A novel N-terminal splice variant of the rat H+-K+-ATPase alpha2 subunit. Cloning, functional expression, and renal adaptive response to chronic hypokalemia.
The H+-K+-ATPase of renal collecting duct mediates K+ conservation during chronic hypokalemia. K+ deprivation promotes H+-K+-ATPase alpha2 (HKalpha2) gene expression in the medullary collecting duct, the principal site of active K+ reabsorption, suggesting that this isozyme contributes to renal K+ reclamation. We report here that alternative transcriptional initiation and mRNA splicing give rise to distinct N-terminal variants of the HKalpha2 subunit. Sequence analysis and in vitro translation revealed that HKalpha2a corresponds to the known HKalpha2 cDNA (Crowson, M. S., and Shull, G. E. (1992) J. Biol. Chem. 267, 13740-13748), whereas HKalpha2b represents a novel variant truncated by 108 amino acids at its N terminus. HKalpha2b mRNA contains a complex 5'-untranslated region with eight upstream open reading frames, features implicated in translational regulation of other genes. Heterologous expression of HKalpha2b with and without the gastric H+-K+-ATPase beta subunit in HEK 293 cells indicated that this variant encodes a K+ uptake mechanism that is relatively Sch 28080-resistant, partially sensitive to ouabain, and appears to require coexpression with the gastric H+-K+-ATPase beta subunit for optimal functional activity. Northern analysis demonstrated that both subtypes (HKalpha2b > HKalpha2a) are expressed abundantly in distal colon and modestly in proximal colon and kidney. Moreover, the abundance of the two mRNAs increases coordinately among the renal zones, but not in colon, with chronic K+ deprivation. These results demonstrate the potential for complex control of HKalpha2 gene expression by transcriptional and posttranscriptional mechanisms not recognized in other members of the Na+-K+-ATPase/H+-K+-ATPase family.
Kone
B C
BC
Department of Internal Medicine, The University of Texas Medical School, Houston, Texas 77030, USA. bkone@heart.med.uth.tmc.edu
Higham
S C
SC
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport / Q000187:drug effects; D003001:Cloning, Molecular; D003106:Colon / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005796:Genes; D014644:Genetic Variation; D017506:H(+)-K(+)-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D007668:Kidney / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D011188:Potassium / Q000378:metabolism*; D011191:Potassium Deficiency; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D014018:Tissue Distribution; D014158:Transcription, Genetic
D018076:DNA, Complementary; D007093:Imidazoles; D007527:Isoenzymes; C035235:Sch 28080; D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium