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+
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+
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
-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+
-ATPase examination. One of the most important methods for monitoring
-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.