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...
a protonatable side chain in E2P-like conformations might allow shuttling of protons across the membrane. We therefore evaluated the role of three key carboxylates, E336 (TM4), E788 (TM5), and D935 (TM8), by mutating C113Y Xenopus pumps. Outward Na/K-pump current was practically abolished in E336Q(C113Y) or E336C(C113Y) pumps, but nonstoichiometric inward current at zero Ko and Nao (replaced by TMAo) was little altered, and was greatly augmented in high Nao. E788C(C113Y) pumps, on the other hand, like parent C113Y pumps, generated robust stoichiometric outward Na/K transport currents (voltage dependent in Nao, but not in TMAo), and little nonstoichiometric inward current in high Nao; but nonstoichiometric current in TMAo was dimin-ished~3-fold compared to parent C113Y pumps. In contrast, D935N(C113Y) pumps generated stoichiometric outward Na/K transport currents with altered voltage dependence that was similar in Nao or in TMAo, but inward nonstoichiometric current was nearly absent both in Nao and in TMAo, and was not augmented by lowering pH to 6 either in Nao and in TMAo. The D935 carboxylate thus seems uniquely required for the nonstoichiometric inward flow of protons through the Na/K-pump. [NIH HL36783].
of Ca 2þ , dissociation of CaM from C28 occurs by a pathway in which Trp 1093, although deeply embedded in a pocket in the C-terminal lobe of CaM, leaves first. The dissociation begins by relatively rapid release of Trp 1093, followed by very slow release of Phe 1110, removal of C28 and return of CaM to its conformation in the free state. The intermediate of dissociation with exposed Trp 1093 has a long lifetime (minutes), and is an activated form of PMCA. This mechanism may explain some unique biological properties of PMCA4b.
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