Binding of the third Na+ ion to the cytoplasmic side of the Na,K-ATPase iselectrogenic

A new experimental setup was constructed to allow parallel measurements of total internal reflection fluorescence and of capacitance changes in Na,K-A TPase-containing membranes. Effects correlated with cytoplasmic sodium bind ing to Na,K-ATPase were investigated. Ion binding-induced fluorescence chan ges of the electrochromic dye RH421 in membrane fragments adsorbed on a tra nsparent capacitative electrode corresponded perfectly to capacitance incre ases detected by a lock-in technique. From these electric measurements it m as possible to estimate a dielectric coefficient of about 0.25 for the elec trogenic binding of the third Na+ ion. Binding of K+ to cytoplasmic sites m as electroneutral. (C) 1999 Federation of European Biochemical Societies.