Na ⁺ /K ⁺ -ATPase E960 and phospholemman F28 are critical for their functional interaction

Abstract

Na + -K + -ATPase (NKA) establishes the transmembrane [Na + ] gradient in cells. In heart, phospholemman (PLM) inhibits NKA activity by reducing its apparent Na + affinity, an effect that is relieved by PLM phosphorylation. The NKA crystal structure suggests regions of PLM-NKA interaction, but the sites important for functional effects in live cells are not known. We tested wild type (WT) and CFP-NKA-α1 point mutants (alanine substitution at F956, E960, L964, and F967) for fluorescence resonance energy transfer (FRET) with WT-PLM-YFP in HEK293 cells. NKA-PLM FRET was unaltered with F956A or F967A, reduced with L964A, and nearly abolished with E960A. Mutating the PLM site (F28A) identified by structural analysis to interact with E960-NKA also nearly abolished NKA-PLM FRET. In contrast, NKA-PLM coimmunoprecipitation was only slightly reduced by E960A-NKA or F28A-PLM mutants, consistent with an additional interaction site. FRET titrations indicate that the additional site has higher affinity than that between E960-NKA and F28-PLM. To test whether the FRET-preventing mutations also prevent PLM functional effects, we measured NKA-mediated Na + -transport in intact cells. For WT-NKA, PLM reduced apparent Na + -affinity of NKA and PLM phosphorylation reversed the effect. In contrast, for E960A-NKA the apparent Na + -affinity was unaltered by either PLM or forskolin-induced PLM phosphorylation. We conclude that E960 on NKA and F28 on PLM are critical for PLM effects on both NKA function and NKA-PLM FRET, but also there is at least one additional site that is critical for tethering PLM to NKA.is critical for electrical excitability and coupled transport. In heart, [Na + ] i closely regulates intracellular Ca 2+ , contraction, and rhythmicity via Na + /Ca 2+ exchange (1, 2). Small changes in [Na + ] i can have major effects on both [Ca 2+ ] i and intracellular pH (via Na + /H + exchange) (2). Therefore, [Na + ] i regulation is very important for understanding basic ion homeostatic mechanisms.There are several Na + entry pathways, whereas the Na + /K + pump (NKA) is the main Na + extrusion pathway (2). NKA is a ubiquitous transmembrane protein that establishes and maintains [Na + ] and [K + ] gradients across the plasma membrane. These gradients ensure osmotic balance, resting membrane potential, and cellular excitability. NKA uses energy derived from hydrolysis of ATP to extrude three Na + ions in exchange for two K + ions.Phospholemman (PLM), a 72-amino acid sarcolemmal protein, is a member of the FXYD protein family, which derives its name from the conserved Phe-X-Tyr-Asp motif in the proximal extracellular domain. FXYDs are tissue-specific NKA regulators that bind to and modulate NKA function by affecting the apparent affinity for internal Na + or external K + (3-5). The [Na + ] i for halfmaximal NKA activation (K 0.5 ) in the heart varies with internal and external ionic conditions and is typically 8-22 mM. This is near the resting [Na + ] i in most cells (6). PLM (FXYD1) is highly expressed in heart, brain, and skele...

Na+/Ca2+ exchange and Na+/K+‐ATPase in the heart

“…The latter may be only one of many physiological conformations but mutational analysis showed that the E960 residue on NKA TM9 and the F28 residue on PLM are critical for both the functional effects on the pump and NKA–PLM FRET (Khafaga et al . ). The co‐immunoprecipitation was not completely abolished by these mutations indicating that additional PLM–NKA interaction sites contribute to the robust physical association of PLM with NKA.…”

Substrate recognition by the cell surface palmitoyl transferase DHHC5

“…Molecular dynamics simulations suggest an interaction between the PLM carboxyl terminus (R65, R66, and R70) and the pump N domain may be responsible for at least part of this functional inhibition (33). The structural imposition of a membrane-anchoring fatty acid on PLM C40 may influence the interaction between elements of the PLM carboxyl terminus and the pump intracellular domains, but it is more probable that PLM palmitoylation influences the pump within the membrane bilayer itself, which is also a site of physical contact between these proteins (34,35). Palmitoylation is well established as regulating protein partitioning to membrane microdomains (36).…”

Na+ transport in the normal and failing heart — Remember the balance

“…The crystal structures identified a number of possible interaction sites between FXYD and NKA-α TM9. In a recent study we demonstrated that interaction between NKA-α-E960 and PLM-F28 sites is critical for the functional effects of PLM on NKA [104]. The conserved FXYD motif is located in the extracellular domain, just prior to the TM domain and seems to interact with both α- and β-subunits.…”