Na⁺-K⁺-ATPase α-subunit containing Q905-V930 of gastric H⁺-K⁺- ATPase α preferentially assembles with H⁺-K⁺-ATPase β

Amino acids N886-A911 of the rat Na⁺-K⁺-ATPase α₃-subunit were replaced by the corresponding region (Q905-V930) of the rat gastric H⁺-K⁺- ATPase α-subunit. The chimera (NGH26) was expressed in yeast with the rat Na⁺-K⁺-ATPase β₁-subunit (rβ₁), the rat H⁺-K⁺-ATPase β-subunit (HKβ), the chimeric β-subunit NHβ₁ (containing the carboxy-terminal ectodomain of HKβ), or the chimeric β-subunit HNβ₁ (containing the carboxy-terminal ectodomain of rβ₁). Increased resistance to trypsin digestion indicated that NGH26 preferentially assembled with HKβ and NHβ₁ rather than with rβ₁ or HNβ₁. Ouabain binding also indicated that more functional complexes were assembled when NGH26 was expressed with HKβ or NHβ₁. These results suggest that the sequence Q905-V930 interacts with the HKβ-subunit on the extracellular side of the cell membrane. The NGH26+HKβ complex is less stable than α₃+HKβ when heated and also has a lower binding affinity for ouabain [dissociation constant (K(d)) = 63 nM] compared with α₃+rβ₁ or α₃+HKβ (K(d)= 5-10 nM). In contrast, the NGH26+NHβ₁ complex is thermally as stable as α₃+rβ₁ complexes, and its ouabain binding affinity (K(d) = 10 nM) is the same as the wild type. These results indicate that the amino acids Q905-V930 of the rat gastric H⁺-K⁺-ATPase α- subunit preferentially associate with the extracellular domain of H⁺-K⁺- ATPase β-subunit to form functional pump complexes and that the cytoplasmic and/or transmembrane region of the β-subunit influences the stability of the αβ complexes.