Use of RH 421 to measure K+ occlusion in the Na,K-ATPase during ATP hydrolysis

PABLO G. SCHVARTZ; JOSÉ LUIS E. MONTI; SERGIO B. KAUFMAN; PATRICIO J. GARRAHAN; ROLANDO C. ROSSI

Aarhus, Dinamarca

Congreso; 12th International ATPase Conference of Na,K-ATPase and Related Transport ATPases of P-type: Structures, Mechanisms, and Roles in Health and Disease; 2008

Several fluorescence probes have been employed for studying the kinetics of partial reactions in the sodium pump. Among these, the styryl dye RH 421 was used to discriminate between different charged states of the enzyme.1 When mixed with membrane suspensions of Na,K-ATPase, the dye shows three main levels of fluorescence: the lowest level corresponds to the enzyme containing 3 Na+ ions bound, the highest level to the E2P phosphoenzyme state and an intermediate level to states with 2 K+ bound or occluded. We have developed a technique to accurately measure occluded cations in the Na+/K+-ATPase under a wide range of experimental conditions.2 To allow a more clear-cut identification of the intermediates reported by fluorescence of RH 421, we measured under identical conditions both fluorescence changes and the amount of occluded 86Rb+ (Rbocc), using a purified preparation of pig-kidney Na+/K+-ATPase. Experiments were performed at 25oC in media with 150 mM Na+, 0.5 mM free Mg2+, and 25 mM Imidazole-HCl, pH 7.4. Results from measurements of fluorescence of RH421 as a function of time at different concentrations of Rb+ show that 3 mM Rb+ suffices to shift the steady-state signal from that due to E2P to that of states with 2 Rb+ bound or occluded. Experiments in steady state carried out in the aforementioned medium plus 3 mM Rb+ explore both the change in fluorescence signal and the amount of occluded Rb+ as a function of the concentrations of ATP (from 0.01 to 1600 uM) and ADP (from 0 to 4 mM, minimal concentration tested 50 uM). Both sets of results show similar responses to ATP and ADP, in such a way that a plot of fluorescence change vs. Rbocc yields a straight line passing through the coordinates origin. To the best of our knowledge, this is the first direct evidence showing that, under appropriate conditions, occluded Rb+ can be estimated by means of changes in RH 421 fluorescence. Results of the effects of ADP can be explained by the Post-Albers model where the nucleotide operates in three ways: (i) competing with ATP for E2(Rb2) and for E1, and (ii) as a product, binding to E1P and regenerating E1ATP.