Electrogenic transport of sodium ions in cytoplasmic and extracellular ion access channels of Na+,K+-ATPase probed by admittance measurement technique

Electrogenic movements of sodium ions in cytoplasmic and extracellular access channel of the Na⁺,K⁺-ATPase have been studied by the admittance measurement technique which allows the detection of small changes of the membrane capacitance and conductance induced by phosphorylation of the ion pump. The measurements were carried out on a model system consisting of a bilayer lipid membrane, to which membrane fragments with ion pumps were adsorbed that contain the ion pumps in high density. Small changes of the membrane capacitance and conductance were induced by a fast release of ATP from caged ATP. The effect was measured at various frequencies and in solutions with different Na⁺ concentrations. The experimentally observed frequency dependences were explained using a theoretical model assuming that Na⁺ movement through the cytoplasmic access channel occurs in one step and through the extracellular access channel, in two steps. The phosphorylation of the protein by ATP leads to a block of the cytoplasmic access channel and an opening the extracellular access channel. The disappearance of electrogenic Na⁺ movements on the cytoplasmic side produces a negative change of capacitance and conductance, while the emergence of extracellular Na⁺ movements generates a positive change. Fitting the experimental dependences of capacitance and conductance by theoretical curves allowed the determination equilibrium and kinetic parameters of sodium transport in the access channels. The text was submitted by the authors in English.