26 Amino acids of an extracellular domain of the Na,K-ATPase α-subunit are sufficient for assembly with the Na,K-ATPase β-subunit

Chimeric cDNAs encoding a sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA1) and regions of the Na,K-ATPase α-subunit were constructed to seek the minimal region of the α-subunit sufficient for assembly with the Na,K- ATPase β-subunit. cDNAs encoding a chimera and the chicken β-subunit were coexpressed in mammalian cells and assembly was assayed by immune precipitation of the chimeric subunit with a monoclonal antibody to the chicken β-subunit. A chimera containing 26 amino acyl residues of the Na,K- ATPase α1-subunit (NDVEDSYGQQWTFEQRKIVEFTCHTA) (Asn⁸⁹⁴ to Ala⁹¹⁹) that replaced the corresponding avian SERCA1 Ca-ATPase amino acyl residues (Thr⁸⁷¹ to Thr⁸⁹⁸) was able to assemble with the chicken β-subunit. This α-subunit region is predicted to be extracellular, located between membrane-spanning domains 7 and 8 (H7-H8). Chimeras that assembled with full- length β-subunit also assembled with a β-subunit chimera that retained only the ectodomain of the chicken β1-subunit. These results suggest that the Na,K-ATPase α-subunit has the same topology in the membrane as the sarcoplasmic reticulum Ca-ATPase, probably with 10 membrane-spanning domains, and that the aminoacyl residues between membrane domains H7 and H8 are involved in assembly with the β-subunit in the extracellular/lumenal space.