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 p athologic RBCs containing hemoglobins S and C and may contribute to th e abnormal dehydration state of these cells, Human K:Cl-CT (gene produ ct KCCl) has been recently sequenced from human (hKCCl), rabbit and ra t tissue by Gillen et al, (J Biol Chem 271:16237, 1996), We report her e the sequence of KCCl from human and mouse erythroleukemic cells (K56 2 and MEL cells, respectively), The cDNA for human erythroid-KCCl is 1 00% identical to hKCCl and the cDNA for mouse erythroid-KCCl shares 89 % identity with hKCCl, which translates to 96% identity at the amino a cid level. Mammalian KCCl is strongly conserved with >95% identity bet ween human, rabbit, rat, and mouse KCCl proteins, We did not detect an y full-length mRNA transcripts of human erythroid-KCCl in circulating reticulocytes. We detected two mRNA isoforms of human erythroid-KCCl t hat resulted in C-terminal truncated proteins (73 amino acid and 17 am ino acids, respectively), Human and mouse erythroid-KCCl differed at s everal consensus sites including a predicted PKC phosphorylation site at (108)threonine and a predicted CK2 phosphorylation site at (51)seri ne, within the predicted cytoplasmic N-terminal, that are present in h uman but not mouse erythroid-KCCl. Expression of MEL-KCCl mRNA increas es substantially upon DMSO-induced differentiation opening the possibi lity that erythroid-KCCl plays a role in early erythroid maturation ev ents, The molecular identification of erythroid-KCCl is an important s tep towards understanding the physiologic role mediated by this protei n in young and pathologic RBCs and during erythropoiesis, as well as p roviding a new tool for the elucidation of pathways and signals involv ed in RBC volume regulation.