(Received for publication, September 26, 1994)
From the
The cardiac calcium release channel (CRC) of sarcoplasmic
reticulum vesicles was incorporated into planar lipid membranes to
evaluate modulation of channel activity by phosphorylation and
dephosphorylation. For this purpose a microsyringe application directly
to the membrane was used to achieve sequential and multiple treatments
of channels with highly purified kinases and phosphatases. Cyclic
application of protein kinase A (PKA) or
Ca/calmodulin-dependent protein kinase II (CalPK) and
potato acid phosphatase or protein phosphatase 1 revealed a channel
block by Mg
(
mM), that is referable to
dephosphorylated states of the channel, and that the Mg
block could be removed by phosphorylation of the CRC by either
PKA or CalPK. By contrast, activation of endogenous CalPK (end CalPK)
led to channel closure which could be reversed by dephosphorylation
using potato acid phosphatase or protein phosphatase 1. Calmodulin by
itself (which activates end CalPK in the presence of MgATP) blocks the
channel in the dephosphorylated state, which can be overcome by
treatment with CalPK but not PKA.
Our findings reveal important
insights regarding channel regulation of the ryanodine receptor: 1) the
calcium release channel must be phosphorylated to be in the active
state at conditions approximating physiological Mg concentrations (
mM); and 2) there are multiple
sites of phosphorylation on the calcium release channel with different
functional consequences, which may be relevant to the regulation of E-C
coupling. Phosphorylation of the CRC may be involved in recruitment of
active channels, and/or it may be directly involved in each
Ca
contraction cycle of the heart. For example,
Ca
release may require phosphorylation of the CRC by
protein kinases at sites which overcome the block by
Mg
. Inactivation may involve CRC block by calmodulin
and/or phosphorylation by endogenous CalPK at the junctional face
membrane.
The macroscopic phenomenology of excitation-contraction coupling
in heart is referred to as calcium-induced calcium release. The initial
event is the activation of voltage gated L-type Ca channels in response to depolarization of the plasmalemma during
each pump cycle(1, 2, 3, 4) . The
Ca
influx into the cardiomyocyte leads to
mobilization of Ca
from intracellular stores via
activation of Ca
release channels (CRC) (
)of the sarcoplasmic reticulum, and thereby to contraction
of the heart (systole). The CRC for both skeletal muscle and heart has
been identified as the ryanodine receptor being morphologically
identical to the foot
structures(1, 5, 6, 7, 8) .
The isolation and characterization of the voltage-gated Ca
channel and the calcium release channel/ryanodine receptor in
molecular terms were major advances in excitation-contracting coupling
in cardiac and skeletal muscle. Yet, the detailed mechanism of
excitation-contraction coupling remains basically unsolved. Modulation
of cell function by protein kinases and phosphatases represent a common
motif in intracellular signaling(9) . The voltage-gated
Ca
channel in heart is a well studied
example(10, 11) . Recent reports have suggested that
the ryanodine receptor from skeletal muscle and heart may be modulated
thusly. Endogenous CalPK has been found in sarcoplasmic reticulum
membranes of cardiac muscle cells (12, 13, 14) . Studies in rat myocytes
indicated that phosphorylation by protein kinase A enhances the early
phase of Ca
release (15) . In another study,
phosphorylation of junctional SR membranes from heart by exogenous
Ca
/calmodulin-dependent protein kinase II (CalPK)
removed the block of the CRC by calmodulin(14) . For frog
skeletal muscle SR, stimulation of endogenous CalPK was shown to
inactivate the CRC(16) . A detailed study on the modulation of
the calcium release channel of heart by
phosphorylation/dephosphorylation is presented and discussed in the
context of excitation-contracting coupling. Preliminary reports have
appeared (17) and a parallel study on skeletal muscle
CRC(18) .
CalPK II, kindly provided by Dr.
Howard Schulman (Department of Pharmacology, Stanford University School
of Medicine, Stanford, CA (24) ) and Dr. Thomas R. Soderling
(Vollum Institute, Oregon Health Science University, Portland,
OR(25) ). Both kinase preparations are highly purified; the
latter is prepared by recombinant DNA technology. The kinase was
applied via the microsyringe. Applied solution contained 7.5 µg/ml
CalPK in CalPK activation solution: 50 µg/ml calmodulin (Sigma),
0.5 mM ATP-Tris, 3 mM MgCl, 1 mM EGTA, 1 mM CaCl
in 115 mM Tris, 250
mM HEPES buffer at pH 7.4. Time intervals of application are
shown by arrows in the figures (with index
``t'' for tube application) after which the
microsyringe was removed and the bath briefly stirred to dilute the 1
µl applied into the 1.3-ml cis solution.
Protein phosphatase 1
(PPT1)(26) , prepared by recombinant DNA technology and hence
highly purified, was kindly provided by Dr. Ernest Lee (University of
Miami Medical Center, Miami, FL). It was applied via the microsyringe
in 1-µl aliquots of solution containing 0.2 µM PPT1,
0.2 mM MnCl, 3 mM MgCl
, 0.5
mM ATP-Tris, 1 mM EGTA in 115 mM Tris, 250
mM HEPES buffer at pH 7.4.
PKA catalytic subunit, purified
to near homogeneity, was kindly provided by Dr. Jackie Corbin,
Vanderbilt University School of Medicine, Nashville, TN(27) .
It was either applied to the bath (available in sufficient amounts) or
via the microsyringe. For bath application of PKA, the catalytic
subunit was dissolved in 115 mM Tris, 250 mM HEPES pH
7.4, with 6 mg/ml dithiothreitol at 0.05 mg of protein/ml and 10 µl
of solution (18 units) were added. For microsyringe application PKA was
present in 3 mM MgCl, 0.5 mM ATP-Tris, 1
mM EGTA, 3 mg/ml dithiothreitol, 115 mM Tris, 250
mM HEPES pH 7.4 at a concentration of 0.025 mg of protein/ml
(1 unit/µl).
Acid phosphatase from potato Type III (PPT),
purchased from Sigma (0.7 ml obtained in 3.2 M (NH)
SO
, 1% serum albumin at pH
6.0) was dialyzed for 6 h with one buffer change after 3 h against 200
ml of solution containing 115 mM Tris, 250 mM HEPES
at pH 7.4 (corresponding to 1 unit of PPT in 13 µl). For bath
application, 65 µl (5 units) were added to the cis buffer and for
microsyringe application PPT was present in 3 mM MgCl
, 0.5 mM ATP-Tris, 1 mM EGTA,
115 mM Tris, 250 mM HEPES pH 7.4 at 0.01 unit of
PPT/µl.
Stock solution used for calmodulin additions contained:
400 µM calmodulin in 115 mM Tris, 250 mM HEPES pH 7.40, and 13 µl were added to the cis side (4
µM final calmodulin concentration). For tube application
of calmodulin the solution contained: 50 µg/ml calmodulin, 1 mM EGTA, 1 mM CaCl in 115 mM Tris, 250
mM HEPES at pH 7.40. The solution for microsyringe application
of calmodulin + ATP was identical to that used for tube
application of CalPK, but without CalPK (see above).
Heart SR microsomes, when fused to planar lipid bilayers
(from the cis side), induced CRC activity by the criteria of unitary
current, Ca selectivity, and sensitivity to ryanodine
or ruthenium red (applied to the cis side and routinely tested at the
end of the described experiments). We investigated qualitative effects
on CRC activities by phosphorylation or dephosphorylation, that is, for
inhibition of active channels or reactivation of channels inhibited by
Mg
or calmodulin. All additions were made to the cis
side (either to the bath, or directly to the membrane by a
microsyringe, cf. ``Materials and Methods'') at
constant conditions of free Ca
(5.5 ± 1.2
µM), of ATP (0.5 mM), and of membrane potential
(0 mV).
Figure 1:
Phosphorylation by CalPK removes
channel block by Mg. Channel activity, initially
sensitive to block by Mg
(see states d
and d
) is recovered by application
of CalPK (state p
) which is
then again abolished by protein phosphatase PPT (state d
). For this figure and all
other figures, the following pertains: holding potential was 0 mV.
``State'' nomenclature: p (phosphorylated), d (dephosphorylated); upper index refers to
absence(-) or presence (+) of 2.6 mM free
Mg
; lower index indicates protein kinase or
phosphatase used (here CalPK and PPT, respectively) or initial
phosphorylation state (init) deduced from the presence (d
) or absence (p
) of
complete block by Mg
. All additions were to the cis
side. Arrows with index t refer to addition by a
microsyringe, which allows placing 1 µl of solution directly to the
membrane for the times indicated by arrows (followed by tube
removal and stirring for 1300
dilution of the applied 1 µl
of solution in the 1.3 ml of cis solution); for more details see
``Materials and Methods.'' Arrowswithout t
refer to bath addition to the cis solution. ``S''
indicates stirring for 20 s. For composition of solutions added, see
``Materials and Methods.'' The cis solution contained 0.5
mM ATP and 5.5 ± 1.2 µM free
Ca
, constant for all data presented in this study.
For evaluation of mean current and for conditions of the representative
traces for each state see ``Materials and
Methods.''
Figure 2:
Channel activation/inactivation during
phosphorylation/dephosphorylation cycles. A, two-channel
activity, initially not sensitive to complete block by Mg (state p
), block is
induced by PPT1 and activity recovered by added CalPK (state p
). After washing out
Mg
by perfusion, activity matches initial activity
(compare p
and p
). This indicates that the
low activity in state p
is
probably due to a low channel open probability in this state rather
than due to incomplete reactivation of only one channel. A second
application of PPT1 reinstalls block by Mg
. The
channel traces indicate cooperative opening and closing events of the
two active channels. This behavior was often seen in this study, mostly
occurring in a modal fashion (Footnote 2). Overall activity is,
therefore, assayed by mean current instead by open probability. B, protocol and conditions were identical to A except
PKA is used instead of CalPK. Evidence for removal of Mg
block by PKA is presented here at the ``single
channel'' (Footnote 2) level by changes of open probability (p
). Single channel traces show that
recovery of activity by PKA from Mg
block leads to an
enhanced residency of the channel in a subconductance state (at about
55% of unitary conductance). The channel resided in this state for
minutes, interrupted with occasional long visits to zero or short
sojourns to unitary conductance. The p
value of 0.45 refers to a discrimination level for open events of
0.35 unitary conductance (A and B). The concept of
open probability becomes debatable due to cooperativity and substates
for which reasons the data from such cyclic phosphorylation
experiments, summarized in Fig. S1, are compared by using
relative mean currents (see also Footnote
2).
Scheme 1:
Scheme 1Summary of cyclic
phosphorylation and dephosphorylation experiments. Numbers on arrows between states give successful attempts out of total
attempts in parentheses; n.d., not done. Relative mean
currents, I(state)/I(p,
are given for each state as average values with maximum deviations (for
CalPK and PKA phosphorylated states) or as upper limits (for PPT and
PPT1 dephosphorylated states). The failures of PPT1 and CalPK
application (one for each) were due to misalignment of the tube syringe
while the one failure of PPT, applied to the bath, remained unresolved.
The data in the scheme are largely from two-channel experiments (see
``Results'') similar to that illustrated in Fig. 2A and from two single channel observations as in Fig. 2B.
Each situation during experimental
protocols in this and other figures is characterized by a symbol, such as d, d
, p
, p
, in Fig. 1, where the
upper index -/+ stands for absence or presence of 2.6 mM free Mg
, and the lower index indicates the
kinase/phosphatase which had been applied to initially untreated
channels (index ``init''). The rationale for choosing
``p'' and ``d'' is given later.
The experimental conditions in Fig. 1are not the most
appropriate choice for establishing significance of the observed effect
of CalPK for two reasons. (i) Conditions of Mg are
not the same: the initially Mg
sensitive activity (d
) is observed in the
absence of Mg
. (ii) There is no clear statement
possible as to the fraction of channels recovered by CalPK due to the
multi or several channel level of analysis. We, therefore, carried out
cycles of phosphorylation and dephosphorylation at conditions as shown
in Fig. 2, A and B. The results are summarized
in Fig. S1. Here we start from one- or two-channel activities
initially insensitive to block by Mg
. Application of
PPT1 induced channel block (see state d
in both Fig. 2, A and B). In Fig. 2A, this is followed by
treatment with CalPK which led to recovery of activity (state p
), and again blocked by a
second application of PPT1. These data show that the activity seen in
response to CalPK was due to phosphorylation which removes the block by
Mg
, and that PPT1 dephosphorylates these sites
leading to complete channel block in the presence of Mg
(state d
). It also
implies that the initial observation of either the presence or absence
of the Mg
block is due to dephosphorylated states (d
) or phosphorylated states (p
) of the CRC, respectively. Fig. 2B shows activities during a similar
phosphorylation cycle as in Fig. 2A only that PKA was
used instead of CalPK. Phosphorylation by PKA also removed channel
block by Mg
(state p
) which was reversed again by
dephosphorylation using PPT1 (d
).
Fig. S1summarizes the results of the cyclic phosphorylation
experiments which were carried out with cycles, following the protocol
of Fig. 2and using different combinations of phosphatases, PPT
or PPT1, and kinases, CalPK or PKA. For each phosphorylation state, the
Scheme shows values of mean current, normalized to the initially
observed mean currents (in state p). This Mg
insensitive activity (p
)
was blocked by either phosphatase (PPT or PPT1). This was followed by
application of CalPK or PKA in all possible combinations, as shown,
which led to recovery of activity. This could again be reversed by
phosphatase treatment (either PPT or PPT1). For interconversion of
phosphorylated and dephosphorylated states only small amounts of
kinases and phosphatases were applied at constant Mg
concentration. The changes in relative current values for each
interconversion step are large (factors 25 to more than 100) and are,
therefore, considered to be qualitative compared with the changes
within phosphorylated states (less than factor 4). The conclusions we
draw relate exclusively to these qualitative changes observed during
the cycles as seen by changes of relative mean currents. This
description is not dependent on the level of activity or the number of
channels. These data from cyclic phosphorylation experiments provide
convincing evidence that both CalPK and PKA remove the Mg
block by protein phosphorylation which is reversed by
dephosphorylation using either phosphatase. Since channel activity
qualitatively and persistently changes upon application of either
kinase, it can be inferred that phosphorylation of some sites took
place (denoted by state index p, or specifically p
or p
). These sites
were dephosphorylated prior to application of the kinases (denoted by
state index d, specifically d
or d
). By analogy, the channel initially
insensitive to Mg
block is in the p state, i.e.p
. It should be added that, in
the absence of Mg
, neither phosphorylation by PKA or
CalPK, nor dephosphorylation by PPT or PPT1 induced significant changes
of CRC activity in at least three independent experiments for each
kinase or phosphatase (cf. examples in Fig. 4B (state d
), 4A (state d
), 2A (state p
). In the presence of
Mg
, recovery of activity by PKA, when assayed by mean
current, was to comparable levels as found initially (see Fig. S1; compare also p
values in states p
and p
in Fig. 2B and 3C), while recovery by CalPK was to lower levels of
activity (see Fig. S1). Quantitation of such differences between
CRC activities in different phosphorylation states by p
values and lifetimes was not attempted due to the following
difficulties: (i) most fusion events led to incorporation of several
channels, mostly of two channels (about 60%), and of <10% single
channels out of more than 100 attempts. This is to be contrasted with
our experience on skeletal muscle, where at least 30% of the membranes
display single channels after vesicle fusion; (ii) two-channel
activities often exhibit coupling or cooperativity (events of
synchronized opening and closing)
; and (iii) some channels,
when phosphorylated by PKA, were found to be locked in subconductance
states as shown in Fig. 2B (compare with trace p
in Fig. 3C).
On the other hand, there was no indication that channel
cooperativity or pronounced substate population had any influence on
the described release from Mg
block by
phosphorylation, or on the induction of block by dephosphorylation.
Figure 4:
Channel block due to phosphorylation by
endogenous CalPK. A, the effect of calmodulin is shown to be
different when applied in the absence of ATP (solution
``Cal'') and its presence (solution ``Cal
+ ATP,'' identical to CalPK activation solution, see
``Materials and Methods''). In the absence of ATP, activity
fully recovered from block by calmodulin upon tube removal and
stirring, while activity remained blocked when ATP had been present.
Solution (1 µl) was placed by the tube syringe directly to the
membrane for the time interval indicated by the arrow lengths.
The persisting block is interpreted to be due to CRC phosphorylation by
endogenous CalPK. The channels had previously been dephosphorylated
using PPT1 and Mg had not been added (state d
) to observe active CRC for
assessment of block by calmodulin and by end CalPK. B, as in A, tube application of CalPK activation solution (Cal
+ ATP) led to persisting block of the activity of
dephosphorylated channels in the absence of Mg
(state d
). The conclusion that the
persisting block is due to phosphorylation of the channels by end CalPK
(state p
), activated by
the applied solution is enforced by the observed recovery of activity
by dephosphorylation using PPT to state d
, which is sensitive to block
by Mg
(state d
). For symbols and other
conditions, see legend to Fig. 1.
Figure 3:
Channel block by calmodulin is removed by
CalPK but not by PKA. A, calmodulin (4 µM) was
applied to a channel in the dephosphorylated state using PPT and in the
absence of Mg (trace d
). The traces for d
are a continuous record
starting 12 s after calmodulin addition from stock solution (see
``Materials and Methods'') to the bath during which time
solution was stirred. The final open probability p
was 0.02. B, calmodulin (4 µM in cis
solution completely blocked activity of two channels in state p
. Upon application of CalPK
(by tube syringe for 90 s) the channels recovered to comparable
activity (average current ratio i(p
)/i(p
)
= 0.7). The two channels do not act completely independently as
seen from coordinated transitions, especially seen in the lowest trace
(graded cooperativity) (see Footnote 2). C, three cycles of
activation/inactivation of a CRC in the presence of Mg
are shown. The first block provides evidence for
dephosphorylation by PPT1 (state d
) with respect to both PKA
and CalPK phosphorylation sites. From the recovery of activity by PKA
(state p
) it is inferred that
the channel is now phosphorylated at sites accessible to PKA. This
activity is blocked by calmodulin. Application of CalPK removed this
block, indicating that it phosphorylates different sites than PKA and
with different effect, i.e. CalPK removes block by calmodulin,
not found for
PKA.
The phosphorylation stoichiometry of the cardiac CRC was measured using exogenously added PKA and CalPK II (Table 1). The stoichiometry is 1.2 using CalPK II and 1.6 with PKA.
Inspection of
unitary currents, at zero membrane potential, revealed that
Mg reduces the value from 2.67 ± 0.05 to 2.47
± 0.06 pA, irrespective of the phosphorylation state (see Table 2). The nature of this reduction was not analyzed.
The microsyringe methodology to treat the channel system
directly at the bilayer surface represents novel technology for the
study of channel modulation. When used in conjunction with highly
purified kinases and phosphatases, it represents a powerful approach to
study channel modulation that is stringent in its own right. The
calcium release channel from cardiac SR, incorporated into planar
bilayers, can exist in two different states. One state (d) is initially sensitive to block by
approximate physiological concentration of Mg
(
1
mM). The other is insensitive (p
) to
block by Mg
. State
``d
'' can be made insensitive to
block by Mg
by treatment with exogenously added PKA
or CalPK II, whereas state ``p
'' can
be treated with phosphatase to confer sensitivity to
Mg
. The interconversion of Mg
sensitivity has been carried out sequentially and repeatedly (Fig. 2, A and B, and Fig. S1) using
highly purified protein kinases and phosphatases. These studies lead to
the conclusion that CRC can be modulated by
phosphorylation/dephosphorylation.
Our studies reveal important
insights into the dimension of channel regulation of the ryanodine
receptor. We find that the CRC activity of heart sarcoplasmic reticulum
can be modified by phosphorylation/dephosphorylation by protein kinases
and phosphatases, respectively. There are two salient findings: 1) the
CRC, which is not phosphorylated at PKA and CalPK sites, is inactive
under physiological Mg concentration
(
millimolar), and this Mg
block can be overcome
by phosphorylation with either PKA or CalPK; 2) inactivation of the CRC
can be achieved by phosphorylation by activating end CalPK; this block
is separate from the reversible block by calmodulin
binding(28) . The essential findings are: (i) channel block by
Mg
is removed by phosphorylation using either
(exogenously added) PKA or CalPK. The evidence for this was achieved by
sequential phosphorylation by protein kinases (PKA or CalPK) and
dephosphorylation by phosphatases (PPT and PPT1) to give channel
activation and inhibition, respectively, in cyclic fashion. (ii)
Activation of endCalPK leads to closure of previously dephosphorylated
channels. (iii) Block induced by endCalPK is reversed by
dephosphorylation using PPT or PPT1. (iv) In parallel with the
requirement of calmodulin for the persistent endCalPK-induced block,
calmodulin also blocks CRC by direct interaction. (v) Channel activity
recovered from Mg
block by phosphorylation with
exogenously added CalPK and ATP is not blocked by calmodulin, whereas
PKA recovered activity is blocked by calmodulin.
These findings for heart are similar in the key conclusions (at least i-iv) to that of the parallel study for the skeletal muscle CRC(18) . Phosphorylation of multiple sites can occur with different functional consequences. The functional consequences are likely involved in the regulation of E-C coupling in both heart and skeletal muscle. At the very least, phosphorylation would be involved in the recruitment of the number of active channels.
The phosphorylation stoichiometry of the
cardiac CRC measured by Witcher et al.(14) was
approximately four for exogenous CalPK II and one for endogenous
CalPK or PKA. In their study phosphorylation with CalPK II overcomes
the block by calmodulin. They did not report on the effect of
phosphorylation with endogenous CalPK or PKA on channel function. The
phosphorylation stoichiometry obtained by us for cardiac SR in the test
tube was 1.2 for exogenous CalPK II and 1.6 for PKA. In our studies,
treatment with CalPK but not PKA overcomes the calmodulin block. A new
finding in our studies is that treatment with exogenously added CalPK
II or PKA overcomes the block by Mg
. Our studies also
show, based on sensitivity to block by Mg
, that the
channel must be phosphorylated at PKA or CalPK II sites to be active in
the presence of physiological Mg
. Based on channel
measurements, approximately 40% of the CRC in our cardiac SR were
already phosphorylated. This means that phosphorylation stoichiometry
values in the future will need to be re-evaluated taking into account
the extent of phosphorylation of the CRC in the SR as isolated. To
achieve this, new methodology will have to be devised.
It has been
recognized for some time that Mg inhibits
Ca
release from SR (28, 29) albeit
not always so. It therefore remained a paradox as to how Ca
can be released from the CRC under physiological Mg
concentration (
1 mM free
Mg
)(30) . The Mg
paradox
can now be explained by the phosphorylation state of the CRC. The
apparent contradictory results in the literature may be due to
differences in the state of phosphorylation of the channel and its
dependence on assay conditions. The insights of multiple
phosphorylation sites with different functional consequences suggest
that modulation of the CRC represents an important dimension in E-C
coupling. Thus far, the literature contains reports that CalPK
activates the CRC in heart (14) and inhibits the CRC in frog
skeletal muscle(16) . In the former study on heart, exogenous
CalPK activated the channel, whereas in the latter study on frog
skeletal muscle, endogenous CalPK inactivated the channel. These two
studies left unresolved whether the apparent difference in the two
systems was referable to heart versus skeletal muscle,
amphibian versus mammalian, and/or to the application of
exogenous versus endogenous CalPK. We confirm both studies for
mammalian heart (this study) and for skeletal muscle(18) . The
multiplicity of action of protein kinases has received only limited
assignments with respect to sites of action. Phosphorylation of the
cardiac CRC by protein kinases had been observed in biochemical studies (13, 14) and for skeletal muscle using a variety of
protein kinases (31) . Exogenously added PKA and CalPK appear
to phosphorylate different residues of the cardiac CRC(13) .
CalPK and endCalPK were found to phosphorylate the same residue, serine
2809, with the difference that CalPK has as substrate all 4 serine 2809
of the homotetrameric CRC, while only 1 serine 2809 appears accessible
for phosphorylation by endCalPK(14) . An analogous region of
the receptor has been reported to be phosphorylated in skeletal muscle
using a variety of protein kinases (31) . On this basis, the
block induced by endCalPK appears to be due to phosphorylation of a
single serine 2809 residue, i.e. only one protomer of the
homotetrameric channel. Such structural asymmetry may perturb
allosteric coupling of the four protomers leading to channel closure.
Phosphorylation of all 4 remaining serine 2809 residues by (exogenous)
CalPK may conserve symmetry leading to channel opening as observed.
This implied cooperativity is analogous to the effect of ryanodine
binding (as well as other ligands such as Ca
and
Adriamycin (32) on the CRC. Binding of one ryanodine locks the
channel in an open subconductance state, which otherwise is observed
only occasionally, whereas the weaker binding (K
0.8 µM) (
4 ryanodine per CRC) at higher
ryanodine concentrations causes the channel to close (33, 34) . Alternatively, the different action of
endogenous versus exogenously added CalPK may be due to
phosphorylation at different sites. The cardiac ryanodine receptor
contains 16 possible positive consensus sequences for multifunctional
calmodulin kinase (38) .
The limited success of activating endCalPK, in 5 out of 9 attempts, may indicate that appropriate co-association of CRC and endCalPK does not always survive membrane isolation and vesicle fusion to planar lipid membranes, since in the four failures, the channels behaved normal otherwise.
As to the
physiological relevance of CRC phosphorylation, it has recently been
shown that the CRC is phosphorylated by PKA during -adrenergic
stimulation of intact myocytes from the newborn rat heart(15) .
The
-adrenergic agonist induced a 2-fold higher level of
phosphorylation of the CRC than found in controls, which is attributed
to phosphorylation by PKA. Since it is well known that
-adrenergic
stimulation enhances Ca
release, it was suggested
that this is due in part to phosphorylation by PKA leading to higher
CRC activity. In this regard, our findings offer insight that
phosphorylation is required for channel activity in the presence of
physiological Mg
concentration. Myoplasmic free
Mg
concentration is in the millimolar
range(30) , and unlike Ca
which serves as a
second messenger, the Mg
concentration does not
change appreciably during the cycle of systole and diastole of the
heart. We find the CRC is blocked in the closed state when
dephosphorylated at sites which are substrates for exogenously added
PKA or CalPK. Such phosphorylation by either kinase was required to
open the CRC. In this regard, the activation of the ryanodine receptor
by phosphorylation using membrane-associated protein kinases has
recently been reported for skeletal muscle(35) .
Inactivation of CRC by endCalPK has, thus far, been reported only
for frog skeletal muscle CRC(16) . Our data on inhibition of
mammalian cardiac CRC activity at conditions favoring phosphorylation
by endCalPK suggest that such inhibition is operative also in cardiac
muscle cells (as well as mammalian skeletal muscle; (17) and (18) ). In the only study thus far of CalPK effects on cardiac
CRC(14) , CRC activity was recovered from block by calmodulin
in the presence of millimolar Mg by the action of
exogenously added CalPK. We were able to confirm the calmodulin block
when the channel had been phosphorylated by PKA. It is important to
note that the CRC activated by PKA is blocked by calmodulin, whereas
CalPK-activated channels are not blocked by calmodulin. Involvement of
endCalPK in CRC inactivation would preclude prior phosphorylation by
(exogenous) CalPK, which suggests that in the myocyte, PKA is operative
to remove block by Mg
. The calmodulin block of
PKA-activated channels may underlie the fast relaxation of the initial
fast release to a steady state since the
Ca
-calmodulin complex appears to be an inhibitory
species(28, 36, 37) . The latter may
represent a temporary block by which calmodulin is relieved by the
decreasing Ca
concentration.
Our studies on
phosphorylation and dephosphorylation as related to channel block by
Mg, the dual action of calmodulin, and the action of
exogenous versus endogenous CalPK, provide novel insights into
activation, regulation, and inactivation of the CRC. The direct
relevance to E-C coupling and to the pump cycle in heart needs to be
tested.