FXYD amino acid sequence alignment and Na/K pump enzymatic cycle. (A) Alignment of the five human FXYD isoforms studied in this work. Residues in the transmembrane (TM), cytoplasmic, and extracellular regions of the isoforms are indicated. (B) Post-Albers kinetic scheme of the Na/K pump cycle (clockwise forward direction). The pump alternates between two major conformations, E1 and E2, which can exist in dephosphorylated or phosphorylated (P) forms. Parentheses indicate ions occluded within the protein. Transitions within the red box produce transient charge movement: the release of the first Na⁺ (forward cycle) or rebinding of the last Na⁺ (backward cycle) during E1P(3Na⁺) ↔ E2P(2Na⁺) + Na⁺_o produces the slow transient charge movement, while the release of the last two Na⁺ ions (forward cycle) or rebinding of the first two Na⁺ ions (backward cycle) during E2P(2Na⁺) ↔ E2P + 2Na⁺_o produces fast charge movement.