Expression and functional characterization of missense mutations in ATP8A2 linked to severe neurological disorders

ATP8A2 is a P4‐ATPase (adenosine triphosphate) that actively flips phosphatidylserine and phosphatidylethanolamine from the exoplasmic to the cytoplasmic leaflet of cell membranes to generate and maintain phospholipid asymmetry. Mutations in the ATP8A2 gene have been reported to cause severe autosomal recessive neurological diseases in humans characterized by intellectual disability, hypotonia, chorea, and hyperkinetic movement disorders with or without optic and cerebellar atrophy. To determine the effect of disease‐associated missense mutations on ATP8A2, we expressed six variants with the accessory subunit CDC50A in HEK293T cells. The level of expression, cellular localization, and functional activity were analyzed by western blot analysis, immunofluorescence microscopy, and ATPase activity assays. Two variants (p.Ile376Met and p.Lys429Met) expressed at normal ATP8A2 levels and preferentially localized to the Golgi‐recycling endosomes, but were devoid of ATPase activity. Four variants (p.Lys429Asn, pAla544Pro, p.Arg625Trp, and p.Trp702Arg) expressed poorly, localized to the endoplasmic reticulum, and lacked ATPase activity. The expression of these variants was increased twofold by the addition of the proteasome inhibitor MG132. We conclude that the p.Ile376Met and p.Lys429Met variants fold in a native‐like conformation, but lack key amino acid residues required for ATP‐dependent lipid transport. In contrast, the p.Lys429Asn, pAla544Pro, p.Arg625Trp, and p.Trp702Arg variants are highly misfolded and undergo rapid proteosomal degradation.