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 _??_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.
