Immanneni C et al. (2024), ATP1A3 Disease Spectrum Includes Paroxysmal Wea...

XB-ART-60710

Neurol Genet 2024 Jun 01;103:e200150. doi: 10.1212/NXG.0000000000200150.

Show Gene links Show Anatomy links

ATP1A3 Disease Spectrum Includes Paroxysmal Weakness and Encephalopathy Not Triggered by Fever.

Immanneni C , Calame D , Jiao S , Emrick LT , Holmgren M , Yano ST .

???displayArticle.abstract???
BACKGROUND AND OBJECTIVES: Heterozygous pathogenic variants in ATP1A3, which encodes the catalytic alpha subunit of neuronal Na+/K+-ATPase, cause primarily neurologic disorders with widely variable features that can include episodic movement deficits. One distinctive presentation of ATP1A3-related disease is recurrent fever-triggered encephalopathy. This can occur with generalized weakness and/or ataxia and is described in the literature as relapsing encephalopathy with cerebellar ataxia. This syndrome displays genotype-phenotype correlation with variants at p.R756 causing temperature sensitivity of ATP1A3. We report clinical and in vitro functional evidence for a similar phenotype not triggered by fever but associated with protein loss-of-function. METHODS: We describe the phenotype of an individual with de novo occurrence of a novel heterozygous ATP1A3 variant, NM_152296.5:c.388_390delGTG; p.(V130del). We confirmed the pathogenicity of p.V130del by cell survival complementation assay in HEK293 cells and then characterized its functional impact on enzymatic ion transport and extracellular sodium binding by two-electrode voltage clamp electrophysiology in Xenopus oocytes. To determine whether variant enzymes reach the cell surface, we surface-biotinylated oocytes expressing N-tagged ATP1A3. RESULTS: The proband is a 7-year-old boy who has had 2 lifetime episodes of paroxysmal weakness, encephalopathy, and ataxia not triggered by fever. He had speech regression and intermittent hand tremors after the second episode but otherwise spontaneously recovered after episodes and is at present developmentally appropriate. The p.V130del variant was identified on clinical trio exome sequencing, which did not reveal any other variants possibly associated with the phenotype. p.V130del eliminated ATP1A3 function in cell survival complementation assay. In Xenopus oocytes, p.V130del variant Na+/K+-ATPases showed complete loss of ion transport activity and marked abnormalities of extracellular Na+ binding at room temperature. Despite this clear loss-of-function effect, surface biotinylation under the same conditions revealed that p.V130del variant enzymes were still present at the oocyte's cell membrane. DISCUSSION: This individual's phenotype expands the clinical spectrum of ATP1A3-related recurrent encephalopathy to include presentations without fever-triggered events. The total loss of ion transport function with p.V130del, despite enzyme presence at the cell membrane, indicates that haploinsufficiency can cause relatively mild phenotypes in ATP1A3-related disease.

???displayArticle.pubmedLink??? 38685976
???displayArticle.pmcLink??? PMC11057438
???displayArticle.link??? Neurol Genet

Genes referenced: atp1a3

???attribute.lit??? ???displayArticles.show???

	Figure 1. Identification of the ATP1A3 p.V130del Variant in an Individual With Recurrent Neurologic Deficits(A) Location of p.V130 within transmembrane helix M2 of the ion-binding domain. The paralogous valine within the crystal structure of the highly homologous ATP1A1 isoform (RCSB PDB 3WGU) is shown in 3D visualization using ChimeraX. The labels indicate adjacent transmembrane helices (TM), nearby side chains numbered according to the human ATP1A3 sequence, and the locations of the 3 bound Na+. The side chain of p.V129 neighbors that of p.M806 in TM6, a residue implicated in AHC. The side chain of p.V130 neighbors that of p.L94 in TM1; the paralog of p.L94 in ATP1A1, p.L104, has also been implicated in disease, and the disease variant at this position (p.L104R) introduces an inward ion leak.23 (B) Conservation of the 2 valines at p.V129-V130. Orthologous ATP1A3 protein sequences from the indicated vertebrates are shown in alignment with the valines in bold beneath the arrow. Owing to a gene duplication event in teleost fish, the zebrafish sequence shown is from Atp1a3a. At the bottom, the invertebrate Drosophila ortholog Atpalpha is shown for comparison along with the 3 human paralogs, ATP1A1, ATP1A2, and ATP1A4.
	Figure 2. p.V130del Causes Loss of Electrogenic Ion Transport by ATP1A3(A) Ouabain complementation assay shows a loss-of-function effect of p.V130del. HEK293 cells were transfected with expression constructs carrying partially ouabain-resistant wildtype or variant ATP1A3 as indicated for 2 days before challenge with ouabain. Cell survival in the presence of ouabain is shown normalized to the cell count in the absence of ouabain from the same transfection. p.V130del rescued significantly less cell survival than wildtype. Error bars shown in all figures are means ± 95% confidence intervals. *p < 0.001. (B) p.V130del causes loss of electrogenic ion transport activity at 0 mV. Steady-state ouabain-sensitive currents generated by ATP1A3-Na+/K+-ATPase in the presence of 5 mM extracellular K+ were measured using a two-electrode voltage clamp in Xenopus oocytes. p = 0.0002 for the difference between V130del and wildtype. *p < 0.001. (C) p.V130del ATP1A3-Na+/K+-ATPase is inactive across the range of tested voltages. Steady-state ouabain-sensitive currents, as in (B), are plotted against voltage. The average of 10 replicates is shown for each construct. (D) Holding currents at 0 mV show that p.V130del ATP1A3-Na+/K+-ATPases are inactive, not ouabain-resistant. The entirety of the representative two-electrode voltage clamp recording is shown for each wildtype or variant ATP1A3 indicated. If the absence of ouabain-sensitive currents with p.V130del ATP1A3-Na+/K+-ATPases in (C) was due to decreased affinity for ouabain, then the enzyme would still generate electrogenic ion transport currents that depend on the presence of extracellular K+. However, holding currents with p.V130del are essentially identical in the presence or absence of K+ (compare before and after the black bar, 5 mM K+), indicating that p.V130del ATP1A3-Na+/K+-ATPases do not generate ion transport currents.
	Figure 3. Extracellular Na+ Transient Currents Are Abnormal With p.V130del(A) Representative tracings of extracellular Na+ transient currents induced by voltage pulses from the holding potential of 0 mV to −160 mV in the absence of extracellular K+. Low-high ouabain subtractions from oocytes expressing untagged ATP1A3-containing Na+/K+-ATPases are shown. The blue line represents the monoexponential decay function obtained by fitting the off-transient current starting 4 ms after the end of the voltage pulse. (B) Charge-voltage plots of off-transient currents. Transient currents induced by the return from each voltage pulse to the holding potential of 0 mV were fit to monoexponential decay curves and integrated to yield the charge moved Q shown on the Y-axis. Owing to the small magnitude of p.V130del transient currents, the same curve is shown at larger scale at the bottom.
	Figure 4. p.V130del ATP1A3 Is Expressed and Reaches the Cell Surface(A) An N-terminal tag does not disrupt ATP1A3 function. N-FLAG-Strep-mCherry-tagged ATP1A3 constructs were expressed in Xenopus oocytes. Na+/K+-ATPase-specific electrogenic ion transport currents in the presence of 5 mM extracellular K+, measured as in Figure 2C, are shown. (B) Representative Western blot of oocytes expressing N-tagged ATP1A3 shows that p.V130del proteins are expressed. A constant amount of total protein was loaded in each lane and visualized by Ponceau stain before blocking (left). The blots were then cut in half (dotted line). The top half was probed using anti-Strep tag primary antibody to detect tagged ATP1A3 (expected size ∼146 kDa including 34 kDa tag), and the bottom half was probed using anti-α-tubulin primary antibody as an additional loading control; the 2 halves are shown together. Lane 1: uninjected negative control; 2: wildtype, 3: p.D801N, 4: p.V130del. (C) Cell surface biotinylation followed by avidin pulldown demonstrates N-tagged p.V130del ATP1A3 at the cell surface. Oocytes expressing the indicated N-tagged construct were treated with a cell surface biotinylation reagent. Avidin chromatography was performed to separate cell surface (bound) and intracellular (unbound) protein fractions for Western blot. More total protein was present in the unbound than the bound fraction, as shown by the Ponceau stain of the blots before blocking at left. The blots were then cut in half and probed against anti-Strep/anti-tubulin; lanes are numbered as in (B). (D) Semiquantitative determination of band intensity from cell surface biotinylation assays, normalized to the total amount of detected ATP1A3 as detailed in the Methods. All 3 ATP1A3 proteins were present at the cell surface.

References [+] :

Arystarkhova, Misfolding, altered membrane distributions, and the unfolded protein response contribute to pathogenicity differences in Na,K-ATPase ATP1A3 mutations. 2021, Pubmed