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

Simple Summary Medicinal treatment of chronic pain prompts finding novel approaches to the creation of safe and effective analgesics. Ouabain, a cardiotonic steroid detected in the human organism at extremely low concentrations, has been previously shown by us to switch off the pain signal produced by peripheral neurons. The current manuscript elucidates the mechanism of ouabain binding to its molecular target, the Na,K-ATPase enzyme. Application of very sensitive physiological techniques demonstrated a complete loss of the ouabain effect upon removal of the rhamnosyl residue, a structural element of the ouabain molecule. Theoretical modeling made it possible to determine the contribution of the rhamnosyl residue to the process of ouabain binding with Na,K-ATPase. It was concluded that intermolecular bonds between the rhamnosyl residue of ouabain and Na,K-ATPase amino acid residues identified through modeling are required for the analgesic effect of ouabain to manifest itself. To facilitate creation of fundamentally new safe and effective analgesics, the mechanisms of their binding to the corresponding molecular receptors should be clarified at the atomic level. 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(V)1.8 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 & alpha;1-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 & alpha;1-Na,K-ATPase amino acid residues, Glu116 and Glu117.