Partial cloning and characterization of Slc12a2: the gene encoding the secretory Na+-K+-2Cl- cotransporter.
The Slc12a2 gene encodes a widely expressed bumetanide-sensitive Na+-K+-2Cl- cotransporter that participates in various functions such as Cl- secretion and cell volume regulation. We isolated and characterized 75 kilobases of the murine gene encoding the cotransporter. The cotransport protein is encoded by 27 exons. Ribonuclease protection assay and primer extension demonstrated tissue-specific transcription initiation sites located within 270 base pairs upstream of the start codon. Nucleotide sequence analysis of the proximal 5'-flanking region revealed the presence of a weak TATA box, multiple Sp1/GC consensus sites, and the consensus sequence of a putative transcriptional initiator. Transfection of luciferase reporter gene constructs in mouse inner medullary collecting duct (mIMCD-3) cells confirmed the location of the minimal promoter within a 120-base pair fragment upstream of the cDNA. We also report the identification of an alternatively spliced variant of the cotransporter, expressed primarily in brain. This new spliced variant lacks exon 21, which encodes a 16-amino acid peptide located in the COOH-terminal tail of the protein. The absence of this exon causes the loss of the single protein kinase A consensus site of the cotransport protein.
Randall
J
J
Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Thorne
T
T
Delpire
E
E
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers; D017930:Genes, Reporter; D015698:Genomic Library; D007679:Kidney Medulla / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000378:metabolism; D007739:L Cells; D008156:Luciferases / Q000096:biosynthesis; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D028021:Sodium-Potassium-Chloride Symporters; D014158:Transcription, Genetic; D014162:Transfection
D002352:Carrier Proteins; D017931:DNA Primers; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D028021:Sodium-Potassium-Chloride Symporters; D008156:Luciferases