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.