Electrogenic Binding of Ions at Cytoplasmic Side of Na,K,ATPase

Abstract

The electrogenic binding of ions from the cytoplasmic side of Na,K,ATPase has been studied by measurements of changes of the membrane capacitance and conductance triggered by a jump of pH or the sodiumion concentration in the absence of ATP. The pH jumps were performed in experiments with membrane fragments containing purified Na,K,ATPase adsorbed to a bilayer lipid membrane (BLM). Protons were released in a sub-millisecond time range from a photosensitive compound (caged-H+) initiated by a UV light flash. The sodium concentration jumps were carried out by a fast solution exchange in experiments with solid-supported membranes which were deposited on a gold electrode. The change of the membrane capacitance triggered by the pH jump depended on sodium-ion concentration. Potassium ions had a similar effect on the capacitance change triggered by the pH jump. The effects of these ions are explained by the their competition with protons in the binding sites on cytoplasmic side of the Na,K,ATPase. The approximation of the experimental data by a theoretical model yields the dissociation constants, K, and the cooperativity coefficients, n, of the binding sites for sodium (K = 2.7 mM, n = 2) and potassium (K = 1.7 mM, n = 2) ions. In the presence of magnesium ions the apparent dissociation constants of sodium increased. As possible reason of the inhibition of sodium-ion binding by magnesium ions is proposed an electrostatic or conformational effect of magnesium ions bound to a separate site of the Na,K,ATPase close to the entrance to the sodium-ion binding sites.