Structural and functional features of the transmembrane domain of the Na,K-ATPase beta subunit revealed by tryptophan scanning

In oligomeric P2-ATPases such as Na,K- and H,K-ATPases, beta subunits play a fundamental role in the structural and functional maturation of the catal ytic alpha subunit. In the present study we performed a tryptophan scanning analysis on the transmembrane alpha -helix of the Na,K-ATPase beta1 subuni t to investigate its role in the stabilization of the alpha subunit, the en doplasmic reticulum exit of alpha-beta complexes, and the acquisition of fu nctional properties of the Na,K-ATPase. Single or multiple tryptophan subst itutions in the beta subunits transmembrane domain had no significant effec t on the structural maturation of alpha subunits expressed in Xenopus oocyt es nor on the level of expression of functional Na,K pumps at the cell surf ace. Furthermore, tryptophan substitutions in regions of the transmembrane alpha -helix containing two GXXXG transmembrane helix interaction motifs or a cysteine residue, which can be cross-linked to transmembrane helix M8 of the alpha subunit, had no effect on the apparent K+ affinity of Na,K-ATPas e. On the other hand, substitutions by tryptophan, serine, alanine, or cyst eine, but not by phenylalanine of two highly conserved tyrosine residues, T yr(40) and Tyr(44), on another face of the transmembrane helix, perturb the transport kinetics of Na,K pumps in an additive way. These results indicat e that at least two faces of the beta subunits transmembrane helix contribu te to inter- or intrasubunit interactions and that two tyrosine residues al igned in the beta subunits transmembrane alpha -helix are determinants of i ntrinsic transport characteristics of Na,K-ATPase.