Electrogenic Transport of Sodium Ions in Cytoplasmic and Extracellular Ion Access Channels of Na+,K+-ATPase Probed by Admittance Measurement Technique~1

摘要

Electrogenic movements of sodium ions in cytoplasmic and extracellular access channel of theNa~+,K~+-ATPase have been studied by the admittance measurement technique which allows the detection ofsmall changes of the membrane capacitance and conductance induced by phosphorylation of the ion pump. Themeasurements were carried out on a model system consisting of a bilayer lipid membrane, to which membranefragments with ion pumps were adsorbed that contain the ion pumps in high density. Small changes of the mem-brane capacitance and conductance were induced by a fast release of ATP from caged ATP. The effect was mea-sured at various frequencies and in solutions with different Na~+concentrations. The experimentally observedfrequency dependences were explained using a theoretical model assuming that Na~+movement through thecytoplasmic access channel occurs in one step and through the extracellular access channel, in two steps. Thephosphorylation of the protein by ATP leads to a block of the cytoplasmic access channel and an opening theextracellular access channel. The disappearance of electrogenic Na~+movements on the cytoplasmic side pro-duces a negative change of capacitance and conductance, while the emergence of extracellular M.+ movementsgenerates a positive change. Fitting the experimental dependences of capacitance and conductance by theoret-ical curves allowed the determination equilibrium and kinetic parameters of sodium transport in the accesschannels.