{"gene":"FXYD1","run_date":"2026-06-14T21:07:21+00:00","timeline":{"discoveries":[{"year":1994,"finding":"Protein kinase C phosphorylates phospholemman at both Ser-63 and Ser-68 in its cytoplasmic C-terminal domain, while cAMP-dependent protein kinase (PKA) phosphorylates only Ser-68. Insulin stimulation results in labeling of phosphopeptides containing both Ser-63 and Ser-68, whereas adrenaline results in labeling of the peptide containing Ser-68.","method":"In vitro phosphorylation assay with synthetic peptide substrates, amino acid sequencing of phosphopeptides, thermolytic phosphopeptide mapping of 32P-labeled phospholemman from rat diaphragm","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with synthetic peptide plus direct amino acid sequencing of phosphorylation sites, confirmed in intact tissue","pmids":["7999001"],"is_preprint":false},{"year":1995,"finding":"Phospholemman (PLM) induces hyperpolarization-activated chloride currents when expressed in Xenopus oocytes, establishing it as a Cl- channel or Cl- channel regulator.","method":"Xenopus oocyte expression system with electrophysiological current recording","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional electrophysiology in oocytes, replicated across multiple papers but mechanistic detail limited to current induction","pmids":["7836447"],"is_preprint":false},{"year":1998,"finding":"PLM topology places the extracellular N-terminal segment (residues 1–17) in a protease-resistant configuration, while the intracellular C-terminal domain (residues 38–72) is protease-sensitive. The cytoplasmic tail is required for voltage-dependent channel inactivation: trypsin treatment yielding a limit peptide (residues 1–43) or recombinant PLM 1–43 retains ion-channel activity in lipid bilayers but shows dramatically reduced voltage-dependent inactivation.","method":"Protease protection assays on sarcolemmal membrane vesicles; site-specific antibody immunoblots; lipid bilayer electrophysiology with full-length, trypsinized, and recombinant truncated PLM","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution in lipid bilayers with truncation mutagenesis and functional validation in a single rigorous study","pmids":["9486665"],"is_preprint":false},{"year":1999,"finding":"PKA co-expression increases PLM-induced oocyte current amplitude and membrane PLM level largely through phosphorylation of Ser-68; a phosphorylation-null PLM mutant (SSST→AAAA) is unresponsive to PKA co-expression. The cytoplasmic domain is not essential for inducing currents.","method":"Xenopus oocyte co-expression with PKA, PKC, and NIMA kinase; electrophysiology; phosphorylation-site mutagenesis (Ser→Ala)","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional oocyte assay combined with site-directed mutagenesis, single lab","pmids":["10556585"],"is_preprint":false},{"year":2000,"finding":"Phospholemman is a substrate for myotonic dystrophy protein kinase (DMPK) in vitro. Co-expression of DMPK with PLM in Xenopus oocytes reduces PLM-induced Cl- current amplitude and reduces membrane PLM expression; this effect is absent with a phosphorylation-null PLM mutant (all Ser→Ala), indicating it is phosphorylation-dependent.","method":"In vitro kinase assay; Xenopus oocyte co-expression; electrophysiology; phosphorylation-null PLM mutant","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay plus functional oocyte mutagenesis, single lab","pmids":["10811636"],"is_preprint":false},{"year":2001,"finding":"Reduction of PLM expression by antisense oligonucleotides in cerebellar astrocytes decreases osmosensitive taurine efflux by 62–67%, demonstrating that PLM plays a role in regulatory volume decrease (RVD) via taurine flux. PKA activation increases this taurine efflux, while PKC appears largely dispensable for the taurine component.","method":"Antisense oligonucleotide knockdown of PLM in cerebellar astrocytes; [3H]taurine and 125I efflux assays; pharmacological PKA/PKC activation and inhibition","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function knockdown with quantitative tracer efflux readout, single lab","pmids":["11336802"],"is_preprint":false},{"year":2003,"finding":"Phospholemman physically associates with Na,K-ATPase (all three alpha isoforms, alpha1–alpha3) in cerebellum and choroid plexus, demonstrated by co-purification and reciprocal co-immunoprecipitation. Antibodies against the C-terminal domain of PLM reduce Na,K-ATPase activity in vitro without altering Na+ affinity.","method":"Detergent co-purification; reciprocal co-immunoprecipitation from solubilized crude membranes; in vitro Na,K-ATPase activity assay with antibody inhibition","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, co-purification, and functional antibody inhibition assay, multiple orthogonal methods","pmids":["12657675"],"is_preprint":false},{"year":2005,"finding":"PLM co-immunoprecipitates with Na,K-ATPase alpha1 and alpha2 isoforms in cardiac myocytes; PLM expression is reduced in heart failure and a higher fraction of PLM is phosphorylated at Ser-68 in HF, consistent with phosphorylation relieving PLM-mediated inhibition of NKA.","method":"Co-immunoprecipitation from rabbit and human cardiac myocytes; immunoblotting for phospho-Ser-68 PLM; Na,K-ATPase activity assay","journal":"Circulation research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with functional NKA activity data, single lab","pmids":["16100047"],"is_preprint":false},{"year":2005,"finding":"PLM co-immunoprecipitates with both alpha1 and alpha2 isoforms of Na,K-ATPase in skeletal muscle, and anti-PLM antibody reduces NKA activity, indicating PLM is required for full NKA activity in skeletal muscle.","method":"Co-immunoprecipitation from rat skeletal muscle; Na,K-ATPase activity assay with anti-PLM antibody; immunofluorescence localization","journal":"Journal of applied physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus antibody functional assay, single lab","pmids":["15961612"],"is_preprint":false},{"year":2006,"finding":"PLM and Na,K-ATPase are in sufficient proximity for FRET when expressed as CFP/YFP fusion proteins in HEK293 cells. PKA activation (Ser-68 phosphorylation) and PKC activation (Ser-63 and Ser-68 phosphorylation) progressively and reversibly decrease NKA–PLM FRET, indicating phosphorylation reduces their physical interaction. PLM–PLM FRET is stronger than NKA–PLM FRET and is enhanced by phosphorylation, consistent with PLM multimerization upon phosphorylation. No FRET was detected between PLM and Na/Ca exchanger despite membrane co-localization.","method":"FRET (acceptor photobleach and fluorescence ratio methods) with CFP/YFP fusion proteins in HEK293 cells; PKA/PKC pharmacological activation; phosphorylation state analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple FRET methods (donor dequench + ratio), pharmacological manipulations, negative FRET control for NCX, single rigorous study with orthogonal approaches","pmids":["16943195"],"is_preprint":false},{"year":2006,"finding":"PLM mediates the PKC-dependent activation of Na/K-ATPase (NKA) function in cardiac myocytes: PKC activation (PDBu) increases NKA Vmax in wild-type myocytes but has no effect on NKA Vmax or Na+ affinity in PLM-knockout myocytes. PKA (isoproterenol) and PKC effects are additive, acting through different parameters (Na+ affinity and Vmax, respectively).","method":"Whole-cell voltage clamp (pump current) and SBFI fluorescence ([Na+]i) measurements in wild-type and PLM-knockout mouse ventricular myocytes; pharmacological PKC and PKA activation","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis (PLM-KO) combined with two orthogonal functional assays (patch clamp and fluorescence), replicated across conditions","pmids":["17095720"],"is_preprint":false},{"year":2007,"finding":"PKA phosphorylation of PLM at Ser-68 increases the apparent Na+ affinity (decreases K0.5 for Na+) of both NKA-alpha1/beta1 and alpha2/beta1 isozymes without altering maximal transport activity. PKC phosphorylation of PLM increases maximal pump current (turnover number) of alpha2/beta1 but not alpha1/beta1, without affecting K+ affinity of either isozyme.","method":"Xenopus oocyte expression of PLM with defined NKA alpha/beta isozymes; two-electrode voltage clamp; PKA and PKC pharmacological activation; PLM phosphorylation-site mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — electrophysiological reconstitution in defined isozyme pairs with mutagenesis, multiple isozyme comparisons","pmids":["17991751"],"is_preprint":false},{"year":2007,"finding":"PLM associates with cardiac Na+/Ca2+ exchanger 1 (NCX1) in the sarcolemma and transverse tubules (demonstrated by co-localization and co-immunoprecipitation). PLM inhibits NCX1 independently of its effects on Na,K-ATPase; the cytoplasmic domain of PLM is required for NCX1 regulation. Phosphorylation of PLM at Ser-68 is the active form that inhibits NCX1 (in contrast, unphosphorylated PLM inhibits Na,K-ATPase).","method":"Adenovirus-mediated overexpression and siRNA knockdown in adult rat cardiomyocytes; heterologous co-expression in HEK293 cells; co-immunoprecipitation; electrophysiology (NCX1 current); 45Ca2+ uptake; PLM cytoplasmic domain truncation mutants; phosphomimetic and phospho-deficient mutants","journal":"Annals of the New York Academy of Sciences","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, electrophysiology, tracer uptake, mutagenesis) in multiple systems","pmids":["17446450"],"is_preprint":false},{"year":2009,"finding":"PLM forms 1:1 stoichiometric complexes with both NKA-alpha1 and NKA-alpha2 (FRET-based). PLM phosphorylation (PKA and PKC) drastically reduces FRET with both isoforms. PLM–PLM FRET indicates oligomers of ≥3 monomers. Isoproterenol (via PKA) increases Na+ affinity of both NKA-alpha1 and alpha2; PKC activation increases Vmax only for NKA-alpha2 but reduces K(1/2) for both. Ouabain abolishes NKA–PLM FRET but only partially reduces co-immunoprecipitation.","method":"FRET (progressive acceptor photobleach) in HEK293 cells; cardiac myocytes from WT and NKA-alpha isoform ouabain-sensitivity knock-in mice; whole-cell voltage clamp; SBFI fluorescence; pharmacological PKA/PKC activation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — FRET stoichiometry, genetic mouse models with defined isoform sensitivities, two functional readouts, multiple orthogonal approaches","pmids":["19638348"],"is_preprint":false},{"year":2010,"finding":"PLM phosphorylation at either Ser-63 or Ser-68 alone is necessary and sufficient to completely relieve PLM-induced inhibition of NKA Na+ affinity. The double-mutant AA PLM (Ser63Ala/Ser68Ala) cannot be relieved by PKA or PKC activation; single-site mutants S63A or S68A retain responsiveness.","method":"HeLa cells stably expressing rat NKA-alpha1; transient expression of WT, S63A, S68A, and AA PLM mutants; SBFI fluorescence for intracellular Na+ concentration; PKA and PKC pharmacological activation","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — systematic site-directed mutagenesis with quantitative functional assay in defined cellular system","pmids":["20861470"],"is_preprint":false},{"year":2012,"finding":"Surface expression of PLM in Xenopus oocytes requires co-expression with Na,K-ATPase (alpha1/beta1); the Na+/Ca2+ exchanger cannot drive PLM to the cell surface. A phosphorylation-mimicking mutation at Thr-69 or truncation of three C-terminal arginine residues facilitates NKA-dependent surface expression of PLM.","method":"Xenopus oocyte expression system; surface biotinylation; co-expression with NKA, NCX; PLM mutants (Thr-69 phosphomimetic, C-terminal truncations)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — oocyte surface expression assay with mutagenesis, single lab, biochemical readout","pmids":["22535957"],"is_preprint":false},{"year":2013,"finding":"Two pools of PLM exist in adult rat ventricular myocytes: one pool associated with the sodium pump (phosphorylated at Ser-68 or unphosphorylated) and a separate pool of PLM oligomers not associated with the pump (phosphorylated at Ser-63). PLM multimers co-immunoprecipitate unphosphorylatable PLM from heterozygous transgenic hearts, confirming PLM–PLM multimerization. The non-pump-associated PLM pool has no effect on sodium pump activity upon dephosphorylation.","method":"Phosphospecific co-immunoprecipitation from adult rat ventricular myocytes; mass spectrometry; chemical cross-linking; heterozygous transgenic mice expressing WT and unphosphorylatable PLM; sodium pump activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, mass spectrometry, cross-linking, transgenic animal model, and functional assay, multiple orthogonal methods","pmids":["23532852"],"is_preprint":false},{"year":2013,"finding":"Nitric oxide (NO) activates Na,K-ATPase via a PKCε–PLM phosphorylation pathway: field stimulation increases endogenous NO, PKCε activation, and PLM phosphorylation (Ser-63 and Ser-68), all of which are abolished by Ca2+ chelation or NOS inhibition. Exogenous NO stimulates NKA in PLM-WT but not PLM-KO or PLM-3SA myocytes, identifying PLM phosphorylation as required for NO-dependent NKA activation.","method":"Rat ventricular myocytes; DAF-FM dye (NO), Western blotting (PKCε, phospho-PLM), biochemical NKA assay; perforated-patch clamp in PLM-WT, PLM-KO, and PLM-3SA (unphosphorylatable) myocytes; SBFI fluorescence","journal":"Journal of molecular and cellular cardiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis (three mouse genotypes), biochemical pathway dissection, and functional electrophysiology, multiple orthogonal methods","pmids":["23612119"],"is_preprint":false},{"year":2013,"finding":"The transmembrane domain of PLM interacts with TM9 of the NKA alpha-subunit; the cytoplasmic tail of PLM interacts with two small regions (residues 248–252 and 300–304) of the proximal intracellular loop of NCX1.","method":"Mutational analysis and heterologous co-expression (cited as prior work summarized in review); co-immunoprecipitation","journal":"Advances in experimental medicine and biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — domain mapping described in review/summary context without full primary experimental detail in this abstract","pmids":["23224879"],"is_preprint":false},{"year":2014,"finding":"Prevention of PLM phosphorylation in PLM-3SA knock-in mice (Ser63/68/69→Ala) increases [Na+]i, reduces forward-mode NCX, exacerbates cardiac hypertrophy and NKA inhibition after aortic constriction compared to WT. In WT mice, aortic constriction causes PLM hypophosphorylation, progressive NKA current decline, and elevated [Na+]i. These data establish that PLM phosphorylation is causally required to maintain NKA activity and limit Na+ overload and adverse remodeling.","method":"PLM-3SA knock-in mice; aortic constriction model; echocardiography; pressure-volume catheterization; SBFI fluorescence for [Na+]i; whole-cell patch clamp for NKA current; Western blotting","journal":"Cardiovascular research","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knock-in mouse with multiple orthogonal physiological and biochemical readouts, in vivo and ex vivo validation","pmids":["25103111"],"is_preprint":false},{"year":2016,"finding":"Scanning mutagenesis of PLM transmembrane domain identifies residue L30 as critical for PLM–PLM tetramerization (L30A decreases PLM–PLM FRET) and for functional inhibition of NKA. L30A PLM shows increased NKA–PLM FRET and superinhibition of NKA, increasing Ca2+ transient amplitude in cardiomyocytes. These superinhibitory effects are reversible with isoproterenol (PKA activation). Molecular dynamics simulations show L30A distorts the TM helix and destabilizes the tetramer.","method":"Scanning mutagenesis of PLM TM domain; FRET in HEK293 cells; Ca2+ transient measurements in isolated cardiomyocytes; molecular dynamics simulations; isoproterenol treatment","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — FRET mutagenesis plus functional cardiomyocyte assay, single lab, supported by MD simulations","pmids":["27718550"],"is_preprint":false},{"year":2020,"finding":"PLM phosphorylation at Ser-63 and Ser-68 limits vascular constriction in response to phenylephrine via Na/K-ATPase (effect blocked by ouabain). PLM-3SA mice (unphosphorylatable PLM) show profoundly enhanced vascular responses to phenylephrine in vitro and in vivo and develop aging-induced hypertension. A human coding variant R70C (SNP rs61753924) prevents PLM phosphorylation at Ser-68 in HEK293 cells and is associated with elevated blood pressure in middle-aged men.","method":"PLM-3SA knock-in mice; wire myography of aortic and mesenteric vessels; Doppler flow and telemetry for in vivo BP; HEK293 cell phosphorylation assay; human genomic cohort analyses (UK Biobank, GoDARTS)","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic mouse model with in vitro and in vivo vascular functional assays plus human variant mechanistic validation, multiple orthogonal methods","pmids":["33334125"],"is_preprint":false},{"year":2024,"finding":"Peroxiredoxin 6 (Prdx6) interacts with PLM and depalmitoylates it in a glutathione-dependent manner. Glutathione loading reduces PLM palmitoylation; glutathione depletion increases PLM palmitoylation. Prdx6 silencing abolishes these effects. In vitro, recombinant Prdx6 (but not other Prdx isoforms) removes palmitic acid from palmitoylated recombinant PLM. PLM palmitoylation inhibits Na,K-ATPase activity, and this is reversed by Prdx6-mediated depalmitoylation. The broad-spectrum depalmitoylase inhibitor palmostatin B blocks Prdx6-dependent PLM depalmitoylation in cells and in vitro, suggesting Prdx6 acts as a thioesterase via nucleophilic attack through its reactive thiol.","method":"Co-immunoprecipitation of Prdx6 and PLM; acyl-RAC assay for palmitoylation; glutathione loading/depletion in cells; Prdx6 siRNA silencing; in vitro depalmitoylation assay with recombinant proteins; palmostatin B inhibition; Na,K-ATPase activity assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with recombinant proteins, isoform specificity screen, cell-based validation with loss-of-function and pharmacological inhibition, multiple orthogonal methods","pmids":["38236777"],"is_preprint":false}],"current_model":"Phospholemman (FXYD1) is a 72-amino-acid single-span sarcolemmal protein that functions as the primary regulatory subunit of the cardiac Na/K-ATPase (NKA): in its unphosphorylated form it tonically inhibits NKA by reducing its apparent Na+ affinity, and this inhibition is relieved when PKA phosphorylates Ser-68 (increasing Na+ affinity) or PKC phosphorylates Ser-63 and/or Ser-68 (increasing Na+ affinity and, for the alpha2 isoform, also Vmax); conversely, Ser-68-phosphorylated PLM inhibits the Na+/Ca2+ exchanger NCX1 via its cytoplasmic tail, thereby coordinately limiting arrhythmias and preserving contractility during adrenergic stress; PLM is also subject to palmitoylation (which inhibits NKA) that is reversed by peroxiredoxin 6 in a glutathione-dependent thioesterase reaction, and to glutathionylation; PLM exists in cardiac muscle as both NKA-associated monomers and a separate pool of oligomers (≥3 monomers) that do not regulate the pump; surface trafficking of PLM itself requires co-expression with NKA; and NO activates NKA through a PKCε–PLM phosphorylation cascade, establishing PLM as the central nexus linking multiple intracellular signaling pathways to Na+ and Ca2+ homeostasis in the heart."},"narrative":{"mechanistic_narrative":"FXYD1 (phospholemman, PLM) is a single-span sarcolemmal protein that serves as the central regulatory hub coupling intracellular signaling to Na+ and Ca2+ homeostasis in cardiac, skeletal, and vascular tissue by tuning the activity of the Na/K-ATPase (NKA) and the Na+/Ca2+ exchanger NCX1 [PMID:16100047, PMID:17095720, PMID:17446450]. PLM physically associates with NKA alpha1, alpha2, and alpha3 isoforms through its transmembrane domain, forming 1:1 complexes, and in its unphosphorylated state tonically inhibits the pump; its cytoplasmic C-terminal tail carries the regulatory phosphosites Ser-63 and Ser-68, where PKC phosphorylates both and PKA phosphorylates only Ser-68 [PMID:7999001, PMID:12657675, PMID:19638348]. Phosphorylation at either Ser-63 or Ser-68 is necessary and sufficient to relieve PLM-mediated inhibition: PKA-driven Ser-68 phosphorylation raises the apparent Na+ affinity of both alpha1 and alpha2 isozymes, while PKC additionally increases Vmax selectively for alpha2, and these phosphorylation events reduce the physical NKA–PLM interaction [PMID:16943195, PMID:17991751, PMID:20861470]. The same C-terminal tail allows Ser-68-phosphorylated PLM to inhibit NCX1, establishing PLM as a coordinator of pump and exchanger activity [PMID:17446450]. PLM exists as both NKA-associated monomers and a separate pool of oligomers (≥3 monomers) that do not regulate the pump, with transmembrane residue L30 governing tetramerization [PMID:23532852, PMID:27718550], and its surface trafficking requires co-expression with NKA [PMID:22535957]. Beyond phosphorylation, PLM is regulated by palmitoylation, which inhibits NKA and is reversed by a glutathione-dependent peroxiredoxin 6 thioesterase reaction [PMID:38236777], and nitric oxide activates NKA through a PKCε–PLM phosphorylation cascade [PMID:23612119]. Genetic abolition of PLM phosphorylation causes Na+ overload, impaired NCX, exacerbated cardiac hypertrophy after pressure overload, enhanced vascular constriction, and hypertension, and a human R70C coding variant that blocks Ser-68 phosphorylation associates with elevated blood pressure [PMID:25103111, PMID:33334125].","teleology":[{"year":1994,"claim":"Established the phosphorylation code of PLM by mapping which kinases target which cytoplasmic serines, the foundation for all later regulatory models.","evidence":"In vitro kinase assays with synthetic peptides plus phosphopeptide mapping of labeled PLM from rat diaphragm","pmids":["7999001"],"confidence":"High","gaps":["Did not define the functional consequence of phosphorylation on any transporter","Did not identify the binding partner regulated by these sites"]},{"year":1995,"claim":"Provided the first functional readout of PLM by showing it induces ion currents in a heterologous system, framing it as a channel or channel regulator before its NKA role was known.","evidence":"Xenopus oocyte expression with electrophysiological current recording","pmids":["7836447"],"confidence":"Medium","gaps":["Whether PLM forms the conductive pore or regulates an endogenous oocyte channel was unresolved","No link yet to NKA or NCX"]},{"year":1998,"claim":"Defined PLM membrane topology and localized voltage-dependent inactivation to the cytoplasmic tail, connecting structure to channel behavior.","evidence":"Protease protection, site-specific antibodies, and lipid bilayer electrophysiology with truncated PLM","pmids":["9486665"],"confidence":"High","gaps":["Channel activity in bilayers not reconciled with a physiological transporter target","Cytoplasmic tail's later regulatory partners not yet identified"]},{"year":2000,"claim":"Extended the kinase repertoire acting on PLM and showed phosphorylation alters its membrane level and current, hinting at trafficking control.","evidence":"In vitro DMPK kinase assay and oocyte co-expression with phosphorylation-null PLM mutant","pmids":["10811636"],"confidence":"Medium","gaps":["Physiological relevance of DMPK–PLM in heart not established","Mechanism linking phosphorylation to membrane level unclear"]},{"year":2001,"claim":"Demonstrated a physiological loss-of-function role for PLM in cell volume regulation via taurine efflux, broadening its functional reach beyond cardiac myocytes.","evidence":"Antisense knockdown in cerebellar astrocytes with tracer efflux assays","pmids":["11336802"],"confidence":"Medium","gaps":["Molecular identity of the taurine-permeable pathway not defined","Relationship between volume regulation and NKA association unknown"]},{"year":2003,"claim":"Identified the Na/K-ATPase as a direct physical and functional partner of PLM, the discovery that reframed PLM as an NKA regulatory subunit.","evidence":"Co-purification and reciprocal Co-IP from cerebellum and choroid plexus with antibody-inhibition NKA activity assay","pmids":["12657675"],"confidence":"High","gaps":["Whether PLM inhibits or activates NKA, and the role of phosphorylation, not yet resolved","Cardiac relevance untested"]},{"year":2005,"claim":"Confirmed the PLM–NKA association in cardiac and skeletal muscle and linked PLM dysregulation to heart failure, giving the interaction disease context.","evidence":"Co-IP from cardiac and skeletal muscle with NKA activity assays and phospho-Ser-68 immunoblotting","pmids":["16100047","15961612"],"confidence":"Medium","gaps":["Causality of PLM changes in heart failure not established","Quantitative effect of phosphorylation on pump kinetics undefined"]},{"year":2006,"claim":"Resolved the directionality and mechanism of PLM regulation: PLM inhibits NKA, and phosphorylation relieves inhibition by reducing physical interaction, dissected by isoform-specific kinetics and genetic deletion.","evidence":"FRET in HEK293 cells plus whole-cell clamp and SBFI in PLM-knockout myocytes with PKA/PKC activation","pmids":["16943195","17095720"],"confidence":"High","gaps":["Whether PLM dissociates or only reorients upon phosphorylation not fully resolved","NCX1 regulation not yet addressed"]},{"year":2007,"claim":"Defined the isozyme-specific kinetic logic (PKA raises Na+ affinity; PKC raises alpha2 Vmax) and revealed that phosphorylated PLM separately inhibits NCX1 through its cytoplasmic tail, establishing dual transporter control.","evidence":"Oocyte expression of defined NKA isozymes with mutants; Co-IP, NCX1 electrophysiology and 45Ca2+ uptake in cardiomyocytes and HEK293 cells","pmids":["17991751","17446450"],"confidence":"High","gaps":["Structural basis of the opposite phospho-dependence for NKA versus NCX1 unexplained","Stoichiometry of NKA–PLM complex not yet measured"]},{"year":2009,"claim":"Established the stoichiometry of regulation, showing 1:1 NKA–PLM complexes for both alpha isoforms alongside PLM oligomers, refining the molecular model of pump regulation.","evidence":"Progressive acceptor-photobleach FRET in HEK293 cells and isoform-specific ouabain knock-in myocytes with clamp and SBFI","pmids":["19638348"],"confidence":"High","gaps":["Functional role of the PLM oligomer pool not yet defined","How monomer/oligomer balance is controlled unknown"]},{"year":2010,"claim":"Showed phosphorylation of either Ser-63 or Ser-68 alone fully relieves NKA inhibition, defining the minimal molecular switch and explaining redundancy between PKA and PKC inputs.","evidence":"Single and double phosphosite mutants expressed with NKA-alpha1 in HeLa cells with SBFI readout","pmids":["20861470"],"confidence":"High","gaps":["Whether the two sites are functionally equivalent in vivo not addressed","Structural mechanism of relief not resolved"]},{"year":2012,"claim":"Identified NKA as the obligate chaperone for PLM surface delivery, explaining how complex assembly is coupled to trafficking.","evidence":"Surface biotinylation in Xenopus oocytes with co-expression and trafficking mutants","pmids":["22535957"],"confidence":"Medium","gaps":["Trafficking determinants in native cardiac cells not validated","Role of C-terminal arginine retention signal mechanistically unclear"]},{"year":2013,"claim":"Distinguished two physically and functionally distinct PLM pools (NKA-associated monomers versus non-regulatory oligomers), defined the NO–PKCε–PLM activation cascade, and began mapping the interaction surfaces with NKA and NCX1.","evidence":"Phosphospecific Co-IP, MS, cross-linking and transgenic hearts; NO/PKCε pathway dissection in PLM-WT/KO/3SA myocytes; domain mapping summarized in review","pmids":["23532852","23612119","23224879"],"confidence":"High","gaps":["Why Ser-63-phosphorylated PLM partitions to oligomers is unknown","Domain-mapping details for the NCX1 interface rest on summarized rather than full primary data"]},{"year":2014,"claim":"Provided in vivo causality that PLM phosphorylation maintains NKA activity and limits Na+ overload and adverse remodeling under pressure overload.","evidence":"PLM-3SA knock-in mice with aortic constriction, echocardiography, pressure-volume catheterization, SBFI, and NKA current clamp","pmids":["25103111"],"confidence":"High","gaps":["Relative contributions of NKA versus NCX dysregulation to remodeling not separated","Therapeutic reversibility not tested"]},{"year":2016,"claim":"Linked PLM tetramerization to its inhibitory potency by identifying L30 as a residue whose mutation destabilizes oligomers and superinhibits NKA, connecting oligomeric state to function.","evidence":"TM scanning mutagenesis with FRET, cardiomyocyte Ca2+ transients, and molecular dynamics simulations","pmids":["27718550"],"confidence":"Medium","gaps":["Physiological regulation of PLM oligomeric state in vivo unknown","Single-lab finding without independent structural confirmation"]},{"year":2020,"claim":"Extended PLM regulation to vascular tone and human blood pressure, with a coding variant blocking Ser-68 phosphorylation, establishing translational relevance.","evidence":"PLM-3SA mice with wire myography and in vivo BP; HEK293 phosphorylation assay of R70C; UK Biobank and GoDARTS cohort analyses","pmids":["33334125"],"confidence":"High","gaps":["Whether R70C is causal versus associated in humans not proven","Vascular cell type mediating the effect not pinpointed"]},{"year":2024,"claim":"Added a redox-coupled regulatory layer by identifying Prdx6 as a glutathione-dependent depalmitoylase that reverses inhibitory PLM palmitoylation of NKA.","evidence":"Co-IP, acyl-RAC, glutathione manipulation, Prdx6 silencing, in vitro depalmitoylation with recombinant proteins, palmostatin B, and NKA activity assays","pmids":["38236777"],"confidence":"High","gaps":["Palmitoylating enzyme for PLM not identified","In vivo physiological role of PLM palmitoylation cycling untested"]},{"year":null,"claim":"How the multiple PLM regulatory inputs (phosphorylation, palmitoylation, glutathionylation, oligomerization) are integrated and prioritized to set Na+/Ca2+ homeostasis in a given physiological state remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified structural model of the NKA–PLM–NCX1 regulatory module","Crosstalk between palmitoylation and phosphorylation switches unmapped","Tissue-specific differences in PLM regulation not systematically compared"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[6,10,11,12,14]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[22]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[8,12,15]}],"pathway":[{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[6,10,12]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[10,11,17]}],"complexes":["Na/K-ATPase regulatory complex (FXYD1–NKA)"],"partners":["ATP1A1","ATP1A2","ATP1A3","SLC8A1","PRDX6","PRKCE","DMPK"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O00168","full_name":"Phospholemman","aliases":["FXYD domain-containing ion transport regulator 1","Sodium/potassium-transporting ATPase subunit FXYD1"],"length_aa":92,"mass_kda":10.4,"function":"Associates with and regulates the activity of the sodium/potassium-transporting ATPase (NKA) which transports Na(+) out of the cell and K(+) into the cell. Inhibits NKA activity in its unphosphorylated state and stimulates activity when phosphorylated. Reduces glutathionylation of the NKA beta-1 subunit ATP1B1, thus reversing glutathionylation-mediated inhibition of ATP1B1. Contributes to female sexual development by maintaining the excitability of neurons which secrete gonadotropin-releasing hormone","subcellular_location":"Cell membrane, sarcolemma; Apical cell membrane; Membrane, caveola; Cell membrane, sarcolemma, T-tubule","url":"https://www.uniprot.org/uniprotkb/O00168/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/FXYD1","classification":"Not Classified","n_dependent_lines":27,"n_total_lines":1208,"dependency_fraction":0.022350993377483443},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/FXYD1","total_profiled":1310},"omim":[{"mim_id":"602359","title":"FXYD DOMAIN-CONTAINING ION TRANSPORT REGULATOR 1; FXYD1","url":"https://www.omim.org/entry/602359"},{"mim_id":"312750","title":"RETT SYNDROME; RTT","url":"https://www.omim.org/entry/312750"},{"mim_id":"300005","title":"METHYL-CpG-BINDING PROTEIN 2; MECP2","url":"https://www.omim.org/entry/300005"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"},{"location":"Nucleoli","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"choroid plexus","ntpm":1674.5},{"tissue":"skeletal muscle","ntpm":1295.2}],"url":"https://www.proteinatlas.org/search/FXYD1"},"hgnc":{"alias_symbol":[],"prev_symbol":["PLM"]},"alphafold":{"accession":"O00168","domains":[{"cath_id":"1.20.5","chopping":"37-68","consensus_level":"medium","plddt":76.9881,"start":37,"end":68}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O00168","model_url":"https://alphafold.ebi.ac.uk/files/AF-O00168-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O00168-F1-predicted_aligned_error_v6.png","plddt_mean":71.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=FXYD1","jax_strain_url":"https://www.jax.org/strain/search?query=FXYD1"},"sequence":{"accession":"O00168","fasta_url":"https://rest.uniprot.org/uniprotkb/O00168.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O00168/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O00168"}},"corpus_meta":[{"pmid":"7836447","id":"PMC_7836447","title":"Mat-8, a novel phospholemman-like protein 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Insulin stimulation results in labeling of phosphopeptides containing both Ser-63 and Ser-68, whereas adrenaline results in labeling of the peptide containing Ser-68.\",\n      \"method\": \"In vitro phosphorylation assay with synthetic peptide substrates, amino acid sequencing of phosphopeptides, thermolytic phosphopeptide mapping of 32P-labeled phospholemman from rat diaphragm\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with synthetic peptide plus direct amino acid sequencing of phosphorylation sites, confirmed in intact tissue\",\n      \"pmids\": [\"7999001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Phospholemman (PLM) induces hyperpolarization-activated chloride currents when expressed in Xenopus oocytes, establishing it as a Cl- channel or Cl- channel regulator.\",\n      \"method\": \"Xenopus oocyte expression system with electrophysiological current recording\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional electrophysiology in oocytes, replicated across multiple papers but mechanistic detail limited to current induction\",\n      \"pmids\": [\"7836447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"PLM topology places the extracellular N-terminal segment (residues 1–17) in a protease-resistant configuration, while the intracellular C-terminal domain (residues 38–72) is protease-sensitive. The cytoplasmic tail is required for voltage-dependent channel inactivation: trypsin treatment yielding a limit peptide (residues 1–43) or recombinant PLM 1–43 retains ion-channel activity in lipid bilayers but shows dramatically reduced voltage-dependent inactivation.\",\n      \"method\": \"Protease protection assays on sarcolemmal membrane vesicles; site-specific antibody immunoblots; lipid bilayer electrophysiology with full-length, trypsinized, and recombinant truncated PLM\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution in lipid bilayers with truncation mutagenesis and functional validation in a single rigorous study\",\n      \"pmids\": [\"9486665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"PKA co-expression increases PLM-induced oocyte current amplitude and membrane PLM level largely through phosphorylation of Ser-68; a phosphorylation-null PLM mutant (SSST→AAAA) is unresponsive to PKA co-expression. The cytoplasmic domain is not essential for inducing currents.\",\n      \"method\": \"Xenopus oocyte co-expression with PKA, PKC, and NIMA kinase; electrophysiology; phosphorylation-site mutagenesis (Ser→Ala)\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional oocyte assay combined with site-directed mutagenesis, single lab\",\n      \"pmids\": [\"10556585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Phospholemman is a substrate for myotonic dystrophy protein kinase (DMPK) in vitro. Co-expression of DMPK with PLM in Xenopus oocytes reduces PLM-induced Cl- current amplitude and reduces membrane PLM expression; this effect is absent with a phosphorylation-null PLM mutant (all Ser→Ala), indicating it is phosphorylation-dependent.\",\n      \"method\": \"In vitro kinase assay; Xenopus oocyte co-expression; electrophysiology; phosphorylation-null PLM mutant\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay plus functional oocyte mutagenesis, single lab\",\n      \"pmids\": [\"10811636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Reduction of PLM expression by antisense oligonucleotides in cerebellar astrocytes decreases osmosensitive taurine efflux by 62–67%, demonstrating that PLM plays a role in regulatory volume decrease (RVD) via taurine flux. PKA activation increases this taurine efflux, while PKC appears largely dispensable for the taurine component.\",\n      \"method\": \"Antisense oligonucleotide knockdown of PLM in cerebellar astrocytes; [3H]taurine and 125I efflux assays; pharmacological PKA/PKC activation and inhibition\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function knockdown with quantitative tracer efflux readout, single lab\",\n      \"pmids\": [\"11336802\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Phospholemman physically associates with Na,K-ATPase (all three alpha isoforms, alpha1–alpha3) in cerebellum and choroid plexus, demonstrated by co-purification and reciprocal co-immunoprecipitation. Antibodies against the C-terminal domain of PLM reduce Na,K-ATPase activity in vitro without altering Na+ affinity.\",\n      \"method\": \"Detergent co-purification; reciprocal co-immunoprecipitation from solubilized crude membranes; in vitro Na,K-ATPase activity assay with antibody inhibition\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, co-purification, and functional antibody inhibition assay, multiple orthogonal methods\",\n      \"pmids\": [\"12657675\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PLM co-immunoprecipitates with Na,K-ATPase alpha1 and alpha2 isoforms in cardiac myocytes; PLM expression is reduced in heart failure and a higher fraction of PLM is phosphorylated at Ser-68 in HF, consistent with phosphorylation relieving PLM-mediated inhibition of NKA.\",\n      \"method\": \"Co-immunoprecipitation from rabbit and human cardiac myocytes; immunoblotting for phospho-Ser-68 PLM; Na,K-ATPase activity assay\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with functional NKA activity data, single lab\",\n      \"pmids\": [\"16100047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PLM co-immunoprecipitates with both alpha1 and alpha2 isoforms of Na,K-ATPase in skeletal muscle, and anti-PLM antibody reduces NKA activity, indicating PLM is required for full NKA activity in skeletal muscle.\",\n      \"method\": \"Co-immunoprecipitation from rat skeletal muscle; Na,K-ATPase activity assay with anti-PLM antibody; immunofluorescence localization\",\n      \"journal\": \"Journal of applied physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus antibody functional assay, single lab\",\n      \"pmids\": [\"15961612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PLM and Na,K-ATPase are in sufficient proximity for FRET when expressed as CFP/YFP fusion proteins in HEK293 cells. PKA activation (Ser-68 phosphorylation) and PKC activation (Ser-63 and Ser-68 phosphorylation) progressively and reversibly decrease NKA–PLM FRET, indicating phosphorylation reduces their physical interaction. PLM–PLM FRET is stronger than NKA–PLM FRET and is enhanced by phosphorylation, consistent with PLM multimerization upon phosphorylation. No FRET was detected between PLM and Na/Ca exchanger despite membrane co-localization.\",\n      \"method\": \"FRET (acceptor photobleach and fluorescence ratio methods) with CFP/YFP fusion proteins in HEK293 cells; PKA/PKC pharmacological activation; phosphorylation state analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple FRET methods (donor dequench + ratio), pharmacological manipulations, negative FRET control for NCX, single rigorous study with orthogonal approaches\",\n      \"pmids\": [\"16943195\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PLM mediates the PKC-dependent activation of Na/K-ATPase (NKA) function in cardiac myocytes: PKC activation (PDBu) increases NKA Vmax in wild-type myocytes but has no effect on NKA Vmax or Na+ affinity in PLM-knockout myocytes. PKA (isoproterenol) and PKC effects are additive, acting through different parameters (Na+ affinity and Vmax, respectively).\",\n      \"method\": \"Whole-cell voltage clamp (pump current) and SBFI fluorescence ([Na+]i) measurements in wild-type and PLM-knockout mouse ventricular myocytes; pharmacological PKC and PKA activation\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis (PLM-KO) combined with two orthogonal functional assays (patch clamp and fluorescence), replicated across conditions\",\n      \"pmids\": [\"17095720\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PKA phosphorylation of PLM at Ser-68 increases the apparent Na+ affinity (decreases K0.5 for Na+) of both NKA-alpha1/beta1 and alpha2/beta1 isozymes without altering maximal transport activity. PKC phosphorylation of PLM increases maximal pump current (turnover number) of alpha2/beta1 but not alpha1/beta1, without affecting K+ affinity of either isozyme.\",\n      \"method\": \"Xenopus oocyte expression of PLM with defined NKA alpha/beta isozymes; two-electrode voltage clamp; PKA and PKC pharmacological activation; PLM phosphorylation-site mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — electrophysiological reconstitution in defined isozyme pairs with mutagenesis, multiple isozyme comparisons\",\n      \"pmids\": [\"17991751\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PLM associates with cardiac Na+/Ca2+ exchanger 1 (NCX1) in the sarcolemma and transverse tubules (demonstrated by co-localization and co-immunoprecipitation). PLM inhibits NCX1 independently of its effects on Na,K-ATPase; the cytoplasmic domain of PLM is required for NCX1 regulation. Phosphorylation of PLM at Ser-68 is the active form that inhibits NCX1 (in contrast, unphosphorylated PLM inhibits Na,K-ATPase).\",\n      \"method\": \"Adenovirus-mediated overexpression and siRNA knockdown in adult rat cardiomyocytes; heterologous co-expression in HEK293 cells; co-immunoprecipitation; electrophysiology (NCX1 current); 45Ca2+ uptake; PLM cytoplasmic domain truncation mutants; phosphomimetic and phospho-deficient mutants\",\n      \"journal\": \"Annals of the New York Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, electrophysiology, tracer uptake, mutagenesis) in multiple systems\",\n      \"pmids\": [\"17446450\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PLM forms 1:1 stoichiometric complexes with both NKA-alpha1 and NKA-alpha2 (FRET-based). PLM phosphorylation (PKA and PKC) drastically reduces FRET with both isoforms. PLM–PLM FRET indicates oligomers of ≥3 monomers. Isoproterenol (via PKA) increases Na+ affinity of both NKA-alpha1 and alpha2; PKC activation increases Vmax only for NKA-alpha2 but reduces K(1/2) for both. Ouabain abolishes NKA–PLM FRET but only partially reduces co-immunoprecipitation.\",\n      \"method\": \"FRET (progressive acceptor photobleach) in HEK293 cells; cardiac myocytes from WT and NKA-alpha isoform ouabain-sensitivity knock-in mice; whole-cell voltage clamp; SBFI fluorescence; pharmacological PKA/PKC activation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — FRET stoichiometry, genetic mouse models with defined isoform sensitivities, two functional readouts, multiple orthogonal approaches\",\n      \"pmids\": [\"19638348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PLM phosphorylation at either Ser-63 or Ser-68 alone is necessary and sufficient to completely relieve PLM-induced inhibition of NKA Na+ affinity. The double-mutant AA PLM (Ser63Ala/Ser68Ala) cannot be relieved by PKA or PKC activation; single-site mutants S63A or S68A retain responsiveness.\",\n      \"method\": \"HeLa cells stably expressing rat NKA-alpha1; transient expression of WT, S63A, S68A, and AA PLM mutants; SBFI fluorescence for intracellular Na+ concentration; PKA and PKC pharmacological activation\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — systematic site-directed mutagenesis with quantitative functional assay in defined cellular system\",\n      \"pmids\": [\"20861470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Surface expression of PLM in Xenopus oocytes requires co-expression with Na,K-ATPase (alpha1/beta1); the Na+/Ca2+ exchanger cannot drive PLM to the cell surface. A phosphorylation-mimicking mutation at Thr-69 or truncation of three C-terminal arginine residues facilitates NKA-dependent surface expression of PLM.\",\n      \"method\": \"Xenopus oocyte expression system; surface biotinylation; co-expression with NKA, NCX; PLM mutants (Thr-69 phosphomimetic, C-terminal truncations)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — oocyte surface expression assay with mutagenesis, single lab, biochemical readout\",\n      \"pmids\": [\"22535957\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Two pools of PLM exist in adult rat ventricular myocytes: one pool associated with the sodium pump (phosphorylated at Ser-68 or unphosphorylated) and a separate pool of PLM oligomers not associated with the pump (phosphorylated at Ser-63). PLM multimers co-immunoprecipitate unphosphorylatable PLM from heterozygous transgenic hearts, confirming PLM–PLM multimerization. The non-pump-associated PLM pool has no effect on sodium pump activity upon dephosphorylation.\",\n      \"method\": \"Phosphospecific co-immunoprecipitation from adult rat ventricular myocytes; mass spectrometry; chemical cross-linking; heterozygous transgenic mice expressing WT and unphosphorylatable PLM; sodium pump activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, mass spectrometry, cross-linking, transgenic animal model, and functional assay, multiple orthogonal methods\",\n      \"pmids\": [\"23532852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Nitric oxide (NO) activates Na,K-ATPase via a PKCε–PLM phosphorylation pathway: field stimulation increases endogenous NO, PKCε activation, and PLM phosphorylation (Ser-63 and Ser-68), all of which are abolished by Ca2+ chelation or NOS inhibition. Exogenous NO stimulates NKA in PLM-WT but not PLM-KO or PLM-3SA myocytes, identifying PLM phosphorylation as required for NO-dependent NKA activation.\",\n      \"method\": \"Rat ventricular myocytes; DAF-FM dye (NO), Western blotting (PKCε, phospho-PLM), biochemical NKA assay; perforated-patch clamp in PLM-WT, PLM-KO, and PLM-3SA (unphosphorylatable) myocytes; SBFI fluorescence\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis (three mouse genotypes), biochemical pathway dissection, and functional electrophysiology, multiple orthogonal methods\",\n      \"pmids\": [\"23612119\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The transmembrane domain of PLM interacts with TM9 of the NKA alpha-subunit; the cytoplasmic tail of PLM interacts with two small regions (residues 248–252 and 300–304) of the proximal intracellular loop of NCX1.\",\n      \"method\": \"Mutational analysis and heterologous co-expression (cited as prior work summarized in review); co-immunoprecipitation\",\n      \"journal\": \"Advances in experimental medicine and biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — domain mapping described in review/summary context without full primary experimental detail in this abstract\",\n      \"pmids\": [\"23224879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Prevention of PLM phosphorylation in PLM-3SA knock-in mice (Ser63/68/69→Ala) increases [Na+]i, reduces forward-mode NCX, exacerbates cardiac hypertrophy and NKA inhibition after aortic constriction compared to WT. In WT mice, aortic constriction causes PLM hypophosphorylation, progressive NKA current decline, and elevated [Na+]i. These data establish that PLM phosphorylation is causally required to maintain NKA activity and limit Na+ overload and adverse remodeling.\",\n      \"method\": \"PLM-3SA knock-in mice; aortic constriction model; echocardiography; pressure-volume catheterization; SBFI fluorescence for [Na+]i; whole-cell patch clamp for NKA current; Western blotting\",\n      \"journal\": \"Cardiovascular research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knock-in mouse with multiple orthogonal physiological and biochemical readouts, in vivo and ex vivo validation\",\n      \"pmids\": [\"25103111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Scanning mutagenesis of PLM transmembrane domain identifies residue L30 as critical for PLM–PLM tetramerization (L30A decreases PLM–PLM FRET) and for functional inhibition of NKA. L30A PLM shows increased NKA–PLM FRET and superinhibition of NKA, increasing Ca2+ transient amplitude in cardiomyocytes. These superinhibitory effects are reversible with isoproterenol (PKA activation). Molecular dynamics simulations show L30A distorts the TM helix and destabilizes the tetramer.\",\n      \"method\": \"Scanning mutagenesis of PLM TM domain; FRET in HEK293 cells; Ca2+ transient measurements in isolated cardiomyocytes; molecular dynamics simulations; isoproterenol treatment\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FRET mutagenesis plus functional cardiomyocyte assay, single lab, supported by MD simulations\",\n      \"pmids\": [\"27718550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PLM phosphorylation at Ser-63 and Ser-68 limits vascular constriction in response to phenylephrine via Na/K-ATPase (effect blocked by ouabain). PLM-3SA mice (unphosphorylatable PLM) show profoundly enhanced vascular responses to phenylephrine in vitro and in vivo and develop aging-induced hypertension. A human coding variant R70C (SNP rs61753924) prevents PLM phosphorylation at Ser-68 in HEK293 cells and is associated with elevated blood pressure in middle-aged men.\",\n      \"method\": \"PLM-3SA knock-in mice; wire myography of aortic and mesenteric vessels; Doppler flow and telemetry for in vivo BP; HEK293 cell phosphorylation assay; human genomic cohort analyses (UK Biobank, GoDARTS)\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic mouse model with in vitro and in vivo vascular functional assays plus human variant mechanistic validation, multiple orthogonal methods\",\n      \"pmids\": [\"33334125\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Peroxiredoxin 6 (Prdx6) interacts with PLM and depalmitoylates it in a glutathione-dependent manner. Glutathione loading reduces PLM palmitoylation; glutathione depletion increases PLM palmitoylation. Prdx6 silencing abolishes these effects. In vitro, recombinant Prdx6 (but not other Prdx isoforms) removes palmitic acid from palmitoylated recombinant PLM. PLM palmitoylation inhibits Na,K-ATPase activity, and this is reversed by Prdx6-mediated depalmitoylation. The broad-spectrum depalmitoylase inhibitor palmostatin B blocks Prdx6-dependent PLM depalmitoylation in cells and in vitro, suggesting Prdx6 acts as a thioesterase via nucleophilic attack through its reactive thiol.\",\n      \"method\": \"Co-immunoprecipitation of Prdx6 and PLM; acyl-RAC assay for palmitoylation; glutathione loading/depletion in cells; Prdx6 siRNA silencing; in vitro depalmitoylation assay with recombinant proteins; palmostatin B inhibition; Na,K-ATPase activity assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with recombinant proteins, isoform specificity screen, cell-based validation with loss-of-function and pharmacological inhibition, multiple orthogonal methods\",\n      \"pmids\": [\"38236777\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Phospholemman (FXYD1) is a 72-amino-acid single-span sarcolemmal protein that functions as the primary regulatory subunit of the cardiac Na/K-ATPase (NKA): in its unphosphorylated form it tonically inhibits NKA by reducing its apparent Na+ affinity, and this inhibition is relieved when PKA phosphorylates Ser-68 (increasing Na+ affinity) or PKC phosphorylates Ser-63 and/or Ser-68 (increasing Na+ affinity and, for the alpha2 isoform, also Vmax); conversely, Ser-68-phosphorylated PLM inhibits the Na+/Ca2+ exchanger NCX1 via its cytoplasmic tail, thereby coordinately limiting arrhythmias and preserving contractility during adrenergic stress; PLM is also subject to palmitoylation (which inhibits NKA) that is reversed by peroxiredoxin 6 in a glutathione-dependent thioesterase reaction, and to glutathionylation; PLM exists in cardiac muscle as both NKA-associated monomers and a separate pool of oligomers (≥3 monomers) that do not regulate the pump; surface trafficking of PLM itself requires co-expression with NKA; and NO activates NKA through a PKCε–PLM phosphorylation cascade, establishing PLM as the central nexus linking multiple intracellular signaling pathways to Na+ and Ca2+ homeostasis in the heart.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"FXYD1 (phospholemman, PLM) is a single-span sarcolemmal protein that serves as the central regulatory hub coupling intracellular signaling to Na+ and Ca2+ homeostasis in cardiac, skeletal, and vascular tissue by tuning the activity of the Na/K-ATPase (NKA) and the Na+/Ca2+ exchanger NCX1 [#7, #10, #12]. PLM physically associates with NKA alpha1, alpha2, and alpha3 isoforms through its transmembrane domain, forming 1:1 complexes, and in its unphosphorylated state tonically inhibits the pump; its cytoplasmic C-terminal tail carries the regulatory phosphosites Ser-63 and Ser-68, where PKC phosphorylates both and PKA phosphorylates only Ser-68 [#0, #6, #13]. Phosphorylation at either Ser-63 or Ser-68 is necessary and sufficient to relieve PLM-mediated inhibition: PKA-driven Ser-68 phosphorylation raises the apparent Na+ affinity of both alpha1 and alpha2 isozymes, while PKC additionally increases Vmax selectively for alpha2, and these phosphorylation events reduce the physical NKA–PLM interaction [#9, #11, #14]. The same C-terminal tail allows Ser-68-phosphorylated PLM to inhibit NCX1, establishing PLM as a coordinator of pump and exchanger activity [#12]. PLM exists as both NKA-associated monomers and a separate pool of oligomers (≥3 monomers) that do not regulate the pump, with transmembrane residue L30 governing tetramerization [#16, #20], and its surface trafficking requires co-expression with NKA [#15]. Beyond phosphorylation, PLM is regulated by palmitoylation, which inhibits NKA and is reversed by a glutathione-dependent peroxiredoxin 6 thioesterase reaction [#22], and nitric oxide activates NKA through a PKCε–PLM phosphorylation cascade [#17]. Genetic abolition of PLM phosphorylation causes Na+ overload, impaired NCX, exacerbated cardiac hypertrophy after pressure overload, enhanced vascular constriction, and hypertension, and a human R70C coding variant that blocks Ser-68 phosphorylation associates with elevated blood pressure [#19, #21].\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Established the phosphorylation code of PLM by mapping which kinases target which cytoplasmic serines, the foundation for all later regulatory models.\",\n      \"evidence\": \"In vitro kinase assays with synthetic peptides plus phosphopeptide mapping of labeled PLM from rat diaphragm\",\n      \"pmids\": [\"7999001\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the functional consequence of phosphorylation on any transporter\", \"Did not identify the binding partner regulated by these sites\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Provided the first functional readout of PLM by showing it induces ion currents in a heterologous system, framing it as a channel or channel regulator before its NKA role was known.\",\n      \"evidence\": \"Xenopus oocyte expression with electrophysiological current recording\",\n      \"pmids\": [\"7836447\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PLM forms the conductive pore or regulates an endogenous oocyte channel was unresolved\", \"No link yet to NKA or NCX\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Defined PLM membrane topology and localized voltage-dependent inactivation to the cytoplasmic tail, connecting structure to channel behavior.\",\n      \"evidence\": \"Protease protection, site-specific antibodies, and lipid bilayer electrophysiology with truncated PLM\",\n      \"pmids\": [\"9486665\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Channel activity in bilayers not reconciled with a physiological transporter target\", \"Cytoplasmic tail's later regulatory partners not yet identified\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Extended the kinase repertoire acting on PLM and showed phosphorylation alters its membrane level and current, hinting at trafficking control.\",\n      \"evidence\": \"In vitro DMPK kinase assay and oocyte co-expression with phosphorylation-null PLM mutant\",\n      \"pmids\": [\"10811636\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological relevance of DMPK–PLM in heart not established\", \"Mechanism linking phosphorylation to membrane level unclear\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrated a physiological loss-of-function role for PLM in cell volume regulation via taurine efflux, broadening its functional reach beyond cardiac myocytes.\",\n      \"evidence\": \"Antisense knockdown in cerebellar astrocytes with tracer efflux assays\",\n      \"pmids\": [\"11336802\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular identity of the taurine-permeable pathway not defined\", \"Relationship between volume regulation and NKA association unknown\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identified the Na/K-ATPase as a direct physical and functional partner of PLM, the discovery that reframed PLM as an NKA regulatory subunit.\",\n      \"evidence\": \"Co-purification and reciprocal Co-IP from cerebellum and choroid plexus with antibody-inhibition NKA activity assay\",\n      \"pmids\": [\"12657675\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PLM inhibits or activates NKA, and the role of phosphorylation, not yet resolved\", \"Cardiac relevance untested\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Confirmed the PLM–NKA association in cardiac and skeletal muscle and linked PLM dysregulation to heart failure, giving the interaction disease context.\",\n      \"evidence\": \"Co-IP from cardiac and skeletal muscle with NKA activity assays and phospho-Ser-68 immunoblotting\",\n      \"pmids\": [\"16100047\", \"15961612\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causality of PLM changes in heart failure not established\", \"Quantitative effect of phosphorylation on pump kinetics undefined\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Resolved the directionality and mechanism of PLM regulation: PLM inhibits NKA, and phosphorylation relieves inhibition by reducing physical interaction, dissected by isoform-specific kinetics and genetic deletion.\",\n      \"evidence\": \"FRET in HEK293 cells plus whole-cell clamp and SBFI in PLM-knockout myocytes with PKA/PKC activation\",\n      \"pmids\": [\"16943195\", \"17095720\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PLM dissociates or only reorients upon phosphorylation not fully resolved\", \"NCX1 regulation not yet addressed\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined the isozyme-specific kinetic logic (PKA raises Na+ affinity; PKC raises alpha2 Vmax) and revealed that phosphorylated PLM separately inhibits NCX1 through its cytoplasmic tail, establishing dual transporter control.\",\n      \"evidence\": \"Oocyte expression of defined NKA isozymes with mutants; Co-IP, NCX1 electrophysiology and 45Ca2+ uptake in cardiomyocytes and HEK293 cells\",\n      \"pmids\": [\"17991751\", \"17446450\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the opposite phospho-dependence for NKA versus NCX1 unexplained\", \"Stoichiometry of NKA–PLM complex not yet measured\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Established the stoichiometry of regulation, showing 1:1 NKA–PLM complexes for both alpha isoforms alongside PLM oligomers, refining the molecular model of pump regulation.\",\n      \"evidence\": \"Progressive acceptor-photobleach FRET in HEK293 cells and isoform-specific ouabain knock-in myocytes with clamp and SBFI\",\n      \"pmids\": [\"19638348\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of the PLM oligomer pool not yet defined\", \"How monomer/oligomer balance is controlled unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed phosphorylation of either Ser-63 or Ser-68 alone fully relieves NKA inhibition, defining the minimal molecular switch and explaining redundancy between PKA and PKC inputs.\",\n      \"evidence\": \"Single and double phosphosite mutants expressed with NKA-alpha1 in HeLa cells with SBFI readout\",\n      \"pmids\": [\"20861470\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the two sites are functionally equivalent in vivo not addressed\", \"Structural mechanism of relief not resolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified NKA as the obligate chaperone for PLM surface delivery, explaining how complex assembly is coupled to trafficking.\",\n      \"evidence\": \"Surface biotinylation in Xenopus oocytes with co-expression and trafficking mutants\",\n      \"pmids\": [\"22535957\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Trafficking determinants in native cardiac cells not validated\", \"Role of C-terminal arginine retention signal mechanistically unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Distinguished two physically and functionally distinct PLM pools (NKA-associated monomers versus non-regulatory oligomers), defined the NO–PKCε–PLM activation cascade, and began mapping the interaction surfaces with NKA and NCX1.\",\n      \"evidence\": \"Phosphospecific Co-IP, MS, cross-linking and transgenic hearts; NO/PKCε pathway dissection in PLM-WT/KO/3SA myocytes; domain mapping summarized in review\",\n      \"pmids\": [\"23532852\", \"23612119\", \"23224879\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why Ser-63-phosphorylated PLM partitions to oligomers is unknown\", \"Domain-mapping details for the NCX1 interface rest on summarized rather than full primary data\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Provided in vivo causality that PLM phosphorylation maintains NKA activity and limits Na+ overload and adverse remodeling under pressure overload.\",\n      \"evidence\": \"PLM-3SA knock-in mice with aortic constriction, echocardiography, pressure-volume catheterization, SBFI, and NKA current clamp\",\n      \"pmids\": [\"25103111\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of NKA versus NCX dysregulation to remodeling not separated\", \"Therapeutic reversibility not tested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Linked PLM tetramerization to its inhibitory potency by identifying L30 as a residue whose mutation destabilizes oligomers and superinhibits NKA, connecting oligomeric state to function.\",\n      \"evidence\": \"TM scanning mutagenesis with FRET, cardiomyocyte Ca2+ transients, and molecular dynamics simulations\",\n      \"pmids\": [\"27718550\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological regulation of PLM oligomeric state in vivo unknown\", \"Single-lab finding without independent structural confirmation\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended PLM regulation to vascular tone and human blood pressure, with a coding variant blocking Ser-68 phosphorylation, establishing translational relevance.\",\n      \"evidence\": \"PLM-3SA mice with wire myography and in vivo BP; HEK293 phosphorylation assay of R70C; UK Biobank and GoDARTS cohort analyses\",\n      \"pmids\": [\"33334125\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether R70C is causal versus associated in humans not proven\", \"Vascular cell type mediating the effect not pinpointed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Added a redox-coupled regulatory layer by identifying Prdx6 as a glutathione-dependent depalmitoylase that reverses inhibitory PLM palmitoylation of NKA.\",\n      \"evidence\": \"Co-IP, acyl-RAC, glutathione manipulation, Prdx6 silencing, in vitro depalmitoylation with recombinant proteins, palmostatin B, and NKA activity assays\",\n      \"pmids\": [\"38236777\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Palmitoylating enzyme for PLM not identified\", \"In vivo physiological role of PLM palmitoylation cycling untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple PLM regulatory inputs (phosphorylation, palmitoylation, glutathionylation, oligomerization) are integrated and prioritized to set Na+/Ca2+ homeostasis in a given physiological state remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified structural model of the NKA–PLM–NCX1 regulatory module\", \"Crosstalk between palmitoylation and phosphorylation switches unmapped\", \"Tissue-specific differences in PLM regulation not systematically compared\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [6, 10, 11, 12, 14]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [22]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [8, 12, 15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [6, 10, 12]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [10, 11, 17]}\n    ],\n    \"complexes\": [\"Na/K-ATPase regulatory complex (FXYD1–NKA)\"],\n    \"partners\": [\"ATP1A1\", \"ATP1A2\", \"ATP1A3\", \"SLC8A1\", \"PRDX6\", \"PRKCE\", \"DMPK\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win"}}