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The [Na.sup.+]/[K.sup.+]-ATPase is a ubiquitous plasma membrane ion pump that utilizes ATP hydrolysis to regulate the intracellular concentration of [Na.sup.+] and [K.sup.+]. It is comprised of at least two subunits, a large catalytic subunit that mediates ATP hydrolysis and ion transport, and an ancillary [beta] subunit that is required for proper trafficking of the holoenzyme. Although processes mediated by the a subunit have been extensively studied, little is known about the participation of the [beta] subunit in conformational changes of the enzyme. To elucidate the role of the [beta] subunit during ion transport, extracellular amino acids proximal to the transmembrane region of the sheep [[beta].sub.1] 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 [[beta].sub.1] 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.sub.+]/[K.sub.+]-ATPase through a fluorophore-labeled [beta] subunit. Simultaneous recording of presteady-state or stationary currents together with fluorescence signals enabled correlation of the observed environmental changes of the [beta] subunit to certain reaction steps of the [Na.sup.+]/[K.sup.+]-ATPase, which involve changes in the occupancy of the two principle conformational states, [E.sub.1]P and [E.sub.2]P. From these experiments, evidence is provided that the [[beta].sub.1]-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 [[beta].sub.1] isoforms, directly influences the effective potential that determines voltage dependence of extracellular cation binding and release. KEY WORDS: voltage-clamp fluorometry * conformational change * kinetics * electrogenic steps * subunit interaction

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-maleilnide (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. [ABSTRACT FROM AUTHOR]