Valine 904, Tyrosine 898, and Cysteine 908 in Na,K-ATPase α Subunits Are Important for Assembly with β Subunits

A 26-amino acid sequence in an extracellular loop of the Na,K-ATPase α subunit between membrane-spanning segments 7 and 8 has been shown to bind to the β subunit of Na,K-ATPase and to promote αβ assembly (Lemas, M. V., Hamrick, M., Takeyasu, K., and Fambrough, D. M. (1994) J. Biol. Chem. 269, 8255–8259) When this 26-amino acid sequence of the rat Na,K-ATPase α3 subunit was replaced by the corresponding sequence of the rat gastric H,K-ATPase α subunit, the chimeric α subunit assembled preferentially with the rat gastric H,K-ATPase β subunit (Wang, S.-G., Eakle, K. A., Levenson, R., and Farley, R. A. (1997)Am. J. Physiol. 272, C923–C930). In the present study, these 26 amino acids (Asn886–Ala911) of rat Na,K-ATPase α3 were replaced by the corresponding amino acids Asn908–Ala933 of rat distal colon H,K-ATPase. Site-directed mutagenesis of the chimeric α subunits and Na,K-ATPase α3 showed that Val904, Tyr898, and Cys908 in the Na,K-ATPase α3 subunit are key residues in αβ subunit interactions. The V904Q mutation in Na,K-ATPase α3 reduced the Bmax for ouabain binding and the ATPase activity of α3β1 complexes by ∼95%, and Y898R reduced theBmax and ATPase activity by ∼60%. The complementary mutations Q904V and R898Y increased the amount of ouabain bound by yeast membranes expressing the chimera with the colon H,K-ATPase sequence. The amount of ouabain bound by complexes assembled between Na,K-ATPase α3 containing the Y898R,C908G mutations and gastric H,K-ATPase β was less than 10% of wild type Na,K-ATPase α3 expressed with the same β subunit. The R898Y,G908C mutations in the chimeric α subunits also increased ouabain binding.