Regulation of the cardiac Na pump by

Regulation of Protein Trafficking and Function by Palmitoylation

Regulation of the cardiac Na + pump by palmitoylation of its catalytic and regulatory subunits Jacqueline Howie*, Lindsay B. Tulloch*, Michael J. Shattock† and William Fuller*1 *Division of Cardiovascular & Diabetes Medicine, Medical Research Institute, College of Medicine Dentistry & Nursing, University of Dundee, Dundee DD1 9SY, U.K., and †Cardiovascular Division, King’s College London, London SE1 7EH, U.K.

Abstract The Na + /K + -ATPase (Na + pump) is the principal consumer of ATP in multicellular organisms. In the heart, the Na + gradient established by the pump is essential for all aspects of cardiac function, and appropriate regulation of the cardiac Na + pump is therefore crucial to match cardiac output to the physiological requirements of an organism. The cardiac pump is a multi-subunit enzyme, consisting of a catalytic α-subunit and regulatory β- and FXYD subunits. All three subunits may become palmitoylated, although the functional outcome of these palmitoylation events is incompletely characterized to date. Interestingly, both β- and FXYD subunits may be palmitoylated or glutathionylated at the same cysteine residues. These competing chemically distinct post-translational modifications may mediate functionally different effects on the cardiac pump. In the present article, we review the cellular events that control the balance between these modifications, and discuss the likely functional effects of pump subunit palmitoylation.

Introduction +

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The ubiquitous Na /K -ATPase (Na pump) links the hydrolysis of ATP to the cellular export of three Na + and import of two K + against their electrochemical gradients. It is an indispensable means of active membrane transport in essentially every eukaryotic single and multicellular organism, and the molecular target of the foxglove extracts digitalis and digoxin, in clinical use for heart failure since the 18th Century. In the last 5 years, the discovery [1] and refinement [2,3] of crystal structures in potassium- and ouabain-bound states has advanced our understanding of the structure–function relationship of this much studied macromolecular complex further.

Quaternary structure +

The Na pump is a multi-subunit enzyme, with a minimum requirement for an α- and β-subunit to form a functional pump. The ∼100 kDa α-subunit is the catalytic core of the enzyme, containing the binding sites for Na + , K + and ATP as well as cardiotonic steroids such as ouabain. It requires obligatory association with a β-subunit to traffic through the secretory pathway to the plasma membrane [4,5]. In a previous study, it became clear that a third protein may also generally form part of the pump complex [6]. Whether this third member of the complex, named as FXYD protein for the conserved extracellular Phe-Xaa-Tyr-Asp motif, is a constant or occasional companion of the pump has not Key words: FXYD, heart, ion transport, Na + pump, palmitoylation, phospholemman. Abbreviations used: H, helix; NCX, Na + /Ca2 + exchanger; P-domain, phosphorylation domain; PK, protein kinase; PLM, phospholemman; SR, sarcoplasmic reticulum. 1 To whom correspondence should be addressed (email [email protected]).

Biochem. Soc. Trans. (2013) 41, 95–100; doi:10.1042/BST20120269

been rigorously investigated to date. The existence of four isoforms of the α-subunit, three isoforms of the β-subunit, and seven FXYD proteins (as well as splice variants of the γ subunit [7]) in mammalian genomes can theoretically support the assembly of over 100 functionally different Na + pumps to fulfil different physiological requirements. Although four isoforms of the α-subunit have been identified, only α1 and α2 are reportedly expressed at significant levels in cardiac myocytes [8,9]. The principal β-subunit found in cardiac muscle is β1, and the principal FXYD protein is PLM (phospholemman).

Importance of pump regulation in the heart In excitable tissues, the activity of the plasmalemmal Na + pump is vital for the maintenance of normal electrical activity and ion gradients. In cardiac muscle, the transarcolemmal Na + gradient established by Na + pump activity is essential not only for generating the rapid upstroke of the action potential but also for driving a number of ion exchange and transport processes critical for normal cellular function, ion homoeostasis and the control of cell volume. These Na + dependent membrane transporters include those responsible for the regulation of other ions {such as NCX (Na + /Ca2 + exchanger), Na + /H + exchanger and Na + –HCO3 − cotransporter [10]}, as well as those involved in the movement of substrates and amino acids [11]. For example, by controlling steady-state intracellular Na + , the pump determines the set point for intracellular Ca2 + via NCX, which in turn determines the SR (sarcoplasmic reticulum) Ca2 + content. Precise control of cytosolic and SR Ca2 + concentrations C The

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are essential in maintaining cardiac output: derangement of Ca2 + handling is a primary cellular cause of contraction abnormalities and heart failure. Interventions that influence either the set point of the Na + pump, or indirectly the transarcolemmal Na + gradient, can therefore profoundly affect myocardial function. In essence, the Na + pump indirectly controls myocardial contractility [12].

Regulation of the cardiac Na + pump by palmitoylation Palmitoylation of PLM PLM (FXYD1) was first identified as an abundant phosphoprotein in the cardiac sarcolemma in 1985 [13], and it was quickly recognized to be the principal sarcolemmal substrate for both PK (protein kinase) A and PKC in the heart [13,14]. PLM is a member of the FXYD family of pump regulators [6]: PLM associates with the Na + pump in the heart [15– 19], and modifies its transport properties. Unphosphorylated PLM inhibits the pump, and PLM phosphorylation leads to pump activation [20]. PLM is palmitoylated at two intracellular cysteine residues, Cys40 and Cys42 , just beyond the transmembrane domain [21]. Notably, these cysteine residues are conserved across species, but also one or both cysteine residues are found in analogous positions throughout the FXYD family: FXYD2, 5 and 7 have one, and FXYD3, 4 and 6 have two [22]. All are predicted to be palmitoylated [21], and, to date, at least one other member of the family (FXYD5) has been found to be palmitoylated [23], suggesting that FXYD protein palmitoylation may be a universal means of regulating the pump. Unpalmitoylatable mutant PLM is degraded with a shorter half-life than wild-type PLM in transiently transfected cells, but the principal functional effect of PLM palmitoylation is inhibition of the Na + pump [21]. The inhibitory effect of PLM on the pump is abolished following application of the pharmacological inhibitor of palmitoyl acyltransferases 2-bromopalmitate. Indeed, given the relatively modest effect of this inhibitor on PLM palmitoylation, it is possible that palmitoylation switches PLM from a pump activator to an inhibitor. Although the DHHC-PATs (palmitoyl acyltransferases) that palmitoylate PLM (or any pump subunits, see below) are yet to be identified, one important regulator of PLM palmitoylation is its phosphorylation status. Paradoxically, phosphorylation of PLM at Ser68 by PKA increases PLM palmitoylation [21]. Hence, one post-translational modification of PLM that activates the Na + pump promotes a second that inhibits it. The molecular basis of phosphorylation promoting palmitoylation can probably be explained by reference to the NMR structures of unphosphorylated and Ser68 -phosphorylated PLM [24,25] (Figure 1A). Ser68 phosphorylation of PLM increases the mobility of PLM H (helix) 4 relative to unphosphorylated PLM, without inducing major changes in the overall C The


structure of the protein [25]. This probably increases the accessibility of the cysteine residues in PLM H3 to the palmitoyl acyltransferase enzyme(s) that palmitoylate PLM, possibly particularly affecting Cys40 , towards which the phosphorylation sites in H4 are oriented. As for the physiological and functional significance of enhanced PLM palmitoylation following PKA activation, this remains to be seen. Site-specific reagents to distinguish which cysteine residue in PLM is palmitoylated following Ser68 phosphorylation of PLM do not exist (neither do they exist for other palmitoylation sites in other proteins). Molecular models of the PLM–Na + pump complex (Figure 1B) suggest that PLM Cys42 could mediate the inhibitory effect of PLM palmitoylation on the pump, as the side chain of this amino acid is oriented towards the pump α-subunit, and Cys40 is oriented away. Palmitoylation of Cys42 (with incorporation of the palmitate into the lipid bilayer) may pull PLM H3 across the intracellular mouth of an Na + -binding site in the α-subunit in order to inhibit the pump. Conversely, palmitoylation of Cys40 on the opposite side of H3 would oppose such a movement by pulling H3 in the opposite direction. This raises the possibility that, although the overall effect of PLM palmitoylation on the pump is inhibitory, the individual palmitoylation sites may have opposing effects on pump activity through their reorienting effects on PLM H3 (Figure 1B).

Palmitoylation of other pump subunits Several independent proteomic screens now report palmitoylation of the Na + pump α1-subunit [23,26–29]. The first to do so used fatty-acyl biotin exchange to purify palmitoylated proteins from a human prostate cancer cell line [26], and identified one high-confidence (Cys374 ) and one medium-confidence (Cys705 ) palmitoylation site in the pump α1-subunit, as well as two low-confidence sites (Cys211 and Cys249 ). The positions of the high- and mediumconfidence sites are highlighted on the crystal structure of the dogfish α1-subunit [2] in Figure 2(A). Both reside within the P-domain (phosphorylation domain) of the α1-subunit, which is transiently phosphorylated at Asp376 during the reaction cycle of the pump [30]. Remarkably, the high-confidence palmitoylation site lies only two amino acids from this catalytic aspartate residue. Both cysteine residues are conserved among species (Figure 2B), and between α-subunit isoforms expressed in the heart (Figure 2C). Palmitoylation of Cys374 and its subsequent recruitment to the bilayer would probably significantly perturb α1-subunit secondary structure and therefore pump activity; however, the functional effect of α-subunit palmitoylation remains to be determined. Another study has identified other P-domain cysteine residues (Cys356 and Cys663 ) as palmitoylated [27], both of which are also conserved among species and cardiac pump isoforms. All regulatory β-subunits of the pump are also reported to be palmitoylated [23,31]. The β-subunits are characterized by the presence of three extracellular disulfide bonds, but curiously β3 has no other cysteine residues in its sequence,


Figure 1 Position of the palmitoylation sites of phospholemman relative to the α-subunit (A) Intracellular region of PLM, indicating the positions of the palmitoylated cysteine residues (Cys40 and Cys42 ) in H3, and the intracellular H4 containing the phosphorylation site Ser68 . (B) Positions of PLM palmitoylation sites relative to the porcine α-subunit. PLM is in green, the α-subunit transmembrane region is in blue, the N (nucleotide-binding) -domain is in cyan, the P-domain is in yellow, and the A (actuator) -domain is in purple. The β-subunit is red. Na + (purple spheres) is shown in its proposed binding sites (reviewed in [3]). H2, H3 and H4 of PLM are labelled. Cys42 of PLM is oriented towards the α-subunit, and Cys40 in oriented away, meaning palmitoylation of Cys42 may pull PLM H3 across the intracellular mouth of an Na + -binding site in the α-subunit. Conversely, palmitoylation of Cys40 would oppose such a movement by pulling H3 in the opposite direction.

whereas Cys46 is conserved in the β1-subunit, and Cys10 is conserved in β2. The fact that Cys46 of β1 may also be glutathionylated (discussed below) adds this protein to the growing list of cases where there is competition between palmitoylation and glutathionylation at the same amino acid.

Palmitoylation dynamics and pump regulation Relatively few studies have investigated the dynamics of palmitoylation turnover in any cell type to date, so it is difficult to comment on the relative contribution of palmitoylation to acute rather than long-term regulation of cardiac Na + pump function. An appreciation is undoubtedly growing that palmitoylation turns over rapidly (minutes) for certain groups of proteins [23,31,32]. In the one study to investigate global palmitoylation turnover dynamics to date, pump α1-subunit palmitoylation was found to turnover slowly in T-cell hybridoma cells, whereas palmitoylation of FXYD5 was found to decline rapidly in pulse– chase experiments (albeit in a manner that could not

be distinguished from protein turnover) [23]. This is in agreement with our own observations that phosphorylation of PLM (FXYD1) rapidly promotes its palmitoylation [21], and suggests that, much as is the case for phospho-regulation of the pump, short-term regulation of pump activity by palmitoylation may be achieved by the dynamic posttranslational modification of the accessory FXYD subunit rather than the catalytic subunit itself.

Oxidant modification as a reversible regulator of the pump It has long been reported that the cardiac Na + pump is sensitive to oxidative stress. For example, superoxide generators reduce pump current in voltage-clamped rabbit ventricular myocytes by approximately 50% at all membrane potentials within 10 min [33]. Pump activity is also significantly reduced by intracellular application of thiolmodifying reagents, or depletion of cellular glutathione [34], C The


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Figure 2 Putative palmitoylation sites in the α-subunit (A) Dogfish α-subunit [2] transmembrane region is in blue, the N-domain is in cyan, the P-domain is in yellow, and the A-domain is in purple. Bound potassium is shown as purple spheres, magnesium is shown as green spheres and magnesium tetrafluoride (phosphate analogue) is shown as green/light blue spheres. The positions of two proposed palmitoylation sites [26] (side chains shown in red) relative to the catalytic aspartate residue (side chain shown in green) strongly suggest that by modifying the structure local to the catalytic site, palmitoylation will alter pump activity. (B) Sequence alignment of α1-subunits from various species showing the palmitoylation sites reported in [26] (underlined), which are conserved among isoforms of the α-subunit expressed in the heart (C).

all manner of signalling and metabolic pathways [36]. Glutathionylation is no longer considered solely to be a consequence of oxidative stress, and occurs under physiological conditions in the absence of overt oxidative burden [37]. Unlike phosphorylation and palmitoylation, glutathionylation is generally non-enzymatic, and occurs by direct reaction between oxidized/modified protein thiols and cellular glutathione [36]. The lack of enzymes to catalyse glutathionylation does not result in a lack of specificity for this modification, as the vast majority of cysteine thiols are not susceptible to glutathionylation because their pK a is above 8.0, such that they remain protonated and hence non-reactive at physiological pH. Redox-sensitive cysteine residues that are susceptible to glutathionylation may be deprotonated to the thiolate anion at physiological pH as a result of a favourable local environment for that particular cysteine residue, usually achieved by the presence of positively charged amino acids nearby to receive the proton. Glutathionylation is reversed enzymatically by glutaredoxins, thioredoxins and sulfiredoxin, and can be removed non-enzymatically by disulfide exchange with glutathione [36]. The Na + pump in the heart is reported to be regulated by glutathionylation of its β-subunit [38]. The cardiac β1subunit is found to be glutathionylated in unstimulated myocytes, and exogenous oxidants such as peroxynitrite or hydrogen peroxide promote additional glutathionylation of the β1-subunit when applied to intact cardiac myocytes [38]. The functional effect of β1-subunit glutathionylation is to destabilize the interaction between cardiac α- and β-subunits, and decrease the V max of the pump. Cys46 , the cysteine residue in the β1-subunit whose glutathionylation leads to pump inhibition [38], is the same site at which this subunit is also reported to be palmitoylated [31].

Competition between cysteine post-translational modifications? confirming a functional link between pump activity and protein thiol status. Remarkably, however, the pump does not require cysteine residues in the α-subunit for activity: the removal of all 23 endogenous cysteine residues from the α-subunit does not inactivate the enzyme [35]. This suggests that, rather than being a requisite for the enzymatic activity of the pump, the α-subunit cysteine residues fulfil a different function: this is possibly to sense the redox state of the environment in which the pump operates, or to transduce post-translational modifications of these cysteine residues into changes in enzymatic activity. Oxidant modification and regulation of the pump has also been the subject of previous investigation. Glutathionylation is the reversible conjugation of the tripeptide glutathione to protein cysteine residues in a mixed disulfide. Many classes of protein have been found to be regulated by glutathionylation (reviewed in [36]). Similar to phosphorylation and palmitoylation, glutathionylation allows dynamic reversible post-translational regulation of C The


Although the consensus understanding of PLM as a regulator of the pump is that it inhibits pump activity, PLM is also proposed to be glutathionylated to relieve oxidantinduced inhibition of the pump following glutathionylation of its β1-subunit [39]. PLM glutathionylation is promoted by the same signalling pathways that promote β1-subunit glutathionylation, but PLM glutathionylation is suggested to be downstream of β1-subunit glutathionylation. The presence of PLM facilitates de-glutathionylation of Cys46 of β1-subunit, with concomitant glutathionylation of PLM at Cys42 [39], one of the cysteine residues in PLM that may also be palmitoylated. Thus the presence of PLM activates the pump in the sense that it is protected from oxidantinduced inhibition as a result of the reduced glutathionylation of Cys46 . This paradigm of PLM as a pump activator during redox signalling in cardiac muscle does not sit well with the consensus regarding the role of PLM in phosphoregulation of the pump. Neither can palmitoylation, redox and phosphorylation regulation by PLM be easily separated since


it is now well established that oxidizing species activate PKA and PKG in ventricular muscle [40–42]. Indeed, hydrogen peroxide treatment of ventricular myocytes, which has been reported to inhibit the pump via β1-subunit glutathionylation [38], leads to substantial phosphorylation of PLM at Ser68 because it activates type 1 PKA (by promoting an interprotein disulfide bond between its two regulatory subunits) [40]. Since phosphorylation of PLM at Ser68 promotes its palmitoylation [21], it is difficult to predict exactly the consequences for the pump. Hence palmitoylation and glutathionylation compete for the same cysteine residues on two of the three subunits of the pump expressed in the heart. The ability of these cysteine residues to receive either glutathione or palmitate will depend on whether they are already modified with the other. So PLM may be a pump activator (through glutathionylation) or inhibitor (through palmitoylation) depending on the state of Cys42 , which is determined by the phosphorylation status of PLM, adrenergic state of the tissue and redox state of the cell. Such competition between posttranslational modifications is likely to not be only restricted to the Na + pump. Indeed, although the structural-sequence determinants of protein palmitoylation remain rather poorly characterized, it is well established that cysteine residues with low pK a values display high acylation rates in vitro [43], and low-pK a solvent-exposed cysteine residues are the very same sites that are also likely to be susceptible to glutathionylation. Competition between palmitoylation and oxidation/glutathionylation has been reported for proteins such as caveolin 1 [44], CD81 (cluster of differentiation 81) [45], H-ras and eNOS (endothelial nitric oxide synthase) [46]. Hence there may exist a set of proteins that are rendered insensitive to oxidation/glutathionylation by palmitoylation of key low-pK a cysteine residues, or even for whom palmitoylation and glutathionylation induce opposite effects in terms of changes in activity, as appears to be the case for the Na + pump.

Funding This work was supported by a grant from the British Heart Foundation to W.F. and M.J.S. [grant number RG/07/001].

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Received 17 October 2012 doi:10.1042/BST20120269