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Biophys J, November 2000, p. 2557-2571, Vol. 79, No. 5

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

Alexandru Babes* and Klaus Fendlerdagger

 dagger Max-Planck-Institut für Biophysik, D-60596 Frankfurt/M, Germany; and  *Department of Physiology and Biophysics, Faculty of Biology, University of Bucharest, Bucharest, Romania

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 left-right-arrow 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 left-right-arrow E2P equilibrium is approximately at a midpoint position at pH 6.2 (equilibrium constant approx  0.8) but moves more to the E1P side at basic pH 8.5 (equilibrium constant approx  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 left-right-arrow E2P, lyotropic anions shift it to E1P.

Biophys J, November 2000, p. 2557-2571, Vol. 79, No. 5
© 2000 by the Biophysical Society   0006-3495/00/11/2557/15  $2.00


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