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(Circulation Research. 2006;98:1506.)
© 2006 American Heart Association, Inc.

Cellular Biology

Maximum Phosphorylation of the Cardiac Ryanodine Receptor at Serine-2809 by Protein Kinase A Produces Unique Modifications to Channel Gating and Conductance Not Observed at Lower Levels of Phosphorylation

Simon Carter, John Colyer, Rebecca Sitsapesan

From the Department of Pharmacology (S.C., R.S.), University of Bristol; and Institute of Membrane & Systems Biology (J.C.), University of Leeds, UK.

Correspondence to Rebecca Sitsapesan, University of Bristol, Department of Pharmacology, School of Medical Sciences, University Walk, Bristol BS8 1TD, UK. E-mail r.sitsapesan{at}bris.ac.uk

	Abstract

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It is suggested that protein kinase A (PKA)-dependent phosphorylation of cardiac ryanodine receptors (RyR2) is linked to the development of heart failure and the generation of fatal cardiac arrhythmias. It is also suggested that RyR2 is phosphorylated to 75% of maximum levels in heart failure resulting in leaky, unregulated channels gating in subconductance states. We now demonstrate that this is unlikely, as RyR2 isolated from nonfailing cardiac muscle is phosphorylated to 75% of maximum at serine-2809, and in this situation, RyR2 displays low open probability (P_o) (0.059±0.010 [SEM]; n=30) and normal regulation of gating by Ca²⁺ and other ligands. However, when serine-2809 is PKA phosphorylated to maximum levels on RyR2, unique changes in channel behavior are observed. The channel displays enhanced single-channel conductance, very long open states causing large increases in P_o, and no evidence of subconductance states. Dephosphorylation of channels by protein phosphatase 1 (from 75% to near 0% at serine-2809) also enhances RyR2 channel activity through abbreviation of closed lifetimes. We propose that channels phosphorylated to 75% of maximum at serine-2809 occupy a natural low point in the RyR2 activity landscape. This optimizes channel control, which can be accomplished either by enhanced or decreased phosphorylation, making the channel particularly sensitive to the kinase:phosphatase balance. Pathological situations such as heart failure might upset this balance and thereby permit prolonged stoichiometric phosphorylation of serine-2809, which would be required for dysregulation of SR Ca²⁺ release.

Key Words: phosphorylation • ryanodine receptor • Ca²⁺ release • excitation–contraction coupling

	Introduction

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Protein kinase A (PKA)-dependent phosphorylation of ryanodine receptors (RyRs) is a controversial topic. Different laboratories present very diverse accounts of the effects of RyR phosphorylation both on SR Ca²⁺ release and on the single-channel behavior of cardiac and skeletal RyR.^1–7 For example, it has been reported that PKA-dependent phosphorylation causes an increase in open probability (P_o,)¹ a decrease in steady-state P_o,⁸ or no change in single-channel behavior.⁷ One model of RyR2 regulation by phosphorylation of serine-2809 has dominated discussions recently and has become something of a paradigm. This model suggests that hyperphosphorylation of RyR2 on serine-2809 (defined as 3 subunits per tetramer phosphorylated at serine-2809¹) occurs in heart failure and is a likely cause of the cardiac dysfunction through the pathological phenotype of hyperphosphorylated RyR2 channels. This phenotype is characterized by an enhanced P_o and deranged control of channel function (subconductance states and loss of coupled gating⁹). These results led to the suggestion that hyperphosphorylation of RyR2 at PKA sites can lead to severe defects in excitation contraction (EC) coupling and the generation of fatal arrhythmias.^1,2 Other investigators, however, have failed to support this model, observing no alteration in serine-2809 phosphorylation in heart failure.^4,5

In view of the controversy surrounding the molecular basis of heart failure, we have investigated the effects of PKA-dependent phosphorylation on the gating and conduction of RyR2 and related the changes in single-channel function to serine-2809 phosphorylation levels. This study has defined clear functional consequences of RyR2 phosphorylation and evaluated whether serine-2809 phosphorylation is a worthwhile marker for these functional states. We show that PKA-dependent phosphorylation of serine-2809 on RyR2 at levels up to and including 75% of maximum is associated with modest changes in P_o with no apparent subconductance state gating. However, phosphorylation above this level causes marked increases in P_o and current amplitude, although channels still do not gate in subconductance states. We suggest that maximum phosphorylation at serine-2809 is normally a short-lived event that occurs in response to adrenergic stimulation. If the substantial changes to channel behavior resulting from maximum phosphorylation at serine-2809 were to be maintained, it is likely that pathological changes to cellular Ca²⁺-handling mechanisms could occur.

	Materials and Methods

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Preparation of Sarcoplasmic Reticulum Vesicles and Planar Lipid Bilayers
Heavy sarcoplasmic reticulum (SR) membrane vesicles were prepared from sheep hearts as described previously,¹⁰ snap frozen in liquid N₂, and stored at –80°C. Vesicles were fused with planar phosphatidylethanolamine lipid bilayers as described previously.¹⁰ SR vesicles fused in a fixed orientation such that the cis chamber corresponded to the cytosolic space and the trans chamber to the SR lumen. The trans chamber was held at ground and the cis chamber held at potentials relative to ground. After fusion, the cis chamber was perfused with 250 mmol/L HEPES, 80 mmol/L Tris, 10 µmol/L free Ca²⁺, pH 7.2, unless stated otherwise. The trans chamber was perfused with 250 mmol/L glutamic acid and 10 mmol/L HEPES, pH to 7.2 with Ca(OH)₂ (free [Ca²⁺], approximately 50 mmol/L). Experiments were performed at room temperature (22±2°C). The free [Ca²⁺] and pH of the solutions were determined at the relevant temperatures using a Ca²⁺ electrode (Orion 93-20) and a Ross-type pH electrode (Orion 81-55) as previously described.¹⁰ Additions of PKA and PP1 were made to the cis chamber as detailed in the relevant results sections.

Data Acquisition and Analysis
Channel recordings were displayed on an oscilloscope and recorded on digital audiotape (DAT). Current recordings were filtered at between 800 Hz and 1 kHz (–3 dB) and digitized at 20 kHz using Pulse (HEKA Elektronik Dr Schulze GmbH, Lambrecht/Pfalz, Germany). Channel open probability (P_o) was determined over 3 minutes of continuous recording, unless otherwise stated, using the method of 50% threshold analysis.¹¹ Lifetime analysis was performed only when a single channel incorporated into the bilayer. Events <1 ms in duration were not fully resolved and were excluded from lifetime analysis. Individual lifetimes were fitted to a probability density function (pdf) by the method of maximum likelihood according to the equation equation

Where ln_I is the logarithm of the ith time constant and a_i is the fraction of the total events represented by that component.¹² Single-channel current amplitudes were measured from digitized data using manually control cursors and the single channel analysis software Windows EDR V2.4.9 (University of Strathclyde, UK).

Alteration of Phosphorylation State at Serine-2809 on RyR2
For alkaline phosphatase dephosphorylation of RyR2, cardiac SR vesicles were incubated with 1 U of alkaline phosphatase per µg protein for 30 minutes at 37°C in a solution containing 50 mmol/L HEPES, 16 mmol/L Tris, pH 7.2. For PP1 dephosphorylation of RyR2, cardiac SR vesicles were incubated with 0.1 U PP1 per microgram of protein for 10 minutes at 37°C in a solution containing 50 mmol/L HEPES, 16 mmol/L Tris, 1 mmol/L MnCl₂, 50 mmol/L DTT, pH 7.2. For PKA phosphorylation of RyR2, cardiac SR vesicles were incubated with 0.5 U of the catalytic subunit of PKA per µg protein for 5 minutes at 37°C in a solution containing 50 mmol/L HEPES, 16 mmol/L Tris, 1 mmol/L ATP, 5 mmol/L MgCl₂, 5 mmol/L NaF, pH 7.2. At the end of the relevant incubation time for PKA, ice-cold buffer containing 250 mmol/L HEPES, 80 mmol/L Tris, 5 mmol/L NaF, pH 7.2, was added to each sample. Samples were centrifuged at 9720g for 10 minutes at 4°C, the supernatant removed, and the samples resuspended in ice-cold buffer. Samples were centrifuged at 9720g for 10 minutes at 4°C and finally resuspended at the relevant protein concentration in the same buffer. Samples were then used for Western blotting.

Western Blotting
Heavy SR proteins were size fractionated by SDS-PAGE on a 6% polyacrylamide gel.¹³ Following separation, proteins were transferred to nitrocellulose membranes and nonspecific binding sites were blocked for 1.5 to 2 hours at room temperature using 5% dried milk and Tris-buffered saline (pH 7.4), 0.1% Tween 20. Membranes were probed over night at 4°C with primary antibodies specific for RyR phosphorylated (RYR2-PS2809) or dephosphorylated (RYR2–2809deP) at serine-2809.¹⁴ A secondary horseradish peroxidase linked anti-rabbit IgG (Amersham Biosciences, Buckinghamshire, UK) was used in combination with an enhanced chemiluminescent detection system (Pierce Biotechnology Inc, Rockford, Ill) to visualize the primary antibodies.

Determination of Phosphorylation State at Serine-2809 on RyR2
PKA treatment resulted in complete phosphorylation at serine-2809 as demonstrated by the increase in staining with the RYR2-PS2809 antibody and the absence of signal from the RYR2–2809deP antibody. RYR2-PS2809 staining of control and PKA-treated samples was quantified by densitometry and the control was expressed as a percentage of the PKA-treated samples. Alkaline phosphatase treatment resulted in complete dephosphorylation at serine-2809, as demonstrated by the increase in staining with RYR2–2809deP and the absence of RYR2-PS2809 signal. Comparing the RYR2–2809deP staining of a control sample with the AP-treated sample staining gives the percentage of dephosphorylated monomers in the sample. It is therefore possible to work out the basal level of phosphorylation. In all cases, control and treated samples were always quantified from the same Western blot. Densitometry was performed using Scion Image (Scion Corporation, Frederick, Md), and the level of staining did not saturate the measurement by the densitometry software in any blot quantified.

Statistics
All values given are mean±SEM. A probability value of 0.05 was taken as significant, as determined by Student t test.

Materials
Alkaline phosphatase was obtained from Roche Diagnostics GmbH (Mannheim, Germany). Protein phosphatase 1 (PP1) was obtained from New England Biolabs (Ipswich, Mass). The catalytic subunit of PKA was obtained from Sigma-Aldrich Company Ltd (Dorset, UK). Other chemicals were AnalaR or the best equivalent grade from BDH (Poole, UK) or Sigma-Aldrich Company Ltd (Dorset, UK). All solutions were made up in deionized water and those for use in bilayer experiments were filtered through a Millipore membrane filter (0.45-µm pore).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
We used antibodies specific for RyR2 phosphorylated (RYR2-PS2809) or dephosphorylated (RYR2–2809deP) at serine-2809 to estimate the level of phosphorylation at this site. The specificity of the antibodies for serine-2809 and their ability to detect phosphorylation at this site has been characterized in detail by Rodriguez et al.¹⁴ By using PKA to maximally phosphorylate and alkaline phosphatase to completely dephosphorylate RyR2 at serine-2809, we can use the pair of antibodies in parallel to estimate the phosphorylation level of serine-2809 in our control heavy SR membrane preparation. Western blot analysis (Figure 1A and 1B) demonstrates that the basal level of phosphorylation at serine-2809 is high in our SR membrane preparations (approximately 75% of maximum). Densitometric analysis provided equivalent results with either the RYR2-PS2809 or RYR2–2809deP antibody, confirming that the antibodies are faithfully reporting on the phosphorylation level at serine-2809. This is not an unexpected result because a high basal level of phosphorylation has also been shown by 3 other independent groups.^4,5,7 It has been suggested by some that this level of phosphorylation (75% of maximum) represents a "hyperphosphorylation" state that occurs in heart failure¹ and produces high P_o and subconductance states. However, our single-channel recordings (Figure 2A) demonstrate that this relatively high phosphorylation level is always associated with low P_o (P_o=0.059±0.010 [SEM]; n=30) with 10 µmol/L cytosolic Ca²⁺ as sole activator). Importantly, as shown in the amplitude histogram in Figure 2B, although there are frequent brief unresolved openings, there is no evidence for subconductance gating states.

View larger version (13K): [in this window] [in a new window]   Figure 1. Basal phosphorylation level. A, For PKA phosphorylation, SR vesicles were incubated for 5 minutes in the presence or absence of 0.5 U of the catalytic subunit of PKA per microgram of SR protein in the presence of 1 mmol/L ATP and 5 mmol/L Mg2+ at 37°C, then washed to remove PKA. For alkaline phosphatase dephosphorylation of RyR2, cardiac SR vesicles were incubated with 1 U of alkaline phosphatase (AP) per microgram of SR protein for 30 minutes at 37°C in a solution containing 50 mmol/L HEPES, 16 mmol/L Tris, pH 7.2, but the alkaline phosphatase was not removed before Western blotting. Western blots were probed with antibodies specific for RyR2 phosphorylated (RyR2-PS2809) or dephosphorylated (RYR2-S2809deP) at serine-2809. B, Histogram illustrating the relative serine-2809 phosphorylation level of our SR preparation.

View larger version (50K): [in this window] [in a new window]   Figure 2. Channel gating at basal phosphorylation levels. A, Representative current fluctuations through a single control RyR2 channel at 0 mV in the presence of 10 µmol/L cytosolic Ca2+. O and C indicate the open- and closed-channel levels, respectively. B, Amplitude histogram of a representative channel activated by 10 µmol/L cytosolic Ca2+ alone.

Figure 3 illustrates the effects of further PKA-dependent phosphorylation of RyR2 to a level we believe equates to stoichiometric phosphorylation of serine-2809. We recorded a 3-minute control period in the presence of physiological levels of cytosolic ATP (10 mmol/L), free Mg²⁺ (0.5 mmol/L), and 10 µmol/L Ca²⁺ (Figure 3, left). Note that because of the stochastic nature of channel gating, the trace is not representative of P_o but has been specifically chosen to include long open events to illustrate control full open-channel level. After 3 minutes of incubation with PKA on the cytosolic side of the channel, PKA was perfused away to rule out the possibility of any protein–protein interactions between RyR2 and PKA. Channel activity was then recorded for a further 3 minutes (Figure 3, right). A large increase in P_o and a small, but significant, increase in single-channel conductance were observed. The mean data are shown in the histograms in Figure 3B and 3C. Amplitude histograms shown in Figure 3D and 3E confirm that resolvable subconductance states were not observed. The increase in P_o (from 0.126±0.066 to 0.673±0.164 [SEM]; n=6) was characterized by a marked change in the gating of the channel, in particular, a large increase in mean open times from 4.06±1.69 to 134±83.9 ms (SEM; n=4) was observed. The change in mean closed times from 30.00±10.01 ms in control to 10.73±6.58 ms after PKA incubation was small in comparison. Lifetime analysis (Figure 4) confirms that phosphorylation of the channel caused an increase in both the frequency and the duration of channel openings but that the increase in open lifetime duration was mainly responsible for the increased P_o.

View larger version (27K): [in this window] [in a new window]   Figure 3. Effects of PKA. A, Current fluctuations through control (left) and PKA treated (right) channels. The cytosolic solutions included ATP (10 mmol/L), free Mg2+ (0.5 mmol/L), and 10 µmol/L Ca2+. The control trace is chosen to include long open events to illustrate the current amplitude level and is not representative of Po. B and C show the changes to Po and current amplitude at 0 mV, respectively. D and E are amplitude histograms from control and PKA treated channels.

View larger version (36K): [in this window] [in a new window]   Figure 4. Effects of PKA on open and closed lifetime durations. The open and closed lifetime distributions and pdfs for a typical single RyR2 in the presence of 10 mmol/L ATP, 0.5 mmol/L free Mg2+, and 10 µmol/L Ca2+ in the cytosolic chamber (A) and after incubation with PKA in the cytosolic chamber for 3 minutes followed by washout of PKA (B). The best fits to the data were obtained by the method of maximum likelihood and the resulting time constants and percentage areas are shown.

To support our assumption that the effects of PKA were manifest by protein phosphorylation, we incubated channels for 5 minutes with PKA in the absence of ATP and Mg²⁺. Under these conditions, PKA did not produce any changes in conductance or gating. In the presence of 50 µmol/L cytosolic Ca²⁺, P_o was 0.141±0.054 before and 0.128±0.084 (SEM; n=5) after PKA incubation. Single-channel current amplitude was 4.53±0.043 pA before and 4.72±0.101 after PKA incubation. These results were confirmed by the use of the inhibitor of PKA, protein kinase inhibitor (PKI). PKI, on its own (in the presence of Mg²⁺ and ATP), had no effect (results not shown), but the peptide was able to completely abolish the effects of PKA (P_o was 0.191±0.104 before and 0.261±0.145 after PKA incubation [SEM]; n=4).

We then wanted to investigate how dephosphorylation of RyR2 would affect the gating and conducting properties of the channel. We used the protein phosphatase PP1 for 2 reasons. Firstly, it has been shown to be associated with RyR2, and, secondly, it proved to be effective at dephosphorylating RyR2 when incubated with hSR for short periods of time (10 minutes) at room temperature. This then allowed us to perform the bilayer experiments with relatively short incubation periods. In contrast, alkaline phosphate could not completely dephosphorylate RyR2 under bilayer conditions, requiring incubation of hSR samples to 37°C and augmentation of its effect by denaturation of RyR2 in Laemmli sample buffer (data not shown). Western blot analysis shown in Figure 5A illustrates that the protein phosphatase PP1 can dephosphorylate RyR2 from the basal level of approximately 75% of maximum to a substantially dephosphorylated level below the detection limits of the RYR2-PS2809 antibody. PP1 could therefore be used to dephosphorylate RyR2 already incorporated into bilayers. We recorded RyR2 current fluctuations for 3 minutes (Figure 5B, top trace), incubated the cytosolic side of RyR2 with 5 U of PP1 for 10 minutes, and then recorded for a further 3 minutes after washing away the PP1 (Figure 5B, bottom trace). Dephosphorylation of the channel by PP1 (5 U) caused a small increase in P_o (from 0.074±0.032 to 0.218±0.028 [SEM]; n=4; Figure 5C) but no change in current amplitude (Figure 5D). Again, amplitude histograms show no evidence of subconductance states (Figure 5E and 5F). Figure 6 illustrates the changes in open and closed lifetime distributions caused by dephosphorylating from approximately 75% of the maximum level at serine-2809 to a situation close to complete dephosphorylation at serine-2809. The figure demonstrates that the increase in P_o results mainly from an increase in the frequency of channel opening. We then performed experiments where we doubled the PP1 levels in the cytosolic chamber to 10 U. The effects on gating and conductance were similar to the effects observed with 5 U of PP1 (mean P_o was 0.059±0.017 before and 0.256±0.108 after) providing evidence that essentially maximum dephosphorylation had already taken place with 5 U of PP1.

View larger version (38K): [in this window] [in a new window]   Figure 5. Effects of PP1. A, SR vesicles were incubated for 10 minutes in the presence or absence of 0.1 U of PP1 per microgram of SR protein in the presence of 50 µmol/L Ca2+ at 21°C, then washed to remove PP1. Western blots were probed with antibodies specific for RyR2 phosphorylated (RYR2-PS2809) or dephosphorylated (RYR2-2809deP) at serine-2809. B, Representative current fluctuations through a single RyR2, before and after 10 minutes incubation with PP1 followed by washout of PP1. C and D, Histograms illustrating Po and current amplitude before and after PP1 treatment. E and F, Amplitude histograms from control and PP1 treated channels.

View larger version (30K): [in this window] [in a new window]   Figure 6. Effects of PP1 on open and closed lifetime durations. The open and closed lifetime distributions and pdfs for a typical single RyR2 in the presence of 50 µmol/L Ca2+ in the cytosolic chamber (A) and after 10 minutes incubation with 5 U of PP1 followed by washout of PP1 (B). The best fits to the data were obtained by the method of maximum likelihood and the resulting time constants and percentage areas are shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study describes the gating and conducting properties of RyR2 channels that are associated with 3 different levels of phosphorylation at serine-2809 (dephosphorylated, 75% phosphorylated, and fully [stoichiometric] phosphorylated). Although currently impossible to monitor phosphorylation status at all phosphorylation sites on the channel, our work provides the first critical evaluation of the use of serine-2809 phosphorylation as a marker of channel activity, which has been a common practice in the literature. The level of phosphorylation was determined using 2 antibodies to the serine-2809 phosphorylation site, 1 of which is wholly specific for the dephosphorylated epitope, and the other, wholly specific for the phosphorylated epitope.¹⁴ RyR2 samples at the extremes of phosphorylation were used to calibrate the immunosignal from sheep hSR preparations. Using this approach, both antibodies indicated a control phosphorylation stoichiometry of approximately 75% of maximum.

We also demonstrated that the coincidence of serine-2809 phosphorylation with changes in channel activity is not described by a simple relationship. Modest changes in channel function are observed at serine-2809 phosphorylation levels up to 75% of maximum, with dramatic changes in channel activity occurring beyond this level of phosphorylation. This nonlinear relationship underscores the need for careful definition of phosphorylation status in studies of RyR2 function and suggests that the term hyperphosphorylation should only be reserved for protein phosphorylated to stoichiometry, rather than its current usage in the literature.

The prevailing message of current literature is that phosphorylation of RyR2 on serine-2809 to 75% of maximum is a state of hyperphosphorylation and that this is seen only in pathological situations in which the hyperphosphorylation results in channel dysregulation sufficient to cause cardiac dysfunction.¹ The present study reveals 2 central findings that do not support this hypothesis. Firstly, we have found that RyR2 exhibits a high level of basal phosphorylation at serine-2809, which is already 75% of maximum. A high level of phosphorylation at serine-2809 in control cardiac SR preparations is not uncommon and has been observed by several groups using protein derived from various species (rabbit, dog, mouse, human),^4,5,7 although 1 group has reported low levels.¹ Secondly, channel activity at this 75% phosphorylation level was low and displayed no sign of dysregulation (subconductance states) as has been reported by 1 group.¹ The functional characterization of channels phosphorylated to 75% of maximum (at serine-2809) was measured comprehensively. Thirty channels displayed a low P_o (0.059±0.01 [SEM]; n=30) with individual values ranging from 0.002 to 0.1943. Furthermore, much of our previous work has been conducted on control sheep cardiac RyR2, and thus one can assume that these channels were also phosphorylated to 75% of maximum at serine-2809, as the procedures for isolation of hSR and reconstitution of RyR2 into bilayers were identical to the present study. The behavior of channels in these previous studies (for example, Sitsapesan and Williams,¹⁵ Sitsapesan et al,¹⁶ and Chan et al¹⁷) is essentially identical to the behavior described for control channels in the present study, which increases the number of observations inconsistent with the previous hyperphosphorylation model of cardiac dysfunction.¹

A large enhancement in channel activity was observed following additional phosphorylation by PKA. This was characterized by an increase in P_o and an increase in the single-channel conductance of RyR2 but was again without the appearance of subconductance states. In parallel studies, the phosphorylation status of serine-2809 was assessed and was shown to be stoichiometric, suggesting that the enhanced activity of the channel measured in the bilayer, requires full (stoichiometric) phosphorylation at serine-2809. These clear changes to RyR2 channel function have not previously been reported, and they differ significantly from the original description of hyperphosphorylated channels.¹ In the original study, hyperphosphorylation of serine-2809 on RyR2 (75% of maximum) was associated with a modest increase in P_o (0.1 to 0.35), no change in mean open time, but the appearance of frequent openings to long-lived subconductance states.¹ RyR2 subconductance states and other forms of dysregulation have been observed following damage to the channel and on solubilization from the SR. Jiang et al⁵ observed subconductance states following limited proteolysis of RyR2, whereas Laver et al¹⁶ observed altered Ca²⁺ regulation of the channel following solubilization of SR with the detergent CHAPS. The present study has minimized hostile treatment of RyR2, avoiding all detergents and using a comprehensive cocktail of protease inhibitors in the isolation of hSR. It is conceivable that this might underlie the difference between the present observations and those of Marks and colleagues,¹ who used, in some studies, RyR2 that was solubilized and immunoprecipitated before functional interrogation.

Our final major finding is that a reduced level of phosphorylation at serine-2809 to below the 75% level is associated with a small increase in P_o, although lifetime analysis demonstrates that the mechanisms underlying this change in gating are very different to that observed when phosphorylating the channel to stoichiometry at serine-2809. The modest enhancement in P_o results from abbreviation of the closed lifetime durations without change to open lifetime durations. The results agree with those of Terentyev et al, who also found that phosphatase treatment increased RyR2 P_o.¹⁸ The use of PP1 to dephosphorylate RyR2 is not, of course, specific for serine-2809; PP1 will dephosphorylate at other sites including other PKA sites and CaMKII sites. Therefore, the rise in P_o that we observed when dephosphorylating from approximately 75% of maximum to approximately zero levels at serine-2809 either could have been attributable solely to dephosphorylation at serine-2809 or could have also included a component resulting from dephosphorylation at another site. As there is controversy over the number of PKA phosphorylation sites, the number of CaMKII sites and the functional effects of changing phosphorylation levels at these sites, at this point, no simple experiment can be performed to address this issue. From the evidence in the literature, however, we can reflect on the most likely situation. A second PKA phosphorylation site on RyR has been reported, serine-2030,⁴ although this has recently been questioned.¹⁹ This phosphorylation site is fully dephosphorylated in the absence of a ß agonist,^4,20 and, thus, it is likely to be dephosphorylated in our hSR preparation. The evidence suggests, therefore, that the effects we observe with PP1 are unlikely to be caused by dephosphorylation of serine-2030.

Could the increase in P_o that we observe after incubation with PP1 partially result from dephosphorylation of a specific CaMKII site? A large number of CaMKII phosphorylation sites have been suggested on RyR2,¹⁴ of which only serine-2815 has been identified to date.²¹ In advance of the identification of these sites, and knowledge of their phosphorylation status before and after PP1 treatment, there is little value in speculating what role they perform in the dephosphorylation-dependent enhancement of RyR2 activity. Instead, we can conclude that the phosphorylation status of serine-2809 at the 3 levels examined coincides with 3 distinct functional states of RyR2. In that capacity, it can be used as a marker of the functional state of the RyR2 protein, although a careful quantitative assessment of the stoichiometry of serine-2809 phosphorylation is required to interpret this accessible marker of channel activity.

It is interesting to note that in the analysis of 30 separate channels from a population phosphorylated on serine-2809 to 75% of maximum, no example of a channel exhibiting the "fully phosphorylated" characteristics (high P_o, long open lifetimes, increased single-channel conductance) was observed. If we assume that serine-2809 phosphorylation is the sole contributor to enhanced channel activity, then one might have expected to observe a fully active channel from this population in a random sample of 30, if phosphorylation status were normally distributed within the population. Our failure to observe a fully active channel in the control population would suggest that phosphorylation is not normally distributed and that the incidence of stoichiometric phosphorylation in this population is low. That would suggest a negative cooperativity in the process of phosphorylation of this site. Alternatively, serine-2809 phosphorylation may not be the sole trigger for enhanced RyR2 activity. One additional PKA phosphorylation site has been described within RyR2 (serine-2030⁴), and this site would be expected to become phosphorylated in response to PKA addition. However, serine-2030 phosphorylation is not associated with increased P_o, as judged by ryanodine binding,⁴ and thus either a combination of serine-2809 and serine-2030 phosphorylation is required or a dramatic activation of the channel accompanies phosphorylation of the final subunit in a tetramer on serine-2809.

The physiological significance of the present findings are considerable. The RyR, as isolated from the cell, has a phosphorylation status that positions the channel at a low point in a complicated activity landscape. Activity of the channel can be raised either by increasing the phosphorylation state of the channel or conversely by decreasing the phosphorylation state of the channel. These changes in activity are not symmetrical (in terms of magnitude) or equivalent (in terms of molecular basis) but, rather, accomplish a substantial increase in channel activity with modest increases in serine-2809 phosphorylation or conversely a modest increase in channel activity with substantial reductions in serine-2809 phosphorylation. This might allow for rapid and dramatic changes in function on PKA activation (a switch from 3 to 4 subunits phosphorylated at serine-2809) but also permits influence on RyR2 function from other pathways able to augment phosphatase activity and effect a more modest enhancement of RyR2 function.

Physiologically, stoichiometric phosphorylation at serine-2809 may be a very transient event that occurs in response to adrenergic stimulation. If stoichiometric phosphorylation at serine-2809 were to become less transitory, this would be expected to have serious consequences on the intracellular Ca²⁺ handling of cardiac cells because the changes to channel gating and conductance are so extreme. Such a situation might exist in heart failure. Circulating catecholamine levels can be elevated in heart failure patients²² and Marx et al¹ have provided evidence indicating that the levels of phosphatases associated with RyR2 are lowered in heart failure. Hence, there is potential for dysregulation of the balance between kinase and phosphatase activity at RyR2 enabling a more readily achievable stoichiometric phosphorylation at serine-2809. Further work is required to determine whether stoichiometric phosphorylation of serine-2809 on RyR2 can occur for prolonged periods in heart disease.

	Acknowledgments

 
Sources of Funding

This work was supported by the British Heart Foundation.

Disclosures

J.C. operates a company that distributes antibodies commercially. These antibodies were used in the present study.

	Footnotes

 
Original received December 20, 2005; resubmission received March 30, 2006; accepted May 9, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
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