Functional Effects of Substitutions Modifying the Hydroxyl or Phenol Ring of Tyrosine 780 at Ion Binding Site-III of the Na, K-Atpase

Abstract

on the cytoplasmic side of the protein, the admittance method has been applied to an experimental setup consisting of membrane fragments with Na,K-ATPase adsorbed to a bilayer lipid membrane (BLM) or a solid supported membrane. The small changes of the membrane admittance (capacitance and conductance) initiated by the fast steps of the pH or Na þ concentration were measured in the absence of ATP. pH jumps were performed by release of protons from a photosensitive compound (caged H þ) triggered by a UV light flash, the Na þ concentration jumps were carried out by fast solution exchange. The changes of the membrane capacitance triggered by a pH jump depended on the initial pH and the concentration of sodium or potassium ions. The effects of these ions are explained by a theoretical model assuming competition of protons with sodium or potassium ions in the binding sites on the cytoplasmic side of the Na,K-ATPase. The approximation of the experimental data by theoretical curves yields the dissociation constants and the cooperativity coefficients of the binding sites for sodium ions and potassium ions. In the presence of magnesium ions the apparent dissociation constants of sodium were increased. The effects of K ions on the pH driven capacitance changes were attenuated in presence of 100 mM of ATP and no magnesium. This observation indicates that the Na,K-ATPase is driven into a conformation with low affinity for the potassium ions presumably because of trapping the enzyme in state with ATP bound in the low-affinity site. Supported by RFBR project 16-04-01162.