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Neurol Genet
2025 Oct 30;115:e200309. doi: 10.1212/NXG.0000000000200309.
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Association of the Recurrent ATP1 A1 Variant p.Gly549Arg With Intermediate CMT and Loss of Na,K-ATPase Function.
Spontarelli Fruit K
,
Olivera JF
,
Colmano N
,
Bird SJ
,
McCray BA
,
Yano ST
,
Scherer SS
,
Artigas P
.
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BACKGROUND AND OBJECTIVES: Charcot-Marie-Tooth (CMT) disease comprises a group of inherited peripheral neuropathies caused by pathogenic variants in various genes, including ATP1A1. This gene encodes the ubiquitous α1 subunit of the sodium pump that generates the Na+ and K+ gradients that are essential for neuronal survival and excitability. We present the clinical cases of 2 unrelated patients with the same ATP1A1 variant causing dominant intermediate CMT disease and the functional characterization of the variant in the heterologous expression system.
METHODS: The patients were evaluated by clinical EMG and by whole-exome sequencing. The function of sodium pump variants was studied with voltage clamp electrophysiology or using ouabain survival curves after heterologous expression in Xenopus oocytes or HEK293 cells, respectively. Localization of the variants was evaluated by fluorescence microscopy of HEK293 cells expressing fluorescently tagged sodium pumps.
RESULTS: We describe the cases of 2 unrelated patients who presented in their second decade with a length-dependent and slowly progressive intermediate neuropathy with both axonal and demyelinating features. Whole-exome sequencing identified a de novo c.1645G>A heterozygous variant in ATP1A1 (p.Gly549Arg) in both patients. The pathogenic nature of the variant was tested through a detailed evaluation of the functional consequences of the Gly549Arg substitution using 2 heterologous expression systems and functional assays that included survival curves of transfected cells and electrophysiology. Patch clamp and 2-electrode voltage clamp electrophysiology experiments showed that the Gly549Arg variant reduced NKA function (≥50%), mainly due to a lower NKA density at the plasma membrane and, to a lesser extent, due to a reduced apparent affinity for intracellular Na+. The reduced plasma membrane density was also observed in HEK293 cells simultaneously expressing wildtype and Gly549Arg variants, marked with fluorescent proteins of different colors, suggesting that the mutant may be partially retained in intracellular membranes. No clear dominant-negative effects were identified in these experimental systems.
DISCUSSION: Our results demonstrate that the pathogenic nature of this variant causes considerable loss of function due to diminished plasma membrane localization and kinetic impairments on the enzyme, without obvious dominant-negative effects. Our findings are similar to those previously reported for other CMT disease-causing ATP1A1 variants.
Figure 1. Gly549Arg (G549R) Causes Loss of NKA Function(A) HEK293 cells transfected with the indicated ouabain-resistant ATP1A1 expression constructs or mock-transfected cells (Mock) were challenged with 10 µM ouabain for 2 days. However, WT constructs supported cell survival and G549R partially but significantly (t test, p < 0.002) decreased cell survival, indicating impaired NKA function. (B) Representative current recordings from Na+-loaded oocytes clamped at −50 mV and expressing WT-α1β1 (top) or G549R-α1β1 (bottom) in external solution containing Na+ and the indicated [K+o]. K+o-induced outward pump current in a concentration-dependent manner. The ramp-like deflections in the trace indicate application of 100 ms-long voltage pulses to measure the voltage dependence of IP at each ion concentration, to obtain the K0.5. (C) Mean K0.5-V from Hill fits to the K+o dependence of IP. Error bars are SD. The t test indicates that the 2 means are significantly different (t test, p < 0.0001). (D) Mean 4.5 mM K+o-induced current at −50 mV, normalized to the average from WT-injected oocytes measured on the same day.
Figure 2. Transient Charge Movement(A) The Post-Albers kinetic scheme describing NKA function. When voltage pulses are applied in the absence of K+, the pumps shuttle back and forth between the states enclosed by the red dashed box, causing transient currents as the ions move within the protein's electric field. (B) Representative ouabain-sensitive currents (current before ouabain − current after ouabain) elicited by voltages pulses between −140 and +40 mV (40-mV increments shown) from oocytes injected with WT (left) or Gly549Arg (G549R, right). (C) Mean charge − voltage (Q-V) for WT and G549R oocytes fitted with a Boltzmann distribution (line plot, see Methods). The inset shows the normalized Q-V to illustrate the leftward shift of the G549R curve (WT V0.5 = −37 mV; G549R V0.5 = −50 mV). (D) Mean Qtot normalized to average value of WT on the same day. Error bars represent SEM. Data from 5 batches with at least 2 WT-injected oocytes recorded. Mean values are significantly different, t test p = 0.01.
Figure 3. Affinity for Intracellular Ligands(A) ATP-activated current traces from inside-out giant patches excised from oocytes expressing WT-α1β1 (top) and G549R-α1β1 (bottom), at 0 mV. The current was induced by changes in [MgATP] at 50 mM Na+i (90 mM K+). The ramp-like deflections in the WT trace absent in the G549R patch correspond to brief 50 ms-long changes in voltage. (B) Bar graph showing the mean Km,ATP overlapped with data from individual experiments. (C) Bar graph with the mean K0.5,Na from Hill fits to dose-response experiments with Na+ (where Na+ was substituted with K+ in the intracellular bath solution Na+ + K+ = 140 mM). The data from individual patches were fitted globally with shared Hill coefficients nH = 2.4 ± 0.4 for WT and nH = 1.65 ± 0.3 for G549R patches (SEM from the global fits to data from 7 patches for each construct). The higher variability in parameters from patches with G549R probably reflects their lower signals (the 50 mM Na+ + MgATP induced currents of 7.4 ± 3.6 pA for WT and 4.1 ± 2.4 for G549R patches, SD, n = 10 in both cases) consistent with the lower current amplitudes observed in whole-oocyte recordings.
Figure 4. Coexpression of G549R and WT(A) Representative current recordings at −50 mV coexpressing WTOR/WTOS (top) or WTOR/G549ROS (bottom). Oocytes were bathed in an extracellular solution of 150 mM Na+. The current elicited by the first application of 4.5 mM K+ is attributed to both the ouabain-sensitive and ouabain-resistant pump. Subsequent application of 10 μM ouabain inhibited the ouabain-sensitive pumps, leaving the ouabain-resistant WT pumps available for activation during the second application of K+. Ten mM ouabain was used to completely inhibit all pumps. (B) Mean pump current (IP) activated by K+ application after adding 10 μM. Data were normalized to the average IP measured in the WTOR/WTOS oocytes on the same day. (C) The 10 mM ouabain–inhibited transient currents were obtained at the end of the experiments as those in A (before the last application of K+); the Qtot of the ouabain-resistant pumps was calculated from Boltzmann plots and normalized to the WTOR/WTOS average measured on the same day.
Figure 5. Localization of G549R and WT in the Same Cell(A, B) Representative images from HEK293 cells cotransfected with CFP-tagged and YFP-tagged α1, overlaid with the transmission detector (left), CFP signal (center), and YFP signal (right). CFP-A WT-α1/YFP-WT-α1 cotransfection is shown in (A) and CFP-WT-α1 and YFP-G549R-α1 in (B). The inset shows a zoom-in of the boxed cell. (C) Intensity in the plasma membrane divided by the intensity in the cytosol for ≥200 images of each transfection pair. Data from stable and transiently expressed CFP were pulled together (see Methods). Box indicates SD and median. The mean and SEM are shown in the center.
