Abstract
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.
Highlights
The H؉-K؉-ATPase of renal collecting duct mediates K؉ conservation during chronic hypokalemia
The HϩKϩ-ATPase ␣2 cDNA was first cloned from rat distal colon (17), where it is abundantly expressed, and lower levels of H؉-K؉-ATPase ␣2 (HK␣2) mRNA were reported in proximal colon (17), uterus (17), and kidney (5– 8)
CDNA Cloning and Structural Analysis of a Truncated Nterminal Variant of the Hϩ-Kϩ-ATPase ␣2 Subunit—The anchor-ligated cDNAs synthesized from rat kidney mRNA were subjected to 5Ј-RACE using adapter primer 2 and HK␣2-specific primer P1 from exon 2 (Fig. 1A)
Summary
HK␣2, Hϩ-Kϩ-ATPase ␣2 subunit, termed the colonic Hϩ-Kϩ-ATPase ␣ subunit; HK␣1, Hϩ-Kϩ-ATPase ␣1 subunit, termed the gastric Hϩ-Kϩ-ATPase ␣ subunit; HKg, gastric Hϩ-Kϩ-ATPase  subunit; OMCD, outer medullary collecting duct; RACE, rapid amplification of cDNA ends; C/EBP, CCAAT enhancer binding protein; NF-B, nuclear factor kappa B; HNF-4, hepatocyte nuclear factor-4; IRF-1, interferon regulatory factor-1; GR, glucocorticoid receptor; UTR, untranslated region; u, upstream; ORF, open reading frame; Sch 28080, 2-methyl,8-(phenylmethoxy)imidazo(1,2-a)pyridine 3-acetonitrile; kb, kilobase pair(s); bp, base pair(s); PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Recent work demonstrating disparate effects of adrenalectomy, dexamethasone treatment (5), and dietary Naϩ depletion (8) on HK␣2 abundance in the rat outer medulla and distal colon indicated that cell type-specific regulatory mechanisms govern HK␣2 gene expression in these tissues Since both transcriptional and translational control mechanisms, as well as alternative mRNA splicing, can lead to regulated, tissue-specific gene expression, we hypothesized that these mechanisms might operate to confer structural and/or regulatory diversity to the HK␣2 subunit gene. We report here that distinct transcription initiation sites in the rat HK␣2 gene and alternative mRNA splicing, combined regulatory mechanisms not known to be utilized by other members of the Xϩ-Kϩ-ATPase ␣ subunit family, direct the synthesis of two N-terminal HK␣2 variants that are expressed principally, if not exclusively, in the kidney and colon and that appear to respond coordinately in kidney to chronic Kϩ deprivation
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