Intracellular Localization of Na,K-ATPase α2 Subunit Mutants

Abstract

The Na,K-ATPase is an essential plasma membrane transporter of mammalian cells composed of two subunits, α and β, of which there are several isoforms. We investigated the effect of a substitution, S364P, on the subcellular localization and enzymatic activity of the wild-type α2 and α2L111R,N122D (α2RD) subunits. The substitutions, L111R and N122D, lower the affinity of the α2 subunit for the inhibitor ouabain roughly one thousand-fold (E. A. Jewell and J.B. Lingrel,J. Biol. Chem.266, 16925–16930, 1991) and were introduced into the α2 subunit to distinguish its enzymatic activity from that of the endogenous α1 subunit of COS-7 cells. The S364P substitution is located in the ATP binding site, only five residues from the aspartyl residue which is phosphorylated during the catalytic cycle of the Na,K-ATPase. This substitution dramatically decreases the amount of enzymatic activity associated with expression of the α2RD subunit. Despite the fact that S364P substitution does not block association of the α2RD subunit with the endogenous β1 subunit, it prevents the α2 and α2RD subunits from accumulating in the plasma membrane and results in their localization in the endoplasmic reticulum.