Osmotic Stress and Viscous Retardation of the Na,K-ATPase Ion Pump

Abstract

The transport function of the Na pump (Na,K-ATPase) in cellular ion homeostasis involves both nucleotide binding reactions in the cytoplasm and alternating aqueous exposure of inward- and outward-facing ion binding sites. An osmotically active, nonpenetrating polymer (poly(ethyleneglycol); PEG) and a modifier of the aqueous viscosity (glycerol) were used to probe the overall and partial enzymatic reactions of membranous Na,K-ATPase from shark salt glands. Both inhibit the steady-state Na,K-ATPase as well as Na-ATPase activity, whereas the K+-dependent phosphatase activity is little affected by up to 50% of either. Both Na,K-ATPase and Na-ATPase activities are inversely proportional to the viscosity of glycerol solutions in which the membranes are suspended, in accordance with Kramers’ theory for strong coupling of fluctuations at the active site to solvent mobility in the aqueous environment. PEG decreases the affinity for Tl+ (a congener for K+), whereas glycerol increases that for the nucleotides ATP and ADP in the presence of NaCl but has little effect on the affinity for Tl+. From the dependence on osmotic stress induced by PEG, the aqueous activation volume for the Na,K-ATPase reaction is estimated to be ∼5–6nm3 (i.e., ∼180 water molecules), approximately half this for Na-ATPase, and essentially zero for p-nitrophenol phosphatase. The change in aqueous hydrated volume associated with the binding of Tl+ is in the region of 9nm3. Analysis of 15 crystal structures of the homologous Ca-ATPase reveals an increase in PEG-inaccessible water space of ∼22nm3 between the E1-nucleotide bound forms and the E2-thapsigargin forms, showing that the experimental activation volumes for Na,K-ATPase are of a magnitude comparable to the overall change in hydration between the major E1 and E2 conformations of the Ca-ATPase.