Structural and Functional Features of the Transmembrane Domain of the Na,K-ATPase β Subunit Revealed by Tryptophan Scanning

Abstract

In oligomeric P2-ATPases such as Na,K- and H,K-ATPases, β subunits play a fundamental role in the structural and functional maturation of the catalytic α subunit. In the present study we performed a tryptophan scanning analysis on the transmembrane α-helix of the Na,K-ATPase β1 subunit to investigate its role in the stabilization of the α subunit, the endoplasmic reticulum exit of α-β complexes, and the acquisition of functional properties of the Na,K-ATPase. Single or multiple tryptophan substitutions in the β subunits transmembrane domain had no significant effect on the structural maturation of α subunits expressed in Xenopus oocytes nor on the level of expression of functional Na,K pumps at the cell surface. Furthermore, tryptophan substitutions in regions of the transmembrane α-helix containing two GXXXG transmembrane helix interaction motifs or a cysteine residue, which can be cross-linked to transmembrane helix M8 of the α subunit, had no effect on the apparent K+ affinity of Na,K-ATPase. On the other hand, substitutions by tryptophan, serine, alanine, or cysteine, but not by phenylalanine of two highly conserved tyrosine residues, Tyr40 and Tyr44, on another face of the transmembrane helix, perturb the transport kinetics of Na,K pumps in an additive way. These results indicate that at least two faces of the β subunits transmembrane helix contribute to inter- or intrasubunit interactions and that two tyrosine residues aligned in the β subunits transmembrane α-helix are determinants of intrinsic transport characteristics of Na,K-ATPase.