BPS2026 – Functional evaluation of ATP1A1 missense variants: Gly903Arg, Ser207Phe, Gly877Ser, and Gly864Arg

The Na + /K + -ATPase (NKA) builds and maintains electrochemical gradients for Na + and K + across the cell membrane. Variants in the gene for NKA’s α1 subunit gene, ATP1A1 , cause various disorders: Gly903Arg was found in a patient with developmental delay with autism; Ser207Phe and Gly877Ser were found in patients with intermediate Charcot-Marie-Tooth disease; and Gly864Arg was found in a patient with borderline learning impairment, sleep disorder, and poor emotional control. Disease mechanisms were examined by comparing function of variant and wild type (WT) NKA in two complementary systems: xenopus oocytes, to evaluate NKA’s kinetic function, and HEK293cells, to evaluate membrane trafficking and potential dominant-negative effects. Evaluation of results from these approaches is underway. NKA is an αβ heterodimer. Oocytes were injected with cRNA for human α1 and β1 subunits. Four days after injection we used two-electrode voltage clamp to measure NKA currents generated when K + was added in the extracellular environment. There was little difference in oocyte expression between WT and Gly903Arg and Ser207Phe mutants while Gly877Ser showed a potential leak in 150 mM Na + solution. When Gly864Arg cRNA was injected in oocytes, most oocytes died on the fourth day after injection. HEK293 cells were co-transfected with plasmids containing WT and mutant human α1 subunits N-terminally tagged with fluorescence proteins to analyze their cellular localization. The CFP tagged WT α1 was co-transfected with a YFP tagged α1 subunit (WT or variants). To quantify protein localization at the plasma membrane, we calculated ratio of fluorescence intensity at the plasma membrane, divided by fluorescence at the cytosol. Preliminary observations indicate that the YFP variant pumps have diminished trafficking to the plasma membrane compared to WT, but that localization of the accompanying WT CFP NKA is not altered by the co-transfected variant NKA. NSF-MCB 2003251.