Binding of the third Na+ ion to the cytoplasmic side of the Na,K-ATPase is electrogenic.

A new experimental setup was constructed to allow parallel measurements of total internal reflection fluorescence and of capacitance changes in Na,K-ATPase-containing membranes. Effects correlated with cytoplasmic sodium binding to Na,K-ATPase were investigated. Ion binding-induced fluorescence changes of the electrochromic dye RH421 in membrane fragments adsorbed on a transparent capacitative electrode corresponded perfectly to capacitance increases detected by a lock-in technique. From these electric measurements it was possible to estimate a dielectric coefficient of about 0.25 for the electrogenic binding of the third Na+ ion. Binding of K+ to cytoplasmic sites was electroneutral.