Stimulation of Human Erythrocyte K-Cl Cotransport and Protein Phosphatase Type 2A by n-Ethylmaleimide: Role of Intracellular Mg ++

Bize, Isabel; Güvenç, Birol; Buchbinder, Guido; Brugnara, Carlo

doi:10.1007/s002320001109

Cited by 54 publications

(37 citation statements)

References 48 publications

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“…Okadaic acid is known to inhibit protein phosphatase 2A at low concentrations, but at 100 nM this agent interferes with other phosphatases as well. 14 The use of calyculin A, a potent inhibitor of protein phosphatase 1, produced results similar to those obtained with okadaic acid (Figure 5A).…”

Section: Effect Of Phosphorylation/dephosphorylation Events On the Acsupporting

confidence: 64%

Abnormal regulation of Mg2+ transport via Na/Mg exchanger in sickle erythrocytes

Rivera

Ferreira

Bertoni

et al. 2005

Blood

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Erythrocyte magnesium (Mg 2؉ ) deficiency has been demonstrated in sickle cell disease to contribute to erythrocyte dehydration, K loss, and thus sickling. No studies have assessed the functional properties of the Na/Mg exchanger in sickle cell disease. Using Mg 2؉ -loaded erythrocytes, we measured Mg 2؉ efflux induced by extracellular Na ؉ . We estimated that the Na/Mg exchanger had higher maximal velocity, higher affinity for Na ؉ , and lower cooperativity for Mg 2؉ in sickle than in normal erythrocytes. The activity of the

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Section: Effect Of Phosphorylation/dephosphorylation Events On the Acsupporting

confidence: 64%

Abnormal regulation of Mg2+ transport via Na/Mg exchanger in sickle erythrocytes

Rivera

Ferreira

Bertoni

et al. 2005

Blood

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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: 90%

“…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%

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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“…KCC activity is regulated by phosphatase/kinase equilibrium, and activation involves net dephosphorylation of the transporter (or other regulatory molecules) via a serine/threonine (S/T) phosphatase. 16,[26][27][28][29][30][31] The S/T kinase which keeps the transporter phosphorylated and inhibited has not been identified. Swelling (as well as other stimuli) appears to activate KCC by inhibiting this regulatory kinase, presumably because of a change in macromolecular crowding resulting from dilution of hemoglobin.…”

Section: Discussionmentioning

confidence: 99%

Urea stimulation of KCl cotransport induces abnormal volume reduction in sickle reticulocytes

Joiner

Rettig

Jiang

et al. 2006

Blood

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KCl cotransport (KCC) activity contributes to pathologic dehydration in sickle (SS) red blood cells (RBCs). KCC activation by urea was measured in SS and IntroductionThe KCl cotransporter (KCC) mediates electroneutral coupled transport of K ϩ and Cl Ϫ in a variety of cell types. 1,2 KCC activation results in net efflux of KCl from cells with high potassium content, with accompanying water loss and volume reduction. KCC is active in reticulocytes and diminishes with red cell maturation. 3,4 It is thought to function physiologically to reduce red blood cell (RBC) volume and establish the high cellular hemoglobin concentration (CHC) characteristic of mature cells. KCC is activated in vitro by cell swelling, acid pH, sulfhydryl oxidation/alkylation, and exposure to urea, but the relative importance of these stimuli in vivo is unknown.In RBCs containing sickle hemoglobin (Hb S), KCC activity is high, in part because of the high percentage of reticulocytes. 4 However, Hb S or Hb C in trait RBCs (AS or AC) with normal mean cell age, or after incorporation into normal RBC ghosts, appears to increase KCC activity. 5,6 Expression of Hb S or C in transgenic mice also increases red cell KCC activity. 7 Recently, we demonstrated an abnormal response to acid pH in SS RBCs that was partially corrected by incubation with the sulfhydryl reducing agent, dithiothreitol. 8 These findings suggested that regulation of KCC is abnormal in SS RBCs.As a volume regulator, KCC activity is responsive to CHC. 9,10 KCC is activated on cell swelling, and, as KCl is lost, cell volume falls, cellular hemoglobin concentration increases, and transport activity diminishes. The resultant CHC thus reflects the physiologic "volume set point" of the system. The inactivation of KCC as the cell shrinks toward its new steady state is most likely a key factor in defining the volume regulatory function of the transporter, which in turn is crucial for establishing the steady state CHC for RBCs in vivo.Volume reduction mediated by KCC has been demonstrated with RBC density measurements. 7,11,12 However, these studies, like flux studies, are complicated by varying numbers of reticulocytes (or young cells) in unfractionated SS blood, which preclude comparisons of KCC-mediated volume reduction in SS and AA cells. We recently compared the RVD mediated by KCC and stimulated by cell swelling and acid pH in SS and AA reticulocytes by tracking the changes in reticulocyte CHC by means of density gradient analysis. 8 Acid-stimulated volume reduction was exaggerated in SS reticulocytes and was diminished by sulfhydryl reduction, parallel to the behavior of KCC flux activity. However, despite the normal response of KCC flux activation to cell swelling in SS RBCs, swelling-stimulated volume reduction was markedly abnormal in SS reticulocytes and resulted in higher final CHC than in AA reticulocytes. Thus, volume reduction and KCC fluxes appear to reflect different functional aspects of the KCC system in RBCs and may be subject to different pathologic influences in SS RBCs...

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Stimulation of Human Erythrocyte K-Cl Cotransport and Protein Phosphatase Type 2A by n-Ethylmaleimide: Role of Intracellular Mg ++

Cited by 54 publications

References 48 publications

Abnormal regulation of Mg2+ transport via Na/Mg exchanger in sickle erythrocytes

Abnormal regulation of Mg2+ transport via Na/Mg exchanger in sickle erythrocytes

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

Urea stimulation of KCl cotransport induces abnormal volume reduction in sickle reticulocytes

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