Differential regulation of renal Na,K-ATPase by splice variants of the gamma subunit.
Sodium and potassium-exchanging adenosine triphosphatase (Na,K-ATPase) in the kidney is associated with the gamma subunit (gamma, FXYD2), a single-span membrane protein that modulates ATPase properties. Rat and human gamma occur in two splice variants, gamma(a) and gamma(b), with different N termini. Here we investigated their structural heterogeneity and functional effects on Na,K-ATPase properties. Both forms were post-translationally modified during in vitro translation with microsomes, indicating that there are four possible forms of gamma. Site-directed mutagenesis revealed Thr(2) and Ser(5) as potential sites for post-translational modification. Similar modification can occur in cells, with consequences for Na,K-ATPase properties. We showed previously that stable transfection of gamma(a) into NRK-52E cells resulted in reduction of apparent affinities for Na(+) and K(+). Individual clones differed in gamma post-translational modification, however, and the effect on Na(+) affinity was absent in clones with full modification. Here, transfection of gamma(b) also resulted in clones with or without post-translational modification. Both groups showed a reduction in Na(+) affinity, but modification was required for the effect on K(+) affinity. There were minor increases in ATP affinity. The physiological importance of the reduction in Na(+) affinity was shown by the slower growth of gamma(a), gamma(b), and gamma(b') transfectants in culture. The differential influence of the four structural variants of gamma on affinities of the Na,K-ATPase for Na(+) and K(+), together with our previous finding of different distributions of gamma(a) and gamma(b) along the rat nephron, suggests a highly specific mode of regulation of sodium pump properties in kidney.
Arystarkhova
Elena
E
Laboratory of Membrane Biology, Neuroscience Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.
Donnet
Claudia
C
Asinovski
Natalya K
NK
Sweadner
Kathleen J
KJ
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic; D007477:Ions; D007668:Kidney / Q000201:enzymology / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D015233:Models, Statistical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009399:Nephrons / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D051381:Rats; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D007477:Ions; D008024:Ligands; D000255:Adenosine Triphosphate; D012964:Sodium; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium