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 α1β1 pump isoform by five human FXYD proteins normally located in muscle, kidney, and neurons. The function of Na/K pump α1β1 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.

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.

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 their 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 other de novo germline or familial mutations (where the mutant must be expressed in numerous tissues) produce a specific disease and not the others. 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.

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.

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 β1 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.

Heterozygous germline variants inATP1A1, 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).ATP1A1variants that cause CMT induce loss-of-function of NKA. This heterodimeric (αβ) 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-nullATP1A1variants. To test whether haploinsufficiency generated by heterozygous protein-null alleles are sufficient to cause disease, we tested the neuromuscular characteristics of heterozygousAtp1a1+/-knockout mice and their wildtype littermates, while also evaluating if exercise increased CMT penetrance. We found thatAtp1a1+/-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 knownATP1A1-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 byATP1A1variants and that if any pathology is associated with protein-null variants, they may display low penetrance or high age of onset.

on the cytoplasmic side of the protein, the admittance method has been applied to an experimental setup consisting of membrane fragments with Na,K-ATPase adsorbed to a bilayer lipid membrane (BLM) or a solid supported membrane. The small changes of the membrane admittance (capacitance and conductance) initiated by the fast steps of the pH or Na þ concentration were measured in the absence of ATP. pH jumps were performed by release of protons from a photosensitive compound (caged H þ) triggered by a UV light flash, the Na þ concentration jumps were carried out by fast solution exchange. The changes of the membrane capacitance triggered by a pH jump depended on the initial pH and the concentration of sodium or potassium ions. The effects of these ions are explained by a theoretical model assuming competition of protons with sodium or potassium ions in the binding sites on the cytoplasmic side of the Na,K-ATPase. The approximation of the experimental data by theoretical curves yields the dissociation constants and the cooperativity coefficients of the binding sites for sodium ions and potassium ions. In the presence of magnesium ions the apparent dissociation constants of sodium were increased. The effects of K ions on the pH driven capacitance changes were attenuated in presence of 100 mM of ATP and no magnesium. This observation indicates that the Na,K-ATPase is driven into a conformation with low affinity for the potassium ions presumably because of trapping the enzyme in state with ATP bound in the low-affinity site. Supported by RFBR project 16-04-01162.

By applying well designed oscillating electrical pulse to a single frog skeletal muscle with voltage-clamp and double vaseline gap techniques, we obtained pump current on both positive and negative half cycle. The frequency of the oscillating pulses remains at 50 Hz which is comparable with the physiological turnover rate of the Na/K pump. Different magnitudes of voltage (from 30mV to 65mV with a 5mV step) were applied to the muscle. The results showed that the pump current as well as the charge mediated by pump moved across the plasma cell membrane were voltage dependent and reached a saturation status when the pulse was greater than 55 mV. In this situation, the total amount of pump-mediated charge remained roughly the same even with the changing magnitude of the activation pulses. The charge on the positive half cycle and negative half cycle which represent the Na extrusion and K intrusion respectively, has a ratio less than but close to 3:2. After adding ouabain, the currents were recorded each 5 minutes. The results showed that the total pump-mediated charge decreased monotonically and reached minimum which was around 0 after 20 minutes. This indicated that this oscillating electrical pulse induced current was highly sensitive to and can be totally eliminated by ouabain, a specific Na/K pump blocker.