Conformational States of Na⁺/K⁺-ATPase Probed with RH-421 and Eosin

Abstract

RH-dyes partition into the lipid bilayer and respond to binding of Na⁺ to Na⁺/K⁺-ATPase (EC.3.6.1.37) by a decrease in their fluorescence (1,5,7). Subsequent addition of K⁺ returns the fluorescence to the “pre-Na⁺ level”. In an attempt to define steps of the reaction cycle or conformational transitions of Na⁺/K⁺-ATPase associated with changes in RH-dyes fluorescence we applied one of the RH-dyes, RH-421, and eosin, a well known conformational probe, to characterize the cation binding to the intracellular sites. Increase in eosin fluorescence reflects a high affinity binding of eosin to the substrate binding site of the enzyme, identifying the E₁-dephosphoforms of Na⁺/K⁺-ATPase (6). RH-421 appeared to be able to distinguish between the Na-bound E₁ form and any other E₁-form in the pool of dephosphoenzymes.