Identification of the RH421-binding site on Na+/K+-ATPase

Martin Kubala1, Miroslav Huličiak1, Václav Bazgier2, Karel Berka2

1Dept. of Biophysics, Centre of Region Hana for Biotechnological and Agricultural Research, Faculty of Science, Palacky University, Slechtitelu 27, 78341 Olomouc, Czech Republic,

2 Dept. of Physical Chemistry, Regional Centre of Advanced Technologies and Materials, Faculty of Science, Palacky University, 17. listopadu 12, 771 46 Olomouc, Czech Republic


Na+/K+-ATPase (sodium pump) plays a privileged role in the metabolism of all animal cells. It maintains the resting value of plasma membrane potential and creates a gradient of sodium ions that is essential for function of numerous secondary active transporters. Consequently, inhibition of this enzyme can result in various diseases, such as hypertension, renal failure or diabetes, to name just few.

Styrylpyrimidium dye RH421 (N-(4-sulphobutyl)-4-(4-(p-dipentylaminophenyl)-butadienyl)-pyrimidium inner salt) became one of the most important tools for monitoring of Na+/K+-ATPase kinetic. It has been used in several laboratories for characterization of ion binding and translocation of ions across the membrane. The RH421 dye responds to changes of electric charge within the membrane dielectric and it is able to detect charge movements associated with cation binding and dissociation at the extracellular and cytoplasmic surfaces of the protein. It was found that this probe specifically responded to the translocation of the third sodium ion by Na+/K+-ATPase. However, structural details of interaction between RH421 and Na+/K+-ATPase are still unclear.

We used six single-tryptophan mutants to identify the binding site in FRET experiments. They revealed that RH421 binds directly into the ATP-binding site. This conclusion was further supported by results from molecular docking and by competitive experiments using ATP. Experiments with protein/DPPC mixture revealed that RH421 can bind to both protein and lipids, but only the former interaction was influenced by the presence of ATP.

 This work was supported by the grant LO1204 from the National Program of Sustainability I.