Meyer DJ , Bijlani S , de Sautu M , Spontarelli K , Young VC , Gatto C , Artigas P .

R03 NS116433 NINDS NIH HHS , R15 GM061583 NIGMS NIH HHS

	Figure 1. FXYD amino acid sequence alignment and Na/K pump enzymatic cycle. (A) Alignment of the five human FXYD isoforms studied in this work. Residues in the transmembrane (TM), cytoplasmic, and extracellular regions of the isoforms are indicated. (B) Post-Albers kinetic scheme of the Na/K pump cycle (clockwise forward direction). The pump alternates between two major conformations, E1 and E2, which can exist in dephosphorylated or phosphorylated (P) forms. Parentheses indicate ions occluded within the protein. Transitions within the red box produce transient charge movement: the release of the first Na+ (forward cycle) or rebinding of the last Na+ (backward cycle) during E1P(3Na+) ↔ E2P(2Na+) + Na+o produces the slow transient charge movement, while the release of the last two Na+ ions (forward cycle) or rebinding of the first two Na+ ions (backward cycle) during E2P(2Na+) ↔ E2P + 2Na+o produces fast charge movement.
	Figure 2. Recording and analysis of an experiment to determine the K0.5 for K+o. (A) Current at −50 mV of a Na+-loaded oocyte overexpressing the α1β1 pump, bathed in Na+o solution. Substituting Na+o with K+o stimulated outward current in a [K+]-dependent manner. Application of 0.5 mM ouabain for 2 min blocked currents in subsequent K+ applications. Vertical deflections along the recording indicate pulses to voltages between −100 and +40 mV. Lowercase letters indicate currents used to obtain ouabain-sensitive currents in B. (B) I-V plot of ouabain-sensitive current (current before ouabain application − current after ouabain application in the same condition) measured during the last 5 ms of 100-ms-long voltage pulses. (C) [K+o] dependence of ouabain-sensitive current at −100 and 0 mV fitted with Hill equations (line plots). (D) Voltage dependence of K0.5 for K+o.
	Figure 3. Effect of FXYD subunits on transient currents. (A–F) Ouabain-sensitive currents elicited by voltage steps from the holding potential to −100 and +40 mV recorded from oocytes bathed in external Na+ solution 3–6 d after injection with α1β1 (A), α1β1FXYD1 (B), α1β1FXYD2 (C), α1β1FXYD4 (D), α1β1FXYD6 (E), or α1β1FXYD7 (F). The oocyte in B was held at −35 mV, and the rest were held at −50 mV. Depolarizing voltage pulses from the holding potential produce transient outward currents, whereas hyperpolarizing voltage pulses produce transient inward currents. The dashed lines are monoexponential fits to the current, beginning 3 ms after the voltage step.
	Figure 4. Effect of FXYDs on charge movement. (A) Mean transient charge-movement rate for α1β1 alone or coexpressed with FXYD proteins. The rates were calculated from monoexponential fits to the integral of transient currents. (B) Mean normalized Q-V curves for α1β1 alone or when coexpressed with FXYD isoforms. Error bars are SEM in both panels. See also Table 2 (note that the α1β1 and α1β1FXYD1 shown here are those that started at a holding potential of −50 mV).
	Figure 5. Effect of FXYD isoforms on turnover rate. Mean turnover rate at 4.5 mM K+o as a function of voltage. Error bars are SEM. Data from Warner amplifier are shown. Values at 0 mV are (± SD in s−1): 14.9 ± 3.0 (n = 47, data from Warner and Dagan amplifiers) for α1β1, 12.9 ± 3.0* for α1β1FXYD1 (n = 20, data from Warner and Dagan amplifiers), 12.8 ± 3.9 for α1β1FXYD2, 14.7 ± 3.3 for α1β1FXYD4, 19.1 ± 3.1 for α1β1FXYD6**, and 13.3 ± 1.7 for α1β1FXYD7. Note that there are more oocytes where we measured pump current at 4.5 mM K+o and charge movement than full dose–response curves and charge movement. Significantly different with respect to α1β1 alone by t test: *, P < 0.05; **, P < 0.001.
	Figure 6. FXYD1 and FXYD6 reduce pump current. (A and B) Mean normalized K+o-induced currents from oocytes expressing α1β1 (n = 40) or α1β1FXYD1 (n = 45; A) and for α1β1 (n = 38) or α1β1FXYD6 (n = 29; B). Each group was normalized to the mean outward current from α1β1 oocytes measured the same day or on subsequent days in seven batches of oocytes in each condition. Error bars are SEM. Normalized data points from individual oocytes are shown as open circles. Asterisks indicate significantly different with respect to α1β1 alone by t test, **, P < 0.001.
	Figure 7. Reduced plasma membrane expression of Na/K pumps with FXYD1 and FXYD6. (A) Confocal image of representative oocytes following incubation in BODIPY FL-ouabain (1 µM). Note that preincubation of uninjected oocytes with unlabeled ouabain (UI+ouab) eliminates the signal observed in uninjected oocytes (UI). Fluorescence intensity was measured in two concentric circles (one including the plasma membrane signal) to subtract background fluorescence. The bar graph at the bottom summarizes results from two batches of oocytes (one batch for FXYD6) normalized to the fluorescence intensity measured in oocytes injected with α1β1 alone. (B) Representative Western blot of plasma membrane preparations from oocytes expressing α1β1 (lane 2), α1β1FXYD1 (lane 3), and FXYD1 alone (lane 4), 3 d after injection (14 oocytes in each condition). The top half was probed with the pan-α-antibody (a5, Developmental Studies Hybridoma Bank) and an anti-mouse secondary antibody (IRDye 800 CW goat anti-mouse; LI-COR). The bottom half was incubated with a polyclonal FXYD1 antibody (ab76597; Abcam) and an anti-rabbit secondary antibody (IRDye 680RD goat anti-rabbit; LI-COR). The bar graphs at the bottom summarize results from five membrane preparations from four oocyte batches, normalized to the intensity of α1β1 alone. Pump current activated by 4.5 mM K+, 145 mM Na+ in 28 α1β1 and 34 α1β1FXYD1 oocytes included in these membrane preparations were 219 ± 19 nA and 100 ± 10 nA (mean ± SEM), respectively.
	Figure 8. Effect of PKA on α1β1FXYD1 expression and kinetics. (A and B) Representative recordings of dose–response for K+o on oocytes expressing α1β1FXYD after injection with PKA (A) or PKI (B). (C) Mean K0.5-V from oocytes expressing α1β1FXYD1 injected with PKA (n = 20) or PKI (n = 19). (D) Mean normalized current induced by 10 mM K+o in two batches of oocytes expressing α1β1 or α1β1FXYD1 after injection with PKA or PKI, measured on the same day. Error bars are SEM. Asterisks indicate significantly different at P < 0.001. (E) Turnover rate in 4.5 mM K+o for oocytes expressing α1β1FXYD1 injected with PKA (n = 10) or PKI (n = 10). Error bars are SEM.
	Figure 9. FXYD effects on Na/K pump interaction with Na+i in the presence of NMDG+i (without K+i). (A) Representative patch from an oocyte expressing α1β1 alone held at 0 mV with extracellular solution containing 5 mM K+o in NMDG+o. The bars on top indicate [Na+]i (in mM), and the bars under the traces indicate application of MgATP (4 mM) to activate outward Na/K pump current. Note the biphasic activation, more obvious in first ATP application. Vertical deflections correspond to application of 25-ms-long voltage pulses. (B) Mean ATP-induced current as a function of [Na+]i. Line plots are Hill equations with the parameters in Table 3. (C) K0.5,Na+ for each FXYD. Error bars are SEM.
	Figure 10. FXYD effects on Na/K pump interaction with Na+i in the presence of K+i. (A) Patches from oocytes expressing each FXYD protein were held at 0 mV with extracellular solution containing 5 mM K+o in NMDG. The bars on top indicate [Na+]i (in mM), and the bars under the traces indicate application of MgATP (4 mM) to activate outward Na/K pump current. Vertical deflections correspond to application of 25-ms-long voltage pulses. Note biphasic activation by ATP. (B) Mean ATP-induced current as a function of [Na+]i, normalized to the value at 50 mM Na+i. Line plots are Hill equations with the parameters in Table 3. (C) K0.5,Na+ for each FXYD, from the number of experiments in parentheses. Error bars are SEM.
	Figure 11. PKA failed to increase Na/K pump current at 25 mM Na+i. (A) Na/K pump current was activated by application of ATP in 50 mM and then 25 mM Na+i. After the current reached steady state, PKA was applied at 25 mM Na+i without effect during 2 min. Subsequent application of 50 mM Na+i minimally increased the current. (B) Bar graph summarizing results from six patches (four at 50 mM Na+i) in which similar maneuvers were performed.
	Figure S1. CFTR recording in a giant patch demonstrating PKA activity. As a positive control for the patch-clamp experiments on α1β1FXYD1, we tested the ability of the same preparation of PKA to activate CFTR, typically a few days after the α1β1FXYD1 recordings. (A) Current recording at 0 mV from a large, inside-out patch excised from an oocyte 1 d after injection with human CFTR cRNA. Endogenous Na/K pumps were inhibited by incubating the oocyte in 10 µM ouabain before the experiment. The pipette contained the same external NMDG+ solution with 5 mM K+ as other patch clamp experiments, and the inside of the patch was perfused with 25 mM Na+ internal solution ([Na+] + [K+] = 140 mM). Under these experimental conditions (∼150 mM external Cl− and ∼30 mM internal Cl−, 0 mV), CFTR currents are outward. Application of 4 mM ATP alone to the inside of the patch produced no change in the baseline current, but simultaneous application of ATP with 10 µM PKA catalytic subunit induced an outward current that returned to baseline upon their removal. The large, vertical current deflections before the initial PKA application are 25-ms I-V pulses. (B) Enlargement of the current trace enclosed by the dashed box in A. Single-channel openings are visible ∼10 s after application of ATP and PKA.

Artigas, 2,3-butanedione monoxime affects cystic fibrosis transmembrane conductance regulator channel function through phosphorylation-dependent and phosphorylation-independent mechanisms: the role of bilayer material properties. 2006, Pubmed, Xenbase

Artigas, 2,3-butanedione monoxime affects cystic fibrosis transmembrane conductance regulator channel function through phosphorylation-dependent and phosphorylation-independent mechanisms: the role of bilayer material properties. 2006, Pubmed , Xenbase
Arystarkhova, Distribution and oligomeric association of splice forms of Na(+)-K(+)-ATPase regulatory gamma-subunit in rat kidney. 2002, Pubmed
Arystarkhova, Impaired AQP2 trafficking in Fxyd1 knockout mice: A role for FXYD1 in regulated vesicular transport. 2017, Pubmed
Attali, A corticosteroid-induced gene expressing an "IsK-like" K+ channel activity in Xenopus oocytes. 1995, Pubmed , Xenbase
Bibert, Phosphorylation of phospholemman (FXYD1) by protein kinases A and C modulates distinct Na,K-ATPase isozymes. 2008, Pubmed , Xenbase
Bibert, FXYD proteins reverse inhibition of the Na+-K+ pump mediated by glutathionylation of its beta1 subunit. 2011, Pubmed , Xenbase
Blanco, Na,K-ATPase subunit heterogeneity as a mechanism for tissue-specific ion regulation. 2005, Pubmed
Blanco, Kinetic properties of the alpha 2 beta 1 and alpha 2 beta 2 isozymes of the Na,K-ATPase. 1995, Pubmed
Bossuyt, Expression and phosphorylation of the na-pump regulatory subunit phospholemman in heart failure. 2005, Pubmed
Bossuyt, Isoform specificity of the Na/K-ATPase association and regulation by phospholemman. 2009, Pubmed
Bradbury, The transport of potassium between blood, cerebrospinal fluid and brain. 1965, Pubmed
Béguin, FXYD7 is a brain-specific regulator of Na,K-ATPase alpha 1-beta isozymes. 2002, Pubmed , Xenbase
Béguin, CHIF, a member of the FXYD protein family, is a regulator of Na,K-ATPase distinct from the gamma-subunit. 2001, Pubmed , Xenbase
Béguin, The gamma subunit is a specific component of the Na,K-ATPase and modulates its transport function. 1997, Pubmed , Xenbase
Capurro, Cellular localization and regulation of CHIF in kidney and colon. 1996, Pubmed , Xenbase
Castillo, Energy landscape of the reactions governing the Na+ deeply occluded state of the Na+/K+-ATPase in the giant axon of the Humboldt squid. 2011, Pubmed
Cornelius, Modulation of FXYD interaction with Na,K-ATPase by anionic phospholipids and protein kinase phosphorylation. 2007, Pubmed
Crambert, FXYD3 (Mat-8), a new regulator of Na,K-ATPase. 2005, Pubmed , Xenbase
Crambert, Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties. 2002, Pubmed , Xenbase
Crambert, Transport and pharmacological properties of nine different human Na, K-ATPase isozymes. 2000, Pubmed , Xenbase
Cserr, Potassium exchange between cerebrospinal fluid, plasma, and brain. 1965, Pubmed
Dascal, Voltage clamp recordings from Xenopus oocytes. 2001, Pubmed , Xenbase
Delprat, FXYD6 is a novel regulator of Na,K-ATPase expressed in the inner ear. 2007, Pubmed , Xenbase
Dempski, Structural arrangement and conformational dynamics of the gamma subunit of the Na+/K+-ATPase. 2008, Pubmed , Xenbase
Despa, Phospholemman-phosphorylation mediates the beta-adrenergic effects on Na/K pump function in cardiac myocytes. 2005, Pubmed
DiFranco, Na,K-ATPase α2 activity in mammalian skeletal muscle T-tubules is acutely stimulated by extracellular K+. 2015, Pubmed
Feschenko, Phospholemman, a single-span membrane protein, is an accessory protein of Na,K-ATPase in cerebellum and choroid plexus. 2003, Pubmed
Fine, Massive endocytosis driven by lipidic forces originating in the outer plasmalemmal monolayer: a new approach to membrane recycling and lipid domains. 2011, Pubmed
Fine, Human-induced pluripotent stem cell-derived cardiomyocytes for studies of cardiac ion transporters. 2013, Pubmed
Forbush, Characterization of a new photoaffinity derivative of ouabain: labeling of the large polypeptide and of a proteolipid component of the Na, K-ATPase. 1978, Pubmed
Friedrich, Na+,K(+)-ATPase pump currents in giant excised patches activated by an ATP concentration jump. 1996, Pubmed
Fuller, FXYD1 phosphorylation in vitro and in adult rat cardiac myocytes: threonine 69 is a novel substrate for protein kinase C. 2009, Pubmed
Gadsby, The dynamic relationships between the three events that release individual Na⁺ ions from the Na⁺/K⁺-ATPase. 2012, Pubmed
Gatto, Kinetic characterization of tetrapropylammonium inhibition reveals how ATP and Pi alter access to the Na+-K+-ATPase transport site. 2005, Pubmed
Geering, Functional roles of Na,K-ATPase subunits. 2008, Pubmed
Geering, FXYD proteins: new regulators of Na-K-ATPase. 2006, Pubmed
Greaves, DHHC palmitoyl transferases: substrate interactions and (patho)physiology. 2011, Pubmed
Han, Extracellular potassium dependence of the Na+-K+-ATPase in cardiac myocytes: isoform specificity and effect of phospholemman. 2009, Pubmed
Han, Role of phospholemman phosphorylation sites in mediating kinase-dependent regulation of the Na+-K+-ATPase. 2010, Pubmed
Hilbers, Tuning of the Na,K-ATPase by the beta subunit. 2016, Pubmed , Xenbase
Hilgemann, Regulation of ion transport from within ion transit pathways. 2020, Pubmed
Hilgemann, Mechanistic analysis of massive endocytosis in relation to functionally defined surface membrane domains. 2011, Pubmed
Hilgemann, Cytoplasmic ATP-dependent regulation of ion transporters and channels: mechanisms and messengers. 1997, Pubmed
Hilgemann, Massive endocytosis triggered by surface membrane palmitoylation under mitochondrial control in BHK fibroblasts. 2013, Pubmed
Hilgemann, Channel-like function of the Na,K pump probed at microsecond resolution in giant membrane patches. 1994, Pubmed
Hilgemann, Control of cardiac contraction by sodium: Promises, reckonings, and new beginnings. 2020, Pubmed
Holmgren, Three distinct and sequential steps in the release of sodium ions by the Na+/K+-ATPase. 2000, Pubmed
Holmgren, Charge translocation by the Na+/K+ pump under Na+/Na+ exchange conditions: intracellular Na+ dependence. 2006, Pubmed , Xenbase
Howie, Substrate recognition by the cell surface palmitoyl transferase DHHC5. 2014, Pubmed
Ino, Dysadherin, a cancer-associated cell membrane glycoprotein, down-regulates E-cadherin and promotes metastasis. 2002, Pubmed
Jaisser, Modulation of the Na,K-pump function by beta subunit isoforms. 1994, Pubmed , Xenbase
Kadowaki, Phosphohippolin expression in the rat central nervous system. 2004, Pubmed
Kanai, Crystal structure of a Na+-bound Na+,K+-ATPase preceding the E1P state. 2013, Pubmed
Keep, Potassium transport at the blood-brain and blood-CSF barriers. 1993, Pubmed
Lansbery, Cytoplasmic targeting signals mediate delivery of phospholemman to the plasma membrane. 2006, Pubmed
Lariccia, Massive calcium-activated endocytosis without involvement of classical endocytic proteins. 2011, Pubmed
Li, Role of the transmembrane domain of FXYD7 in structural and functional interactions with Na,K-ATPase. 2005, Pubmed , Xenbase
Lifshitz, Purification of the human alpha2 Isoform of Na,K-ATPase expressed in Pichia pastoris. Stabilization by lipids and FXYD1. 2007, Pubmed
Lin, Massive palmitoylation-dependent endocytosis during reoxygenation of anoxic cardiac muscle. 2013, Pubmed
Lindzen, Structure-function relations of interactions between Na,K-ATPase, the gamma subunit, and corticosteroid hormone-induced factor. 2003, Pubmed
Lindzen, Domains involved in the interactions between FXYD and Na,K-ATPase. 2003, Pubmed
Lu, Profound regulation of Na/K pump activity by transient elevations of cytoplasmic calcium in murine cardiac myocytes. 2016, Pubmed
Lu, Na/K pump inactivation, subsarcolemmal Na measurements, and cytoplasmic ion turnover kinetics contradict restricted Na spaces in murine cardiac myocytes. 2017, Pubmed
Lubarski, Interaction with the Na,K-ATPase and tissue distribution of FXYD5 (related to ion channel). 2005, Pubmed , Xenbase
Mercer, Molecular cloning and immunological characterization of the gamma polypeptide, a small protein associated with the Na,K-ATPase. 1993, Pubmed
Meyer, Na/K Pump Mutations Associated with Primary Hyperaldosteronism Cause Loss of Function. 2019, Pubmed , Xenbase
Meyer, On the effect of hyperaldosteronism-inducing mutations in Na/K pumps. 2017, Pubmed , Xenbase
Mishra, Molecular Mechanisms and Kinetic Effects of FXYD1 and Phosphomimetic Mutants on Purified Human Na,K-ATPase. 2015, Pubmed
Moorman, Phospholemman expression induces a hyperpolarization-activated chloride current in Xenopus oocytes. 1992, Pubmed , Xenbase
Moreno, Transient Electrical Currents Mediated by the Na+/K+-ATPase: A Tour from Basic Biophysics to Human Diseases. 2020, Pubmed
Morrison, Mat-8, a novel phospholemman-like protein expressed in human breast tumors, induces a chloride conductance in Xenopus oocytes. 1995, Pubmed , Xenbase
Morth, Crystal structure of the sodium-potassium pump. 2007, Pubmed
Mounsey, Modulation of Xenopus oocyte-expressed phospholemman-induced ion currents by co-expression of protein kinases. 1999, Pubmed , Xenbase
Nakao, Voltage dependence of Na translocation by the Na/K pump. , Pubmed
Palmer, Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium. 1991, Pubmed
Pavlović, The intracellular region of FXYD1 is sufficient to regulate cardiac Na/K ATPase. 2007, Pubmed
Price, Structure-function relationships in the Na,K-ATPase alpha subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme. 1988, Pubmed
Pu, Functional role and immunocytochemical localization of the gamma a and gamma b forms of the Na,K-ATPase gamma subunit. 2001, Pubmed
Silverman, Serine 68 phosphorylation of phospholemman: acute isoform-specific activation of cardiac Na/K ATPase. 2005, Pubmed
Stanley, External Ion Access in the Na/K Pump: Kinetics of Na+, K+, and Quaternary Amine Interaction. 2018, Pubmed , Xenbase
Stanley, Importance of the Voltage Dependence of Cardiac Na/K ATPase Isozymes. 2015, Pubmed , Xenbase
Stanley, Intracellular Requirements for Passive Proton Transport through the Na+,K+-ATPase. 2016, Pubmed , Xenbase
Stoica, The α2β2 isoform combination dominates the astrocytic Na+ /K+ -ATPase activity and is rendered nonfunctional by the α2.G301R familial hemiplegic migraine type 2-associated mutation. 2017, Pubmed , Xenbase
Tokhtaeva, Subunit isoform selectivity in assembly of Na,K-ATPase α-β heterodimers. 2012, Pubmed
Tulloch, The inhibitory effect of phospholemman on the sodium pump requires its palmitoylation. 2011, Pubmed
Vedovato, The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions. 2010, Pubmed , Xenbase
Wypijewski, A separate pool of cardiac phospholemman that does not regulate or associate with the sodium pump: multimers of phospholemman in ventricular muscle. 2013, Pubmed
Yaragatupalli, Altered Na+ transport after an intracellular alpha-subunit deletion reveals strict external sequential release of Na+ from the Na/K pump. 2009, Pubmed , Xenbase
Zhang, Phospholemman regulates cardiac Na+/Ca2+ exchanger by interacting with the exchanger's proximal linker domain. 2009, Pubmed