ATP1A1 encodes the α1 subunit of the Na + /K + -ATPase (NKA). Its protein variants (L302R, G303R, M859R, and W931R) cause hypomagnesemia with seizures that are resistant to treatment with magnesium supplementation. NKA normally builds the electrochemical gradient across the plasma membrane. Utilizing NKAs with reduced ouabain sensitivity, we previously showed that these variant NKA are leaky, allowing ions to flow down the electrochemical gradient and causing a channel-like current; these currents were insensitive to ouabain application. We used two electrode voltage clamps to evaluate the function of ouabain-sensitive NKAs expressed in Xenopus oocytes. K-induced normal outward NKA current was observed only with wildtype and W931R. All variants had larger-than-wildtype Na-dependent inward current (current in Na-Current in NMG) at negative voltages. Inward current from oocytes expressing M859R were unaffected by ouabain but were reduced by ∼10% and ∼50%, respectively, in L302R and G303R oocytes. Surprisingly, ouabain doubled the inward current in W931R oocytes indicating that the channel-like conformation of this variant is favored in the ouabain-bound E2P state. We expressed variant and wildtype α1 with β1 in HEK293S GnT1 cells and solved their ouabain-bound E2P (BeF 4 -inhibited) cryo-EM structures. The variant and wildtype structures were nearly identical except for the disordered intracellular half of TM9 in W931R, which distorted the micelle and exposed a cytosolic ion pathway reaching Y778, in ion-binding site 3, as well as R931. The presence of a positive charge in the membrane in all these leaky variants made us re-evaluate their ion selectivity. Ouabain alteration of G303R and W931R currents response allowed us to demonstrate that these variants also permeate Cl - . Our results suggest that disruption in Cl - gradients by the leaky mutants may participate in the generation of epileptic seizures.
Colmano et al. (2026) studied this question.