The β Subunit of the Na⁺/K⁺-ATPase Follows the Conformational State of the Holoenzyme

The Na⁺/K⁺-ATPase is a ubiquitous plasma membrane ion pump that utilizes ATP hydrolysis to regulate the intracellular concentration of Na⁺ and K⁺. It is comprised of at least two subunits, a large catalytic α subunit that mediates ATP hydrolysis and ion transport, and an ancillary β subunit that is required for proper trafficking of the holoenzyme. Although processes mediated by the α subunit have been extensively studied, little is known about the participation of the β subunit in conformational changes of the enzyme. To elucidate the role of the β subunit during ion transport, extracellular amino acids proximal to the transmembrane region of the sheep β₁ subunit were individually replaced for cysteines. This enabled sulfhydryl-specific labeling with the environmentally sensitive fluorescent dye tetramethylrhodamine-6-maleimide (TMRM) upon expression in Xenopus oocytes. Investigation by voltage-clamp fluorometry identified three reporter positions on the β₁ subunit that responded with fluorescence changes to alterations in ionic conditions and/or membrane potential. These experiments for the first time show real-time detection of conformational rearrangements of the Na⁺/K⁺-ATPase through a fluorophore-labeled β subunit. Simultaneous recording of presteady-state or stationary currents together with fluorescence signals enabled correlation of the observed environmental changes of the β subunit to certain reaction steps of the Na⁺/K⁺-ATPase, which involve changes in the occupancy of the two principle conformational states, E₁P and E₂P. From these experiments, evidence is provided that the β₁-S62C mutant can be directly used to monitor the conformational state of the enzyme, while the F64C mutant reveals a relaxation process that is triggered by sodium transport but evolves on a much slower time scale. Finally, shifts in voltage dependence and kinetics observed for mutant K65C show that this charged lysine residue, which is conserved in β₁ isoforms, directly influences the effective potential that determines voltage dependence of extracellular cation binding and release.