Domain swapping between Na,K- and H,K-ATPase identifies regions that specify Na,K-ATPase activity.
We have used expression of chimeras between the structurally related Na,K- and H,K-ATPase alpha subunits to localize regions that determine Na,K-ATPase activity. Segments of the rat Na,K-ATPase alpha1 subunit were replaced by the corresponding portions of the rat gastric H,K-ATPase alpha subunit, and the constructs were transfected into ouabain-sensitive human HEK 293 cells. Using the ability to transfer ouabain resistance as a measure of sodium pump activity, we identified segments within the sodium pump that could be replaced with proton pump sequences without the loss of biological activity. These functionally interchangeable segments encompassed approximately 75% of the amino acid differences between the two transporters. Segments that could not be exchanged mapped to three discrete regions. One region spans residues 63-117 and includes the first transmembrane (TM) segment and a portion of the amino-terminal cytoplasmic domain. The second, from residue 320 to residue 413, encompasses TM 4 and a portion of the third cytoplasmic domain, while the third region (encompassing residues 735-861 and 898-953) includes several TM domains in the carboxyl-terminal portion of the ATPase. Our results suggest that functional differences between Na,K- and H,K-ATPase, including differences in ion transport specificity, are likely to reside within these noninterchangeable segments.
Canfield
V A
VA
Department of Pharmacology, Penn State College of Medicine, Milton S. Hershey Medical Center, Pennsylvania 17033, USA.
Levenson
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation / Q000235:genetics; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016623:Ion Pumps / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007668:Kidney; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism
D016623:Ion Pumps; D011993:Recombinant Fusion Proteins; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase