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Molecular identification and expression of erythroid K:Cl cotransporter in human and mouse erythroleukemic cells
C M Pellegrino1, A C Rybicki, S Musto
1Division of Hematology, Albert Einstein College of Medicine-Montefiore Medical Center, 111 East 210th Street, Bronx, NY, USA.
Insights
The K+Cl cotransporter (KCC1) is crucial for red blood cell (RBC) volume regulation. This study identifies and characterizes KCC1 in human and mouse erythroid cells, revealing its conservation and role in erythropoiesis.
Area of Science:
- Molecular biology
- Cell physiology
- Hematology
Background:
- K+Cl cotransport (K:Cl-CT) is a primary K+ efflux pathway in human reticulocytes and young red blood cells (RBCs).
- Elevated K:Cl-CT activity in pathologic RBCs with hemoglobins S and C may contribute to cellular dehydration.
- Human K:Cl-CT (gene product KCC1) has been previously sequenced from human, rabbit, and rat tissues.
Purpose of the Study:
- To report the sequence of KCC1 from human and mouse erythroleukemic cells.
- To investigate the conservation and potential functional differences of erythroid KCC1 between species.
- To explore the role of erythroid KCC1 in erythropoiesis and RBC volume regulation.
Main Methods:
- Sequencing of KCC1 cDNA from human (K562) and mouse (MEL) erythroleukemic cells.
- Comparison of human and mouse KCC1 sequences at cDNA and amino acid levels.
- Analysis of KCC1 mRNA transcripts in human reticulocytes and during erythroid differentiation.
Main Results:
- Human and mouse erythroid KCC1 sequences were determined, showing high amino acid identity (96%) between species.
- No full-length human erythroid KCC1 mRNA was detected in circulating reticulocytes; truncated isoforms were identified.
- Differences in predicted phosphorylation sites were observed between human and mouse erythroid KCC1.
- Mouse erythroid KCC1 mRNA expression increased during DMSO-induced differentiation.
Conclusions:
- Mammalian KCC1 is highly conserved, suggesting a fundamental role in RBC physiology.
- Truncated KCC1 isoforms and species-specific phosphorylation sites may influence KCC1 function in erythropoiesis.
- Erythroid KCC1 is a potential key player in RBC volume regulation and may be a target for understanding RBC disorders.
Abstract:
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 KCC1) has been recently sequenced from human (hKCC1), rabbit and rat tissue by Gillen et al. (J Biol Chem 271:16237, 1996). We report here the sequence of KCC1 from human and mouse erythroleukemic cells (K562 and MEL cells, respectively). The cDNA for human erythroid-KCC1 is 100% identical to hKCC1 and the cDNA for mouse erythroid-KCC1 shares 89% identity with hKCC1, which translates to 96% identity at the amino acid level. Mammalian KCC1 is strongly conserved with >95% identity between human, rabbit, rat, and mouse KCC1 proteins. We did not detect any full-length mRNA transcripts of human erythroid-KCC1 in circulating reticulocytes. We detected two mRNA isoforms of human erythroid-KCC1 that resulted in C-terminal truncated proteins (73 amino acid and 17 amino acids, respectively). Human and mouse erythroidKCC1 differed at several consensus sites including a predicted PKC phosphorylation site at 108threonine and a predicted CK2 phosphorylation site at 51serine, within the predicted cytoplasmic N-terminal, that are present in human but not mouse erythroid-KCC1. Expression of MEL-KCC1 mRNA increases substantially upon DMSO-induced differentiation opening the possibility that erythroid-KCC1 plays a role in early erythroid maturation events. The molecular identification of erythroid-KCC1 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.