-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 effect of choline iodide, bromide and chloride on the kinetics of the electrogenic sodium transport by the Na,K-ATPase was investigated in a model system of ATPase-containing membrane fragments adsorbed on the lipid bilayer membrane. The kinetic parameters of Na + transport were determined from short circuit currents after fast release of ATP from its caged precursor. The falling phase of the current transients could be fitted by a single exponential with the time constant, s 2 . Its temperature dependence allowed an estimation of the activation energy of the rate-limiting reaction step, the conformation transition E 1 /E 2 . Choline iodide and bromide caused a decrease of the activation energy as well as the overall rate of the process expressed as the pre-exponential factor A of the Arrhenius equation. If choline iodide or bromide were present on the cytoplasmic and extracellular sides of the protein, the temperature dependent changes were more pronounced than when present on the cytoplasmic side only. These results can be explained by an effect of the anions on water structure on the extracellular surface of the protein, where a deep access channel connects the ion-binding sites with the solution. Chloride ions also caused a deceleration of the electrogenic transport, however, in contrast to iodide or bromide, they did not affect the activation energy, and were more effective when added on the cytoplasmic side. This effect can be explained by asymmetric screening of the negative surface charges which leads to a transmembrane electric potential that modifies the ion transfer.

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The effect of pH on electrogenic sodium transport by the Na+ ,K+-ATPase has been studied. Experiments were carried out by admittance recording in a model system consisting of a bilayer lipid membrane with adsorbed membrane fragments containing purified Na+ ,K+-ATPase. Changes in the membrane admittance (capacitance and conductance increments in response to photo-induced release of ATP from caged ATP) were measured as function of AC voltage frequency, sodium ion concentration, and pH. In solutions containing 150 m M Na+, the frequency dependence of capacitance increments was not significantly dependent on pH in the range between 6 and 8. At a low NaCl concentration (3 mM), the capacitance increments at low frequencies decreased with the increasing pH. In the absence ofNaCl, the frequency-dependent capacitance increment at low frequencies was similar to that measured in the presence of 3 mM NaCI. These results may be explained by involvement of protons in the Na+, K+-ATPase pump cycle, i.e., electroneutral exchange of sodium ions for protons under physiological conditions, electrogenic transport of sodium ions at high pH, and electrogenic transport of protons at low concentrations (and in the absence) of sodium ions.