Molecular identification and expression of erythroid K:Cl cotransporter in human and mouse erythroleukemic cells

A major pathway for K+ efflux in human reticulocytes and young RBCs is K:Cl cotransport (K:Cl-CT), The activity of K:Cl-CT is increased in pathologic RBCs containing hemoglobins S and C and may contribute to the abnormal dehydration state of these cells, Human K:Cl-CT (gene product KCCl) has been recently sequenced from human (hKCCl), rabbit and rat tissue by Gillen et al, (J Biol Chem 271:16237, 1996), We report here the sequence of KCCl from human and mouse erythroleukemic cells (K562 and MEL cells, respectively), The cDNA for human erythroid-KCCl is 100% identical to hKCCl and the cDNA for mouse erythroid-KCCl shares 89% identity with hKCCl, which translates to 96% identity at the amino acid level. Mammalian KCCl is strongly conserved with >95% identity between human, rabbit, rat, and mouse KCCl proteins, We did not detect any full-length mRNA transcripts of human erythroid-KCCl in circulating reticulocytes. We detected two mRNA isoforms of human erythroid-KCCl that resulted in C-terminal truncated proteins (73 amino acid and 17 amino acids, respectively), Human and mouse erythroid-KCCl differed at several consensus sites including a predicted PKC phosphorylation site at (108)threonine and a predicted CK2 phosphorylation site at (51)serine, within the predicted cytoplasmic N-terminal, that are present in human but not mouse erythroid-KCCl. Expression of MEL-KCCl mRNA increases substantially upon DMSO-induced differentiation opening the possibility that erythroid-KCCl plays a role in early erythroid maturation events, The molecular identification of erythroid-KCCl is an important step towards understanding the physiologic role mediated by this protein in young and pathologic RBCs and during erythropoiesis, as well as providing a new tool for the elucidation of pathways and signals involved in RBC volume regulation.