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IntroductionThe KCl cotransporter (KCC) is a member of the cation-chloride cotransporter family of proteins that mediate electroneutral net transport of salt to effect cell volume regulation, epithelial solute secretion and absorption, and control of intracellular ion concentrations. 1 In red blood cells (RBCs), KCC functions during reticulocyte maturation to establish the steady-state volume and mean corpuscular hemoglobin concentration (MCHC) of the mature RBC. 2,3 RBC KCC activity is stimulated in vitro by cell swelling (low MCHC), 4,5 acid pH, 6,7 and urea, 8 but the physiologic stimuli that effect reticulocyte volume reduction in vivo are not clear.KCC activation is controlled by phosphatase/kinase equilibria. Activation involves dephosphorylation of a serine (threonine) residue, probably by protein phosphatase 1 (PP1) 9,10 or PP2A. 11 There is also evidence for control by tyrosine phosphorylation; in fact, certain tyrosine kinase inhibitors activate KCC, 12,13 whereas others inhibit, 14,15 suggesting multiple control points, including PP1 itself. 16,17 Activation by cell swelling appears to involve inhibition of a putative volume-sensitive serine/threonine kinase, which maintains the system in a phosphorylated, quiescent state. 18 It is unknown whether control is mediated through phosphorylation of the transporter itself or other regulatory protein(s).KCC is also activated by sulfhydryl alkylation with Nethylmaleimide (NEM) 5,[19][20][21] and oxidation by diamide 22 or hydrogen peroxide. 23 CDNB (chloro-dinitrobenzine) stimulates KCC without a direct oxidant effect by enzymatic coupling to reduced glutathione (GSH). [24][25][26][27] Although the target of these agents is not known, it is clear that KCC activity and regulation are modulated by the oxidation state of RBC sulfhydryls.KCC activity in sickle (SS) RBCs is greatly elevated compared with normal (AA) RBCs, regardless of the activating stimulus. Some of this elevated activity is a consequence of the high percentage of reticulocytes in SS blood. 2,28 Nevertheless, there is evidence that the presence of sickle hemoglobin (Hb S, ␤ 6glu3val ) or hemoglobin C (Hb C, ␤ 6glu3lys ) directly affects the activity and properties of KCC in RBCs and RBC ghosts. [29][30][31]7 demonstrated acid stimulation of KCC in SS RBCs and showed that acidification of SS RBCs produced an increase in cell density. 6,7 Bookchin et al 32 showed that SS reticulocytes were susceptible to acid-induced dehydration and suggested that most of the dense cells in sickle blood arose from a subpopulation of SS reticulocytes with high sensitivity to KCC-mediated dehydration. Franco et al 33 found that SS reticulocytes that were dense in vivo were more susceptible to acid-induced dehydration via KCC than reticulocytes that were normally hydrated in vivo. These studies demonstrated the capacity of KCC to mediate SS RBC dehydration in vitro, but since direct comparisons with AA reticulocytes have not been made, the abnormal volume regulatory response of KCC in SS reticulocytes has not been f...

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...