Fig 1: Pump current recordings of Na+/K+-ATPase transfected in HEK293T cells. a and b Continuous recordings with wild-type pumps in HEK293T cells. 1 µM ouabain was added to block endogenous pumps. The pump current was activated by superfusion of 20 mM K+ and inhibited by 10 µM ouabain. c Representative raw currents of WT (blue), G762S (yellow), R938P (green) and G615R (red) in response to 200 ms steps with + 20 mV increments from − 100 to + 100 mV. d Coexpression of ATP1A2 (green) and ATP1B1 (red) in transfected HEK293T cells. e Ouabain-sensitive pump currents measured in the last 50 ms of test pulses (two-way ANOVA: P = 0.040) (amplitude at + 40 mV, one-way ANOVA compared with WT, n = 9; G762S, n = 10, P = 0.018; R938P, n = 8, P = 0.142; G615R, n = 10, P = 0.008)
Fig 2: Expression and cell viability of mutant Na+/K+-ATPase. a Western blot of total membrane and plasma membrane proteins expressed in transfected HeLa cells (ATP1A2: 102 kDa; Tubulin: 50 kDa). The colored labels represented different groups: blue, WT; yellow, FHM; green, FHME; red, FHMEI. b The ATP1A2 expression of mutants was equal to that of the WT (compared with WT, TM: P = 0.3822, PM: P = 0.5507, Kruskal-Wallis test). c and d Cell survival assays. All mutants showed a significantly reduced survival rate under 1 µM ouabain in both HEK293T (c) and HeLa (d) cells (P < 0.0001, two-way ANOVA)
Fig 3: Structural location of ATP1A2 mutations. a Phenotypic distribution of ATP1A2 missense mutations (colored circles, n = 83). Ten transmembrane segments are shown as blue cylinders. b ATP1A2 mutant residues are shown as spheres and divided into four groups: pure HM (green), HM with epilepsy (magenta), HM with ataxia (yellow) and HM with intellectual disability (with or without epilepsy) (red) according to the clinical symptoms of patients. The intracellular loops are shown in the top view, indicating that mutations causing severe HM (accompanied by intellectual disability) were closer to Mg2+ (blue) near the phosphorylation site. c The structural locations and phenotypes of the ten mutations investigated in this study
Fig 4: Phenotypic effects on total Na+/K+-ATPase activity. The phenotype of the ATP1A2 mutations: red, FHM/AHC with epilepsy and intellectual disability; green, FHM with epilepsy; blue, pure FHM. a The activity of mutant Na+/K+-ATPase associated with intellectual disability showed lower activity than that of pure FHM or with epilepsy. GTCS, generalized tonic-clonic seizure; ID, intellectual disability. b-d Among mutations associated with epilepsy and intellectual disability, patients had a younger age of first hemiplegic migraine attack, but there was no statistical correlation with Na+/K+-ATPase activity (no ouabain: P = 0.597, 10 µM ouabain: P = 0.463, 100 µM ouabain: P = 0.169). HM, hemiplegic migraine
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