Na + Transport, and the E 1P-E 2P Conformational Transition of the Na +/K +-ATPase

Abstract

We have used admittance analysis together with the black lipid membrane technique to analyze electrogenic reactions within the Na + branch of the reaction cycle of the Na +/K +-ATPase. ATP release by flash photolysis of caged ATP induced changes in the admittance of the compound membrane system that are associated with partial reactions of the Na +/K +-ATPase. Frequency spectra and the Na + dependence of the capacitive signal are consistent with an electrogenic or electroneutral E 1P ↔ E 2P conformational transition which is rate limiting for a faster electrogenic Na + dissociation reaction. We determine the relaxation rate of the rate-limiting reaction and the equilibrium constants for both reactions at pH 6.2–8.5. The relaxation rate has a maximum value at pH 7.4 (∼320 s −1), which drops to acidic (∼190 s −1) and basic (∼110 s −1) pH. The E 1P ↔ E 2P equilibrium is approximately at a midpoint position at pH 6.2 (equilibrium constant ≈ 0.8) but moves more to the E 1P side at basic pH 8.5 (equilibrium constant ≈ 0.4). The Na + affinity at the extracellular binding site decreases from ∼900 mM at pH 6.2 to ∼200 mM at pH 8.5. The results suggest that during Na + transport the free energy supplied by the hydrolysis of ATP is mainly used for the generation of a low-affinity extracellular Na + discharge site. Ionic strength and lyotropic anions both decrease the relaxation rate. However, while ionic strength does not change the position of the conformational equilibrium E 1P ↔ E 2P, lyotropic anions shift it to E 1P.