Rb+ occlusion stabilized by vanadate in gastric H+/K+-ATPase at 25 ºC

MONTES MR; SPIAGGI AJ; MONTI, JLE; CORNELIUS F; OLESEN C; GARRAHAN PJ; ROSSI, RC

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2011 vol. 1808 p. 316 - 322

0005-2736

Abstract: Despite its similarity with the Na+/K+ ATPase, it has not been possible so far to isolate a K+-occluded state in the H+/K+ ATPase at room temperature. We report here results on the time course offormation of a state containing occluded Rb+ (as surrogate for K+) in H+/K+ ATPase from gastricvesicles at 25 ºC. Alamethicin (a pore-forming peptide) showed to be a suitable agent to open vesicles,allowing a more efficient removal of Rb+ ions from the intravesicular medium than C12E8 (a non-ionicdetergent). In the presence of vanadate and Mg2+, the time course of [86Rb]Rb+ uptake displayed afast phase due to Rb+ occlusion. The specific inhibitor of the H+/K+-ATPase SCH28080 significantlyreduces the amount of Rb+ occluded in the vanadate-H+/K+-ATPase complex. Occluded Rb+ varieswith [Rb+] according to a hyperbolic function with K0.5 = 0.29 ± 0.06 mM. The complex between theRb+-occluded state and vanadate proved to be very stable even after removal of free Mg2+ with EDTA.Our results yield a stoichiometry lower than one occluded Rb+ per phosphorylation site, which mightbe explained assuming that, unlike for the Na+/K+ ATPase, Mg2+-vanadate is unable to recruit all theRb+-bound to the Rb+-occluded form of the Rb+-vanadate-H+/K+ ATPase complex.