©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation Modulates the Function of the Calcium Release Channel of Sarcoplasmic Reticulum from Cardiac Muscle (*)

(Received for publication, September 26, 1994)

Jürgen Hain (1) Hitoshi Onoue (2)(§) Martin Mayrleitner (2) Sidney Fleischer (2)(¶) Hansgeorg Schindler (1)(¶)

From the  (1)Institute for Biophysics, University of Linz, A-4040 Linz, Austria and the (2)Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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) .


MATERIALS AND METHODS

Preparation of Cardiac SR

The sarcoplasmic reticulum fraction from canine heart muscle was isolated as described previously (19) .

Stoichiometry of Phosphorylation of CRC in Cardiac SR Vesicles

Phosphorylation with protein kinases was carried out at room temperature in a 50-µl assay volume using conditions similar to Witcher et al.(14) , but optimized to achieve higher phosphorylation stoichiometry. Among a number of differences in our protocol, the 10 mM NaF is especially important to inhibit phosphatase activity. Cardiac SR (19) (1 mg/ml) were phosphorylated with catalytic subunit of PKA (0.42 µg) (provided by Dr. Jackie Corbin) for 5 min in the phosphorylation buffer containing 300 µM [P]ATP (DuPont NEN), 25 mM MOPS, pH 7.0, 5 mM MgCl(2), 10 mM NaF, 1 mM EGTA, and 1 mM CaCl(2). Protein phosphorylation with Ca/calmodulin-dependent protein kinase II (CalPK II) (0.13 µg) was performed under similar conditions using 1.5 mM CaCl(2) instead of 1 mM CaCl(2) and calmodulin (2 µg) was added additionally. The reaction was stopped after 15 min by adding 25 µl of SDS dissociation buffer (1% SDS, 5% beta-mercaptoethanol, 1 mM EDTA, and 10 mM Tris-HCl, pH 8.0). The entire sample was then subjected to SDS-PAGE in 6% gels, 1.5-mm thick (Laemmli, 39), followed by autoradiography, to identify radioactive ryanodine receptor protomer, using Kodak X-Omat AR film after the gels had been stained with Coomassie Brilliant Blue. The amount of P incorporation into the cardiac muscle ryanodine receptor protomer was determined by counting the radioactivity of the gel bands containing the phosphorylated band referable to the CRC protomer. The molar ratio of P/CRC was calculated by dividing the P-phosphorylation (picomoles) by the equivalent amount of CRC as determined from the measured B(max) of ryanodine binding. The latter was determined for each SR preparation (7 pmol/mg protein).

Planar Bilayer Measurements

Cardiac SR microsomes were fused with Mueller-Rudin planar lipid bilayers following the protocol described by Smith et al.(20) with minor modifications. Planar bilayers were formed across a 0.25-mm hole in a 6-µm PTFE-Teflon sheet with boundary irregularities less than 1 µm, which rendered bilayer thinning fast and reproducible and gave high bilayer stability as a function of time and voltage. The lipid mixture applied was phosphatidylethanolamine, phosphatidylserine (both from bovine brain), and synthetic diphytanoyl phosphatidylcholine (all from Avanti Polar Lipids, Inc., Alabaster, AL) in a weight ratio of 5:3:2, dissolved in decane at 50 mg/ml. During bilayer formation cis and trans chambers contained 53 mM Ca(OH)(2), 250 mM HEPES, pH 7.4. For fusion, 600 mM KCl (21) was added to the cis chamber and a 10-µl microsome suspension (3.5 µg of total protein) was applied near the bilayer. Fusion was monitored by Cl specific currents. After the first fusion event, the cis chamber was stirred and perfused for 4 min at 3 ml/min with 115 mM Tris, 250 mM HEPES, pH 7.4. Then 0.5 mM EGTA and 0.5 mM ATP-Tris were added. The resulting free Ca concentration was 5.5 ± 1.2 µM in all experiments as measured by a Ca electrode (Orion SA720) after each experiment. The chambers always contained 1.3 ml of solution. All additions or treatments in this study started from this condition of cis solution and perfusions during experiments mimic this initial condition by perfusion for 4 min at 3 ml/min with 115 mM Tris, 250 mM HEPES, pH 7.4, 0.5 mM ATP-Tris, 0.5 mM EGTA, and 5.5.µM free Ca adjusted by the Orion electrode. Electrical contact was made by Ag/AgCl electrodes via agar bridges (1% agar in 1 M KCl). Voltage is expressed as the voltage applied to the cis chamber. The Ca channel currents shown were observed at 0 mV holding potential. The currents are therefore negative and are shown as downward deflections. The voltage signal across the feedback resistance (10 ohms) of the current-measuring operational amplifier was filtered at 1 kHz and stored on a pulse code modulated audio tape recorder modified to accept direct current signals. For fast acquisition and analysis of channel data, the data were transferred to a hard disk of a personal computer, at a sampling rate of 1 kHz and analyzed using programs: AXOTAPE and PCLAMP, version 5.5, from Axon Instrument. Channel activities during time intervals specified in the figures are characterized by either mean current i (pA) or by channel open probability, p(o). p(o) values were determined from areas of Gaussian distribution at best fit to peaks of amplitude histograms. Unitary channel currents were determined from one- and two-channel traces using maxima of Gaussian distribution at best fit. Mean current values for the often observed two-channel activities are baseline corrected values, that is, mean baseline current above baseline, estimated from Gaussian fits, was subtracted from the total mean current above baseline. Mean current values for channels blocked by Mg were too low to be determined from deviations from Gaussian fits to baseline currents. They were estimated from basal widths of all negative current spikes reaching at least half the unitary value of channel current and is, therefore, given as an upper limit. For presentation of channel traces the data were filtered at 300 Hz.

Additions to the cis Chamber

Times of additions of Mg, protein kinases, and protein phosphatases, calmodulin, and calmodulin + ATP are specified in the figures. Additions of Mg refer always to additions of 3 mM MgCl(2). Free Mg was 2.6 mM as calculated from Robertson and Potter(22) . Additions were either to the bath or using a microsyringe(23) . The microsyringe, of inner diameter 0.85 mm, was adjusted with its end to the membrane (center to center) at a distance of 0.15 mm. The microsyringe places a droplet of reagent directly to the bilayer surface. It can be removed for refilling and accurately replaced to the same position. Delivery of 1 µl of solution displaces the solution between tube and membrane and remains effectively undiluted for more than 5 min, as assayed by valinomycin. The microsyringe affords a number of advantages: 1) reagent is applied directly to the membrane without interfering with the composition of the bath solution; 2) application of small amounts of reagents which otherwise would be prohibitive by bath applications; 3) reagents or enzymes applied to the membrane can readily be diluted (>1000-fold) to assess whether application is reversible on dilution or has a persistent effect; 4) sequential application of different reagents to achieve cycles of phosphorylation and dephosphorylation, or other treatments such as by Ca/calmodulin.

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(2), 1 mM EGTA, 1 mM CaCl(2) 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(2), 3 mM MgCl(2), 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(2), 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(4))(2)SO(4), 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(2), 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(2) 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).


RESULTS

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).

Phosphorylation Removes CRC Block by Mg

We found two classes of channels with regard to sensitivity to block by Mg (2.6 mM free Mg). That is, addition of Mg blocked CRC activity in 10 of 16 experiments (see Fig. 1). In the other 6 of 16 experiments, Mg had little or no effect as shown in Fig. 2, A and B. The Mg block, when observed, was fully reversible by removing Mg by perfusion with Mg free cis solution (not shown). Based on this observation of two distinct responses of the Ca channels to Mg, we investigated whether this is referable to the state of phosphorylation. Fig. 1shows an example where multichannel activity was blocked by Mg. Application of CalPK II led to recovery of activity. The block was restored by dephosphorylation using PPT.


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 times 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 pvalue 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.



CRC Block by Calmodulin Is Removed by CalPK but Not by PKA

The scope of the study was next extended to include effects of calmodulin, in view of a report of channel block by calmodulin and its reversal by action of exogenous CalPK(14) . For a closer investigation of this finding in the context of the present study, we first investigated for possible relationship between block of CRC by Mg and calmodulin. Calmodulin (always applied in concentration of 50 µg/ml corresponding to 4 µM) blocked channel activities, both in the absence and presence of Mg. Fig. 3A shows that activity in state d is blocked by calmodulin (see legend for details), which was found also in the other dephosphorylated states, i.e.d and d (not shown). Initial activity in the presence of Mg (state p) was also blocked by calmodulin (see Fig. 3B), as well as activity in state p (not shown). Since the study by Witcher et al.(14) started with open channels in the presence of 3 mM Mg, the initial condition p in Fig. 3B appeared appropriate for attempts to repeat this finding. After block by calmodulin (see traces p) application of CalPK led to recovery of activity (see traces p). These data confirm the observation of Witcher et al.(14) that block by calmodulin is removed by phosphorylation using CalPk. They also imply, however, that in state p the phosphorylation sites for CalPK are in a dephosphorylated state so that other phosphorylated sites render the CRC insensitive to Mg block but sensitive to block by calmodulin. These properties were, indeed, found for CRC phosphorylated by PKA as shown in Fig. 3C. Initially, block of Mg insensitive activity (state p) by PPT1 is used to assure that both PKA and CalPK phosphorylation sites are in the dephosphorylated state. Then, recovery from Mg block by PKA is taken as evidence that phosphorylation took place (p). Calmodulin blocked the activity in the PKA phosphorylated state (p) while further phosphorylation by CalPK removed the block by calmodulin (p). The final PPT1 effect provides further evidence that phosphorylation took place (d). Taken together, complete block by 4 µM calmodulin was neither dependent on the presence or absence of Mg, nor on whether the channel was dephosphorylated or phosphorylated by PKA; only phosphorylation by CalPK overcomes this block. The data suggest different phosphorylation sites for PKA and CalPK.

Phosphorylation by EndCalPK Blocks CRC Activity

Block by calmodulin in Fig. 3was observed in the presence of ATP. Channel block could, therefore, be referable to: 1) direct inhibition of the CRC by calmodulin; and/or 2) activation of endogenous CalPK (endCalPK) which may be associated with the CRC in the planar bilayer. To discern between these possibilities, experiments as shown in A and B of Fig. 4were carried out. Both experiments start with activities of dephosphorylated channels in the absence of Mg using PPT1 in A and PPT in B of Fig. 4. Part A shows two different responses to calmodulin, applied by the tube syringe in CalPK activation solution (see Cal+ATP) and in the same solution but devoid of ATP (see Cal). In both cases, activity was blocked as long as 1 µl of solution remained in place at the membrane. In the absence of ATP the activity was recovered upon tube removal and stirring (dilution of the 1 µl into the 1.3-ml cis solution), see second state d. Full recovery was observed in 4 out of 4 independent attempts. This is expected for a block caused by reversible calmodulin binding requiring micromolar concentration. However, when the applied solution also contained ATP, the activity remained blocked after microsyringe removal and stirring. This persistent block of the channel suggests involvement of endCalPK. Part B of Fig. 4starts out with d and obtaining persistent block by application of CalPK activation solution (state p). This block could be removed by a second application of PPT (second state d) to channel activity which was sensitive to block by Mg (state d). These data provide clear evidence that the observed block was due to phosphorylation by endCalPK. EndCalPK induced channel block, as in B of Fig. 4, was observed in 5 out of 9 independent attempts. In the four failures, channel activity was recovered after tube removal and stirring indicating unsuccessful activation of end CalPK. Reversal of the endCalPK induced block by phosphatases was found in 4 out of 4 attempts, 2 each for PPT and PPT1.


DISCUSSION

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(d) 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 beta-adrenergic stimulation of intact myocytes from the newborn rat heart(15) . The beta-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 beta-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.


FOOTNOTES

*
This investigation was supported in part by Grants S-45/04 and 07 from the Austrian Research Fonds (to J. H. and H. S.) and National Institutes of Health Grants HL32711 and HL 46681 (to S. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Fellow of the Muscular Dystrophy Association of America.

Address reprint requests to: H. Schindler, Institute for Biophysics, University of Linz, Altenbergerstrasse 69, 4040 Linz, Austria. Tel.: 43-732-2468-9269; Fax: 43-732-2468-822 or S. Fleischer, Dept. of Molecular Biology, Vanderbilt University, Box 1820 Station B, Nashville, TN 37235. Tel.: 615-322-2132; Fax: 615-343-6833; fleiscs{at}ctrvax.vanderbilt.edu.

(^1)
The abbreviations used are: CRC, calcium release channel; CalPK, Ca/calmodulin-dependent protein kinase II; endCalPK, endogenous CalPK; PKA, protein kinase A; PPT, potato acid phosphatase; PPT1, protein phosphatase 1; SR, sarcoplasmic reticulum; MOPS, morpholinepropanesulfonic acid.

(^2)
The predominantly observed two-channel activities showed at least two types of ``modal'' behavior. One relates to channel cooperativity. There were periods with sychronous opening and closing events of the two channels (positive cooperativity), which, however, occurred in bursts too fast to be clearly resolved except of some longer lasting events (cf. Fig. 2A and Fig. 3B; see, for example, the lower traces for p and for p in Fig. 3B). The same records show other periods with seemingly one channel activity (see, for example, the upper traces for p and p in Fig. 3B). Periods of random overlapping of unitary currents of two channels are also seen (cf. traces in Fig. 4B). The second modal behavior relates to the occurrence of subconductance states (see top trace in Fig. 2B). There are indications that the occurrence of substates enhances the overall open probability and that the population of substates may be influenced by phosphorylation using PKA (see lowest trace in Fig. 2B). In view of these two types of modal behavior we preferred to describe activity changes during phosphorylation cycles (Fig. S1) by relative mean currents since comparison by open probabilities in the conventional definition is not applicable.


ACKNOWLEDGEMENTS

We are grateful to Dr. Howard Schulman and Dr. Thomas Soderling who kindly provided us with CalPK; to Dr. Ernest Lee for his gift of PPT1. We thank Dr. Barbara Ehrlich (Division of Cardiology, University of Connecticut Health Center, Farmington, CT) for her advice on the incorporation of cardiac SR into planar bilayers.


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