title: Molecular identification of K-CL cotransporter in dog erythroid progenitor cells.
abstract: KCC1 cDNA was cloned in dog erythroblasts that had differentiated from peripheral mononuclear cells. The size of the cDNA was 3,258 bp, the same as in pigs, but 3 bp longer than in humans and rodents. The dog KCC1 cDNA encodes for 1,086 amino acid residues with a calculated molecular mass of 120 kDa. The 560 bp cDNA fragment from position 679 to 1,238 in the full length cDNA from the dog erythroblasts was 100% identical to that in the kidney. Hydropathy analysis showed that the structure of dog KCC1 was similar to in other species; 12 trans membrane domains, four glycosylation sites in loop 5, and 17 consensus phosphorylation sites in the cytosol. However, there were variations in dog KCC1 compared to in other species; there was one CK2 phosphorylation site that was found only in dog KCC1. There were also substitutions of amino acids that affect pH sensitivity (His) and change acidic/basic residues or charged residues. In HEK 293 cells transfected with dog KCC1 cDNA (HEK-dKCC1), the Rb influx, which was ouabain-resistant, Cl-dependent, N-ethyl maleimide (NEM)- stimulative and Na-independent, was measured as for K-Cl cotransport, and the influx was found to be increased approximately 3 fold in HEK-dKCC1 compared to in the control. This ouabain-resistant Cl-dependent Rb influx was also volume-sensitive in hyposmotic medium, and the volume-sensitive component was inhibited by furosemide. Thus, the KCC1 cDNA cloned in dog erythroblasts encodes a volume-sensitive K-Cl cotransporter.
authors.affiliation: Institute of Biosciences, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 229-8501.
authors.forename: Hideharu
authors.initials: H
authors.lastname: Ochiai
authors.forename: Kazunari
authors.initials: K
authors.lastname: Higa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Fujise
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004285:Dogs; D015672:Erythroid Precursor Cells / Q000378:metabolism*; D005665:Furosemide / Q000494:pharmacology; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010042:Ouabain / Q000494:pharmacology; D012413:Rubidium / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D027981:Symporters / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000096922:K Cl- Cotransporters
chemical_list: D005665:Furosemide; D010042:Ouabain; D012413:Rubidium; D027981:Symporters; D000096922:K Cl- Cotransporters