The sodium/potassium-ATPase (NKA) is the enzyme that establishes gradients of sodium and potassium across the plasma membrane. NKA activity is tightly regulated for different physiological contexts through interactions with single-span transmembrane peptides, the FXYD proteins. This diverse family of regulators has in common a domain containing a Phe-X-Tyr-Asp (FXYD) motif, two conserved glycines, and one serine residue. In humans, there are seven tissue-specific FXYD proteins that differentially modulate NKA kinetics as appropriate for each system, providing dynamic responsiveness to changing physiological conditions. Our understanding of how FXYD proteins contribute to homeostasis has benefitted from recent advances described in this review: biochemical and biophysical studies have provided insight into regulatory mechanisms, genetic models have uncovered remarkable complexity of FXYD function in integrated physiological systems, new posttranslational modifications have been identified, high-resolution structural studies have revealed new details of the regulatory interaction with NKA, and new clinical correlations have been uncovered. In this review, we address the structural determinants of diverse FXYD functions and the special roles of FXYDs in various physiological systems. We also discuss the possible roles of FXYDs in protein trafficking and regulation of non-NKA targets.

The ATP-dependent ion pump sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) sequesters Ca²⁺ in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, the activity of this transporter is tightly controlled via direct interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, the micropeptide phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA. These competing interactions determine cardiac performance by modulating the amplitude of Ca²⁺ signals that drive the contraction/relaxation cycle. We hypothesized that the functions of these peptides may relate to their reciprocal preferences for SERCA binding; SERCA binds PLB more avidly at low cytoplasmic [Ca²⁺] but binds DWORF better when [Ca²⁺] is high. In the present study, we demonstrated this opposing Ca²⁺ sensitivity is due to preferential binding of DWORF and PLB to different intermediate states that SERCA samples during the Ca²⁺ transport cycle. We show PLB binds best to the SERCA E1-ATP state, which prevails at low [Ca²⁺]. In contrast, DWORF binds most avidly to E1P and E2P states that are more populated when Ca²⁺ is elevated. Moreover, FRET microscopy revealed dynamic shifts in SERCA–micropeptide binding equilibria during cellular Ca²⁺ elevations. A computational model showed that DWORF exaggerates changes in PLB–SERCA binding during the cardiac cycle. These results suggest a mechanistic basis for inhibitory versus stimulatory micropeptide function, as well as a new role for DWORF as a modulator of dynamic oscillations of PLB–SERCA regulatory interactions.

The discovery of allosteric modulators is an emerging paradigm in drug discovery, and signal transduction is a subtle and dynamic process that is challenging to characterize. We developed a time-correlated single photon counting (TCSPC) imaging approach to investigate the structural mechanisms for small-molecule activation of the cardiac sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a), a pharmacologically important pump that transports Ca²⁺ at the expense of ATP hydrolysis. We first tested whether the dissociation of SERCA2a from its regulatory protein phospholamban (PLN) is required for small-molecule activation. We found that CDN1163, a validated SERCA2a activator, does not have significant effects on the stability of the SERCA2a–PLN complex. TCSPC imaging experiments using the non-hydrolyzable ATP analog AMP-PCP showed ATP is an allosteric modulator of SERCA2a, increasing the fraction of catalytically competent structures at physiologically relevant Ca²⁺ concentrations. Unlike ATP, CDN1163 alone has no significant effects on the Ca²⁺-dependent shifts in the structural populations of SERCA2a, and it does not increase the pump’s affinity for Ca²⁺ ions. However, we found that CDN1163 enhances the ATP-mediated modulatory effects to increase the population of catalytically competent SERCA2a structures. Importantly, this structural shift occurs within the physiological window of Ca²⁺ concentrations at which SERCA2a operates. We demonstrated that ATP is both a substrate and modulator of SERCA2a and showed that CDN1163 and ATP act synergistically to populate SERCA2a structures that are primed for phosphorylation. This study provides novel insights into the structural mechanisms for SERCA2a activation by its substrate and a synthetic allosteric modulator.

Background: The Na⁺/K⁺-ATPase (NKA) maintains electrochemical gradients by exporting Na+ and importing K⁺ at the expense of ATP hydrolysis. Although NKA inhibition is a well-established strategy for increasing cardiac contractility, existing inhibitors such as cardiotonic steroids (CTS) are limited by serious adverse effects. N106 is a small molecule previously shown to enhance cardiac lusitropy by promoting SERCA2a SUMOylation and, intriguingly, also exerts positive inotropic effects, suggesting additional mechanisms of action. Methods: To test whether N106 directly modulates NKA, we combined ATPase activity assays with molecular docking and microsecond-scale molecular dynamics simulations. Results: Biochemical measurements showed that N106 partially inhibits NKA, achieving ~80% maximal inhibition with an IC50 of 7 ± 1 µM, while leaving the pump’s apparent affinity for Na⁺, K⁺, and ATP unchanged. Computational analyses suggest that N106 binds within the canonical CTS-binding pocket but undergoes intermittent unbinding events, consistent with the partial inhibition observed experimentally. Conclusions: These findings identify N106 as a first-in-class dual modulator of cardiac ion pumps, partially inhibiting NKA while previously shown to activate SERCA2a through enhanced SUMOylation. This combined mechanism likely underlies its positive inotropic and lusitropic effects and positions the N106 scaffold as a promising lead for developing next-generation dual-target therapeutics for heart failure.

We recently developed a time-correlated single photon counting (TCSPC) spectroscopy approach to investigate the activation mechanisms of the calcium pump SERCA (sarcoplasmic reticulum Ca ²⁺ -ATPase). Here, we apply this approach to characterize the effects of two chemically distinct SERCA inhibitors, thapsigargin (TG) and cyclopiazonic acid (CPA), and to determine how they differentially modulate SERCA conformational dynamics. Although TG and CPA appear to stabilize similar SERCA states in structural studies, TCSPC reveals fundamentally distinct mechanisms of inhibition. TG stabilizes an inactive conformation that prevents Ca ²⁺ - and ATP-dependent transitions, effectively trapping SERCA in a nonproductive ‘dead-end’ state. In contrast, CPA attenuates, rather than abolishes, Ca ²⁺ - and nucleotide-dependent structural transitions, producing graded, concentration-dependent effects that reduce the population of the closed, activation-associated state. Notably, CPA decreases apparent Ca ²⁺ affinity only in the presence of a non-hydrolyzable ATP analog, consistent with an ATP-dependent, allosteric mechanism that redistributes conformational populations rather than directly occluding Ca ²⁺ binding sites. These findings demonstrate that TCSPC resolves mechanistic differences between inhibitors that appear structurally similar and provide a framework for understanding how distinct allosteric ligands modulate SERCA function. Graphical

The sodium-potassium ATPase (NKA) establishes ion gradients that facilitate many physiological processes. In the heart, NKA activity is regulated by its interaction with phospholemman (PLM, FXYD1). Here we used a novel fluorescence lifetime-based assay to investigate the structure, stoichiometry, and affinity of the NKA-PLM regulatory complex. We observed concentration dependent association of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit followed by lower affinity alpha-alpha and alpha-PLM interactions. The data provide the first evidence that the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits in intact cells. Docking and molecular dynamics simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with stoichiometry (alpha-beta-PLM) ₂ .