Benzyltriethylammonium Ions Inhibit the Na,K-ATPase at Extracellular Potassium Binding Sites

GONZÁLEZ-LEBRERO, R. M.; PELUFFO, R. D.; KAUFMAN, S. B.; ROSSI, R. C.; BERLIN, J. R.

Baltimore, Maryland, EEUU

Congreso; Biophysical Society 48th Annual Meeting; 2004

American Biophysical Society

Organic quaternary amines inhibit the Na,K-ATPase in a manner that is competitive with extracellular K+ activation of ion transport. To investigate the mechanism of enzyme inhibition by quaternary amines in more detail, the effects of these amines on the kinetics and steady state levels of 86Rb+ occlusion were determined in pig kidney microsomal preparations of the Na,K-ATPase. In the presence of Na+, Mg2+ and micromolar concentrations of ATP (physiologic route), para-nitrobenzyltriethylammonium ions (pNBTEA) decreased steady state levels of 86Rb+ occlusion to a degree that was inversely proportional to Rb+ concentration. By analyzing the Rb+ and pNBTEA concentration dependence of 86Rb+ occlusion, we found that the apparent K0.5 for Rb+ increased linearly with pNBTEA concentration without changing maximal 86Rb+ occlusion levels. Likewise, the pNBTEA concentration for half-maximal inhibition of 86Rb+ occlusion increased with Rb+ concentration. These results suggest that pNBTEA and Rb+ interact competitively to bind to the Na,K-ATPase. Rb+ occlusion experiments conducted without additional enzyme ligands (direct route) showed similar results. pNBTEA also had little effect on the initial rate of 86Rb+ occlusion whether or not the amine was preincubated with the enzyme. These data could be described by a model in which quaternary amines compete with Rb+ for binding to a K+ site of the Na,K-ATPase without becoming occluded. Comparing these data to the effects of pNBTEA on Na,K-pump current suggests that quaternary amines inhibit the enzyme by interacting with an extracellular K+ binding site in the enzyme´s E2P conformation. These data combined with the VM dependence of Na,K-pump current inhibition and the ability of pNBTEA to support ouabain-inhibitable transient charge movements suggest that benzyltriethylamine compounds inhibit the Na,K-ATPase by binding to extracellular K+ sites responsible for activating ion transport.