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Cardiovascular Division, The Rayne Institute, King’s College London, St. Thomas Hospital, London, UK
1Correspondence: Cardiovascular Division, The Rayne Institute, King’s College London, St. Thomas Hospital, London, SE1 7EH, UK. E-mail: michael.shattock{at}kcl.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 subunit. We conclude that unphosphorylated FXYD1 inhibits Na/K ATPase, whereas S68 phosphorylated FXYD1 stimulates Na/K ATPase to a level above that seen in the absence of FXYD1.—Pavlovi, D., Fuller, W., and Shattock, M. J. The intracellular region of FXYD1 is sufficient to regulate cardiac Na/K ATPase.
Key Words: ion pumps • Na/K pump • protein kinase A • protein phosphorylation • phospholemman • FXYD
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit that couples ATP hydrolysis to ion transport and a glycoprotein ß subunit responsible for maturation, assembly, and membrane targeting of the Na/K ATPase. Of the 4 -isoforms, 1, predominates in cardiac cells, especially in rodent hearts.
Changes in ionic cellular conditions and physiological stimuli can lead to profound changes in the Na/K ATPase activity, thus affecting normal cellular function. Activation of the sympathetic nervous system and cardiac ß-adrenergic receptors causes cAMP formation and activation of protein kinase A (PKA). Regulation of Na/K ATPase by cAMP-PKA signaling cascade has been widely reported (1)
. Although PKA may phosphorylate Na/K ATPase directly (1
, 2)
, in isolated guinea pig myocytes PKA regulation of Na/K ATPase occurs via an associated protein FXYD1, (phospholemman) (3)
, which is a 72-amino acid membrane-spanning protein with a 20-amino acid cleavable signal sequence (4)
. FXYD1 is a major sarcolemmal substrate for PKA in the heart (4
, 5)
and belongs to the FXYD1 family of low MW proteins that act as tissue-specific regulators of Na/K ATPase (6)
. FXYD1 is unique in the FXYD1 family in having multiple phosphorylation sites at the carboxyl-terminus. We have previously shown in cardiac myocytes that PKA phosphorylates FXYD1 at serine 68 (3
, 7)
, and this phosphorylation event is associated with Na/K ATPase activation. However, the effects of PKA phosphorylation of FXYD1 on Na/K ATPase activity are not fully characterized.
The aims of the present study were to characterize Na/K ATPase currents in wild-type (WT) and FXYD1 knockout (KO) isolated mouse ventricular myocytes, and investigate the acute effects of serine 68 phosphorylation of FXYD1 on Na/K ATPase activity. Using whole-cell voltage clamping we show that a peptide corresponding to the C-terminus of FXYD1 (FXYD154–72) induces substantial changes in Na/K ATPase pump current and the nature of the change in Na/K ATPase pump current is determined by the phosphorylation state of FXYD1 peptide at serine 68.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 9
). Mice were congenic on a pure C57B/6 background. Heterozygous breeding pairs were used to generate FXYD1 KO and WT littermates. Mice of 3 months of age were used in this study and all have 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 U.S. National Institutes of Health (NIH Publication No. 85–23, revised 1996).
Cell isolation
Mouse ventricular myocytes were isolated by Langendorff perfusion and enzymatic dispersion following a modified version of the Zhou et al. protocol (10)
. Briefly, FXYD1 KO mice and age matched WT littermates were anesthetized by intraperitoneal (i.p.) injection of sodium pentobarbitone (60 mg/kg) plus heparin (100 IU). Hearts were excised, Langendorff perfused, and exposed to 0.3 mg/ml collagenase (Type 2, Worthington Biomedicals, Lakewood, NJ, USA) for 13 min. Ventricular tissue was removed, dispersed, and filtered before calcium reintroduction. The cell suspension was allowed to stabilize at room temperature for 30 min before use. The yield of rod-shaped cells was 70% to 80%. Cell size was similar in WT and KO myocytes, based on membrane capacitance (141±38 n=36 vs. 172±46 pF n=36, respectively).
Peptide synthesis
Two peptides representing the 19 C-terminal residues of FXYD1 and its scrambled analog were synthesized by Alta Bioscience (University of Birmingham, Birmingham, AL, USA): DEEEGTFRSSIRRLSTRRR, GRIERDTRSRFETRSSLRE, respectively. The purity and identity of the peptides was assessed to be > 90% by high-performance liquid chromatography (HPLC) and MALDI-Mass Spectrometry (MALDI-MS), respectively.
Peptide phosphorylation/purification
FXYD154–72 (30 µM) was phosphorylated with purified PKA (Sigma, St. Louis, MO, USA) in vitro at 37°C for 1.5 h in a buffer containing 20 mM Tris, 15 mM MgCl2, 1 mM dithiothreitol, 10 mM ATP, pH 7.4. The reaction was stopped by the addition of 10% acetonitrile. Phosphorylated peptide was subsequently HPLC purified on a C18 reverse phase column (Jones Chromatography, Hengoed, UK) using a Shimadzu 10A Chromatograph collecting 1 ml fractions and its purity and identity was confirmed by MALDI-MS (Bruker, Autoflex, Billerica, MA, USA). Solvent A was 0.1% TFA in Milli-Q water. Solvent B was 0.1% TFA and 90% acetonitrile in Milli-Q water. Gradient conditions for FXYD154–72 analysis were as follows: TIME = 0 min (t=0), 100% solvent A (1 ml/min); t = 5, 100% solvent A (1 ml/min); t = 30, 0% solvent A (1 ml/min); t = 35, 0% solvent A (1 ml/min); t = 40, 100% solvent A (1 ml/min).
Whole-cell voltage-clamping
Single isolated mouse ventricular myocytes were voltage-clamped using the whole cell-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
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 (5 ìs duration) from a holding potential of 0 mV. Access resistance on gaining access to the inside of the cell was <6 M. 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 pipette and extracellular solutions were designed to inhibit all voltage-gated channels and the Na/Ca exchanger. All studies were performed at 35°C. Solutions were made up using deionized water with Analar Grade chemicals (BDH, Leicestershire, UK). The standard pipette solution contained (in mmol/l) NaCl 15, MgCl2 1, CsCl 8, HEPES 10, EGTA 5, MgATP 5, creatine phosphate 5, CsCH3O3S 90, NaCH3O3S 35, pH 7.16. The standard extracellular solution contained (in mmol/l) NaCl 140, KCl 5, MgCl2 1, NiCl2 2, BaCl2 1, glucose 10, HEPES 10, pH 7.4. K-free extracellular solutions were made by removing KCl from the solution, with no correction for osmolarity. Under these conditions, the Na/K pump current (Ip) can be defined as that inhibited by the removal of extracellular K. Pump current was normalized to cell size (capacitance) and expressed as pA/pF. Approximately 1 min after the seal was established, currents were recorded continuously and were quantified every 5 min for 20 min.
Immunoprecipitation protocol
Cells were resuspended in modified radio-immuno-precipitation assay (RIPA) buffer (50 mM Tris-HCl pH 7.4, 1% TWEEN 20, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, and 1 mM NaF) supplemented with Protease Inhibitor Cocktail (Roche, Basel, Switzerland) and agitated at 4°C for 15 min. Insoluble material was removed by centrifugation at 14,000 g in a precooled centrifuge for 15 min. Cell lysate supernatant was precleared with Protein G sepharose (Amersham, Freiburg, Germany) for 10 min at 4°C and incubated with 10 µM of FXYD154–72 for 30 min at 37°C. Samples were then agitated for 4 h in the presence of anti-Na/K ATPase 1 subunit IgG (clone C464.6, Upstate Biotechnology, Lake Placid, NY, USA). Immune complexes were harvested with Protein G sepharose (Amersham) overnight at 4°C. The agarose beads were washed four times, for 5, 5, 15, and 15 min with PBS (1% TWEEN 20), after which beads were resuspended in 2x SDS-PAGE sample buffer. SERCA2 subunit was immunoprecipitated under identical conditions by using a monoclonal anti-SERCA2 antibody (Lot #077–126, Affinity Bioreagents, Golden, CO, USA). To ensure complete dissociation of the heavy and light chains of the immunoprecipitating antibody, prior to electrophoresis samples were heated for 15 min at 60°C in 2x SDS PAGE sample buffer supplemented with 5% ß- mercaptoethanol and 100 mmol/L dithiothreitol.
Dot blots
For detection of FXYD154–72 samples were dot blotted (0.5 µl) onto nitrocellulose membranes (Amersham), fixed with 0.2% glutaraldehyde in PBS for 20 min and blocked with 5% milk in PBS (0.1% TWEEN 20) overnight at 4°C. Membranes were further probed with antibodies as described previously (7)
.
Statistical analysis
Quantitative data are shown as mean ± SE of the means (SEM). 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A, top panel). FXYD154–72, PKA phosphorylated for 1 h, was eluted as two single peaks at 19.5 and 21 min (Fig. 1B
, top panel), whereas 1.5 h reaction caused all of the peptide to elute as a single peak at 19.5 min (Fig. 1C
, top panel). The collected peptides were dot blotted onto nitrocellulose membranes and probed with C2 (recognizes unphosphorylated FXYD154–72) and CP68 antibody (recognizes S68 phosphorylated FXYD154–72). The unphosphorylated 21 min fraction reacted with C2 antibody (Fig. 1A
, bottom panel), whereas the 19.5 min fraction only reacted with CP68 antibody (Fig. 1C
, bottom panel). Both peptides were subjected to MALDI-MS analysis and in each case a single species was observed at 2351.4 and 2433 m/z ratio (data not shown) corresponding to unphosphorylated and a singly phosphorylated FXYD154–72, respectively.
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Na/K ATPase pump current (Ip) whole cell measurements
Na/K ATPase pump currents were measured at 0 mV using the ruptured-patch technique.
Ip (normalized to cell capacitance pA/pF) in WT and KO mouse ventricular myocytes was measured at 5 min intervals and showed stability for 20 min (Fig. 2
A). The average Ip over 20 min of recording was higher in KO myocytes (2.9±0.1 pA/pF; n=4) compared to WT (1.9±0.1 pA/pF; n=4) as shown in Fig. 2B
.
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To investigate the effects of S68 phosphorylation of FXYD1 on Na/K ATPase Ip FXYD154–72 was delivered to the intracellular environment of the mouse myocytes through the patch pipette, and its effects were expressed as the Ip recorded at 1 min (ionic dialysis is complete but peptide diffusion is limited) and 11 min (peptide dialysis complete) after patching onto the cell. Unphosphorylated FXYD154–72 at a concentration of 4 µM reduced Ip in both WT (Fig. 3
A) and KO (data not shown). Na/K pump currents were reduced in WT (from 2.1±0.1 to 1.3±0.1 pA/pF; P<0.05, n=7) and KO (from 2.9±0.1 to 1.7±0.1 pA/pF; P<0.05, n=5) myocytes, as shown in Fig. 3B
. In contrast, 1 µM of S68 phosphorylated FXYD154–72 increased Ip in both WT (Fig. 3C
) and KO (data not shown). Ip was increased in WT (from 1.9±0.1 to 3.1±0.5 pA/pF; P<0.05, n=6) and KO (from 2.7±0.1 to 3.8±0.2 pA/pF; P<0.05, n=6) myocytes, as shown in Fig. 3D
. These effects were usually complete within 10 min of the start of recording and remained stable for the remainder of the recording period. This suggests that the interacting peptides are neither degraded nor dephosphorylated within this timeframe or that the vast excess of peptide within the patch-pipette can continually replace any proteolysed or dephosphorylated peptide. No detectable changes in Na/K ATPase currents were observed in the presence of 4 µM of the scrambled peptide (Fig. 3E and F
).
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Prior to undertaking the studies described above, a series of pilot dose-ranging studies were undertaken (Fig. 4
) with both the phosphorylated and unphosphorylated peptide in WT and KO cells. These studies confirm that the unphosphorylated and phosphorylated peptides both influence Ip in a dose-dependent manner. For the unphosphorylated peptide, the IC50 in WT and KO were estimated to be 0.95 ± 0.21 µM and 0.68 ± 0.16 µM, respectively. For the phosphorylated peptide, the EC50s in WT and KO were 0.69 ± 0.0.19 µM and 0.43 ± 0.28 µM, respectively.
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Immunoprecipitation of Na/K ATPase 1 subunit
We have previously reported the association of FXYD1 with the 1 subunit of Na/K ATPase (3
, 7)
. To assess the ability of FXYD154–72 to interact with the 1 subunit of Na/K ATPase, 1 subunit was immunoprecipitated from the KO cells using the 1 antibody and the immunoprecipitated protein was subsequently dot blotted onto nitrocellulose membranes. Membranes were probed with C2 and CP68 antibodies. When 1 subunit was immunoprecipitated (Fig. 5
, top panel) phosphorylated and unphosphorylated FXYD154–72 were coprecipitated (Fig. 5
, bottom panel). As a control, the calcium ATPase of the SR was immunoprecipitated (Fig. 6
, top panel), however, phosphorylated and unphosphorylated FXYD154–72 were not coprecipitated (Fig. 6
, bottom panel).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
Several groups (3
, 12
, 13)
have suggested that FXYD1 regulates Na/K ATPase in a manner analogous to phospholamban regulation of calcium ATPase of the SR (SERCA). PKA and calcium calmodulin (CaM)-dependent protein kinase phosphorylate phospholamban leading to its partial dissociation from SERCA. This in turn activates SERCA by increasing its affinity for calcium (14)
. Whether FXYD1 exerts its effects on Na/K ATPase through changes in Na affinity, K affinity, Vmax, or a combination of these is controversial. On the one hand, overexpression of FXYD1 in adult rat ventricular myocytes decreased Na/K ATPase Ip by 27–40%, with no effect on Na or K affinity (15)
. On the other hand, expression of FXYD1 and 1-ß1 Na/K ATPase in Xenopus oocytes resulted in decreased affinities for Na or K, yet no detectable effect on the Vmax (13)
. Similar effects on Na affinity were observed in in vitro Na/K ATPase enzyme assays purified from shark rectal gland (12)
in which the protein FXYD10 is expressed. Possibly the reason for differences observed in the effects on Vmax/Na affinity between the three studies can be attributed to FXYD1 being in a different state, i.e., different processing in different cell types. A recent study by Lifshitz and colleagues using reconstituted FXYD1 and porcine and rat /ß complexes from HeLa and Pichia pastoris cells clearly shows that the phosphorylation state of FXYD1 and therefore the functional effects it has on Na/K ATPase activity are highly dependent on the expression system. Interestingly, Lifshitz and colleagues also found that the transmembrane domain of FXYD1 is directly responsible for changes in Na affinity (16)
.
In our previous study, we showed correlation between PKA phosphorylation of FXYD1 and increased sarcolemmal Na/K ATPase activity in ischemic rat heart tissue (7)
. This was confirmed in our second study, whereby Na/K ATPase Ip in guinea pig myocytes increased following treatment with the PKA activator forskolin. The presence of PKA inhibitor H89 completely blocked the effect of forskolin, which was correlated with PKA phosphorylation of FXYD1 at S68 (3)
. In both studies we showed that 1 subunit of Na/K ATPase associated with FXYD1. Thus, we have previously shown that FXYD1 is a functional component of the cardiac Na/K ATPase complex and that PKA phosphorylation of FXYD1 correlates with substantial increases in Na/K ATPase activity (7)
. In 2003, we proposed a model (3
, 7)
, which was more recently adopted by others (17)
, that the 1 subunit of cardiac Na/K ATPase and FXYD1 form a close association and the phosphorylation of FXYD1 releases a functional inhibition on the Na/K ATPase pump. Original data from our laboratory (7)
suggested that phosphorylation of FXYD1 causes a change in the relationship between FXYD1 and the 1 subunit such that, while phosphorylated FXYD1 remains associated with the 1 subunit (and can be immunoprecipitated) its orientation in the pump complex changes. This model has recently been elegantly confirmed using FRET (17)
.
Initially, Jia and colleagues reported that maximal Na/K ATPase activity was reduced by 50% in FXYD1-deficient cardiac sarcolemma (9)
, which is in conflict with a study by Despa et al., where increased Na/K ATPase activities in KO were reported (8)
. In both studies, the level of 1 expression was found to be decreased by 20–25% in KO compared to WT (9)
. We also see decreased Na/K ATPase subunit expression in KO myocytes (not shown). Considering that in the KO cells Ip is 34% higher than in WT, even though there is 20–25% less Na/K ATPase protein suggests that, in the KO, normalized unitary pump current is almost double (180%) of that in WT myocytes. This is in agreement with previous estimates in other studies (18)
, giving further evidence that FXYD1 acts as a brake on the Na/K ATPase. However, the peptide data suggest a more complex scenario. Whereas unphosphorylated peptide reduces Ip in both WT and KO cells, FXYD1 phosphorylated at S68 stimulates Na/K ATPase Ip above the basal levels even in KO cells. This implies that FXYD1 may not simply act as a limiter of Na/K ATPase activity (with inhibition relieved following phosphorylation) but that it also stimulates the Na/K ATPase activity on phosphorylation by PKA. The analogy between FXYD1 and phospholamban breaks down with this observation, as PLB does not influence the Vmax of SERCA2a, and PLB phosphorylation merely disinhibits SERCA.
The advantage of the method used in the current study is that we are able to compare the effect of unphosphorylated FXYD1 with 100% S68-phosphorylated FXYD154–72. We, and others, have observed significant (30–40%) basal S68 phosphorylation of FXYD1 in unstimulated cells (unpublished data). Hence, comparison of unstimulated cells with those in which PKA is activated actually compares partially phosphorylated FXYD1 with phosphorylated FXYD1. Likewise, investigations using FXYD1 WT and KO mice measure a mixed population in the case of WT. Basal phosphorylation levels of FXYD1 are likely to vary according to the myocyte preparation methods in different laboratories. Unlike previous studies in which a PKA agonist has been used to stimulate phosphorylation of FXYD1 (as well as other potential PKA targets such as phospholamban), in this study, prior in vitro PKA phosphorylation ensures that we can attribute the effects on Na/K ATPase activity solely to the FXYD1. Here, we show a direct effect of S68 FXYD1 phosphorylation on Na/K ATPase activity. Interestingly, these effects were not only observed in FXYD1 deficient mice but also in WT mice, suggesting that a 19 amino acid peptide could successfully and rapidly compete out endogenous FXYD1.
We have preliminary and unpublished data showing that, in rat cardiac myocytes, there is a pool of 1 subunits that is not associated with FXYD1. Immunoprecipitation of all of the FXYD1 coprecipitates only 44 ± 8% of the 1 subunit while immuprecipitation of 1 subunit coimmunoprecipitates 100% of FXYD1 (unpublished results). It is unclear whether this 1 subunit that is not associated with FXYD1 reflects a subpopulation of functional pump units at the cell surface that contribute to pump current or a subpopulation of nonfunctional 1 subunits inside the cell. To complicate matters further, a significant fraction of FXYD1, in basal unstimulated conditions, is phosphorylated (3
, 8)
. We have recently estimated that 
30% of FXYD1 is basally phosphorylated at S68. Thus, in WT cells, when unphosphorylated peptide is included in the pipette, not only may the peptide displace endogenous phosphorylated FXYD1 from the pump units that are associated with FXYD1, but may also inhibit 1 subunits which have no FXYD1 associated with them (if this population is functional—see above). Conversely, phosphorylated peptide may displace endogenous unphosphorylated FXYD1 from the pump and/or stimulate 1 subunits from the pool of pump protein that is not associated with endogenous FXYD1 (again, assuming that this pool is functionally active).
Unphosphorylated FXYD154–72 (4 µM) decreased Ip in WT by 34% and by 41% in KO. On the other hand, 1 µM of FXYD154–72 phosphorylated at S68 increased Ip in WT by 40% and by 27% in KO. Interestingly, S68 phosphorylated FXYD154–72 required four times less peptide than the unphosphorylated FXYD154–72 in order to achieve similar time-dependent effect on the Na/K ATPase activity. Whether this is due to a change in the FXYD1 secondary structure, which ensures more stable interaction with the Na/K ATPase, or due to the reduction in hydrophobicity (addition of a single phosphate at S68) and thus more efficient entry into the cell is not known.
Little is known about the interaction between Na/K ATPase and the FXYD1. Experiments based on thermal denaturation and site-directed mutagenesis of Na/K ATPase suggest that the association of some FXYD1 proteins, occurs with transmembrane domains 8–10 of Na/K ATPase (19
, 20)
. Transmembrane segments of FXYD2 and FXYD4 were modeled within a groove between M2, M6 and M9 (21)
, however, which domains of FXYD1 are important for efficient association between these two proteins and transmittance of functional effects of FXYD1 on Na/K ATPase activity is still not clear. Our data imply that the C-terminal 19 residues are sufficient for Na/K ATPase Vmax regulation, although the transmembrane domain of FXYD1 has been shown to have an effect on Na/K ATPase Na affinity (16)
. What portions of FXYD1 are necessary for other physiological functions attributed to FXYD1 such as its reported ion channel function (22
23
24
25
26
27
28)
is not known.
Despa and colleagues, using the same mouse model, have not observed any differences in Vmax between the KO and WT mice, as we have observed in the present study. Furthermore, in their study isoproterenol stimulated the pump in the WT mice by reducing the Km for internal Na and phosphorylated FXYD1 at S68, however, isoproterenol had no significant effect in the KO mice (8)
. Using perforated patch to study the effects of knocking out FXYD1 in mouse cardiac myocytes, we have observed similar increases in FXYD1 KO in Vmax, as shown in this study, as well as an increase in Na affinity (18)
. Whether Na/K ATPase activity is measured by whole cell patch clamp, perforated patch clamp in cardiac myocytes or ouabain-sensitive ATPase activity of cardiac homogenates, we consistently observe a higher Vmax in KO animals. This effect on Vmax is in agreement with the observations of both Jia et al. (9)
and Zhang et al. (15)
It is worth noting that the functional measurements of Na/K ATPase presented in this study, and those of Jia et al. (9)
and Zhang et al. (15)
, were made at 35–37°C. The studies of Despa et al. (8)
were at room temperature; thus, it is possible that at this temperature the effects of FXYD1 on enzyme kinetics may be underestimated. This may explain the discrepancy between this study and the previous studies in terms of an effect of FXYD1 on Na/K ATPase Vmax.
This study showed that in adult mouse ventricular myocytes FXYD1 plays an important role in Na/K ATPase regulation. In particular, the PKA mediated phosphorylation of the FXYD1 S68 residue was found to be directly responsible for the substantial increase in Na/K ATPase activity, whereas, the unphosphorylated FXYD1 had an inhibiting effect on the Na/K ATPase activity. These findings provide an important insight into the regulation of the Na/K ATPase by PKA and are in agreement with our previous studies (3
, 7)
. Further work is required to highlight the roles of the other kinases known to phosphorylate FXYD1.
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ACKNOWLEDGMENTS |
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Received for publication September 11, 2006. Accepted for publication December 25, 2006.
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