A sublethal ATP11A mutation associated with neurological deterioration causes aberrant phosphatidylcholine flipping in plasma membranes
Katsumori Segawa, … , Shigeo Kure, Shigekazu Nagata
Katsumori Segawa, … , Shigeo Kure, Shigekazu Nagata
Published August 17, 2021
Citation Information: J Clin Invest. 2021;131(18):e148005. https://doi.org/10.1172/JCI148005.
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A sublethal ATP11A mutation associated with neurological deterioration causes aberrant phosphatidylcholine flipping in plasma membranes

Abstract

ATP11A translocates phosphatidylserine (PtdSer), but not phosphatidylcholine (PtdCho), from the outer to the inner leaflet of plasma membranes, thereby maintaining the asymmetric distribution of PtdSer. Here, we detected a de novo heterozygous point mutation of ATP11A in a patient with developmental delays and neurological deterioration. Mice carrying the corresponding mutation died perinatally of neurological disorders. This mutation caused an amino acid substitution (Q84E) in the first transmembrane segment of ATP11A, and mutant ATP11A flipped PtdCho. Molecular dynamics simulations revealed that the mutation allowed PtdCho binding at the substrate entry site. Aberrant PtdCho flipping markedly decreased the concentration of PtdCho in the outer leaflet of plasma membranes, whereas sphingomyelin (SM) concentrations in the outer leaflet increased. This change in the distribution of phospholipids altered cell characteristics, including cell growth, cholesterol homeostasis, and sensitivity to sphingomyelinase. Matrix-assisted laser desorption ionization–imaging mass spectrometry (MALDI-IMS) showed a marked increase of SM levels in the brains of Q84E-knockin mouse embryos. These results provide insights into the physiological importance of the substrate specificity of plasma membrane flippases for the proper distribution of PtdCho and SM.

Authors

Katsumori Segawa, Atsuo Kikuchi, Tomoyasu Noji, Yuki Sugiura, Keita Hiraga, Chigure Suzuki, Kazuhiro Haginoya, Yasuko Kobayashi, Mitsuhiro Matsunaga, Yuki Ochiai, Kyoko Yamada, Takuo Nishimura, Shinya Iwasawa, Wataru Shoji, Fuminori Sugihara, Kohei Nishino, Hidetaka Kosako, Masahito Ikawa, Yasuo Uchiyama, Makoto Suematsu, Hiroshi Ishikita, Shigeo Kure, Shigekazu Nagata

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Figure 3

Q84E heterozygous–knockin mice and their lethality with neurological deficits.

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Q84E heterozygous–knockin mice and their lethality with neurological def...
(A) Body weights of the pups (Atp11aWT/WT, n = 38; Atp11aQ84E/WT, n = 5). (B) Percentage of surviving pups (Atp11aWT/WT, n = 38; Atp11aQ84E/WT, n = 7). (C) Gait analysis by the footprint test. After coating the paws with ink, 17-week-old female Atp11aQ84E/WT and WT littermate mice were allowed to walk. (D) Clasping of the hind limbs during tail suspension by a 20-week-old Atp11aQ84E/WT female mouse, a sign of a neurological defect. The same mutant mouse is shown in Supplemental Video 3. (E) Midaxial T2-weighted MRI of 11-week-old female WT and Atp11aQ84E/WT littermate mice. Red arrowheads indicate the dilated lateral ventricle. The calculated brain volume is shown. Scale bars: 5 mm. (F) Sections of the medulla oblongata from E14 embryos were stained with toluidine blue or observed by electron microscopy. Arrowheads indicate degenerated neurons with pyknotic nuclei. Scale bars: 10 μm. (G) TUNEL-stained brain sections around the ventricle of 2-week-old male Atp11aWT/WT and Atp11aQ84E/WT neonates. Scale bar: 300 μm. TUNEL-positive foci of 6–7 sections from different parts of the entire brain were computationally counted and are represented as TUNEL-positive foci per mm2. P = 0.00697, by Student’s t test. (H) Presence of Q84E-containing peptide in mouse Atp11aQ84E/WT placenta. Proteins with a MW of 100–130 kDa in the E15 placenta membrane fraction of female Atp11aWT/WT and Atp11aQ84E/WT mice were digested with chymotrypsin. ATP11A peptides containing Q84 (left) and E84 (right) were analyzed by PRM by using the data in Supplemental Figure 4. Extracted ion chromatograms of N-terminal b-ions (b5+, b8+, and b17+) and C-terminal y-ions (y11+, y13+, and y14+) are shown.