RH421 binds into the ATP-binding site on the Na⁺/K⁺-ATPase

The Na⁺/K⁺-ATPase plays a key role in ion transport across the plasma membrane of all animal cells. The voltage-sensitive styrylpyrimidium dye RH421 has been used in several laboratories for monitoring of Na⁺/K⁺-ATPase kinetics. It is known, that RH421 can interact with the enzyme and it can influence its activity at micromolar concentrations, but structural details of this interaction are only poorly understood. Experiments with isolated large cytoplasmic loop (C45) of Na⁺/K⁺-ATPase revealed that RH421 can interact with this part of the protein with dissociation constant 1μM. The Trp-to-RH421 FRET performed on six single-tryptophan mutants revealed that RH421 binds directly into the ATP-binding site. This conclusion was further supported by results from molecular docking, site-directed mutagenesis and by competitive experiments using ATP. Experiments with C45/DPPC mixture revealed that RH421 can bind to both C45 and lipids, but only the former interaction was influenced by the presence of ATP.