Molecular Identification and Expression of Erythroid K:Cl Cotransporter in Human and Mouse Erythroleukemic Cells

Pellegrino, C.; Rybicki, Anne C.; Musto, Sylvia; Nagel, Ronald L.; Schwartz, Robert S.

doi:10.1006/bcmd.1998.0168

Cited by 70 publications

(44 citation statements)

References 9 publications

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“…The widespread expression pattern of KCC1 overlaps with that of KCC3, including in rbc (9)(10)(11). Immunoblot analysis of WT rbc ghost membranes demonstrated the presence of both KCC1 and KCC3b, but not of KCC3a, KCC2, and KCC4 ( Figure 1E).…”

Section: Resultsmentioning

confidence: 89%

“…Early Northern blot analysis studies revealed that KCC1 transcripts are ubiquitously expressed, leading investigators to suggest a fundamental role in cell volume regulation (29), in particular in rbc (9). To address the physiological roles of Kcc1, we disrupted the Kcc1 gene in mice.…”

Section: Discussionmentioning

confidence: 99%

“…Specific roles of KCC1 have remained unknown in the absence of a Kcc1 -/-mouse or a KCC1-associated human disease. The ubiquitous expression of KCC1 suggested a general role in cell volume regulation (9)(10)(11). In addition to KCC1, KCC3 has also been detected in rbc (11).…”

Section: Introductionmentioning

confidence: 99%

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Disruption of erythroid K-Cl cotransporters alters erythrocyte volume and partially rescues erythrocyte dehydration in SAD mice

Rust

Alper²,

Rudhard³

et al. 2007

J. Clin. Invest.

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K-Cl cotransport activity in rbc is a major determinant of rbc volume and density. Pathologic activation of erythroid K-Cl cotransport activity in sickle cell disease contributes to rbc dehydration and cell sickling. To address the roles of individual K-Cl cotransporter isoforms in rbc volume homeostasis, we disrupted the Kcc1 and Kcc3 genes in mice. As rbc K-Cl cotransport activity was undiminished in Kcc1 -/-mice, decreased in Kcc3 -/-mice, and almost completely abolished in mice lacking both isoforms, we conclude that K-Cl cotransport activity of mouse rbc is mediated largely by KCC3. Whereas rbc of either Kcc1 -/-or Kcc3 -/-mice were of normal density, rbc of Kcc1 -/-Kcc3 -/-mice exhibited defective volume regulation, including increased mean corpuscular volume, decreased density, and increased susceptibility to osmotic lysis. K-Cl cotransport activity was increased in rbc of SAD mice, which are transgenic for a hypersickling human hemoglobin S variant. Kcc1 -/-Kcc3 -/-SAD rbc lacked nearly all K-Cl cotransport activity and exhibited normalized values of mean corpuscular volume, corpuscular hemoglobin concentration mean, and K + content. Although disruption of K-Cl cotransport rescued the dehydration phenotype of most SAD rbc, the proportion of the densest red blood cell population remained unaffected.

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Section: Resultsmentioning

confidence: 89%

Section: Discussionmentioning

confidence: 99%

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Disruption of erythroid K-Cl cotransporters alters erythrocyte volume and partially rescues erythrocyte dehydration in SAD mice

Rust

Alper²,

Rudhard³

et al. 2007

J. Clin. Invest.

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“…A potential molecular basis for transporter regulation differences between species has been identified in the case of the K-Cl cotransporter. Human and mouse K-Cl cotransporters share 96% amino acid conservation, but important consensus binding sites for PKC and CKII phosphorylation that are present in human cDNA are missing in the mouse (51).…”

Section: Discussionmentioning

confidence: 99%

Mild spherocytosis and altered red cell ion transport in protein 4.2–null mice

Peters¹,

Jindel²,

Gwynn³

et al. 1999

J. Clin. Invest.

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IntroductionProtein 4.2 is a 72-kDa component of the red blood cell (RBC) membrane skeleton (200,000 copies per RBC) (1). An intact membrane skeleton attached to the lipid bilayer by vertical interactions between spectrin, ankyrin, protein 4.2, and band 3 (anion exchanger 1 [AE1]) is required for normal RBC membrane integrity. Defects in all of these components have been identified in hereditary spherocytosis (HS) (2). In humans, protein 4.2 defects are most common in people of Japanese descent and give rise to pleiotropic RBC morphological changes and hemolytic anemias of varying severity (3).In RBCs, protein 4.2 binds to the cytoplasmic domain of band 3 (4). In solution, protein 4.2 binds directly to spectrin, ankyrin, and protein 4.1, and promotes binding of spectrin to ankyrin-depleted inside-out vesicles (4, 5). The significance of these interactions in vivo is unknown. However, mice totally deficient in band 3 are also completely protein 4.2 deficient, suggesting that band 3 is critical for stable incorporation of protein 4.2 into the membrane skeleton (6).Hereditary spherocytes are characterized by increased Na + and decreased K + content compared with normal RBCs (2). Studies in mice and humans suggest that the cation alterations are the result of increased passive RBC membrane permeability and that the increased passive flux reflects the overall loss of membrane skeleton integrity, not the specific protein defect (7,8). Protein 4.2 has been proposed to negatively regulate band 3-mediated RBC anion transport (9), but its role in regulating RBC cation transport and intracellular cation content is unknown.We targeted the erythrocyte protein 4.2 gene (Epb4.2) in embryonic stem (ES) cells to create a null mutation (4.2 -/-) in mice. Deficiency of protein 4.2 in mice results in mild HS. Normal amounts of spectrin and ankyrin are assembled onto the membrane, and the membrane skeleton architecture is intact. The band 3 content of the membrane is decreased. Contrary to previous reports, band 3-mediated anion transport in not increased in 4.2 -/-RBCs, but is decreased. Protein 4.2-deficient RBCs show significant changes in RBC cation transporter activities. These changes are in marked contrast to those obtained in spectrin-and ankyrin-deficient mouse and human red cells (7,8), suggesting that specific membrane skeleton defects differentially influence cation transporter activities. Our results show that protein 4.2 is important in the maintenance of normal surface area in RBCs and is required for normal RBC cation transport. resulting in dehydration. The passive Na + permeability and the activities of the Na-K-2Cl and K-Cl cotransporters, the Na/H exchanger, and the Gardos channel in 4.2 -/-RBCs are significantly increased. Protein 4.2 -/-RBCs demonstrate an abnormal regulation of cation transport by cell volume. Cell shrinkage induces a greater activation of Na/H exchange and Na-K-2Cl cotransport in 4.2 -/-RBCs compared with controls. The increased passive Na + permeability of 4.2 -/-RBCs is also dependent on c...

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“…K-Cl cotransporters also serve to regulate the cellular electrochemical equilibrium potential for Cl Ϫ and can regulate [K ϩ ] of the interstitial space, especially in the nervous system. KCC1 cDNAs have been cloned from rabbits (5), rats (5), humans (5), pigs (6), and mice (7,8), with gene structures reported for humans (6), mice (9), and Caenorhabditis elegans (6). KCC2 cDNA has been cloned from rat (10).…”

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confidence: 99%