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Na+ Transport, and the E1P-E2P Conformational Transition of the Na+/K+-ATPase

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Fendler,  Klaus
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Babes, A., & Fendler, K. (2000). Na+ Transport, and the E1P-E2P Conformational Transition of the Na+/K+-ATPase. Biophysical Journal, 79(5), 2557-2571. doi:10.1016/S0006-3495(00)76496-4.


Cite as: http://hdl.handle.net/21.11116/0000-0007-6E78-8
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 E1P ↔ E2P 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 E1P ↔ E2P equilibrium is approximately at a midpoint position at pH 6.2 (equilibrium constant ≈ 0.8) but moves more to the E1P 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 E1P ↔ E2P, lyotropic anions shift it to E1P.