Role of β-subunit domains in the assembly, stable expression, intracellular routing, and functional properties of Na,K-ATPase

Abstract

The β-subunit of Na,K-ATPase (βNK) interacts with the catalytic α- subunit (αNK) in the ectodomain, the transmembrane, and the cytoplasmic domain. The functional significance of these different interactions was studied by expressing αNK in Xenopus oocytes along with N-terminally modified βNK or with chimeric βNK/βH,K-ATPase (βHK). Complete truncation of the βNK N terminus allows for cell surface-expressed, functional Na,K- pumps that exhibit, however, reduced apparent K+ and Na+ affinities as assessed by electrophysiological measurements. A mutational analysis suggests that these functional effects are not related to a direct interaction of the β N terminus with the αNK but rather that N-terminal truncation induces a conformational change in another functionally relevant β domain. Comparison of the functional properties of αNK·βNK, αNK·βHK, or αNK·βNK/βHK complexes shows that the effect of the βNK on K+ binding is mainly mediated by its ectodomain. Finally, βHK/NK containing the transmembrane domain of βHK produces stable but endoplasmic reticulum-retained αNK·β complexes, while αNK/βHK complexes can leave the ER but exhibit reduced ouabain binding capacity and transport function. Thus, interactions of both the transmembrane and the ectodomain of βNK with αNK are necessary to form correctly folded Na,K-ATPase complexes that can be targeted to the plasma membrane and/or become functionally competent. Furthermore, the β N terminus plays a role in the β-subunit's folding necessary for correct interactions with the α-subunit.