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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 3, 2003 as doi:10.1096/fj.03-0213fje. |
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Cardiac Physiology, The Centre for Cardiovascular Biology and Medicine, King’s College London, The Rayne Institute, St. Thomas’ Hospital, London SE1 7EH, UK
2Correspondence: Cardiac Physiology, The Centre for Cardiovascular Biology and Medicine, The Rayne Institute, St. Thomas’ Hospital, London SE1 7EH, UK. E-mail: michael.shattock{at}kcl.ac.uk
SPECIFIC AIMS
Acute and chronic regulation of the cardiac Na/K-ATPase is essential in maintaining cardiac output as the ion gradients this enzyme establishes are crucial for driving membrane transport processes and controlling the set point for other ions. The aim of this investigation was to define the mechanisms behind regulation of the Na/K-ATPase during cardiac ischemia and identify the roles of partner proteins in this regulation.
PRINCIPAL FINDINGS
1. Rat cardiac Na/K-ATPase is profoundly activated after cardiac ischemia
We determined the activity of the Na/K-ATPase by measuring ouabain-sensitive phosphate production from ATP by cardiac homogenates and subcellular fractions purified from cardiac homogenates. In unfractionated homogenates from ischemic hearts (15 and 30 min), Na/K-ATPase activity was depressed by the accumulation of a labile, soluble inhibitor of cardiac Na/K-ATPase (Fig. 1
, filled bars). Separation of a sarcolemma/particulate fraction (SLP) from the bulk cardiac homogenate revealed a substantial underlying activation of the Na/K-ATPase due to cardiac ischemia, usually concealed by the production of this inhibitor (Fig. 1
, open bars). After 30 min ischemia, Na/K-ATPase activity in the SLP fraction increased by threefold compared with aerobic controls.
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Signaling mechanisms underlying this activation of Na/K-ATPase in ischemic SLP were investigated by perfusing hearts with the nonspecific kinase inhibitor staurosporine and the specific PKA inhibitor H89. After 30 min of cardiac ischemia, activation of sarcolemmal Na/K-ATPase was abolished by 100 nmol/L staurosporine and 1 µmol/L H89.
2. The principal catalytic subunit of the Na/K-ATPase is not phosphorylated during cardiac ischemia, but phospholemman is
Given the sensitivity of the ischemia-induced activation of the Na/K-ATPase to a kinase inhibitor, we investigated the effect of ischemia on the net charge of the Na/K-ATPase 
1 subunit using 1-dimensional isoelectric focusing (IEF) gels and antibodies specific for consensus phosphorylation sites on the 1 subunit.
IEF gels indicated no change in the net charge of the 1 subunit after ischemia, suggesting it is not phosphorylated. This conclusion was supported by findings using phosphospecific antibodies showing that neither the PKA consensus phosphorylation site at ser938 nor a PKC consensus phosphorylation site at ser18 in the 1 subunit was phosphorylated during ischemia.
Phospholemman (PLM), the primary substrate for PKA and PKC in cardiac sarcolemma, has been proposed to associate with cardiac Na/K-ATPase. PLM is unsuitable for analysis by IEF because of its high hydrophobicity, and no phosphospecific antibodies are available. The phosphorylation status of PLM was investigated by measuring its association with recombinant 14-3-3. The amount of PLM pulled down by 14-3-3 from 30 min ischemic SLP was increased by >300% compared with aerobic SLP, but this association was absent if wild-type 14-3-3 was replaced with a mutant 14-3-3 (K49Q), which does not bind phosphoserine. This suggests that ischemia induces significant phosphorylation of PLM.
3. Phospholemman associates with the Na/K-ATPase 1, but not 2, subunit in aerobic and ischemic tissue and increases its association with the catalytic subunit of PKA during ischemia
PLM and the Na/K-ATPase 1 subunit were immunoprecipitated under nondenaturing conditions from cardiac homogenates (Fig. 2
). PLM was found to be associated with Na/K-ATPase 1 and ß1 subunits and the catalytic subunit of PKA, but no Na/K-ATPase 2 subunit was detected in association with PLM (Fig. 2A, B
). Association of PLM with the catalytic subunit of PKA was significantly increased after 30 min ischemia, but not if the heart had been perfused with staurosporine prior to ischemia (Fig. 2C
).
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Cross-linking reagent BS3 was used to investigate the association of the Na/K-ATPase 1 subunit with partner proteins. A low molecular mass protein (
8 kDa, similar to PLM) was cross-linked to the 1 subunit by BS3, and cross-linking was significantly reduced after ischemia (Fig. 2D
). Our PLM antibody was unable to identify this cross-linked protein as PLM, which may reflect concealment of its epitope after cross-linking.
CONCLUSIONS AND SIGNIFICANCE
Members of the FXYD family have recently been identified as tissue-specific regulators of the Na/K-ATPase. The effect of PLM on the transport properties of 1 and 2 subunits of the Na/K-ATPase has been described in Xenopus oocytes. The effect of PLM phosphorylation on Na/K-ATPase activity has not yet been described, and no studies have examined the relationship between PLM and Na/K-ATPase in mammalian cells or tissues. This is crucial because PLM is unique in the FXYD family in having several phosphorylation sites at its carboxyl terminus. While researchers have investigated the effects of the presence or absence of PLM on the Na/K-ATPase, the more important question in cardiac physiology may actually be, What is the effect of phosphorylation or dephosphorylation of PLM on Na/K-ATPase activity?
The data reported above describe the consequences of phosphorylation of PLM on Na/K-ATPase activity. We have identified a profound activation of the Na/K-ATPase that is usually masked by the production of a soluble inhibitor of the enzyme during cardiac ischemia. This activation is the result of phosphorylation of PLM during ischemia (PLM is an integral part of the Na/K-ATPase enzyme complex in the heart).
Other researchers have identified PKC as a regulator of the Na/K-ATPase through phosphorylation of a homologue of PLM, but we detected no increase in the association of any PKC isoforms with PLM after ischemia. However, we did observe a significant increase in the association of PKA with PLM after ischemia; this increase was abolished by treatment with staurosporine. Given the substantial literature regarding the effects of PKA on Na/K-ATPase activity yet the reported inaccessibility of the single PKA consensus phosphorylation site on the enzyme, the mechanism by which PKA regulates Na/K-ATPase has been controversial. Our data provide a link between these observations in tissues in which PLM is expressed. PKA does not directly phosphorylate the Na/K-ATPase: the effects we observe are mediated by phosphorylation of PLM. A similar mechanism is likely to explain stimulation of the Na/K-ATPase by catecholamines via ß-adrenoceptor stimulation in ventricular myocytes.
This study has identified PLM as an important modulator of the function of a P-type ATPase. PLM regulates Na/K-ATPase in a manner analogous to the regulation of SERCA by phospholamban (PLB): phosphorylation of PLM leads to a change in the association between PLM and the Na/K-ATPase and an isoform-specific increase in Na/K-ATPase activity (Fig. 3
). Researchers have suggested that PLM phosphorylation may simply lead to disinhibition of the Na/K-ATPase through a decrease in the affinity of PLM for the enzyme. However, we see no change in the affinity of PLM for the Na/K-ATPase after phosphorylation, and the increase in the Vmax of the Na/K-ATPase that accompanies this phosphorylation is substantialCzmore so than could be accounted for by disinhibition of the enzyme that other researchers have reported. This is a key difference between the PLM–Na/K-ATPase relationship and the PLB–SERCA relationship. Phosphorylation of PLB leads to an increase in the affinity of SERCA for its substrate calcium, but phosphorylation of PLM in our model leads to an increase in the Vmax of the Na/K-ATPase. Although PLM is a type I membrane protein, PLB is orientated with its amino terminus cytoplasmic and carboxyl terminus within the SR membrane. PLB is not a member of the FXYD family. However, there is homology between PLM and PLB, most notably in the cytoplasmic region surrounding the PKA consensus phosphorylation site (phosphorylated serine underlined): RSXIRRXST (residues 61-69 in PLM and 9-17 in PLB), so the proteins may share some evolutionary heritage.
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Taken together, the present findings uncover a new mechanism by which activity of the cardiac Na/K-ATPase may be regulated in vivo. The sodium gradient established by the Na/K-ATPase is crucial in countless membrane transport processes, particularly in determining calcium handling by the sodium/calcium exchanger. Regulation of the cardiac Na/K-ATPase by phospholemman is therefore of great importance in cardiac physiology and pathophysiology.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0213fje; 
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