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Charcot-Marie-Tooth (CMT) disease comprises a group of inherited peripheral neuropathies caused by pathogenic variants in various genes, including ATP1A1. This gene encodes the ubiquitous α1 subunit of the sodium pump that generates the Na + and K + gradients that are essential for neuronal survival and excitability. We present the clinical cases of 2 unrelated patients with the same ATP1A1 variant causing dominant intermediate CMT disease and the functional characterization of the variant in the heterologous expression system. 

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. 

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 (αβ) pump that usually exports 3Na⁺in exchange for 2 K⁺per hydrolyzed ATP.Artemiaexpress several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2_KK, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines (Xenopusα1’s Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found thatArtemiaexpress two salinity-independent canonical α subunits (α1_NNand α3_NN), as well as two β variants, in addition to the salinity-controlled α2_KK. These β subunits permitted heterologous expression of the α2_KKpump 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 α2_KKpumps and compared it to that ofXenopusα1 (and its α2_KK-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2_KK-like characteristics toXenopusα1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2_KK’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⁺-congener⁸⁶Rb⁺, 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 allowingArtemiato maintain steeper Na⁺gradients in hypersaline environments. 

Abstract 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. 

Abstract BackgroundCharcot–Marie–Tooth disease (CMT) is a genetically and clinically heterogeneous group of inherited neuropathies. Monoallelic pathogenic variants inATP1A1were associated with axonal and intermediate CMT.ATP1A1encodes 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 novelATP1A1variant. MethodsWhole-exome sequencing on the patient’s genomic DNA and Sanger sequencing to validate and confirm the segregation of the identified p.P600RATP1A1variation were performed. To evaluate functional effects, blood-derived mRNA and protein levels ofATP1A1and the auxiliary β1 subunit encoded byATP1B1were 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. ResultsThe 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 thatATP1A1^p.P600Rinjected Xenopus oocytes have reduced Na⁺/ K⁺ATPase function. Moreover, HEK cells transfected with a construct encodingATP1A1^p.P600Rharbouring 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. ConclusionOur results further confirm the causative role ofATP1A1in peripheral neuropathy and broaden the mutational and phenotypic spectrum ofATP1A1-associated CMT.