Na+-K+-ATPase alpha-subunit containing Q905-V930 of gastric H+-K+-ATPase alpha preferentially assembles with H+-K+-ATPase beta

Amino acids N886-A911 of the rat Na+-K+-ATPase alpha(3)-subunit were replaced by the corresponding region (Q905-V930) of the rat gastric H+-K+-ATPase alpha-subunit. The chimera (NCH26) was expressed in yeast with the rat Na+-K+-ATPase beta(1)-subunit (r beta(1)), the rat H+-K+-ATPase beta-subunit (HK beta), the chimeric beta-subunit NH beta(1) (containing the carboxy-terminal ectodomain of HK beta), or the chimeric beta-subunit HN beta(1) (containing the carboxy-terminal ectodomain of r beta(1)). Increased resistance to trypsin digestion indicated that NGH26 preferentially assembled with HK beta and NH beta(1) rather than with r beta(1) or HN beta(1). Ouabain binding also indicated that more functional complexes were assembled when NGH26 was expressed with HK beta or NH beta(1). These results suggest that the sequence Q905-V930 interacts with the HK beta-subunit on the extracellular side of the cell membrane. The NGH26+HK beta complex is less stable than alpha(3)+HK beta when heated and also has a lower binding affinity for ouabain [dissociation constant (K-d) = 63 nM] compared with alpha(3)+r beta(1) or alpha(3)+HK beta (K-d = 5-10 nM). In contrast, the NGH26+NH beta(1) complex is thermally as stable as alpha(3)+r beta(1) 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 alpha-subunit preferentially associate with the extracellular domain of H+-K+-ATPase beta-subunit to form functional pump complexes and that the cytoplasmic and/or transmembrane region of the beta-subunit influences the stability of the alpha beta complexes.