Isoform-specific interactions of Na,K-ATPase subunits are mediated via extracellular domains and carbohydrates

The functional unit of the Na,K-ATPase consists of a catalytic alpha subunit noncovalently linked with a glycoprotein subunit, beta. Using ouabain binding assays and immunoprecipitation of rodent alpha/beta complexes, we show here that all six possible isozymes between three alpha and two beta isoforms fan be formed in Xenopus oocytes. Two isoform-specific differences in alpha/beta interactions are observed: (i) alpha 1/beta 1 and alpha 2/beta 2 complexes, in contrast to alpha 1/beta 2 complexes, are stable against Triton X-100-mediated dissociation, and (ii) beta 2 subunits must carry N-glycans to combine with alpha 1 but not with alpha 2. The interacting surfaces are mainly exposed to the extracellular side because coexpression of a truncated beta 1 subunit comprising the ectodomain results in assembly with alpha 1 and alpha 2, but not with alpha 3; the beta 2 ectodomain combines with alpha 2 only, A chimera consisting of 81% and 19% of the alpha 1 N terminus and alpha 2 C terminus, respectively, behaves like alpha 2 and coprecipitates with the beta 2 ectodomain, In contrast, the reciprocal chimera does not coprecipitate with the beta 2 ectodomain. These results provide evidence for a selective interaction of Na,K-ATPase alpha and beta subunits.