Skip Navigation

Cardiovascular Research 2005 65(1):93-103; doi:10.1016/j.cardiores.2004.09.005
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Silverman, B. d.Z.
Right arrow Articles by Shattock, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Silverman, B. d.Z.
Right arrow Articles by Shattock, M. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2004, European Society of Cardiology

Serine 68 phosphorylation of phospholemman: acute isoform-specific activation of cardiac Na/K ATPase

Benjamin d.Z. Silvermana,1, William Fullera,1, Philip Eatona, Juelin Dengb, J. Randall Moormanc, Joseph Y. Cheungd, Andrew F. Jamese and Michael J. Shattocka,*

aCardiovascular Division, King's College London, The Rayne Institute, St Thomas' Hospital, London SE1 7EH, UK
bWest China Hospital, Sichuan University, People's Republic of China
cDepartment of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA
dWeis Center for Research, Geisinger Medical Center, Danville, PA 17822, USA
eDepartment of Physiology, School of Medical Sciences, University of Bristol, Bristol, United Kingdom

* Corresponding author. Tel.: +44 20 7188 0945; fax: +44 20 7188 3902. Email address: michael.shattock{at}kcl.ac.uk

Received 15 July 2004; revised 3 September 2004; accepted 6 September 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: The mechanism by which the cardiac Na/K ATPase (NKA) is regulated by phosphorylation is controversial. We have used the perforated-patch technique to limit cell dialysis and maintain conditions as near physiological as possible.

Methods: NKA pump current (Ip) was measured in isolated guinea pig ventricular myocytes, and its components (I{alpha}1 and I{alpha}2) defined by their differing dihydroouabain sensitivities.

Results: Treatment with 1 µmol/l forskolin for 4 min at 35 °C caused a significant increase in I{alpha}1 of 36 ± 15% (P<0.05, n=6), but no change in I{alpha}2. The presence of the PKA selective inhibitor H89 (50 µmol/l) throughout the protocol blocked the effect of the forskolin on I{alpha}1. Treatment with H89 alone did not change I{alpha}1 or I{alpha}2. Isoelectric focusing gels of the NKA {alpha}1 subunit demonstrated six charge states, which were unaltered following treatment with forskolin. Western blots using an antibody specific for the PKA phosphorylation consensus site on the {alpha}1 subunit showed no change in the phosphorylation status of this residue following forskolin treatment. The sarcolemmal protein phospholemman (PLM) was found associated with NKA {alpha}1 but not {alpha}2 subunits by immunoprecipitation and immunofluorescence. PLM was phosphorylated at serine 68, but not 63, following treatment with forskolin.

Conclusions: PKA-dependent, {alpha}1-specific NKA activation may be mediated through phosphorylation of the accessory protein PLM, rather than direct {alpha}1 subunit phosphorylation.

KEYWORDS Ion pumps; Na/K pump; Protein kinase A; Protein phosphorylation

Abbreviations: NKA, Na/K ATPase • PLM, phospholemman • DIDS, 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid

This article is referred to in the Editorial by P. Fransen (pages 13–15) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
In excitable cells, the transmembrane sodium (Na) gradient, established by the Na/K ATPase (NKA), is essential for a plethora of cellular functions. In cardiac cells, approximately 40% of the resting ATP consumption has been attributed to the turnover of the NKA [1]. The metabolic energy invested in establishing the Na gradient is then used to drive numerous ionic and metabolic transport processes and is a key prerequisite for the control of cell excitability and volume. Alterations in the activity and regulation of the NKA can therefore have a profound effect on normal cellular function.

In many tissues, the cAMP–PKA signaling cascade is involved in NKA regulation (for review, see Ref. [2]); however, the effect of PKA activation on NKA activity is tissue- and model-specific. In the kidney, PKA activation increases NKA activity in rat proximal convoluted tubule cells through insertion of additional pump units in the plasma membrane [3]. However, other researchers report that forskolin has an inhibitory effect on NKA activity in COS-7 cells, with no effect on the amount of pump protein at the membrane [4].

There is no evidence that the PKA cascade alters NKA expression at the cell surface in ventricular myocytes. In guinea-pig ventricular myocytes, isoprenaline has a Ca2+-modulated effect on NKA [5]. PKA-dependent, isoprenaline-induced inhibition of NKA at low intracellular Ca2+ has been described in guinea-pig myocytes [6,7]. However, other researchers have reported that, in the same cell type, forskolin activates NKA at relatively low intracellular Ca2+ [8]. It has also been reported that PKA stimulation in guinea-pig ventricular myocytes alters the activity of the predominant (low ouabain-affinity) {alpha}1 isoform of NKA but not the {alpha}2 isoform [9]. All these studies used the whole-cell voltage-clamp technique, which dialyses the cytoplasm and may alter relevant intracellular messenger cascades, while clamping the intracellular ion concentrations to those in the pipette. In the present study, the perforated patch technique has been employed to minimize these limitations.

While PKA activation has been demonstrated to cause phosphorylation of the {alpha}1 subunit of NKA in studies on purified protein [10,11], there is limited evidence for phosphorylation of NKA by PKA in living cells or tissues [12,13]. Indeed, crystallographic data suggest that the postulated PKA phosphorylation site in the {alpha}1 subunit is inaccessible to the enzyme [14]. If PKA stimulation does not act via direct phosphorylation of the NKA {alpha} subunit, then alternative accessory protein targets must be considered. We have recently reported a substantial, ischemia-induced, PKA-dependent activation of NKA activity in sarcolemmal membranes purified from rat hearts [15]. This coincided with phosphorylation of phospholemman (PLM, FXYD1) in this tissue. PLM is the major sarcolemmal substrate for PKA and PKC in the heart [16–18] and belongs to the FXYD family of low molecular mass proteins that act as tissue-specific regulators of NKA [19].

The aim of the present study was therefore to investigate phosphoregulation of NKA in cardiac myocytes under aerobic conditions. We have investigated the immediate effects of PKA activation on NKA. Using whole-cell voltage clamping and the perforated-patch technique, we show that acute activation of PKA with forskolin induces an isoform-specific increase in {alpha}1 but not {alpha}2 pump current, and this activation is associated with the phosphorylation of PLM at serine 68. The implications of this for cardiac function will be discussed.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Animals
All animals used in this study received humane care in accordance with "Guidance on the Operation of the Animals (Scientific Procedures) Act of 1986" published by HM Stationery Office, London UK and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). This study was also subjected to local ethical review by the Ethical Review Process Committee of King's College London.

2.2. Cell isolation
Guinea-pig and rat ventricular myocytes were isolated, as described previously [20,21]. The yield of rod-shaped cells was typically ~70%. The cell suspension was allowed to stand at room temperature for 1 h before use.

2.3. cAMP assay procedure
Cells (treated with forskolin, 1 µmol/l or vehicle, 4 min at 35 °C) were resuspended in phosphate buffer from a commercially available cAMP enzyme immunoassay kit (Cayman Chemical, MI, USA) supplemented with 10% trichloroacetic acid, at a volume of 100 µl buffer per mg protein. The cAMP assay was used according to the manufacturer's directions. cAMP content of cells was normalized to cell protein content, which was determined using the BioRad protein assay reagent.

2.4. Voltage clamping
Single isolated guinea-pig ventricular myocytes were voltage-clamped using the perforated-patch technique. Patch electrodes were made from borosilicate glass capillaries (Clark Electromedical Instruments, Reading, UK) and were fire-polished (DMG Universal Puller, Zeitz-Instrumente Ventreibs GmbH, Germany). The electrodes had a resistance of 1–2 M{Omega} when filled with the standard pipette solution. Current signals were recorded using an Axopatch 1-C single electrode voltage-clamp amplifier (Axon Instruments, USA) controlled by a microcomputer running pClamp software (v.6, Axon Instruments). A gigaohm seal was rapidly established between the electrode and the cell surface, and series resistance was monitored by a repetitive +5 mV pulse (5msec duration) from a holding potential of –40 mV. The membrane patch was allowed to permeabilize, and series resistance typically fell to <20 M{Omega} within 10 min. Current signals were filtered at 1 kHz and sampled at 3 kHz and converted into conductance units by normalizing current records to cell capacitance. The typical cell size used in these experiments, as defined by cell capacitance, was 106 ± 7 pF (n=22).

The pipette and extracellular solutions were designed to inhibit all voltage-gated channels and the Na/Ca exchanger. Under these conditions, the Na/K pump current (Ip) can be defined as that inhibitable by the removal of extracellular K or the addition of 1 mmol/l dihydroouabain (DHO). Inasmuch as the DHO-sensitive and K-sensitive pump currents are identical (data not shown), we have defined the whole-cell Na/K pump current as that sensitive to the removal of extracellular K.

The IC50 for DHO block of {alpha}2-mediated pump current (I{alpha}2) is 0.75 µmol/l and, for {alpha}1-mediated current (I{alpha}1), the IC50 is 75 µmol/l. The contributions of the {alpha}1 and {alpha}2 isoforms of NKA to total pump current were therefore defined by their respective sensitivities to DHO. To block I{alpha}2 5 µmol/l DHO was used, while 1 mmol/l DHO or K-free extracellular solution blocks both components of Ip. The use of DHO to distinguish the contributions of NKA isoforms to Ip in guinea-pig ventricular myocytes is well established and has been characterized in detail elsewhere [9].

2.4.1. Solutions
All studies were performed at 35 °C. Solutions were made up using deionised water with Analar Grade chemicals (BDH, UK). The standard pipette solution contained (in mmol/l) NaCl 140, KCl 5, MgCl2 1, aspartic acid 25, HEPES 10, pH 7.16. Amphotericin B (from Streptomyces, Sigma, UK; 30 mg/ml) in DMSO was added to the pipette solution on the day of use to a final concentration of 225 µg/ml (DMSO=0.74% v/v). The standard extracellular solution contained (in mmol/l) NaCl 140, KCl 5, MgCl2 1, NiCl2 2, BaCl2 1, glucose 10, HEPES 5, pH 7.36. In some experiments where forskolin was used to raise cAMP, a chloride-balanced extracellular test solution was used to limit cAMP-activated Cl currents. In this chloride-balanced solution, NaCl was lowed to 9.5 mmol/l and replaced with 130.5 mmol/l Na isethionate. KCl was replaced with 5 mmol/l K isethionate, and glibenclamide (200 µmol/l) and 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 1 mmol/l) were also added to further limit Cl currents. K-free extracellular solutions were made by removing KCl in the solution with no correction for osmolarity.

2.5. Quantitative immunoblotting
Myocytes were treated with forskolin or PMA, and aliquots of cell suspension mixed with SDS-PAGE or isoelectric focusing loading buffer immediately in order to ensure phosphatases were unable to dephosphorylate proteins prior to analysis. SDS-PAGE, isoelectric focusing, and quantitative immunoblotting were carried out as described previously [15].

2.6. Antibodies
All NKA subunits and secondary antibodies were as previously described [15]. We developed antibodies to distinguish four forms of PLM: total PLM, dephosphorylated PLM, PLM phosphorylated at S68 (the PKA site), and PLM phosphorylated at S63 (which, along with S68, is phosphorylated by PKC). Total PLM was detected using an antibody raised in chickens that we have described previously [15], which we have called N1. We call the antibody to the dephosphorylated form C2 [22].

The peptide antigen that mimicked the C-terminus of PLM was DEEEGTFRSSIRRLS(68)TRRR. To make phosphorylation-specific antibodies, phosphoserines were substituted at S63 and S68. Antibodies were generated in rabbits and affinity purified using peptides linked to agarose (Bethyl Labs). Antibodies to the phosphorylated peptides were first cleared of antibodies to the dephosphorylated peptide. We call the antibodies to the phosphorylated forms CP68 and CP63 respectively.

FITC-labelled antimouse and antirabbit IgG was supplied by Vector Laboratories Inc (Burlingame, CA). Texas Red-labelled anti-chicken IgY was supplied by Abcam (Cambridge, UK).

2.7. Immunoprecipitation of PLM
Immunoprecipitation studies were carried out exactly as described previously [15] but using the detergent n-dodecyl octaethylene glycol monoether (C12E8) at a concentration of 10 mmol/l (5.4 mg/ml, a concentration reported to preserve interactions between NKA subunits [23]) to solubilize proteins and in all washes.

2.8. Immunofluorescence
Cells were plated onto laminin-coated coverslips for 30 min at room temperature, washed once with phosphate-buffered saline (PBS), fixed for 20 min at room temperature with 0.5% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS, blocked with 2% defatted powdered milk in PBS, and immunolabelled. Anti-PLM IgY and anti-NKA {alpha}1 or {alpha}2 IgG were diluted 1:200 and detected with FITC-linked anti-IgG and Texas Red-linked anti-IgY.

Fluorescent images were collected with a Hamamatsu Orca ER camera on a Zeiss Axiovert 100 microscope using Improvision (Warwick, UK) OpenLab 2.0.5 software directing a piezo z-axis drive in 200-nm steps, with filter wheel control of excitation and Pinkel filter set (Omega Optical) to handle emitted wavelengths. Images were volume deconvolved and passed to Vococity (Improvision) software for three-dimensional analysis.

2.9. Statistical analysis
Quantitative data are shown as means ± standard errors of the means (S.E.M.). Differences in mean measurements between experimental groups were tested by ANOVA followed by a t test, and differences were considered significant at the p<0.05 level.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Measurement of NKA current (IP)
Whole-cell currents were measured at 0 mV using the perforated-patch technique to minimize cell dialysis. Currents due to the {alpha}1 and {alpha}2 forms of NKA (I{alpha}1 and I{alpha}2) were distinguished according to their sensitivity to dihydroouabain (DHO). Hence, we were able to quantify the currents of the two isoforms individually. Inhibition of Ip by DHO occurred rapidly (<1 min), and recovery from inhibition was also very rapid, with an "overshoot" of Ip followed by a return to the preinhibition level within 3 min.

The acute effect of the agonist forskolin on Ip was assessed using the protocol described in Fig. 1A. I{alpha}1 and I{alpha}2 were measured twice with 7 min between measurements. Forskolin was applied for 4 min before measurement of Ip for the second time. A current record is shown in Fig. 1B. In the absence of agonist, successive measurements gave identical values of I{alpha}1 and I{alpha}2 (Fig. 1C).


Figure 1
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Experimental protocol. Panel (A) shows the protocol distinguishing high (I{alpha}2) and low (I{alpha}1) affinity NKA currents. I{alpha}2 is blocked by 5 µmol/l DHO, and both currents are blocked by zero K. A typical current record from guinea-pig ventricular myocytes is shown in panel (B). The reproducibility of current records during the protocol is demonstrated in the bar graph in panel (C). All recordings were made in the standard test solution (see Materials and methods; n=6 cells from five animals).

 
3.2. Forskolin treatment elevates cAMP and stimulates I{alpha}1 but not I{alpha}2
Forskolin (1 µmol/l) increased cAMP from 15.0 ± 1.6 to 27.0 ± 3.3 pmol/mg protein, a significant increase (p<0.05, paired t test) of 79.7 ± 12.7%. These values are in line with cellular cAMP concentrations measured in cell lines using this method [24].

The effect of forskolin treatment on the {alpha}1 and {alpha}2 components of Ip is shown in Fig. 2A. Forskolin elicited an outward current, which is likely to reflect residual cAMP-dependent chloride current that is incompletely blocked by glibenclamide and DIDS. This current is K-independent and did not interfere with the measurement of components of Ip (not shown). A significant increase in I{alpha}1 followed the addition of 1 µmol/l forskolin (p<0.05, paired t test). No change in I{alpha}2 was seen during the protocol (p>0.05). Total Ip also increased significantly (p<0.05, paired t tests) during treatment with forskolin (not shown). The specific increase in I{alpha}1 with forskolin treatment was 36 ± 15%. The increase in the total NKA pump current was 31 ± 7%.


Figure 2
View larger version (10K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Forskolin (1 µmol/l) stimulates I{alpha}1 but not I{alpha}2. Forskolin (1 µmol/l) was applied in the second pulse: mean data are shown in panel (A). There was a significant increase in I{alpha}1 in guinea-pig ventricular myocytes with forskolin treatment (*: p<0.05, paired t test) but no effect on I{alpha}2 (n=6 cells from five animals). The effect of H89 (50 µmol/l) on the response to forskolin is shown in panel (B). H89 blocked the stimulatory effect of forskolin on I{alpha}1 (n=5 cells from two animals). All recordings were made in the chloride-balanced test solution (see Materials and methods).

 
3.3. Effect of forskolin is blocked by H89, but H89 has no effect alone
H89 (50 µmol/l) blocked the stimulatory effect of forskolin on I{alpha}1 (Fig. 2B). H89 also inhibited forskolin-activated ouabain-insensitive outward currents (not shown). In addition, H89 blocked the changes in the total Ip induced by treatment with forskolin, (p>0.05, paired t tests, not shown).

Fig. 2B shows that the initial I{alpha}1 measured with H89 present was 284 ± 34 fA/pF, while Fig. 2A shows an initial I{alpha}1 of 383 ± 51 fA/pF in the absence of H89. A significant difference caused by H89 treatment might indicate a significant acute basal regulation of NKA by PKA. However, these results were obtained in different cells from different animals, and statistical analysis indicates that I{alpha}1 was not significantly lower in the H89 exposed group (p>0.05, unpaired t test). However, to systematically investigate whether in unstimulated myocytes, I{alpha}1 is under acute basal regulation by endogenous PKA, the effects of H89 on unstimulated Ip were studied. Fig. 3 shows the effect of applying H89 (50 µmol/l) as the "agonist" during the second measurement of Ip in our standard protocol. Mean data indicate no significant change in the components of Ip or total Ip (not shown).


Figure 3
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 H89 alone is without effect on components of Ip. The effect of applying H89 (50 µmol/l) alone on components of Ip in guinea-pig ventricular myocytes was assessed. Mean data are shown (n=6 cells from two animals). H89 was without effect on components of Ip over this time scale. All recordings were made in the chloride-balanced test solution (see Materials and methods).

 
3.4. Forskolin treatment does not phosphorylate {alpha}1 but phosphorylates PLM at serine 68
We investigated the phosphorylation status of NKA subunits following treatment of guinea-pig myocytes with forskolin using isoelectric focusing gels and phosphospecific antibodies. NKA {alpha}1 subunit was separated on an IEF gel in pH range 4–7, as described previously [15]. Fig. 4A indicates that the distribution of the {alpha}1 protein between different charge states was not altered following treatment with forskolin, indicating no change in its gross phosphorylation status. We routinely observe multiple charge states for the {alpha}1 subunit by IEF [15], however the relative distribution between these states was identical in the two treatment groups.


Figure 4
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Forskolin treatment phosphorylates PLM at S68 but does not phosphorylate {alpha}1. Panel (A) shows a typical section of an IEF gel immunoblotted for NKA {alpha}1 subunit from guinea-pig ventricular myocytes. The {alpha}1 subunit exists in six charge states in both the control [label (C)] and forskolin-treated [label (F)] samples. The proportion of {alpha}1 subunit detected in each band was unchanged following treatment with 1 µmol/l forskolin (n=3). Panel (B) shows immunoblots for NKA {alpha}1 subunit from guinea-pig ventricular myocytes. A typical immunoblot with an antibody specific for phosphorylated serine 938 (uppermost immunoblots) shows faint bands in both the control [label (C)] and forskolin-treated [label (F), 1 µmol/l] samples, which are not significantly different (n=3). Probing the same samples with an anti-{alpha}1 antibody (central immunoblots) demonstrated the presence of NKA {alpha}1 subunit: the amount detected in the two groups was not significantly different. A positive control was prepared by incubating NKA {alpha}1 subunit with PKA in the presence of 0.02% SDS (lower immunoblots). PKA treatment (+) led to a sixfold increase in phosphospecific antibody binding. Immunoblots are quantitated on the right side of panel (B): graphs show mean normalized in each case to the control values. Panel (C) shows immunoblots with antibodies phosphospecific for PLM (left side), which are quantitated on the right side of panel (C). While the total amount of PLM is not different between control [label (C)] and forskolin-treated [label (F)] samples (not shown), serine 68 on PLM is significantly phosphorylated, while serine 63 is not following treatment with forskolin (n=3).

 
The phosphorylation status of serine 938 (in the PKA consensus phosphorylation site on the NKA {alpha}1 subunit) was investigated using a phosphospecific antibody to this site [25]. Fig. 4B indicates that treatment of cells with forskolin was without effect on the phosphorylation status of this residue, indicating that NKA {alpha}1 subunit is not likely to be the target for PKA in the isoform-specific stimulation of NKA that we have observed. Like other researchers [13], we are unable to detect phosphorylation of this residue by PKA in the absence of low concentrations of detergent to unfold the {alpha}1 subunit (Fig. 4B).

We [15], and others [19,26], have recently proposed that phospholemman (PLM) forms part of the NKA enzyme complex in the heart. We therefore investigated the phosphorylation status of PLM using antibodies specific for the PKA (serine 68) and PKC (serines 63 and 68) consensus phosphorylation sites [27] on PLM. Fig. 4C indicates that, while forskolin treatment was without effect on serine 63, there was an approximately fivefold increase in the phosphorylation status of serine 68 following treatment with forskolin.

3.5. Antibody characterization
We characterized antibodies to dephosphorylated (C2) [22], serine 63 phosphorylated (CP63), and serine 68 phosphorylated (CP68) PLM by immunoblotting samples from rat ventricular myocytes treated with forskolin (100 µmol/l) and PMA (100 nmol/l) to activate PKA and PKC, respectively. PKA is reported to phosphorylate serine 68, and PKC both serines 63 and 68, of PLM [27]. We characterized the phosphospecific antibodies in rat cardiomycoytes because rat PLM has been cloned whereas guinea-pig PLM has not. However, given the high amino acid identity between cloned mammalian PLMs (the intracellular regions of rat, mouse, human, and canine PLM are 100% identical except for a single conserved substitution in murine PLM), we believe it is reasonable to extend the use of these phosphospecific antibodies to guinea-pig samples once we have established their specificity. Immunoblots are shown in Fig. 5.


Figure 5
View larger version (36K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Antibody characterization. Antibodies C2, CP63, and CP68 were characterized using PMA and forskolin-treated rat ventricular myocytes. Cells were treated with forskolin [label (F), 1 µmol/l] or PMA [label (P), 100 nmol/l] for the times indicated (in minutes) at 37 °C, immediately mixed with SDS-PAGE loading buffer, proteins separated by SDS-PAGE, and blotted using the antibody indicated. Representative blots are shown. Signals were quantitated using the NIH Image software package and normalized to the signal at time zero. Mean data (n=3 animals) are shown in bar graphs. The N1 (total PLM) antibody indicated there was equal loading of PLM in all lanes (not shown).

 
Binding of the C2 antibody is reduced when PLM is phosphorylated (Fig. 5, upper panel). This is particularly pronounced in cells treated with PMA, which might suggest that the C2 epitope includes serine 63 but possibly not serine 68; the binding patterns of C2 and CP63 are reciprocal.

The CP63 antibody shows a substantial, sustained increase in binding following treatment with PMA (Fig. 5, middle panel). CP68 reports phosphorylation of serine 68 in cells treated with either forskolin or PMA (Fig. 5, bottom panel). Serine 68 phosphorylation is better maintained in cells treated with PMA than in cells treated with forskolin. CP68 cross-reacts with another protein in cells treated with forskolin, which we believe to be serine 16 phosphorylated phospholamban. The amino acid sequences surrounding the PKA phosphorylation sites in PLM and phospholamban are very similar.

In addition to data generated with forskolin and PMA-treated rat ventricular myocytes, antibodies N1, C2, CP63, and CP68 detected no bands in immunoblots of samples from PLM-deficient mice (not shown). Using purified, unphosphorylated recombinant PLM [28], we find that CP68 is specific for PLM phosphorylated at S68 (manuscript under preparation), but CP63 cross-reacts with unphosphorylated PLM to a small extent.

3.6. NKA {alpha}1, β1, and PKA but not {alpha}2 coprecipitate with PLM
We have recently reported an association between NKA {alpha}1 subunit and PLM in sarcolemmal preparations from rat hearts [15]. In the current study, we investigated the association between PLM and other NKA subunits by immunoprecipitation with PLM antibodies under conditions known to preserve the association between these NKA subunits [23]. When PLM was immunoprecipitated, NKA {alpha}1 but not {alpha}2 subunits were coprecipitated, as was the catalytic subunit of PKA (Fig. 6). Treatment of cells with forskolin substantially increased the amount of PKA that copurified with PLM.


Figure 6
View larger version (50K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 NKA {alpha}1 and PKA but not {alpha}2 coprecipitate with PLM. PLM was immunoprecipitated from guinea-pig ventricular myocytes under conditions that preserve interactions between NKA subunits, and immunoprecipitated complexes immunoblotted for the proteins indicated. NKA {alpha}1 but not {alpha}2 was found associated with PLM. A positive control for the {alpha}2 subunit (unfractionated guinea-pig cardiac myocytes) is shown (+). PKA was also associated with PLM, and this association was increased in cells treatment with forskolin [label (F); 1 µmol/l] compared to vehicle [label (C)]. Results are representative of independent immunoprecipitations from cells from three different animals.

 
3.7. Immunofluorescent colocalization of PLM and NKA subunits
We investigated the cellular localization of NKA subunits by immunofluorescence microscopy. NKA {alpha}1 and {alpha}2 are shown in green in Fig. 7, PLM in red, and overlap in yellow. NKA {alpha}1 and PLM are predominantly located in the peripheral sarcolemma, while NKA {alpha}2 is mainly distributed in the t-tubules. There is substantial overlap between PLM and {alpha}1 in guinea-pig ventricular myocytes; approximately 90% of PLM staining on the peripheral sarcolemma is found associated with {alpha}1 staining. Substantially less overlap is observed between PLM and {alpha}2. The subcellular distribution of NKA subunits was not altered following treatment with forskolin (not shown).


Figure 7
View larger version (27K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Immunofluorescent colocalization of PLM and NKA subunits. Guinea-pig ventricular myocytes were fixed, permeabilized, and stained for NKA {alpha}1, {alpha}2 (both shown in green), and PLM (shown in red). There is substantial overlap (shown in yellow) between PLM and {alpha}1, but not PLM and {alpha}2. Nuclei in the plane of focus are shown in blue.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
This study indicates that, in guinea-pig ventricular myocytes, a forskolin-activated increase in cAMP is associated with a PKA-mediated {alpha}1 isoform-specific increase in NKA activity. Isoelectric focusing gels and a phosphospecific antibody for serine 938 on NKA {alpha}1 subunit suggest that PKA regulation of NKA in guinea-pig ventricular myocytes is not mediated through direct {alpha}1 subunit phosphorylation. Instead, we propose that phosphorylation of the small accessory protein phospholemman (PLM) by PKA at serine 68 is responsible for the observed isoform-specific activation of NKA.

We have specifically investigated the acute effect of forskolin on cardiac NKA. Under physiological conditions, the stimulation of NKA we observe may shorten the action potential duration and balance the increased sarcolemmal Na and K fluxes during adrenergic stimulation of the heart. However, the effect of prolonged activation of PKA on components of Ip remains to be established.

Our striking isoform-specific observation is in agreement with other reports of a similar PKA-mediated {alpha}1 isoform-specific effect of isoprenaline in whole-cell voltage-clamped guinea-pig myocytes [9]. In addition, this study provides a second model in which PLM is found to regulate NKA activity. Previously, we established that the ouabain-sensitive ATPase activity of NKA (Vmax) was activated in concert with phosphorylation of PLM by PKA in the rat heart during ischemia [15]. We now report that the ion transporting function of NKA, measured as a membrane current under aerobic conditions, is also activated concomitant with a PKA-mediated phosphorylation of PLM. The concentration of the H89 used in this study (50 µmol/l) is somewhat higher than that used by other investigators. H89 at 10 µmol/l will not discriminate between PKA and other kinases such as MSK1, S6K1, and ROCK-II [29]. While it is possible that these kinases may be involved, they have not previously been implicated in PLM phosphorylation. The combination of the effect of H89, the immunoprecipitation of PKA with PLM, the fivefold increase in phosphorylation of the PKA consensus sequence in PLM, and the known robust activation of PKA by forskolin leads us to suggest that these effects are mediated via PKA.

Intracellular Ca is critical in determining whether PKA activation causes inhibition (at Ca less than 150 nmol/l) or stimulation of NKA in guinea-pig ventricular myocytes [5–7]. Our study has avoided dialyzing cell contents by using the perforated patch technique, so Ca is not buffered. It is noteworthy that, in cardiac myocytes, PKC (among other kinases) is regulated in part by Ca. PLM is a substrate for PKC, and it has been reported that phosphorylation of PLM by PKA at serine 68 limits the ability of PKC to phosphorylate it at serine 63, indicating that PLM may be able to integrate signaling from different kinases [30]. This may explain the dependence on Ca of PKA stimulation of NKA that other authors have reported: the effect of PKA may be determined by the degree to which PLM is PKC-phosphorylated. In this study, we saw no change in the phosphorylation status of PLM at serine 63, indicating that changes in PKC activity are not relevant to the NKA stimulation we observe.

We do not observe an interaction between PLM and NKA {alpha}2 subunit. This is in contrast to other reports, which have observed functional [26] and physical [23] interactions between these proteins. We cannot rule out the possibility that NKA {alpha}2 does copurify with PLM in our immunoprecipitation experiments but in an amount that is below the level of detection by immunoblotting. However, our immunofluorescence data may shed some light on the contradiction between our data and other reports. NKA {alpha}2 subunit is targeted predominantly to the t-tubules in guinea-pig ventricular myocytes, while approximately 75% of PLM is found in the peripheral sarcolemma. Targeting of these proteins to predominantly different cellular compartments renders an interaction between them impossible. Hence, our data do not rule out that NKA {alpha}2 and PLM may associate when expressed in the same membrane compartment (for example in Xenopus oocytes [26] or regions of the CNS [23]), or that PLM phosphorylation may effect the activity of NKA {alpha}2 when they are able to interact. In guinea-pig ventricular myocytes, PLM only interacts with NKA {alpha}1 because this is the only isoform of NKA in the same subcellular location.

The ability to regulate the activity of NKA {alpha}1 independent of {alpha}2 has important implications for cardiac function. It has been proposed that the {alpha}1 and {alpha}2 forms of NKA have distinct functional roles in cardiac physiology. Specifically, {alpha}2 NKA has been proposed to regulate Ca signaling through a functional association with the sodium–calcium exchanger [31], although this hypothesis has been challenged [32]. Our study sheds no light on this controversy directly. However, if NKA {alpha}1 and {alpha}2 are indeed playing different roles in the physiology of the heart, it is natural to assume that the regulatory mechanisms controlling the two isoforms may be distinct. The specific functional and physical colocalization of PLM with NKA {alpha}1 but not {alpha}2 provides the means to achieve differential regulation of these NKA isoforms. The physiological significance of our observations is therefore clear: PLM is a functional component of the NKA enzyme complex, and phosphorylation of PLM modulates the ATPase and pumping function of NKA. We propose that PLM is the functional link between PKA and NKA in the heart. Given that PLM is also a substrate for PKC, it is tempting to speculate that PLM may also provide a functional link between PKC and NKA. Experiments are underway in PLM-deficient mice to test these hypotheses. The clinical significance of this observation is also clear: inhibition of PKA (through blockade of β-adrenergic receptors) and PKC (through inhibition of angiotensin-converting enzyme) improves the prognosis of patients in heart failure. A common path may be the reduced phosphorylation of PLM. Hypophosphorylation of PLM in heart failure may contribute to the elevation of intracellular Na and contractile dysfunction by modifying NKA activity. This is currently under investigation.

In summary, we propose that isoform-specific activation of NKA by PKA in guinea-pig ventricular myocytes is through phosphorylation of PLM rather than the catalytic subunits of NKA. PLM is an integral regulator of NKA in the heart.


   Acknowledgements
 
This work was supported by grants from the British Heart Foundation, The Wellcome Trust, the NIH, the American Heart Association, and the Trustees of St. Thomas' Hospital.

We thank Angela Jen and Roger Morris at the Molecular Neurobiology Group, MRC Centre for Developmental Neurobiology (King's College London, Guy's Campus) for assistance with immunofluorescence.

No authors have conflicts of interest to disclose.


   Notes
 
1 These authors contributed equally to this work. Back

Time for primary review 18 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

  1. Ismail-Beigi F., Edelman I.S. The mechanism of the calorigenic action of thyroid hormone. Stimulation of Na+,K+-activated adenosine triphosphatase activity. J. Gen. Physiol. (1971) 57:710–722.[Abstract/Free Full Text]
  2. Therien A.G., Blostein R. Mechanisms of sodium pump regulation. Am. J. Physiol. (2000) 279:C541–C566.[Web of Science]
  3. Carranza M.L., Rousselot M., Chibalin A.V., Bertorello A.M., Favre H., Feraille E. Protein kinase A induces recruitment of active Na+,K+-ATPase units to the plasma membrane of rat proximal convoluted tubule cells. J. Physiol. (1998) 511:235–243.[Abstract/Free Full Text]
  4. Andersson R.M., Cheng S.X., Aperia A. Forskolin-induced down-regulation of Na+,K(+)-ATPase activity is not associated with internalization of the enzyme. Acta Physiol. Scand. (1998) 164:39–46.[CrossRef][Web of Science][Medline]
  5. Gao J., Mathias R.T., Cohen I.S., Baldo G.J. Isoprenaline, Ca2+ and the Na(+)–K+ pump in guinea-pig ventricular myocytes. J. Physiol. (1992) 449:689–704.[Abstract/Free Full Text]
  6. Gao J., Mathias R.T., Cohen I.S., Shi J., Baldo G.J. The effects of beta-stimulation on the Na(+)–K+ pump current–voltage relationship in guinea-pig ventricular myocytes. J. Physiol. (1996) 494:697–708.[Abstract/Free Full Text]
  7. Gao J., Cohen I.S., Mathias R.T., Baldo G.J. The inhibitory effect of beta-stimulation on the Na/K pump current in guinea pig ventricular myocytes is mediated by a cAMP-dependent PKA pathway. Pflugers Arch. (1998) 435:479–484.[CrossRef][Web of Science][Medline]
  8. Kockskamper J., Erlenkamp S., Glitsch H.G. Activation of the cAMP-protein kinase A pathway facilitates Na+ translocation by the Na+–K+ pump in guinea-pig ventricular myocytes. J. Physiol. (2000) 523:561–574.[Abstract/Free Full Text]
  9. Gao J., Wymore R., Wymore R.T., Wang Y., McKinnon D., Dixon J.E., et al. Isoform-specific regulation of the sodium pump by alpha- and beta-adrenergic agonists in the guinea-pig ventricle. J. Physiol. (1999) 516:377–383.[Abstract/Free Full Text]
  10. Bertorello A.M., Aperia A., Walaas S.I., Nairn A.C., Greengard P. Phosphorylation of the catalytic subunit of Na+,K+-ATPase inhibits the activity of the enzyme. Proc. Natl. Acad Sci. U. S. A. (1991) 88:11359–11362.[Abstract/Free Full Text]
  11. Fisone G., Cheng S.X., Nairn A.C., Czernik A.J., Hemmings H.C. Jr., Hoog J.O., et al. Identification of the phosphorylation site for cAMP-dependent protein kinase on Na+,K(+)-ATPase and effects of site-directed mutagenesis. J. Biol. Chem. (1994) 269:9368–9373.[Abstract/Free Full Text]
  12. Beguin P., Beggah A.T., Chibalin A., Burgener-Kairuz P., Jaisser F., Mathews P.M., et al. Phosphorylation of the Na,K-ATPase {alpha}-subunit by protein kinase A and C in vitro and in intact cells. Identification of a novel motif for PKC-mediated phosphorylation. J. Biol. Chem. (1994) 269:24437–24445.[Abstract/Free Full Text]
  13. Feschenko M.S., Sweadner K.J. Conformation-dependent phosphorylation of Na,K-ATPase by protein kinase A and protein kinase C. J. Biol. Chem. (1994) 269:30436–30444.[Abstract/Free Full Text]
  14. Sweadner K.J., Feschenko M.S. Predicted location and limited accessibility of protein kinase A phosphorylation site on Na–K-ATPase. Am. J. Physiol. (2001) 280:C1017–C1026.[Web of Science]
  15. Fuller W., Eaton P., Bell J.R., Shattock M.J. Ischemia-induced phosphorylation of phospholemman directly activates rat cardiac Na/K-ATPase. FASEB J. (2004) 18:197–199.[Abstract/Free Full Text]
  16. Presti C.F., Scott B.T., Jones L.R. Identification of an endogenous protein kinase C activity and its intrinsic 15-kilodalton substrate in purified canine cardiac sarcolemmal vesicles. J. Biol. Chem. (1985) 260:13879–13889.[Abstract/Free Full Text]
  17. Presti C.F., Jones L.R., Lindemann J.P. Isoproterenol-induced phosphorylation of a 15-kilodalton sarcolemmal protein in intact myocardium. J. Biol. Chem. (1985) 260:3860–3867.[Abstract/Free Full Text]
  18. Palmer C.J., Scott B.T., Jones L.R. Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium. J. Biol. Chem. (1991) 266:11126–11130.[Abstract/Free Full Text]
  19. G. Crambert, K. Geering, FXYD proteins: new tissue-specific regulators of the ubiquitous Na,K-ATPase. Sci. STKE 2003;2003:RE1.
  20. Silverman B.Z., Warley A., Miller J.I., James A.F., Shattock M.J. Is there a transient rise in sub-sarcolemmal Na and activation of Na/K pump current following activation of I(Na) in ventricular myocardium? Cardiovasc. Res. (2003) 57:1025–1034.[Abstract/Free Full Text]
  21. James A.F., Ramsey J.E., Reynolds A.M., Hendry B.M., Shattock M.J. Effects of endothelin-1 on K(+) currents from rat ventricular myocytes. Biochem. Biophys. Res. Commun. (2001) 284:1048–1055.[CrossRef][Web of Science][Medline]
  22. Song J., Zhang X.Q., Carl L.L., Qureshi A., Rothblum L.I., Cheung J.Y. Overexpression of phospholemman alters contractility and Cai2+ transients in adult rat myocytes. Am. J. Physiol. (2002) 283:H576–583.[Web of Science]
  23. Feschenko M.S., Donnet C., Wetzel R.K., Asinovski N.K., Jones L.R., Sweadner K.J. Phospholemman, a single-span membrane protein, is an accessory protein of Na,K-ATPase in cerebellum and choroid plexus. J. Neurosci. (2003) 23:2161–2169.[Abstract/Free Full Text]
  24. Feschenko M.S., Stevenson E., Sweadner K.J. Interaction of protein kinase C and cAMP-dependent pathways in the phosphorylation of the Na,K-ATPase. J. Biol. Chem. (2000) 275:34693–34700.[Abstract/Free Full Text]
  25. Cheng X.J., Fisone G., Aizman O., Aizman R., Levenson R., Greengard P., et al. PKA-mediated phosphorylation and inhibition of Na+–K+-ATPase in response to β-adrenergic hormone. Am. J. Physiol. (1997) 273:C893–901.[Web of Science][Medline]
  26. Crambert G., Fuzesi M., Garty H., Karlish S., Geering K. Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties. Proc Natl Acad Sci U S A (2002) 99:11476–11481.[Abstract/Free Full Text]
  27. Walaas S.I., Czernik A.J., Olstad O.K., Sletten K., Walaas O. Protein kinase C and cyclic AMP-dependent protein kinase phosphorylate phospholemman, an insulin and adrenaline-regulated membrane phosphoprotein, at specific sites in the carboxy terminal domain. Biochem. J. (1994) 304:635–640.[Web of Science][Medline]
  28. Crowell K.J., Franzin C.M., Koltay A., Lee S., Lucchese A.M., Snyder B.C., et al. Expression and characterization of the FXYD ion transport regulators for NMR structural studies in lipid micelles and lipid bilayers. Biochim. Biophys. Acta (2003) 1645:15–21.[Medline]
  29. Davies S.P., Reddy H., Caivano M., Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. (2000) 351:95–105.[CrossRef][Web of Science][Medline]
  30. Lu K.P., Kemp B.E., Means A.R. Identification of substrate specificity determinants for the cell cycle-regulated NIMA protein kinase. J. Biol. Chem. (1994) 269:6603–6607.[Abstract/Free Full Text]
  31. James P.F., Grupp I.L., Grupp G., Woo A.L., Askew G.R., Croyle M.L., et al. Identification of a specific role for the Na,K-ATPase {alpha}2 isoform as a regulator of calcium in the heart. Mol. Cell (1999) 3:555–563.[CrossRef][Web of Science][Medline]
  32. Schwartz A., Petrashevskaya N.N. The importance of calcium in interpretation of Na,K-ATPase isoform function in the mouse heart. Cardiovasc. Res. (2001) 51:9–12.[Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
W. Fuller, J. Howie, L. M. McLatchie, R. J. Weber, C. J. Hastie, K. Burness, D. Pavlovic, and M. J. Shattock
FXYD1 phosphorylation in vitro and in adult rat cardiac myocytes: threonine 69 is a novel substrate for protein kinase C
Am J Physiol Cell Physiol, June 1, 2009; 296(6): C1346 - C1355.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
X.-Q. Zhang, J. Wang, L. L. Carl, J. Song, B. A. Ahlers, and J. Y. Cheung
Phospholemman regulates cardiac Na+/Ca2+ exchanger by interacting with the exchanger's proximal linker domain
Am J Physiol Cell Physiol, April 1, 2009; 296(4): C911 - C921.
[Abstract] [Full Text] [PDF]

Home page
 Exp PhysiolHome page
J. R. Bell, D. Lloyd, C. L. Curl, L. M. D. Delbridge, and M. J. Shattock
Cell volume control in phospholemman (PLM) knockout mice: do cardiac myocytes demonstrate a regulatory volume decrease and is this influenced by deletion of PLM?
Exp Physiol, March 1, 2009; 94(3): 330 - 343.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Biol.Home page
P.-J. Wang, C.-H. Lin, H.-H. Hwang, and T.-H. Lee
Branchial FXYD protein expression in response to salinity change and its interaction with Na+/K+-ATPase of the euryhaline teleost Tetraodon nigroviridis
J. Exp. Biol., December 1, 2008; 211(23): 3750 - 3758.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
J. A. Armitage, S. Gupta, C. Wood, R. I. Jensen, A.-M. Samuelsson, W. Fuller, M. J. Shattock, L. Poston, and P. D. Taylor
Maternal dietary supplementation with saturated, but not monounsaturated or polyunsaturated fatty acids, leads to tissue-specific inhibition of offspring Na+,K+-ATPase
J. Physiol., October 15, 2008; 586(20): 5013 - 5022.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
J. Davis, M. V. Westfall, D. Townsend, M. Blankinship, T. J. Herron, G. Guerrero-Serna, W. Wang, E. Devaney, and J. M. Metzger
Designing Heart Performance by Gene Transfer
Physiol Rev, October 1, 2008; 88(4): 1567 - 1651.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. Song, X.-Q. Zhang, J. Wang, E. Cheskis, T. O. Chan, A. M. Feldman, A. L. Tucker, and J. Y. Cheung
Regulation of cardiac myocyte contractility by phospholemman: Na+/Ca2+ exchange versus Na+-K+-ATPase
Am J Physiol Heart Circ Physiol, October 1, 2008; 295(4): H1615 - H1625.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
S. Despa, A. L. Tucker, and D. M. Bers
Phospholemman-Mediated Activation of Na/K-ATPase Limits [Na]i and Inotropic State During {beta}-Adrenergic Stimulation in Mouse Ventricular Myocytes
Circulation, April 8, 2008; 117(14): 1849 - 1855.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
P. Fransen
Phospholemman, a chaperone of Na+,K+-ATPase?
Cardiovasc Res, January 1, 2005; 65(1): 13 - 15.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Silverman, B. d.Z.
Right arrow Articles by Shattock, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Silverman, B. d.Z.
Right arrow Articles by Shattock, M. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?