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Comparative analysis of alternating hemiplegia of childhood and rapid-onset dystonia-parkinsonism ATP1A3 mutations reveals functional deficits, which do not correlate with disease severity.
Heterozygous mutations in the ATP1A3 gene, coding for an alpha subunit isoform (α3) of Na+/K+-ATPase, are the primary genetic cause for rapid-onset dystonia-parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). Recently, cerebellar ataxia, areflexia, pes cavus, optic atrophy and sensorineural hearing loss (CAPOS), early infantile epileptic encephalopathy (EIEE), childhood rapid onset ataxia (CROA) and relapsing encephalopathy with rapid onset ataxia (RECA) extend the clinical spectrum of ATP1A3 related disorders. AHC and RDP demonstrate distinct clinical features, with AHC symptoms being generally more severe compared to RDP. Currently, it is largely unknown what determines the disease severity, and whether severity is linked to the degree of functional impairment of the α3 subunit. Here we compared the effect of twelve different RDP and AHC specific mutations on the expression and function of the α3 Na+/K+-ATPase in transfected HEK cells and oocytes. All studied mutations led to functional impairment of the pump, as reflected by lower survival rate and reduced pump current. No difference in the extent of impairment, nor in the expression level, was found between the two phenotypes, suggesting that these measures of pump dysfunction do not exclusively determine the disease severity.
Fig. 1. Variations in the expression of ATP1A3 with different AHC and RDP mutations in HEK cells. (A) Example of western blot demonstrating the ATP1A3 (~112 kDa) and GAPDH (~37 kDa) bands for all constructs examined in this study. (B) Quantitative analysis of band intensities relative to wild type, averaged over 7 repetitions (mean ± SEM). Differences in the expression were significant within the AHC mutations group (one way ANOVA, p = .0045), but not within the RDP group (p = .95).
Fig. 2. RDP and AHC mutations reduce cell survival in an ouabain challenge test. Shown is the average increase in cell death relative to ouabain resistant, wild type ATP1A3 control (n = 6; mean ± SEM). The wild type (ouabain sensitive) ATP1A3 construct increases the death rate by 87% compared to the ouabain resistant wild type construct, killing more than 96% of the cells (Table S3). All mutant constructs increase the death rate relative to ouabain resistant wild type, with the I274T mutation showing a milder effect compared to the other mutations.
Fig. 3. Crystal structures of the Na+/K+ -ATPase heterotrimer. (A) The [Na3]E1P-ADP state (PDB ID 3WGV) with the location of the residues studied here. The Na+ ions are shown as yellow spheres. (B) Close up view of Na+ binding sites. (C) Close up view of K+ (Rb+) binding sites in the [Rb2]E2-Pi (PDB ID 3KDP) crystal structure. Rb+ ions are depicted as purple spheres. Figure was created using PyMOL (The PyMOL Molecular Graphics System, Version 2.3.0 Schrödinger, LLC). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. RDP and AHC mutations reduce the forward cycling current in oocytes. (A) Immunoprecipitation analysis of oocytes injected with myc-tagged ouabain resistant wild type ATP1A3 and ATP1B1 cRNA. Unmanipulated oocytes were used as control (left lane). The blot shows bands at ~110 kDa and ~ 40 kDa, corresponding to precipitated ATP1A3 and ATP1B1, respectively. An additional 55 kDa-band represents the heavy chain of the antibody used for the precipitation. (B) Representative current traces measured in oocytes in response to the application of 10 mM K+ to the extracellular solution. (C) Average steady state currents measured in oocytes for the different mutant constructs, demonstrating significant differences compared to the wild type construct (mean ± SEM, *p > .05, **p > .001, ***p > .0001). (D) Representative current traces demonstrating that the K+-stimulated currents were blocked by the application of 10 mM ouabain.