Change in Expressional Profile of KCl Cotransporter Genes during Human Erythroid Differentiation.
Authors: Pan Dao , Kalfa Theodosia A. , Wang Daren , Risinger Mary , Crable Scott , Ciraolo Peter , Franco Robert S. , Joiner Clinton H. 
Published: April 2020

Abstract
Maintenance of cell volume by regulated cation transport is a fundamental cellular process. The KCl cotransporter (KCC) contributes to red blood cell (RBC) volume regulation, especially in reticulocytes. Erythroid K-Cl cotransport activity is increased in sickle cells (SS RBC) and contribute to SS RBC dehydration, which potentiates sickling. Three cation cotransporter genes, SLC12A4 (KCC1), SLC12A6 (KCC3) and SLC12A7 (KCC4), and several splicing variants, mediate KCC activity in non-neuronal tissues. To determine which KCC isoform(s) predominates in human RBC we examined the quantitative expression patterns of KCC isoforms during erythroid differentiation. We developed a set of real-time RT-QPCR assays specific for KCC1, KCC1b, KCC3a, KCC3b or KCC4, over a 7-log quantitation range and sensitivity of 10 copies per reaction using multiplex amplification of GAPDH as internal controls. In human reticulocytes isolated by magnetic separation using anti-transferrin receptor coated beads, KCC3a mRNA levels were consistently the highest (4–24 fold of KCC1), while KCC4 levels varied from 1 to 7-fold of KCC1 levels (n=8). Message levels for KCC3b were relatively low (20–80% of KCC1), and for KCC1b were negligible (1–2% of KCC1). Substantial variability in the relative levels of KCC1, KCC3a, and KCC4 mRNA was observed among individual samples, but no consistent difference was apparent comparing sickle and normal reticulocytes. Western blot analysis of sickle and normal RBC ghost membranes confirmed the presence of KCC1, KCC3 and KCC4 at the protein level. To evaluate cells at various erythroid differentiation stages, human CD34+ cells were cultured under conditions favoring erythroid differentiation for 26 days. During early in vitro differentiation, KCC1 was the main mRNA species, followed by KCC4, with similar levels of KCC3a and KCC3b. RNA levels for KCC3a and KCC4 increased during maturation and became the most abundant at later stages. KCC1b mRNA remained low, and KCC3b levels decreased during erythroid development. To further define this temporal sequence of KCC expression, cells cultured for 10–17 days were sorted by FACS into four subpopulations (I to IV), characterized by immunostaining for relative expression of CD71 and glycophorin A, with enrichment of pronormoblasts
basophilic normoblasts polychromatophilic normoblasts or orthrochromatic normoblasts and reticulocytes KCC1, KCC3a, KCC3b, and KCC4 were expressed in these populations, with KCC1 as the main KCC species in early precursors (I), KCC3b decreasing &gt;80% during maturation, KCC4 and KCC3a becoming the most abundant in the most differentiated subpopulation (IV).
In summary, we identified KCC3a as the predominant KCC isoform in human reticulocytes, followed by KCC4 and KCC1, consistent with the presence of KCC1, KCC3 and KCC4 proteins in RBC membranes. The expression of KCC3a and KCC4 increased during erythriod differentiation in vitro. Variations in relative expression of KCC species are a potential source of inter-individual differences in KCC function for volume regulation. These results also provide the foundation for the possibility of improving SS RBC hydration by reducing gene expression of the major KCC isoforms in RBC.