Rb+ deocclusion from the vanadate-bound Na,K-ATPase is not parallel to the E2-E1 transition

SPIAGGI AJ; MONTI JLE; ROSSI RC; MONTES MR

Pacific Grove, CA

Congreso; 13th International P-type ATPase meeting; 2011

ASMBM

Vanadate is a well-known inhibitor of the activity of P-type ATPases (1) that is currently being used in structural studies to mimic the bipyramidal transition state of the E2P form (2). The present study explores the effect of vanadate on the transition between the E2 and E1 conformations and its relation with the intermediate with Rb+ occluded by the ?direct route? in the Na,K-ATPase. Conformational transitions were measured through eosin fluorescence changes and occluded Rb+ was quantified by a rapid filtration technique (3), under presteady-state and equilibrium conditions. Control experiments performed in the absence of vanadate showed that the time course of the E2-E1 conformational change promoted by Na+ is parallel to the release of Rb+ from the occluded state. The addition of vanadate (0.1 mM) in the presence of Mg2+ (1.75 mM) cancels the transition from E2 to E1 induced by Na+ whereas Rb+ deocclusion still occurs at a significant rate. Mg2+-vanadate increased the K0.5 of Rb+ for the occluded state from 5 to 40 micromolar reaching the same maximal level. Our results suggest that even when Mg2+-vanadate stabilizes an E2-like state, this is not a guarantee of a more stable Rb+-occluded form. This work was supported by grants from Agencia Nacional de Promoción Científica y Tecnológica, Consejo Nacional de Investigaciones Científicas y Técnicas and Universidad de Buenos Aires, Argentina. 1- Cantley L.C. Cantley L.G. and Josephson L., J.Biol.Chem. (1978) 253:7361-7368. 2- Clausen J.D., McIntosh D., Woolley D. and Andersen J.P., J.Biol.Chem. (2011) 286: 11792-11802 3- Rossi R.C., Kaufman S.B., González-Lebrero R.M., Nørby J.G. and Garrahan P.J., Anal. Biochem. (1999) 270:276-285.172