On the mechanism of hypomagnesemia with treatment-resistant seizures caused by variants of the Na+,K+-ATPase α1 subunit (ATP1A1).

Various dominant disease phenotypes are caused by germline variants of ATP1A1, the gene for the ubiquitously expressed Na+,K+-ATPase (NKA) α1 subunit. What characteristics link specific variants to a particular disease phenotype remains unclear. We used heterologous expression in mammalian cells and Xenopus oocytes to evaluate the characteristics of α1 variants causing hypomagnesemia with treatment-resistant seizures: L302R, G303R, M859R, and W931R. Cell survival in the presence of micromolar ouabain was reduced in cells expressing ouabain-resistant versions of these variants, indicating defective function of the variants relative to WT. Fluorescently tagged WT-α1 expressed in HEK293 cells showed plasmalemma localization, while all variants displayed increased cytosolic signal. In variant-expressing oocytes under voltage clamp, each variant showed passive "leak" currents, which were unaffected by ouabain in L302R and M859R, partially inhibited by ouabain in G303R and enhanced by ouabain in W931R. Na+ and Cl‒ permeate through G303R and W931R. M859R and W931R presented small K+-activated ouabain-inhibitable NKA currents that were absent in L302R and G303R. Growth of cells in ouabain and enzymatic studies demonstrated Na+/K+-dependent active transport for W931R, albeit with reduced Na+ affinity. We solved the cryo-EM structure of W931R-α1β1 with bound ouabain at 3 Å resolution. The structure showed a distorted transmembrane segment M9, which causes a concavity in the detergent micelle, suggesting that similar distortions may provide a leak pathway at the protein-lipid interface. Our results demonstrate that besides a loss of NKA function, all four variants carry an aberrant leak current that probably underlies the seizures and the hypomagnesemia characterizing this phenotype.