Fig 2: Validation of NKA binding to PrP.(A) Western blot-based validation of co-immunoprecipitation of NKA subunits with PrP. (B) Evidence of partial cellular co-localization of PrPC and ATP1A1 by co-immunocytochemical analysis of ReN VM cells, with Hoechst stain serving as the nuclear counter stain. Scale bar: 10 nm. (C) Chemical structure of the NKA inhibitor ouabain. (D) Parallel ouabain concentration-dependent effects on NKA and PrPC levels. Exposure of differentiated ReN VM cells to ouabain at concentrations up to 50 nM causes a biphasic effect on PrPC and ATP1A1 levels, with concentrations up to 30 nM leading to a ouabain concentration-dependent reduction in steady-state levels of both proteins, and exposure to 50 nM provoking a reproducible slowing of PrPC during SDS-PAGE separation that parallels an increase in steady-state ATP1A1 levels.

Fig 3: Molecular and cellular architecture appears normal in SGNs from KNa1 DKO mice. The expression and distribution of various proteins shaping SGN excitability were examined using immunofluorescence in the isolated preparation of the organ of Corti and SGNs from (6-week-old) WT and DKO mice. (A) The expression of the Na+, K+-ATPase a3 (ATP1A3, green) and patterns of myelination were similar in both WT and DKO mice. (B) The expression and distribution of the low voltage-activated KV1.1 (green) was similar in both WT and DKO mice. Tubulin J (TuJ, red) marks the SGN afferent dendrites and is provided for reference. (C) The expression and distribution of the high voltage-activated KV3.1 (green) and the voltage-gated NaV1.6 (red) were similar in both WT and DKO mice. All images are presented as Z-projections through a stack of confocal micrographs from the 16 kHz region. Expression patterns of ATP1A3, MBP, KV1.1, KV3.3 and NaV1.6 were similarly expressed at other regions as well as in the somata of the SGNs from WT and DKO mice.

Fig 4: Retinoschisin binding to ATP1B2 patch II mutants.HEK293 cells were transfected with expression constructs for ATP1A3 in combination with expression constructs for different ATP1B2 variants mutated in patch II regions depicted within the figure. (A) 48 h after transfection, cells were subjected to recombinant retinoschisin for 1 h, followed by intensive washing. Cells transfected with expression constructs for only ATP1B2 served as a negative control, cells transfected with expression constructs for ATP1A3 and normal ATP1B2 served as a positive control in the retinoschisin binding assay [13]. Heterologous protein expression as well as retinoschisin binding was investigated by Western blot analyses with antibodies against retinoschisin, ATP1A3, and ATP1B2. The ACTB staining served as loading control. (B) 48 h after transfection, cell surface proteins were biotinylated, purified by streptavidin affinity chromatography and analysed by Western blotting using antibodies against ATP1B2. The ATP1A1 immunoblot was performed as loading control. (C) 48 h after transfection, cells were subjected to FACS analyses applying an anti-ATP1B2 antibody. Representative histograms of cells transfected with selected ATP1B2 patch II mutants are given in this figure. Light grey: histogram of untransfected HEK293 cells depicting unspecific background signals. Dark grey: histogram of transfected HEK293 cells.

Fig 5: PGC-1a4 Overexpression Induces Na-K ATPase Expression and Leads to a Drastic Increase in Its Current in Neonatal Cardiomyocytes. (a) Representative action potentials from cultured neonatal cardiomyocytes. (b) Action potential duration (APD) plotted against repolarization phase. (c) Resting membrane potential (RMP, left, n[ctrl] = 4/15 (animals/cells), n[a4+] = 3/11), amplitude (middle, n[ctrl] = 4/12, n[a4+] = 3/8) and rise time (right, n[ctrl] = 4/11, n[a4+] = 3/8) of the action potentials. (d) mRNA expression of sodium–potassium ATPase isoforms a1 (Atp1a1), a2 (Atp1a2) and a3 (Atp1a3) in ventricles of neonatal control (ctrl) and Pgc-1a4 overexpressing (a4+) (n = 6). (e) Protein expression of sodium–potassium ATPase isoforms in neonatal ventricles. (f) Representative Na+/K+ ATPase current traces under application of a saturating concentration of ouabain (left) and statistics of the ouabain sensitive current (right) in isolated neonatal cardiomyocytes (n[ctrl] = 3/11, n[a4+] = 2/13). (g) Representative relative Na+/K+ ATPase current traces under consecutive applications of 10 µM and 1 mM ouabain (left) and statistics of relative proportion of the current sensitive to 10 µM ouabain (right) in neonatal cardiomyocytes (n[ctrl] = 1/7, n[a4+] = 1/10). (h) Changes in glycolytic rate of isolated neonatal cardiomyocytes induced application of 10 µM ouabain (n[ctrl] = 3/14 (animals/wells), n[a4+] = 3/17). p-value from hierarchical statistical test (or Student’s t-test in panel d): *, p < 0.05; **, p < 0.01; ***, p < 0.001.