Functional Significance of E2 State Stabilization by Specific α/β-Subunit Interactions of Na,K- and H,K-ATPase (original) (raw)

2008, Journal of Biological Chemistry

The ␤-subunits of Na,K-ATPase and H,K-ATPase have important functions in maturation and plasma membrane targeting of the catalytic ␣-subunit but also modulate the transport activity of the holoenzymes. In this study, we show that tryptophan replacement of two highly conserved tyrosines in the transmembrane domain of both Na,K-and gastric H,K-ATPase ␤-subunits resulted in considerable shifts of the voltagedependent E 1 P/E 2 P distributions toward the E 1 P state as inferred from presteady-state current and voltage clamp fluorometric measurements of tetramethylrhodamine-6-maleimidelabeled ATPases. The shifts in conformational equilibria were accompanied by significant decreases in the apparent affinities for extracellular K ؉ that were moderate for the Na,K-ATPase ␤-(Y39W,Y43W) mutation but much more pronounced for the corresponding H,K-ATPase ␤-(Y44W,Y48W) variant. Moreover in the Na,K-ATPase ␤-(Y39W,Y43W) mutant, the apparent rate constant for reverse binding of extracellular Na ؉ and the subsequent E 2 P-E 1 P conversion, as determined from transient current kinetics, was significantly accelerated, resulting in enhanced Na ؉ competition for extracellular K ؉ binding especially at extremely negative potentials. Analogously the reverse binding of extracellular protons and subsequent E 2 P-E 1 P conversion was accelerated by the H,K-ATPase ␤-(Y44W,Y48W) mutation, and H ؉ secretion was strongly impaired. Remarkably tryptophan replacements of residues in the M7 segment of Na,K-and H,K-ATPase ␣-subunits, which are at interacting distance to the ␤-tyrosines, resulted in similar E 1 shifts, indicating their participation in stabilization of E 2. Thus, interactions between selected residues within the transmembrane regions of ␣and ␤-subunits of P 2C-type ATPases exert an E 2-stabilizing effect, which is of particular importance for efficient H ؉ pumping by H,K-ATPase under in vivo conditions. * This work was supported by the Max-Planck-Gesellschaft zur Fö rderung der Wissenschaften e.V. and the Deutsche Forschungsgemeinschaft (Grants SFB 472 and SFB 740 and Cluster of Excellence "Unifying Concepts in Catalysis").