Poster Presentation 2014 International Biophysics Congress

RH421 binding to Na+/K+-ATPase analyzed using FRET and molecular docking (#456)

Martin Kubala 1 , Miroslav Huliciak 1 , Vaclav Bazgier 2 , Karel Berka 2
  1. Dept. of Biophysics, Faculty of Science, Palacky University, Olomouc, Czech Republic
  2. Dept. of Physical Chemistry, Faculty of Science, Palacky University, Olomouc, Czech Republic
The Na+/K+-ATPase belongs to P-type ATPase superfamily of active cation transport proteins, which use ATP as a fuel for transport of various ions across biological membranes. The Na+/K+-ATPase transports three sodium ions out of the cell and two potassium ions into the cell using energy from hydrolysis of one ATP molecule. This active transport creates electrochemical gradient, which is essential for electrical excitability, cellular uptake of ions, nutrients and neurotransmitters, and regulation of cell volume and intracellular pH.
The minimal functional unit of the Na+/K+-ATPase consists of the α- and β-subunits assembly. The α-subunit contains the sites for binding of cations and ATP and it is homologous to single-subunit P-type ATPases like e.g. the Ca2+-ATPase. Recently, our understanding to the functioning of Na+/K+-ATPase increased after the high-resolution crystal structures were published. However, crystallography provides only static pictures, and kinetic studies performed under physiological-like conditions are still an important tool for Na+/K+-ATPase examination. One of the most important methods for monitoring of Na+/K+-ATPase kinetic became the fluorescence spectroscopy of the styrylpyrimidium dye RH421.

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 5.4 ± 1.1 mM. 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 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.


This work was supported by grant OPVK, CZ.1.07/2.3.00/20.0057 from Czech Ministry of Schools, Youth and Sports and by grant LO1204 from the National program of Sustainability.