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Abstract

The β-subunits of Na,K-ATPase and H,K-ATPase have important functions in maturation and plasma membrane targeting of the catalytic α-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 β-subunits resulted in considerable shifts of the voltage-dependent E₁P/E₂P distributions toward the E₁P state as inferred from presteady-state current and voltage clamp fluorometric measurements of tetramethylrhodamine-6-maleimide-labeled 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 β-(Y39W,Y43W) mutation but much more pronounced for the corresponding H,K-ATPase β-(Y44W,Y48W) variant. Moreover in the Na,K-ATPase β-(Y39W,Y43W) mutant, the apparent rate constant for reverse binding of extracellular Na⁺ and the subsequent E₂P-E₁P 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 E₂P-E₁P conversion was accelerated by the H,K-ATPase β-(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 α-subunits, which are at interacting distance to the β-tyrosines, resulted in similar E₁ shifts, indicating their participation in stabilization of E₂. Thus, interactions between selected residues within the transmembrane regions of α- and β-subunits of P_2C-type ATPases exert an E₂-stabilizing effect, which is of particular importance for efficient H⁺ pumping by H,K-ATPase under in vivo conditions.