Cinarli Yuksel F , Nicolaou P , Spontarelli K , Dohrn MF , Rebelo AP , Koutsou P , Georghiou A , Artigas P , Züchner SL , Kleopa KA , Christodoulou K .

R01 NS105755 NINDS NIH HHS

	Fig. 1 Genetic findings of the Cypriot proband. A Electropherogram of the control ATP1A1 sequence (upper) versus patient ATP1A1: c.1799C > G (p.P600R) sequence (lower). B In silico analysis of the ATP1A1: c.1799C > G (p.P600R). C Conversation analysis of amino acid sequences of ATP1A1 (NM_000701.8) across species compared to p.P600R
	Fig. 2 mRNA and protein expression analysis of the wild type and mutant ATP1A1. A mRNA expression of ATP1A1 exons in the patient peripheral blood cells compared to healthy controls (Patient: 58.04 ± 2.16%, Control 1: 100.25 ± 2.32%, Control 2: 122.32 ± 6.54%, Control 3: 111.76 ± 2.77%. P = 0.00002). Three independent qPCRs were performed and samples were run as triplicates in each experiment. B Western blot analysis results of ATP1A1 expression normalized against β-ACTIN and GAPDH. C Quantification of protein expression of ATP1A1 in the patient versus healthy control LCLs (Patient: 45.49 ± 10.02%, Control 1: 99.01 ± 14.84%, Control 2: 94.15 ± 9.10%, Control 3: 123.94 ± 17.00%. P = 0.002). WB was replicated eight times for quantification. Statistical analysis was performed by one-way ANOVA. P < 0.05 considered statistically significant. D The expression of mutant ATP1A1 (c.1799 C > G, P600R) transcript in patient blood confirmed by Sanger sequencing
	Fig. 3 mRNA and protein expression analysis of ATP1B1. A mRNA expression analysis of ATP1B1 cDNA in the patient peripheral blood cells compared to healthy controls (Patient: 44.71 ± 3.63%, Control 1: 100.04 ± 3.40%, Control 2: 94.63 ± 4.31%, Control 3: 103.99 ± 3.21%. P = 0.00001). Four independent qPCRs were performed and samples were analysed as triplicates in each run. B Western blot analysis results of ATP1B1 expression normalized against β-ACTIN and GAPDH. C) ATP1B1 protein expression quantification in patient versus healthy control LCLs (Patient: 30.22 ± 6.43%, Control 1: 118.20 ± 13.21%, Control 2: 110.05 ± 10.23%, Control 3: 103.64 ± 11.04%. P = 0.0002). WB was replicated four times for quantification. Statistical analysis was performed by one-way ANOVA. P < 0.05 considered statistically significant
	Fig. 4 Electrophysiological findings. A Representative traces from an oocyte expressing WT (that is the RD ouabain resistant version) and P600R pumps. Oocytes were held at − 50 mV and exposed to the indicated [K+] (in mM) to activate pump current. Vertical deflections indicate application of 100 ms-long pulses to measure voltage-dependent parameters. B K0.5 for K+ activation of pump current obtained from Hill fits (“Methods”) to the [K+] dependencies at each voltage, as a function of the applied voltage. C Current, normalized to the mean from oocytes expressing WT pumps on the same day under the same conditions (results from 3 batches of oocytes, 11 WT and 13 P600R oocytes total. D Ouabain-sensitive transient currents elicited by pulses from − 50 mV to voltages ranging from − 140 mV to + 40 mV (in 20 mV increments). E Charge (integral of current transients) as a function of voltage normalized to the total charge moved in the whole voltage range (Qtot = 21.6 ± 10.6 for WT and 15.3 ± 11.1 for P600R). Line plots are Boltzmann distributions fitted to the data (mean V0.5 = − 47.5 ± 4.3 mV for WT and − 38.1 ± 4.3 mV for P600R, n = 29 each). F Mean total charge (measured from Boltzmann distributions in individual experiments) normalized to the mean from oocytes expressing WT pumps on the same day
	Fig. 5 Ouabain survival (luciferase) assay. Viability of ouabain insensitive HEK cells transfected with wild type ATP1A1, p.P600R and, positive controls p.G509D and p.G718S normalized to untreated cells transfected with the same plasmid
	Fig. 6 ATP1A1 protein and respective locations of the pathogenic variants reported up to date. Variants written in black represent CMT related variants identified in other studies, red represents the ATP1A1 variant identified in this study, blue represent hypomagnesaemia and intellectual disability or spastic paraplegia, green represent developmental delay, orange represent complex neurodevelopmental syndrome

Araki, ZNRF proteins constitute a family of presynaptic E3 ubiquitin ligases. 2003, Pubmed

Araki, ZNRF proteins constitute a family of presynaptic E3 ubiquitin ligases. 2003, Pubmed
Argente-Escrig, Pediatric inherited peripheral neuropathy: a prospective study at a Spanish referral center. 2021, Pubmed
Arystarkhova, Tissue-specific expression of the Na,K-ATPase beta3 subunit. The presence of beta3 in lung and liver addresses the problem of the missing subunit. 1997, Pubmed
Auld, Transcriptional regulation by the proteasome as a mechanism for cellular protein homeostasis. 2006, Pubmed
Bienfait, Late onset axonal Charcot-Marie-Tooth phenotype caused by a novel myelin protein zero mutation. 2006, Pubmed
Biondo, Diseases caused by mutations in the Na+/K+ pump α1 gene ATP1A1. 2021, Pubmed
Blanco, Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function. 1998, Pubmed
Boerkoel, Charcot-Marie-Tooth disease and related neuropathies: mutation distribution and genotype-phenotype correlation. 2002, Pubmed
Chen, Phosphorylation of adaptor protein-2 mu2 is essential for Na+,K+-ATPase endocytosis in response to either G protein-coupled receptor or reactive oxygen species. 2006, Pubmed
Chibalin, Dopamine-induced endocytosis of Na+,K+-ATPase is initiated by phosphorylation of Ser-18 in the rat alpha subunit and Is responsible for the decreased activity in epithelial cells. 1999, Pubmed
Comellas, Hypoxia-mediated degradation of Na,K-ATPase via mitochondrial reactive oxygen species and the ubiquitin-conjugating system. 2006, Pubmed
Coppi, Ubiquitination of Na,K-ATPase alpha1 and alpha2 subunits. 1997, Pubmed
Dada, Hypoxia-induced endocytosis of Na,K-ATPase in alveolar epithelial cells is mediated by mitochondrial reactive oxygen species and PKC-zeta. 2003, Pubmed
Dohrn, De Novo ATP1A1 Variants in an Early-Onset Complex Neurodevelopmental Syndrome. 2022, Pubmed
Eakle, High-affinity ouabain binding by yeast cells expressing Na+, K(+)-ATPase alpha subunits and the gastric H+, K(+)-ATPase beta subunit. 1992, Pubmed
Fambrough, Analysis of subunit assembly of the Na-K-ATPase. 1994, Pubmed
Geering, Functional roles of Na,K-ATPase subunits. 2008, Pubmed
Geering, The functional role of beta subunits in oligomeric P-type ATPases. 2001, Pubmed
Havrilla, A map of constrained coding regions in the human genome. 2019, Pubmed
He, ATP1A1 mutations cause intermediate Charcot-Marie-Tooth disease. 2019, Pubmed
Helenius, Role of ubiquitination in Na,K-ATPase regulation during lung injury. 2010, Pubmed
Hicke, Ubiquitin-dependent internalization and down-regulation of plasma membrane proteins. 1997, Pubmed
Hicke, Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins. 2003, Pubmed
Himoro, New mutation of the myelin P0 gene in a pedigree of Charcot-Marie-Tooth neuropathy 1. 1993, Pubmed
Hoxhaj, ZNRF2 is released from membranes by growth factors and, together with ZNRF1, regulates the Na+/K+ATPase. 2012, Pubmed
Jaisser, Modulation of the Na,K-pump function by beta subunit isoforms. 1994, Pubmed , Xenbase
Lassuthova, Mutations in ATP1A1 Cause Dominant Charcot-Marie-Tooth Type 2. 2018, Pubmed , Xenbase
Lavoie, The molar ratios of alpha and beta subunits of the Na+-K+-ATPase differ in distinct subcellular membranes from rat skeletal muscle. 1997, Pubmed
Lecuona, Regulation of Na,K-ATPase during acute lung injury. 2007, Pubmed
Lecuona, Ubiquitination participates in the lysosomal degradation of Na,K-ATPase in steady-state conditions. 2009, Pubmed
Lerat, Hearing loss in inherited peripheral neuropathies: Molecular diagnosis by NGS in a French series. 2019, Pubmed
Lin, ATP1A1 de novo Mutation-Related Disorders: Clinical and Genetic Features. 2021, Pubmed
Manganelli, Insights into the pathogenesis of ATP1A1-related CMT disease using patient-specific iPSCs. 2019, Pubmed
Martín-Vasallo, Oligodendrocytes in brain and optic nerve express the beta3 subunit isoform of Na,K-ATPase. 2000, Pubmed
Meyer, On the effect of hyperaldosteronism-inducing mutations in Na/K pumps. 2017, Pubmed , Xenbase
Mohan, Regulation of Neuronal Na+/K+-ATPase by Specific Protein Kinases and Protein Phosphatases. 2019, Pubmed
Nicolaou, Charcot-Marie-Tooth disease in Cyprus: epidemiological, clinical and genetic characteristics. 2010, Pubmed
Nicolaou, A Novel CLN6 Variant Associated With Juvenile Neuronal Ceroid Lipofuscinosis in Patients With Absence of Visual Loss as a Presenting Feature. 2021, Pubmed
Nicolaou, A novel LRSAM1 mutation is associated with autosomal dominant axonal Charcot-Marie-Tooth disease. 2013, Pubmed
Noguchi, Expression of functional (Na+ + K+)-ATPase from cloned cDNAs. 1987, Pubmed , Xenbase
Peng, Isoforms of Na,K-ATPase alpha and beta subunits in the rat cerebellum and in granule cell cultures. 1997, 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
Schlingmann, Germline De Novo Mutations in ATP1A1 Cause Renal Hypomagnesemia, Refractory Seizures, and Intellectual Disability. 2018, Pubmed
Shamraj, A putative fourth Na+,K(+)-ATPase alpha-subunit gene is expressed in testis. 1994, Pubmed
Silander, Spectrum of mutations in Finnish patients with Charcot-Marie-Tooth disease and related neuropathies. 1998, Pubmed
Skre, Genetic and clinical aspects of Charcot-Marie-Tooth's disease. 1974, Pubmed
Spontarelli, Role of a conserved ion-binding site tyrosine in ion selectivity of the Na+/K+ pump. 2022, Pubmed , Xenbase
Stregapede, Hereditary spastic paraplegia is a novel phenotype for germline de novo ATP1A1 mutation. 2020, Pubmed
Suryadevara, Walk the Line: The Role of Ubiquitin in Regulating Transcription in Myocytes. 2019, Pubmed
Tokhtaeva, Assembly with the Na,K-ATPase alpha(1) subunit is required for export of beta(1) and beta(2) subunits from the endoplasmic reticulum. 2009, Pubmed
Vagin, The role of the beta1 subunit of the Na,K-ATPase and its glycosylation in cell-cell adhesion. 2006, Pubmed
Vavlitou, Axonal pathology precedes demyelination in a mouse model of X-linked demyelinating/type I Charcot-Marie Tooth neuropathy. 2010, Pubmed
Wallace, Proteasome-mediated glucocorticoid receptor degradation restricts transcriptional signaling by glucocorticoids. 2001, Pubmed
Wetzel, Immunocytochemical localization of NaK-ATPase isoforms in the rat and mouse ocular ciliary epithelium. 2001, Pubmed