Role of the Rhamnosyl Residue of Ouabain in the Activation of the Na,K-ATPase Signaling Function

The signaling or non-pumping Na,K-ATPase function was first observed by us in the nociceptive neuron; Na,K-ATPase transduced the signals from the opioid-like receptors to Na_V1.8 channels. This study elucidates the role of the rhamnosyl residue of ouabain in the activation of the Na,K-ATPase signaling function. The effects resulting from activation of Na,K-ATPase signaling by the Ca²⁺ chelate complex of ouabain (EO) are not manifested upon removal of the rhamnosyl residue, as demonstrated in viable cells by the highly sensitive patch-clamp and organotypic cell culture methods. Docking calculations show that the rhamnosyl residue is involved in five intermolecular hydrogen bonds with the Na,K-ATPase α1-subunit, which are fundamentally important for activation of the Na,K-ATPase signaling function upon EO binding. The main contribution to the energy of EO binding is provided by its steroid core, which forms a number of hydrogen bonds and hydrophobic interactions with Na,K-ATPase that stabilize the ligand-receptor complex. Another critically important role in EO binding is expected to be played by the chelated Ca²⁺ cation, which should switch on strong intermolecular ionic interactions between the EO molecule and two α1-Na,K-ATPase amino acid residues, Glu116 and Glu117.