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

Abstract

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 NaV1.8 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.

Sensational site: the sodium pump ouabain-binding site and its ligands

“… 357 , this appears to be the first claim that the sugar moiety in ouabain has a specific role in signaling. The same authors also suggest, based upon modeling studies of the polyhydroxylated steroid cores in ouabain and ouabagenin, that both steroids might chelate Ca 2+ ions and that the resultant NKA bound complexes lead to calcium-related ionic interactions with Glu-116 and Glu-117 ( 366 ). We suspect, however, that Ca 2+ chelates in the circulation are likely to be relatively unstable.…”

“…On a related topic, in primary sensory neurons, the ouabain-NKA-Src complex apparently is involved in transducing signals from opioid-like receptors to NaV1.8 channels ( 364 ). In this system, ouabain has an antinociceptive effect ( 365 ) and it has been suggested that the rhamnosyl residue of ouabain is critical because ouabagenin did not activate signaling ( 366 ). Although it has long been recognized that the rhamnose moiety contributes to the high-affinity binding of ouabain to NKA, e.g., Ref.…”