Kinetics of conformational changes of transport intermediates in the Na+/K+ ATPase

RC. ROSSI; MR. MONTES; PG. SCHVARTZ; JLE MONTI; RM. GONZÁLEZ-LEBRERO; SB. KAUFMAN; PJ. GARRAHAN

Montevideo

Congreso; 6th International Conference on Biological Physics and the 5th Southern Cone Biophysics Congress; 2007

According to the currently accepted model, during active transport of Na+ and K+ the Na+/K+-ATPase undergoes phosphorylation and dephosphorylation reactions, as well as conformational changes accompanying the formation and breakdown of intermediates containing occluded (trapped) cations. Occlusion of K+ can occur either during hydrolysis of ATP (physiological route) or spontaneously after mixing the enzyme with K+ without formation of phosphorylated states (direct route).In our laboratory, we have developed a technique to accurately measure occluded cations in the Na+/K+-ATPase under a wide range of experimental conditions (Rossi et al., 1999, Anal.Biochem. 270, 276-285).Several fluorescence probes have been employed to the study of the kinetics of partial reactions in the sodium pump. Among these, eosin and the styryl dye RH 421 were respectively used to detect E1 <=> E2 conformational changes (Skou and Esmann, 1983, Biochim.Biophys. Acta 746, 101-113) and to discriminate between different charged states of the enzyme (Stürmer et al., 1991, J.Membr.Biol 121, 141-161). Changes in fluorescence levels of these probes are usually assigned to changes in the concentration of K+-occluded states, an assumption that has been poorly tested.To allow a more precise identification of the steps reported by fluorescence of eosin and RH 421 and eventually detect new intermediates of the reaction cycle, we performed experiments under identical conditions measuring both fluorescence changes and the amount of occluded (86Rb)Rb+ in a purified preparation of pig-kidney Na+/K+-ATPase.We found that, despite eosin fluorescence change seems to be a good reporter of Rb+ occlusion through the direct route for the initial stages of the reaction, a second phase of occlusion persists after completion of the eosin-fluorescence change. This is not only of mechanistic importance but also indicates that caution has to be exerted in taking fluorescence changes as a marker of occlusion.Concerning RH 421, at sufficiently high concentration of Rb+ the dye shows a very good correlation with the steady-state amount of occluded cation. To the best of our knowledge, this is the first direct evidence showing that occluded Rb+ can be estimated by means of changes in RH 421 fluorescence.With grants from CONICET, ANPCyT and UBACyT, Argentina.