filtered at 2,000 or 1,000 Hz
by a filter with Bessel characteristics (octave attenuation, 48 dB) and
analyzed off-line on a computer (P5-200, Gateway 2000). For
single-channel analysis, the threshold used to judge to open state was
set at a half-amplitude of the single-channel currents (19).
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RESULTS |
Permeation of Various Anions--
We have reported that a 116 pS
Cl
channel in cardiac SR is activated via
PKA-dependent phosphorylation or in the presence of MgATP
(5, 6). Fig. 1, panel A,
a, shows a continuous recording of Cl
channel
openings with 500 mM CsCl in the cis and 50 mM CsCl in the trans chamber solutions. Because
SR membrane contained not only Cl
channels, but also
ryanodine receptor Ca2+ release channels (RyRs) and
K+ channels (2, 7), these channels were blocked by
replacement of K+ with Cs+ and the application
of 10 µM ryanodine to the cis solution. The current amplitude of the channel remaining after this treatment was
15 pA at
40 mV. In the absence of MgATP, channel activity ran down
spontaneously within several minutes after incorporation into the lipid
bilayer. In the presence of 2 mM MgATP, however, channel
openings were maintained until the experiments were interrupted by a
break of bilayer (Fig. 1, panel A, b). The
open-time and closed-time histograms could be fitted by the sum of two
exponentials (Fig. 5, panel A). The time
constants for the open-time histogram were 1.5 ± 0.2 (mean ± S.D.) ms (n = 6) and 43 ± 4 ms
(n = 6). The time constants for the closed-time
histograms were 0.7 ± 0.1 ms (n = 6) and 5.0 ± 0.5 ms (n = 5). Thus, the kinetic properties and
channel conductance were identical to those of the 116 pS Cl
channel reported previously (5, 6). These channel
activities could be restored in the presence of 2 mM or
higher MgATP or by adding of PKA with 0.05 mM MgATP.
Therefore, we performed the following experiments with the
cis solution containing 2 mM MgATP.

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Fig. 1.
Single-channel activity of Cl
channel. Panel A, tracings showing the
single-channel activity of the SR Cl channels
incorporated into planar lipid bilayers. The continuous recording of
Cl channel activity is shown in a. The
cis solution contains 500 mM Cl
and the trans solution contains 50 mM
Cl . Voltage was held at 40 mV. Channel activity ran
down soon after being incorporated into the planar lipid bilayer in the
absence of MgATP in the cis solution. Shown in b,
in the presence of 2 mM MgATP in the cis
solution, the Cl channel openings were sustained until
the break of bilayer. o, open channel; c, closed
channel. Downward deflection indicates current flow from the
trans to cis side. Panel B, current
traces with various anions through Cl channel.
Cl ions in both cis and trans
solutions were replaced with various anions: Br ,
I , NO3 , and F ,
respectively. The concentration of each anion is 500 mM in
the cis solution and 50 mM in the
trans solution. In all experiments, the cis
solutions contained 2 mM MgATP. Holding potentials are
indicated on the left side of each tracing. Panel
C, the plot of unitary current amplitude (mean ± S.D.) with
five different anions as a function of voltages. All currents reached 0 current level at around +60 mV. The slope conductance of 500 mM Cl (n = 18) and
Br (n = 5) is about 116 pS and those of
500 mM NO3 (n = 6),
I (n = 6), and F
(n = 6) are about 40 pS.
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To test the anion selectivity, current-voltage relationships were
obtained with various anions in both the cis and
trans solutions. When Cl
was replaced with
equimolar Br
, the unit amplitude of the current was not
changed significantly (Fig. 1, panel B). The
slope conductance with Br
was about 116 pS, which was
identical to that with Cl
. When Cl
was
replaced with equimolar I
, NO3
, or
F
, the unit amplitudes were decreased (Fig. 1,
panel C). The slope conductances were 40 pS under
these conditions. The reversal potential (Erev) was
approximately equal to the calculated equilibrium potential, which was
+58 mV (Fig. 1, panel C). From these results, we
concluded that this SR-Cl
channel was highly permeable to
these anions.
To determine the anion permeability ratio to Cl
,
Erev was obtained under bi-ionic conditions with 500 mM anion in the cis solution and 50 mM Cl
in the trans solution (Fig.
2). The permeability ratios were calculated from the Goldman-Hodgkin-Katz equation.
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(Eq. 1)
|
where [X]cis and [X]trans are the
cis and trans concentrations of anion,
respectively. The values of Erev and the
permeability ratios were summarized in Table
I. The order of anion permeability
(Px) through the Cl
channel was
determined to be Br
> Cl
> I
> NO3
> F
.

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Fig. 2.
Anion selectivity. Current traces
were recorded with 500 mM various anions (Br ,
I , NO3 , or F ) in the
cis solution and 50 mM Cl in the
trans solution. Holding potentials are indicated on the
left side of each trace.
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ATP Conduction through the Cl
Channel--
We
examined whether adenine nucleotides permeated this Cl
channel. After incorporation of Cl
channel into the lipid
bilayers, 500 mM Cl
in the cis
solutions was replaced with 200 mM ATP (198 mM
Tris ATP and 2 mM MgATP). Fig.
3 shows a continuous recording of channel openings before and after replacement of the cis solution,
where the membrane potential was held at
80 mV. After replacement of 500 mM Cl
with 200 mM ATP, the
current amplitudes decreased (Fig. 3, panel A).
We have often observed a subconductance level of the
SR-Cl
channel in the Cl
solution and
similar subconductance levels were recognized in the ATP solution as
shown in Fig. 3, panel B.

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Fig. 3.
ATP conduction through Cl
channel in cardiac SR. Panel A, continuous recordings
of Cl channel openings before and after replacement of
the cis solution with 200 mM ATP (198 mM Tris ATP and 2 mM MgATP) from 500 mM CsCl. This membrane contained 3 channels. C, closed
levels of the channels; O-1, O-2, O-3, open levels. Voltage
was held at 80 mV. The conductance of these channels became small
after changing the cis solution to 200 mM ATP.
The expanded time scale of records from the points indicated in the
upper trace are shown in a and b. Panel
B, subconductance of ATP channel. Voltage was held at 120
mV. The continuous recording of channel activities is shown.
Lower panels a and b: expanded time scale of
recordings of the upper panel A. subconductance level of
open channel is indicated by s. Amplitude histogram is shown
on the right. All amplitude histograms were fitted with
Gaussian fit.
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To confirm the ATP conduction, current-voltage relationships were
obtained at two different ATP concentrations in the cis solution and 50 mM Cl
in the trans
solutions (Fig. 4). With 200 mM ATP, the slope conductance of the inward current was 83 pS (n = 15), and Erev was +62.6 mV.
With 100 mM ATP, the slope conductance decreased to 68 pS
(n = 9) and Erev shifted to +40.5 mV
(Fig. 4, panel B). Under these experimental
conditions, the only anion present in the cis solution was
ATP. Therefore, inward currents could only be carried by ATP at
negative potentials. It is difficult to estimate the theoretical
Erev for the ATP current, because we do not know the
actual free and complexed ATP concentrations under our experimental
conditions. If ATP was assumed to exist as a divalent anion,
PATP/PCl was 0.54 at 200 mM ATP and
was 0.5 at 100 mM ATP calculated by the following equation
(20),
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(Eq. 2)
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The value of Erev at each condition was almost
identical to the theoretical value. From these results, we concluded that the currents were mostly carried by ATP2
.

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Fig. 4.
ATP currents at different concentrations of
ATP. Panel A shows single-channel activity at different
holding potentials indicated on the left of each tracing in
200 and 100 mM ATP in the cis solution.
Panel B, the plot of unitary current amplitude (mean ± S.E.) with different ATP concentrations in the cis solution
as a function of holding potential. Open circles ( ) indicate current
with 100 mM ATP (n = 9 experiments) and
closed circles ( ) indicate current with 200 mM ATP
(n = 15 experiments).
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The Kinetics of ATP Currents--
As reported previously (5, 6),
the channel open-time and closed-time histograms could be fitted with
two exponentials when currents were carried by Cl
(Fig.
5, panel A). By
analyzing the currents carried by ATP, the open-time histograms could
be fitted by a single exponential with time constants of 26 ± 11 ms at
60 mV and 23 ± 10 ms at
80 mV (n = 4).
The fast component of the open-time histogram observed when chloride
was charge carrier seemed to disappear when ATP was the charge carrier.
The closed-time histogram analyzed with a single-active channel could
be fitted by a single exponential with time constants of 0.4 ± 0.4 ms at
60 mV and
80 mV (n = 4). The fast
component was not different from those in the Cl
currents. Thus, the slow component of the closed-time histogram with
chloride as charge carrier disappeared with ATP as charge carrier (Fig.
5, panel B). In this analysis we chose the data in which only one channel existed in the bilayer and no subconductance levels were observed.

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Fig. 5.
Kinetics of Cl channel activity
with Cl and ATP. Panel A, open-time
and closed-time histograms with 500 mM Cl in
the cis solution are shown at 60 mV holding potential. The
open-time histogram was fitted by double exponential curves with time
constants of the fast (T1) and the slow
component (T2) of 1.5 and 43 ms, respectively.
The closed-time histogram was fitted by double-exponentials with
T1 and T2 of 0.6 and 5.0 ms, respectively. Panel B, when all Cl ions
were replaced with 100 mM ATP, open-time and closed-time
histograms at 60 mV and 80 mV are shown. At 60 mV both open-time
and closed-time histograms are fitted by single exponential with time
constants of 28 and 0.4 ms, respectively. At 80 mV, both histograms
were fitted by a single exponential with time constant of 21 and 0.4 ms, respectively. All cis bath solutions contained 2 mM Mg-ATP.
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ADP and AMP Conduction through the Cl
Channels--
We examined whether SR-Cl
channel might
conduct other adenine nucleotides besides ATP. ADP and AMP were tested
using the same approach as shown in Fig. 3, panel
A. When the cis solution was replaced with 100 mM Na2ADP or Na2AMP for 500 mM CsCl, the channel activities were maintained (Fig.
6). Thus,
the SR-Cl
channel also conducts these adenine
nucleotides. We compared the slope conductances and relative
permeabilities in bi-ionic conditions with 100 mM ATP, ADP,
or AMP in the cis and 50 mM Cl
in
the trans solution. The slope conductance was 68 pS in 100 mM ATP, 87 pS in ADP, or 115 pS in AMP. The reversal
potentials were +41 mV, +20.6 mV, and +3.0 mV, respectively (Fig. 7).
The relative permeabilities
(Px/PCl) are summarized in
Table II.

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Fig. 6.
ADP and AMP conduction through
Cl channel. Panel A, a
shows single-channel activities in 100 mM ADP in the
cis solution and 50 mM Cl in the
trans solution at 80 mV. Amplitude histogram with Gaussian
fit is shown in b. In c, open- and closed-time
histograms are shown. Both histograms were fitted by double exponential
curves with time constants of 4.5 and 49 ms in open time and 0.2 and
163 ms in closed time. Panel B, a shows
single-channel activities in 100 mM AMP in the
cis and 50 mM Cl in the
trans solutions at 100 mV. In b, the amplitude
histogram is shown. In both ADP and AMP current measurements, the
cis solution contained 2 mM MgATP.
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Fig. 7.
ADP and AMP currents, through
Cl channel. Panel A shows
single-channel activities in 100 mM AMP in the
cis solution (left) and 100 mM ADP
(right) at different holding potentials indicated at the
left of each tracing. The cis solution contained
2 mM MgATP. Panel B, the plot of unitary current
amplitude (mean ± S.D.) with 100 mM
Na2ATP (n = 9), 100 mM
Na2ADP (n = 8), and 100 mM
Na2AMP (n = 5) as a function of holding
potential.
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Modulation of the Currents by Protein Kinase
A-dependent Phosphorylation--
If these currents carried
by adenine nucleotides were activated via PKA-mediated phosphorylation
as reported previously (5, 6), currents should be blocked by removal of
MgATP from the cis solution or by the application of PKI.
After the ADP currents were activated in the presence of 2 mM MgATP, MgATP in the cis solution was removed.
The ADP current was quickly and completely blocked (Fig.
8A), and restored by the
reapplication of 2 mM MgATP (Fig. 8B). The
application of PKI completely blocked the channel openings (Fig.
8C). Therefore we conclude that the anion channel-conducting adenine nucleotides can be activated via PKA-dependent
phosphorylation.

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Fig. 8.
Properties of nucleotides channel.
Current traces with 100 mM ADP in the presence
(A) and absence (B) of 2 mM
MgATP. Membrane potential was held at 40 mV. In the absence of MgATP,
no channel activities were observed (A), although the
addition of 2 mM MgATP restored the channel activities
within 2 min (B). The further application of 10 µM PKI completely blocked ADP channel (C).
These traces were continuous records in the same experiment. The
trans solution contained 50 mM
Cl .
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DISCUSSION |
Anion Selectivity of Cl
channel in the cardiac
SR--
The order of anion selectivity of this Cl
channel is consistent with the predicted Eisenman's sequence III (21).
These results suggest that the electrodiffusion through this
Cl
channel may be controlled by a so-called "weak field
strength" selectivity site (21). The selectivity sequence of other
Cl
channels in cardiac or skeletal muscle SR are
different from this (3, 8, 22). The Ca2+- and
voltage-sensitive Cl
channel in cardiac SR displayed the
order of SCN
> I
> NO3
,
Br
> Cl
> F
> HCO3
, which is consistent with sequence 1 of Eisenman
(8). In skeletal muscle SR, the Cl
channels displayed the
sequence of NO3
> Br
> Cl
(1) or NO3
> SCN
> I
> Br
= Cl
(22). Therefore,
we speculate that SR-Cl
channel in this study may be a
different type of Cl
channel than others previously
described in SR. The cystic fibrosis transmembrane regulator (CFTR)
expressed in epithelial cells or cardiac sarcolemma has an anion
selectivity sequence similar to the 116 pS SR Cl channel (23, 24). In
addition, both CFTR and the 116 pS SR Cl
channel are
activated by PKA-dependent phosphorylation (Fig. 1) (5, 6),
and exhibit voltage-independent activation (Figs. 1 and 2; Table I)
(25). However, the conductance of the Cl
channel in
cardiac SR is much larger than that of CFTR (Fig. 1). Therefore, it
seems unlikely that the SR-Cl
channel is CFTR.
Permeation Pathway for Cl
and Adenine
Nucleotides--
The data in this report first demonstrate ATP
currents in cardiac SR by recording single-channel activities (Figs. 3
and 4). Our results suggest that ATP is permeable through the same
channel, which mediates Cl
permeation. First, the
continuous channel activity before and after replacement of
Cl
with ATP (Fig. 3, panel A) is
consistent with this interpretation. The alternative, that new channels
are incorporated into the bilayer as a consequence of ATP addition,
seems highly unlikely. Furthermore, we could always detect the channel
openings in the ATP solutions, whenever the activity of
Cl
channels incorporated into the bilayer was recorded
(15/15). On the other hand, ATP currents were never observed when
Cl
channels were not incorporated into the bilayer
(10/10). Second, the anion channel requires phosphorylation to conduct
either Cl
or ATP (Fig. 8). Third, the pharmacological
properties are similar for both currents. We have reported that the
SR-Cl
channel is insensitive to DIDS (5). Likewise, DPC
or DIDS did not block ATP or ADP currents (data not shown). Therefore, we conclude that the 116 pS Cl
channel in cardiac SR can
conduct both Cl
and adenine nucleotides.
Our data are in agreement with other reports indicating the existence
of ATP conduction pathway in intracellular membranes (26-31). It is
known that VDAC (mitochondrial porin) is responsible for most of the
metabolite flux across the mitochondrial outer membrane and also
provides a pathway for nucleotide transport (28-31). A working model
of the VDAC pore proposes a barrel with a diameter of 2.4-3.0
nM, and it has been reported that VDAC is sufficient to
mediate ATP flux through the mitochondrial membrane (32, 33). The
molecular structures of ATP channels in a variety of intracellular
membranes, including SR, have not been identified. Further studies are
required to clarify the structural basis for the ATP conduction through
the inner membrane channels.
In contrast to the inner membrane channels, there is no consensus
regarding the existence of any ATP conduction pathway in sarcolemmal
membranes. Although recent studies have suggested ATP conduction
through CFTR or the multidrug resistance channel (9-11), conflicting
results have also been presented (12-14). Furthermore, it is suggested
that the size of the ATP anion is much larger than the estimated size
of the CFTR pore (34). A different view of these controversial findings
have been presented by Pasyk and Foskett (26) and Sugita et
al. (34), showing the existence of the CFTR-associated ATP
channels in the plasma membrane by patch-clamp technique. Thus, an ATP
conducting pathway appears to exist in the plasma membrane, but the
identity of the responsible channels, whether they are the same or
different from CFTR, are not clear.
Selectivity of Adenine Nucleotides--
Based on the theoretical
value of Erev at different ATP concentrations, our
results were consistent with the assumption that ATP might move
predominantly as a divalent anion (Fig. 4). Observed
PATP/PCl ratios did not
fit with calculations based on ATP as a monovalent (calculated
PATP/PCl
ratio was 0.73 at 200 mM ATP and 1.01 at 100 mM
ATP), tetravalent (PATP/P Cl = 0.527 at 100 mM ATP and 1.168 at 200 mM ATP) or trivalent (PATP/PCl is 0.462 at 100 mM ATP and 0.71 at 200 mM ATP) anion. This
suggests that ATP permeation as ATP4
, ATP3
,
or ATP
is unlikely. Sugita et al. (35) showed
the ATP currents through CFTR-associated ATP channels with
PATP/PCl = 0.4. This
value is almost identical to our results (0.5). We have also
demonstrated the permeation of ADP and AMP through this
Cl
channel (Figs. 6 and 7) and tried to determine the
selectivity of adenine nucleotides (Tables I and II). It was quite
difficult, however, to estimate precisely free concentrations of these
anions in solution or those valences as charge carriers. If ADP and AMP were assumed to be present as a monovalent anion,
PADP/PCl was 0.71 and
PAMP/PCl was 0.527. The
order of apparent permeability became ADP > ATP > AMP. If
they were assumed to be divalent anions, PADP/PCl was 0.25, and
PAMP/PCl was 0.14. Then the order of apparent permeability became ATP > ADP > AMP.
Physiological Implication--
In the lumen of intracellular
organelles, many processes may require adenine nucleotides for the
functional source, and intraluminal ATP may be physiologically
regulated. ATP is needed for energy-requiring processes (36) and is
also the substrate for phosphorylation of intraluminal proteins in ER
(37-39). Luminal ATP is required for protein translocation in the ER
(40-42). Therefore, we speculate that the SR Cl
channel
may mediate the transport of ATP between lumen and cytosol, which may
be responsible for important regulatory functions in cardiac
excitation-contraction coupling.