Na+,K+-ATPase with Disrupted Na+ Binding Sites I and III Binds Na+ with Increased Affinity at Site II and Undergoes Na+-Activated Phosphorylation with ATP.

Na+,K+-ATPase actively extrudes three cytoplasmic Na+ ions in exchange for two extracellular K+ ions for each ATP hydrolyzed. The atomic structure with bound Na+ identifies three Na+ sites, named I, II, and III. It has been proposed that site III is the first to be occupied and site II last, when Na+ binds from the cytoplasmic side. It is usually assumed that the occupation of all three Na+ sites is obligatory for the activation of phosphoryl transfer from ATP. To obtain more insight into the individual roles of the ion-binding sites, we have analyzed a series of seven mutants with substitution of the critical ion-binding residue Ser777, which is a shared ligand between Na+ sites I and III. Surprisingly, mutants with large and bulky substituents expected to prevent or profoundly disturb Na+ access to sites I and III retain the ability to form a phosphoenzyme from ATP, even with increased apparent Na+ affinity. This indicates that Na+ binding solely at site II is sufficient to promote phosphorylation. These mutations appear to lock the membrane sector into an E1-like configuration, allowing Na+ but not K+ to bind at site II, while the cytoplasmic sector undergoes conformational changes uncoupled from the membrane sector.

ATP1A1-linked diseases require a malfunctioning protein product from one allele.

Heterozygous germline variants in ATP1A1, the gene encoding the α1 subunit of the Na+/K+-ATPase (NKA), have been linked to diseases including primary hyperaldosteronism and the peripheral neuropathy Charcot-Marie-Tooth disease (CMT). ATP1A1 variants that cause CMT induce loss-of-function of NKA. This heterodimeric (αß) enzyme hydrolyzes ATP to establish transmembrane electrochemical gradients of Na+ and K+ that are essential for electrical signaling and cell survival. Of the 4 catalytic subunit isoforms, α1 is ubiquitously expressed and is the predominant paralog in peripheral axons. Human population sequencing datasets indicate strong negative selection against both missense and protein-null ATP1A1 variants. To test whether haploinsufficiency generated by heterozygous protein-null alleles are sufficient to cause disease, we tested the neuromuscular characteristics of heterozygous Atp1a1+/- knockout mice and their wildtype littermates, while also evaluating if exercise increased CMT penetrance. We found that Atp1a1+/- mice were phenotypically normal up to 18 months of age. Consistent with the observations in mice, we report clinical phenotyping of a healthy adult human who lacks any clinical features of known ATP1A1-related diseases despite carrying a plasma-membrane protein-null early truncation variant, p.Y148*. Taken together, these results suggest that a malfunctioning gene product is required for disease induction by ATP1A1 variants and that if any pathology is associated with protein-null variants, they may display low penetrance or high age of onset.

A Na pump with reduced stoichiometry is up-regulated by brine shrimp in extreme salinities.

Brine shrimp (Artemia) are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na+,K+-ATPase (NKA), a heterodimeric (αß) pump that usually exports 3Na+ in exchange for 2 K+ per hydrolyzed ATP. Artemia express several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2KK, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines (Xenopus α1's Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found that Artemia express two salinity-independent canonical α subunits (α1NN and α3NN), as well as two ß variants, in addition to the salinity-controlled α2KK. These ß subunits permitted heterologous expression of the α2KK pump and determination of its CryoEM structure in a closed, ion-free conformation, showing Lys758 residing within the ion-binding cavity. We used electrophysiology to characterize the function of α2KK pumps and compared it to that of Xenopus α1 (and its α2KK-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2KK-like characteristics to Xenopus α1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2KK's Lys308 helps to maintain high affinity for external K+ when Lys758 occupies an ion-binding site. By measuring uptake under voltage clamp of the K+-congener 86Rb+, we prove that double-lysine-substituted pumps transport 2Na+ and 1 K+ per catalytic cycle. Our results show how the two lysines contribute to generate a pump with reduced stoichiometry allowing Artemia to maintain steeper Na+ gradients in hypersaline environments.

An unusual conformation from Na+-sensitive non-gastric proton pump mutants reveals molecular mechanisms of cooperative Na+-binding.

The Na+,K+-ATPase (NKA) and non-gastric H+,K+- ATPase (ngHKA) share ~65 % sequence identity, and nearly identical catalytic cycles. These pumps alternate between inward-facing (E1) and outward-facing (E2) conformations and differ in their exported substrate (Na+ or H+) and stoichiometries (3 Na+:2 K+ or 1 H+:1 K+). We reported that structures of the NKA-mimetic ngHKA mutant K794S/A797P/W940/R949C (SPWC) with 2 K+ occluded in E2-Pi and 3 Na+-bound in E1·ATP states were nearly identical to NKA structures in equivalent states. Here we report the cryo-EM structures of K794A and K794S, two poorly-selective ngHKA mutants, under conditions to stabilize the E1·ATP state. Unexpectedly, the structures show a hybrid with both E1- and E2-like structural features. While transmembrane segments TM1-TM3 and TM4's extracellular half adopted an E2-like conformation, the rest of the protein assumed an E1 configuration. Two spherical densities, likely bound Na+, were observed at cation-binding sites I and III, without density at site II. This explains the E2-like conformation of TM4's exoplasmic half. In NKA, oxygen atoms derived from the unwound portion of TM4 coordinated Na+ at site II. Thus, the lack of Na+ at site II of K794A/S prevents the luminal portion of TM4 from taking an E1-like position. The K794A structure also suggests that incomplete coordination of Na+ at site III induces the halfway rotation of TM6, which impairs Na+-binding at the site II. Thus, our observations provide insight into the molecular mechanism of E2-E1 transition and cooperative Na+-binding in the NKA and other related cation pumps.

ATP1A1 -linked diseases require a malfunctioning protein product from one allele.

Heterozygous germline variants in ATP1A1 , the gene encoding the α1 subunit of the Na + /K + -ATPase (NKA), have been linked to diseases including primary hyperaldosteronism and the peripheral neuropathy Charcot-Marie-Tooth disease (CMT). ATP1A1 variants that cause CMT induce loss-of-function of NKA. This heterodimeric (αß) enzyme hydrolyzes ATP to establish transmembrane electrochemical gradients of Na + and K + that are essential for electrical signaling and cell survival. Of the 4 catalytic subunit isoforms, α1 is ubiquitously expressed and is the predominant paralog in peripheral axons. Human population sequencing datasets indicate strong negative selection against both missense and protein-null ATP1A1 variants. To test whether haploinsufficiency generated by heterozygous protein-null alleles are sufficient to cause disease, we tested the neuromuscular characteristics of heterozygous Atp1a1 +/- knockout mice and their wildtype littermates, while also evaluating if exercise increased CMT penetrance. We found that Atp1a1 +/- mice were phenotypically normal up to 18 months of age. Consistent with the observations in mice, we report clinical phenotyping of a healthy adult human who lacks any clinical features of known ATP1A1 -related diseases despite carrying a protein-null early truncation variant, p.Y148*. Taken together, these results suggest that a malfunctioning gene product is required for disease induction by ATP1A1 variants and that if any pathology is associated with protein-null variants, they may display low penetrance or high age of onset.

The phenotypic spectrum of pathogenic ATP1A1 variants expands: the novel p.P600R substitution causes demyelinating Charcot-Marie-Tooth disease.

BACKGROUND: Charcot-Marie-Tooth disease (CMT) is a genetically and clinically heterogeneous group of inherited neuropathies. Monoallelic pathogenic variants in ATP1A1 were associated with axonal and intermediate CMT. ATP1A1 encodes for the catalytic α1 subunit of the Na+/ K+ ATPase. Besides neuropathy, other associated phenotypes are spastic paraplegia, intellectual disability, and renal hypomagnesemia. We hereby report the first demyelinating CMT case due to a novel ATP1A1 variant. METHODS: Whole-exome sequencing on the patient's genomic DNA and Sanger sequencing to validate and confirm the segregation of the identified p.P600R ATP1A1 variation were performed. To evaluate functional effects, blood-derived mRNA and protein levels of ATP1A1 and the auxiliary ß1 subunit encoded by ATP1B1 were investigated. The ouabain-survival assay was performed in transfected HEK cells to assess cell viability, and two-electrode voltage clamp studies were performed in Xenopus oocytes. RESULTS: The variant was absent in the local and global control datasets, falls within a highly conserved protein position, and is in a missense-constrained region. The expression levels of ATP1A1 and ATP1B1 were significantly reduced in the patient compared to healthy controls. Electrophysiology indicated that ATP1A1p.P600R injected Xenopus oocytes have reduced Na+/ K+ ATPase function. Moreover, HEK cells transfected with a construct encoding ATP1A1p.P600R harbouring variants that confers ouabain insensitivity displayed a significant decrease in cell viability after ouabain treatment compared to the wild type, further supporting the pathogenicity of this variant. CONCLUSION: Our results further confirm the causative role of ATP1A1 in peripheral neuropathy and broaden the mutational and phenotypic spectrum of ATP1A1-associated CMT.

Structure and function of H+/K+ pump mutants reveal Na+/K+ pump mechanisms.

Ion-transport mechanisms evolve by changing ion-selectivity, such as switching from Na+ to H+ selectivity in secondary-active transporters or P-type-ATPases. Here we study primary-active transport via P-type ATPases using functional and structural analyses to demonstrate that four simultaneous residue substitutions transform the non-gastric H+/K+ pump, a strict H+-dependent electroneutral P-type ATPase, into a bona fide Na+-dependent electrogenic Na+/K+ pump. Conversion of a H+-dependent primary-active transporter into a Na+-dependent one provides a prototype for similar studies of ion-transport proteins. Moreover, we solve the structures of the wild-type non-gastric H+/K+ pump, a suitable drug target to treat cystic fibrosis, and of its Na+/K+ pump-mimicking mutant in two major conformations, providing insight on how Na+ binding drives a concerted mechanism leading to Na+/K+ pump phosphorylation.

Role of a conserved ion-binding site tyrosine in ion selectivity of the Na+/K+ pump.

The essential transmembrane Na+ and K+ gradients in animal cells are established by the Na+/K+ pump, a P-type ATPase that exports three Na+ and imports two K+ per ATP hydrolyzed. The mechanism by which the Na+/K+ pump distinguishes between Na+ and K+ at the two membrane sides is poorly understood. Crystal structures identify two sites (sites I and II) that bind Na+ or K+ and a third (site III) specific for Na+. The side chain of a conserved tyrosine at site III of the catalytic α-subunit (Xenopus-α1 Y780) has been proposed to contribute to Na+ binding by cation-π interaction. We substituted Y780 with natural and unnatural amino acids, expressed the mutants in Xenopus oocytes and COS-1 cells, and used electrophysiology and biochemistry to evaluate their function. Substitutions disrupting H-bonds impaired Na+ interaction, while Y780Q strengthened it, likely by H-bond formation. Utilizing the non-sense suppression method previously used to incorporate unnatural derivatives in ion channels, we were able to analyze Na+/K+ pumps with fluorinated tyrosine or phenylalanine derivatives inserted at position 780 to diminish cation-π interaction strength. In line with the results of the analysis of mutants with natural amino acid substitutions, the results with the fluorinated derivatives indicate that Na+-π interaction with the phenol ring at position 780 contributes minimally, if at all, to the binding of Na+. All Y780 substitutions decreased K+ apparent affinity, highlighting that a state-dependent H-bond network is essential for the selectivity switch at sites I and II when the pump changes conformational state.

Effects of a high-intensity interval training program on body composition and physical fitness in female field hockey players / Efectos de un programa de entrenamiento intervalado de alta intensidad sobre la composición corporal y condición física en jugadoras de hockey césped

SUMMARY: High-intensity interval training (HIIT) is zed by achieving similar effects to conventional physical and physiological training in a shorter time, allowing its dissemination in the sports field. The present study was aimed to analyze the effects of a HIIT program on body composition and general and specific physical fitness in Chilean female field hockey players. Experimental, repeated measures, simple blind, parallel groups, and a quantitative approach were used. The participants were randomized, and distributed into a control group (CG; n= 10) that maintained regular field hockey training and an experimental group (EG; n= 10) that also received complementary training with HIIT. Body composition (muscle mass and adipose mass), general physical fitness (jump performance with countermovement jump [CMJ] and maximum oxygen consumption [VO2max] were evaluated with the test Course-Navette), and specific physical fitness (pushing speed, dribbling speed, and shooting accuracy) were assessed with established protocols. Pre- and post-intervention comparisons were made with Student's t and Wilcoxon tests, considering p<0.05. The main results indicate that the EG presented a significant increase in muscle mass (p = 0.024; d = 0.62), CMJ (p = 0.005; d = 1.10), VO2max (p = 0.001; d = 1.58) and a significant reduction in adipose mass (p = 0.023; d = 0.36) and time in pushing speed (p = 0.028; d = 0.79). The CG did not present significant changes in any of the variables analyzed, and no significant differences were reported between the groups. In conclusion, eight weeks of HIIT significantly increases muscle mass, jump performance, and VO2max and significantly reduces adipose mass and time in pushing speed in Chilean female field hockey players.

RESUMEN: El entrenamiento intervalado de alta intensidad (EIAI) se caracteriza por conseguir en un menor tiempo efec- tos similares al entrenamiento convencional a nivel físico y fisiológico, lo que ha permitido su difusión en el ámbito deportivo. El objetivo del presente estudio fue analizar los efectos de un programa de EIAI sobre la composición corporal, condición física general y específica en mujeres chilenas que practican hockey césped. Estudio experimental, de medidas repetidas, simple ciego, grupos paralelos y enfoque cuantitativo. Las participantes fueron aleatorizadas y distribuidas en grupo control (GC; n=10) que mantuvo los entrenamientos regulares de hockey césped y grupo experimental (GE; n=10) que además recibió de complemento EIAI. Se evaluó la composición corporal (masa muscular y masa adiposa), condición física general (capacidad de salto con el salto contra movimiento [CMJ] y consumo máximo de oxígeno [VO2máx] con la prueba Course de Navette) y condición física específica (velocidad de empuje, velocidad de dribling y precisión de tiro) con protocolos establecidos. Se realizaron comparaciones pre y post intervención con las pruebas t de Student y Wilcoxon, considerando un p<0,05. Los principales resultados indican que el GE presentó un aumento significativo de la masa muscular (p=0,024; d=0,62), CMJ (p=0,005; d=1,10), VO2máx (p=0,001; d=1,58) y, una reducción significativa, de la masa adiposa (p=0,023; d=0,36) y del tiempo en la velocidad de empuje (p=0,028; d=0,79). El GC no presentó cambios significativos en ninguna de las variables analizadas, mientras que no se reportaron diferencias significativas entre los grupos. En conclusión, ocho semanas de EIAI aumentan significativamente la masa muscular, capacidad de salto y VO2máx, además de reducir significativamente la masa adiposa y el tiempo en la velocidad de empuje en mujeres chilenas que practican hockey césped.

Diseases caused by mutations in the Na+/K+ pump α1 gene ATP1A1.

Human cell survival requires function of the Na+/K+ pump; the heteromeric protein that hydrolyzes ATP to extrude Na+ and import K+ across the plasmalemma, thereby building and maintaining these ions' electrochemical gradients. Numerous dominant diseases caused by mutations in genes encoding for Na+/K+ pump catalytic (α) subunit isoforms highlight the importance of this protein. Here, we review literature describing disorders caused by missense mutations in ATP1A1, the gene encoding the ubiquitously expressed α1 isoform of the Na+/K+ pump. These various maladies include primary aldosteronism with secondary hypertension, an endocrine syndrome, Charcot-Marie-Tooth disease, a peripheral neuropathy, complex spastic paraplegia, another neuromuscular disorder, as well as hypomagnesemia accompanied by seizures and cognitive delay, a condition affecting the renal and central nervous systems. This article focuses on observed commonalities among these mutations' functional effects, as well as on the special characteristics that enable each particular mutation to exclusively affect a certain system, without affecting others. In this respect, it is clear how somatic mutations localized to adrenal adenomas increase aldosterone production without compromising other systems. However, it remains largely unknown how and why some but not all de novo germline or familial mutations (where the mutant must be expressed in numerous tissues) produce a specific disease and not the other diseases. We propose hypotheses to explain this observation and the approaches that we think will drive future research on these debilitating disorders to develop novel patient-specific treatments by combining the use of heterologous protein-expression systems, patient-derived pluripotent cells, and gene-edited cell and mouse models.

FXYD proteins and sodium pump regulatory mechanisms.

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.

Displacement of the Na+/K+ pump's transmembrane domains demonstrates conserved conformational changes in P-type 2 ATPases.

Cellular survival requires the ion gradients built by the Na+/K+ pump, an ATPase that alternates between two major conformations (E1 and E2). Here we use state-specific engineered-disulfide cross-linking to demonstrate that transmembrane segment 2 (M2) of the pump's α-subunit moves in directions that are inconsistent with distances observed in existing crystal structures of the Na+/K+ pump in E1 and E2. We characterize this movement with voltage-clamp fluorometry in single-cysteine mutants. Most mutants in the M1-M2 loop produced state-dependent fluorescence changes upon labeling with tetramethylrhodamine-6-maleimide (TMRM), which were due to quenching by multiple endogenous tryptophans. To avoid complications arising from multiple potential quenchers, we analyzed quenching of TMRM conjugated to R977C (in the static M9-M10 loop) by tryptophans introduced, one at a time, in M1-M2. This approach showed that tryptophans introduced in M2 quench TMRM only in E2, with D126W and L130W on the same helix producing the largest fluorescence changes. These observations indicate that M2 moves outward as Na+ is deoccluded from the E1 conformation, a mechanism consistent with cross-linking results and with proposals for other P-type 2 ATPases.

FXYD protein isoforms differentially modulate human Na/K pump function.

Tight regulation of the Na/K pump is essential for cellular function because this heteromeric protein builds and maintains the electrochemical gradients for Na+ and K+ that energize electrical signaling and secondary active transport. We studied the regulation of the ubiquitous human α1ß1 pump isoform by five human FXYD proteins normally located in muscle, kidney, and neurons. The function of Na/K pump α1ß1 expressed in Xenopus oocytes with or without FXYD isoforms was evaluated using two-electrode voltage clamp and patch clamp. Through evaluation of the partial reactions in the absence of K+ but presence of Na+ in the external milieu, we demonstrate that each FXYD subunit alters the equilibrium between E1P(3Na) and E2P, the phosphorylated conformations with Na+ occluded and free from Na+, respectively, thereby altering the apparent affinity for Na+. This modification of Na+ interaction shapes the small effects of FXYD proteins on the apparent affinity for external K+ at physiological Na+. FXYD6 distinctively accelerated both the Na+-deocclusion and the pump-turnover rates. All FXYD isoforms altered the apparent affinity for intracellular Na+ in patches, an effect that was observed only in the presence of intracellular K+. Therefore, FXYD proteins alter the selectivity of the pump for intracellular ions, an effect that could be due to the altered equilibrium between E1 and E2, the two major pump conformations, and/or to small changes in ion affinities that are exacerbated when both ions are present. Lastly, we observed a drastic reduction of Na/K pump surface expression when it was coexpressed with FXYD1 or FXYD6, with the former being relieved by injection of PKA's catalytic subunit into the oocyte. Our results indicate that a prominent effect of FXYD1 and FXYD6, and plausibly other FXYDs, is the regulation of Na/K pump trafficking.

Distinct effects of Q925 mutation on intracellular and extracellular Na+ and K+ binding to the Na+, K+-ATPase.

Three Na+ sites are defined in the Na+-bound crystal structure of Na+, K+-ATPase. Sites I and II overlap with two K+ sites in the K+-bound structure, whereas site III is unique and Na+ specific. A glutamine in transmembrane helix M8 (Q925) appears from the crystal structures to coordinate Na+ at site III, but does not contribute to K+ coordination at sites I and II. Here we address the functional role of Q925 in the various conformational states of Na+, K+-ATPase by examining the mutants Q925A/G/E/N/L/I/Y. We characterized these mutants both enzymatically and electrophysiologically, thereby revealing their Na+ and K+ binding properties. Remarkably, Q925 substitutions had minor effects on Na+ binding from the intracellular side of the membrane - in fact, mutations Q925A and Q925G increased the apparent Na+ affinity - but caused dramatic reductions of the binding of K+ as well as Na+ from the extracellular side of the membrane. These results provide insight into the changes taking place in the Na+-binding sites, when they are transformed from intracellular- to extracellular-facing orientation in relation to the ion translocation process, and demonstrate the interaction between sites III and I and a possible gating function of Q925 in the release of Na+ at the extracellular side.

Na/K Pump Mutations Associated with Primary Hyperaldosteronism Cause Loss of Function.

Primary hyperaldosteronism (Conn's syndrome), a common cause of secondary hypertension, is frequently produced by unilateral aldosterone-producing adenomas that carry mutations in ion-transporting genes, including ATP1A1, encoding the Na/K pump's α1 subunit. Whether Na/K pump mutant-mediated inward currents are required to depolarize the cell and increase aldosterone production remains unclear, as such currents were observed in four out of five mutants described so far. Here, we use electrophysiology and uptake of the K+ congener 86Rb+, to characterize the effects of eight additional Na/K pump mutations in transmembrane segments TM1 (delM102-L103, delL103-L104, and delM102-I106), TM4 (delI322-I325 and I327S), and TM9 (delF956-E961, delF959-E961, and delE960-L964), expressed in Xenopus oocytes. All deletion mutants induced abnormal inward currents of different amplitudes at physiological voltages, while I327S lacked such currents. A detailed functional characterization revealed that I327S significantly reduces intracellular Na+ affinity without altering affinity for external K+. 86Rb+-uptake experiments show that I327S dramatically impairs function under physiological concentrations of Na+ and K+. Since Na/K pumps in the adrenal cortex may be formed by association of α1 with ß3 instead of ß1 subunits, we evaluated whether G99R (another mutant without inward currents when associated with ß1) would show inward currents when associated with ß3. We found that the kinetic characteristics of either mutant or wild-type α1ß3 pumps expressed in Xenopus oocytes to be indistinguishable from those of α1ß1 pumps. The observed functional consequences of each hyperaldosteronism mutant point to the loss of Na/K pump function as the common feature of all mutants, which is sufficient to induce hyperaldosteronism.

External Ion Access in the Na/K Pump: Kinetics of Na+, K+, and Quaternary Amine Interaction.

Na/K pumps build essential ion gradients across the plasmalemma of animal cells by coupling the extrusion of three Na+, with the import of two K+ and the hydrolysis of one ATP molecule. The mechanisms of selectivity and competition between Na+, K+, and inhibitory amines remain unclear. We measured the effects of external tetrapropylammonium (TPA+) and ethylenediamine (EDA2+) on three different Na/K pump transport modes in voltage-clamped Xenopus oocytes: 1) outward pump current (IP), 2) passive inward H+ current at negative voltages without Na+ or K+ (IH), and 3) transient charge movement reporting the voltage-dependent extracellular binding/release of Na+ (QNa). Both amines competed with K+ to inhibit IP. TPA+ inhibited IH without competing with H+, whereas EDA2+ did not alter IH at pH 7.6. TPA+ competed with Na+ in QNa measurements, reducing Na+-apparent affinity, evidenced by a ∼-75 mV shift in the charge-voltage curve (at 20 mM TPA+) without reduction of the total charge moved (Qtot). In contrast, EDA2+ and K+ did not compete with Na+ to inhibit QNa; both reduced Qtot without decreasing Na+-apparent affinity. EDA2+ (15 mM) right-shifted the charge-voltage curve by ∼+50 mV. Simultaneous occlusion of EDA2+ and Na+ by an E2P conformation unable to reach E1P was demonstrated by voltage-clamp fluorometry. Trypsinolysis experiments showed that EDA2+-bound pumps are much more proteolysis-resistant than Na+-, K+-, or TPA+-bound pumps, therefore uncovering unique EDA2+-bound conformations. K+ effects on QNa and IH were also evaluated in pumps inhibited with beryllium fluoride, a phosphate mimic. K+ reduced Qtot without shifting the charge-voltage curve, indicating noncompetitive effects, and partially inhibited IH to the same extent as TPA+ in non-beryllium-fluorinated pumps. These results demonstrate that K+ interacts with beryllium-fluorinated pumps inducing conformational changes that alter QNa and IH, suggesting that there are two external access pathways for proton transport by IH.

38. CIA: cierre transcatéter. Valor agregado del ETE 3D en la toma de decisiones. Caso clínico

Historia clínica: paciente de 36 años de edad, sexo femenino, HTA de reciente diagnóstico. Asintomática. Del examen físico cardiovascular se destaca: R2 desdoblado. ECG: bloqueo incompleto de rama derecha. ETT: comunicación interauricular (CIA) tipo ostium secundum (OS). Dilatación de cavidades derechas. Qp/Qs: 1,6. Presión pulmonar sistólica 35 mmHg. Pruebas complementarias: se realiza ETE bidimensional (ETE2D) que evidencia CIA tipo OS con extensión anterior de 15 por 13 mm, con borde aórtico de 2 mm y presencia de buenos bordes en el resto de sus sectores. Septum impresiona firme, no aneurismático. ETE tridimensional (ETE3D) evidencia CIA de forma oval con eje mayor anteroposterior de 17 mm, en posición descrita, de 13 mm de eje menor y borde aórtico de 2,7 mm. Evolución clínica: se decide cierre transcatéter con dispositivo Amplatzer bajo anestesia general y monitoreo con ETE2D. Presión arteria pulmonar 26/7/13 mmHg. Por oximetría Qp/Qs 2,4. Por técnica de balón oclusor diámetro máximo de CIA de 17 mm. A pesar del borde aórtico subóptimo y tomando en consideración la buena calidad del resto de los bordes, la forma oval de orificio y el arco relativamente escaso del mismo en la topografía yuxta-aórtica, se decide proceder al implante, seleccionándose dispositivo no 19. Luego de desplegar ambos discos se corrobora posición con ecocardiografía. Se libera dispositivo luego de maniobra de Minnesota. Ecografía de control del dispositivo: mínimo shunt residual central, sin cabalgamiento sobre aorta. Diagnóstico: CIA OS con extensión anterior, con relevancia hemodinámica y anatomía favorable para cierre transcatéter. Discusión: el cierre percutáneo de CIA tipo OS es de elección cuando la anatomía lo permite. Las medidas exactas, forma, posición y relaciones anatómicas del defecto son imprescindibles. La vista "in face" del septum interauricular con ETE3D permite una valoración complementaria más precisa de su morfología. Presentamos un caso clínico en el que el ETE3D agregó información adicional a la aportada por el ETE2D, la cual fue determinante para planificar un exitoso procedimiento de cierre.

On the effect of hyperaldosteronism-inducing mutations in Na/K pumps.

Primary aldosteronism, a condition in which too much aldosterone is produced and that leads to hypertension, is often initiated by an aldosterone-producing adenoma within the zona glomerulosa of the adrenal cortex. Somatic mutations of ATP1A1, encoding the Na/K pump α1 subunit, have been found in these adenomas. It has been proposed that a passive inward current transported by several of these mutant pumps is a "gain-of-function" activity that produces membrane depolarization and concomitant increases in aldosterone production. Here, we investigate whether the inward current through mutant Na/K pumps is large enough to induce depolarization of the cells that harbor them. We first investigate inward currents induced by these mutations in Xenopus Na/K pumps expressed in Xenopus oocytes and find that these inward currents are similar in amplitude to wild-type outward Na/K pump currents. Subsequently, we perform a detailed functional evaluation of the human Na/K pump mutants L104R, delF100-L104, V332G, and EETA963S expressed in Xenopus oocytes. By combining two-electrode voltage clamp with [3H]ouabain binding, we measure the turnover rate of these inward currents and compare it to the turnover rate for outward current through wild-type pumps. We find that the turnover rate of the inward current through two of these mutants (EETA963S and L104R) is too small to induce significant cell depolarization. Electrophysiological characterization of another hyperaldosteronism-inducing mutation, G99R, reveals the absence of inward currents under many different conditions, including in the presence of the regulator FXYD1 as well as with mammalian ionic concentrations and body temperatures. Instead, we observe robust outward currents, but with significantly reduced affinities for intracellular Na+ and extracellular K+ Collectively, our results point to loss-of-function as the common mechanism for the hyperaldosteronism induced by these Na/K pump mutants.

Arginine substitution of a cysteine in transmembrane helix M8 converts Na+,K+-ATPase to an electroneutral pump similar to H+,K+-ATPase.

Na+,K+-ATPase and H+,K+-ATPase are electrogenic and nonelectrogenic ion pumps, respectively. The underlying structural basis for this difference has not been established, and it has not been revealed how the H+,K+-ATPase avoids binding of Na+ at the site corresponding to the Na+-specific site of the Na+,K+-ATPase (site III). In this study, we addressed these questions by using site-directed mutagenesis in combination with enzymatic, transport, and electrophysiological functional measurements. Replacement of the cysteine C932 in transmembrane helix M8 of Na+,K+-ATPase with arginine, present in the H+,K+-ATPase at the corresponding position, converted the normal 3Na+:2K+:1ATP stoichiometry of the Na+,K+-ATPase to electroneutral 2Na+:2K+:1ATP stoichiometry similar to the electroneutral transport mode of the H+,K+-ATPase. The electroneutral C932R mutant of the Na+,K+-ATPase retained a wild-type-like enzyme turnover rate for ATP hydrolysis and rate of cellular K+ uptake. Only a relatively minor reduction of apparent Na+ affinity for activation of phosphorylation from ATP was observed for C932R, whereas replacement of C932 with leucine or phenylalanine, the latter of a size comparable to arginine, led to spectacular reductions of apparent Na+ affinity without changing the electrogenicity. From these results, in combination with structural considerations, it appears that the guanidine+ group of the M8 arginine replaces Na+ at the third site, thus preventing Na+ binding there, although allowing Na+ to bind at the two other sites and become transported. Hence, in the H+,K+-ATPase, the ability of the M8 arginine to donate an internal cation binding at the third site is decisive for the electroneutral transport mode of this pump.

Intracellular Requirements for Passive Proton Transport through the Na+,K+-ATPase.

The Na+,K+-ATPase (NKA or Na/K pump) hydrolyzes one ATP to exchange three intracellular Na+ (Na+i) for two extracellular K+ (K+o) across the plasma membrane by cycling through a set of reversible transitions between phosphorylated and dephosphorylated conformations, alternately opening ion-binding sites externally (E2) or internally (E1). With subsaturating [Na+]o and [K+]o, the phosphorylated E2P conformation passively imports protons generating an inward current (IH), which may be exacerbated in NKA-subunit mutations associated with human disease. To elucidate the mechanisms of IH, we studied the effects of intracellular ligands (transported ions, nucleotides, and beryllium fluoride) on IH and, for comparison, on transient currents measured at normal Na+o (QNa). Utilizing inside-out patches from Xenopus oocytes heterologously expressing NKA, we observed that 1) in the presence of Na+i, IH and QNa were both activated by ATP, but not ADP; 2) the [Na+]i dependence of IH in saturating ATP showed K0.5,Na = 1.8 ± 0.2 mM and the [ATP] dependence at saturating [Na+]i yielded K0.5,ATP = 48 ± 11 µM (in comparison, Na+i-dependent QNa yields K0.5,Na = 0.8 ± 0.2 mM and K0.5,ATP = 0.43 ± 0.03 µM; 3) ATP activated IH in the presence of K+i (∼15% of the IH observed in Na+i) only when Mg2+i was also present; and 4) beryllium fluoride induced maximal IH even in the absence of nucleotide. These data indicate that IH occurs when NKA is in an externally open E2P state with nucleotide bound, a conformation that can be reached through forward Na/K pump phosphorylation of E1, with Na+i and ATP, or by backward binding of K+i to E1, which drives the pump to the occluded E2(2K), where free Pi (at the micromolar levels found in millimolar ATP solutions) promotes external release of occluded K+ by backdoor NKA phosphorylation. Maximal IH through beryllium-fluorinated NKA indicates that this complex mimics ATP-bound E2P states.