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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 α2KK, 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 (α1NNand α3NN), as well as two β variants, in addition to the salinity-controlled α2KK. These β subunits permitted heterologous expression of the α2KKpump 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 α2KKpumps and compared it to that ofXenopusα1 (and its α2KK-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2KK-like characteristics toXenopusα1, 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+-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.
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The phenotypic spectrum of pathogenic ATP1A1 variants expands: the novel p.P600R substitution causes demyelinating Charcot–Marie–Tooth disease
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 thatATP1A1p.P600Rinjected Xenopus oocytes have reduced Na+/ K+ATPase function. Moreover, HEK cells transfected with a construct encodingATP1A1p.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.
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- Award ID(s):
- 2003251
- PAR ID:
- 10395354
- Publisher / Repository:
- Springer Science + Business Media
- Date Published:
- Journal Name:
- Journal of Neurology
- Volume:
- 270
- Issue:
- 5
- ISSN:
- 0340-5354
- Page Range / eLocation ID:
- p. 2576-2590
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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