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1
Holtzman, Eli J., Sumit Kumar, Carol A. Faaland, Fern Warner, Paul J. Logue, Sara J. Erickson, Gesa Ricken, Jeremy Waldman, Shiv Kumar, and Philip B. Dunham. "Cloning, characterization, and gene organization of K-Cl cotransporter from pig and human kidney and C. elegans." American Journal of Physiology-Renal Physiology 275, no. 4 (October 1, 1998): F550—F564. http://dx.doi.org/10.1152/ajprenal.1998.275.4.f550.
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We isolated and characterized the cDNAs for the human, pig, and Caenorhabditis elegansK-Cl cotransporters. The pig and human homologs are 94% identical and contain 1,085 and 1,086 amino acids, respectively. The deduced protein of the C. elegans K-Cl cotransporter clone (CE-KCC1) contains 1,003 amino acids. The mammalian K-Cl cotransporters share ∼45% similarity with CE-KCC1. Hydropathy analyses of the three clones indicate typical KCC topology patterns with 12 transmembrane segments, large extracellular loops between transmembrane domains 5 and 6 (unique to KCC), and large COOH-terminal domains. Human KCC1 is widely expressed among various tissues. This KCC1 gene spans 23 kb and is organized in 24 exons, whereas the CE-KCC1 gene spans 3.5 kb and contains 10 exons. Transiently and stably transfected human embryonic kidney cells (HEK-293) expressing the human, pig, and C. elegans K-Cl cotransporter fulfilled two (pig) or five (human and C. elegans) criteria for increased expression of the K-Cl cotransporter. The criteria employed were basal K-Cl cotransport; stimulation of cotransport by swelling, N-ethylmaleimide, staurosporine, and reduced cell Mg concentration; and secondary stimulation of Na-K-Cl cotransport.
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Race, Joanne E., Fadi N. Makhlouf, Paul J. Logue, Frederick H. Wilson, Philip B. Dunham, and Eli J. Holtzman. "Molecular cloning and functional characterization of KCC3, a new K-Cl cotransporter." American Journal of Physiology-Cell Physiology 277, no. 6 (December 1, 1999): C1210—C1219. http://dx.doi.org/10.1152/ajpcell.1999.277.6.c1210.
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We isolated and characterized a novel K-Cl cotransporter, KCC3, from human placenta. The deduced protein contains 1,150 amino acids. KCC3 shares 75–76% identity at the amino acid level with human, pig, rat, and rabbit KCC1 and 67% identity with rat KCC2. KCC3 is 40 and 33% identical to two Caenorhabditis elegans K-Cl cotransporters and ∼20% identical to other members of the cation-chloride cotransporter family (CCC), two Na-K-Cl cotransporters (NKCC1, NKCC2), and the Na-Cl cotransporter (NCC). Hydropathy analysis indicates a typical KCC topology with 12 transmembrane domains, a large extracellular loop between transmembrane domains 5 and 6 (unique to KCCs), and large NH2 and COOH termini. KCC3 is predominantly expressed in kidney, heart, and brain, and is also expressed in skeletal muscle, placenta, lung, liver, and pancreas. KCC3 was localized to chromosome 15. KCC3 transiently expressed in human embryonic kidney (HEK)-293 cells fulfilled three criteria for increased expression of K-Cl cotransport: stimulation of cotransport by swelling, treatment with N-ethylmaleimide, or treatment with staurosporine.
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Gillen, Christopher M., and Bliss Forbush. "Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells." American Journal of Physiology-Cell Physiology 276, no. 2 (February 1, 1999): C328—C336. http://dx.doi.org/10.1152/ajpcell.1999.276.2.c328.
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We have studied the regulation of the K-Cl cotransporter KCC1 and its functional interaction with the Na-K-Cl cotransporter. K-Cl cotransporter activity was substantially activated in HEK-293 cells overexpressing KCC1 (KCC1-HEK) by hypotonic cell swelling, 50 mM external K, and pretreatment with N-ethylmaleimide (NEM). Bumetanide inhibited 86Rb efflux in KCC1-HEK cells after cell swelling [inhibition constant ( K i) ∼190 μM] and pretreatment with NEM ( K i ∼60 μM). Thus regulation of KCC1 is consistent with properties of the red cell K-Cl cotransporter. To investigate functional interactions between K-Cl and Na-K-Cl cotransporters, we studied the relationship between Na-K-Cl cotransporter activation and intracellular Cl concentration ([Cl]i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activity than controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells was activated <2-fold by low-Cl hypotonic prestimulation, compared with 10-fold activation in HEK-293 cells and >20-fold activation in cells overexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cells had lower resting [Cl]i than HEK-293 cells; cell volume was not different among cell lines. We found a steep relationship between [Cl]i and Na-K-Cl cotransport activity within the physiological range, supporting a primary role for [Cl]iin activation of Na-K-Cl cotransport and in apical-basolateral cross talk in ion-transporting epithelia.
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Crable, Scott C., Suzan M. Hammond, Richard Papes, R. Kirk Rettig, Guo-Ping Zhou, Patrick G. Gallagher, Clinton H. Joiner, and Kathleen P. Anderson. "Multiple isoforms of the KC1 cotransporter are expressed in sickle and normal erythroid cells." Experimental Hematology 33, no. 6 (June 2005): 624–31. http://dx.doi.org/10.1016/j.exphem.2005.02.006.
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Strange, Kevin, Thomas D. Singer, Rebecca Morrison, and Eric Delpire. "Dependence of KCC2 K-Cl cotransporter activity on a conserved carboxy terminus tyrosine residue." American Journal of Physiology-Cell Physiology 279, no. 3 (September 1, 2000): C860—C867. http://dx.doi.org/10.1152/ajpcell.2000.279.3.c860.
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K-Cl cotransporters (KCC) play fundamental roles in ionic and osmotic homeostasis. To date, four mammalian KCC genes have been identified. KCC2 is expressed exclusively in neurons. Injection of Xenopus oocytes with KCC2 cRNA induced a 20-fold increase in Cl−-dependent, furosemide-sensitive K+ uptake. Oocyte swelling increased KCC2 activity 2–3 fold. A canonical tyrosine phosphorylation site is located in the carboxy termini of KCC2 (R1081–Y1087) and KCC4, but not in other KCC isoforms. Pharmacological studies, however, revealed no regulatory role for phosphorylation of KCC2 tyrosine residues. Replacement of Y1087 with aspartate or arginine dramatically reduced K+ uptake under isotonic and hypotonic conditions. Normal or near-normal cotransporter activity was observed when Y1087 was mutated to phenylalanine, alanine, or isoleucine. A tyrosine residue equivalent to Y1087 is conserved in all identified KCCs from nematodes to humans. Mutation of the Y1087 congener in KCC1 to aspartate also dramatically inhibited cotransporter activity. Taken together, these results suggest that replacement of Y1087 and its congeners with charged residues disrupts the conformational state of the carboxy terminus. We postulate that the carboxy terminus plays an essential role in maintaining the functional conformation of KCC cotransporters and/or is involved in essential regulatory protein-protein interactions.
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Pan, Dao, Theodosia A. Kalfa, Daren Wang, Mary Risinger, Scott Crable, Peter Ciraolo, Robert S. Franco, and Clinton H. Joiner. "Change in Expressional Profile of KCl Cotransporter Genes during Human Erythroid Differentiation." Blood 110, no. 11 (November 16, 2007): 1709. http://dx.doi.org/10.1182/blood.v110.11.1709.1709.
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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 >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.
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Delpire, Eric, and Jiangtao Guo. "Cryo-EM structures of DrNKCC1 and hKCC1: a new milestone in the physiology of cation-chloride cotransporters." American Journal of Physiology-Cell Physiology 318, no. 2 (February 1, 2020): C225—C237. http://dx.doi.org/10.1152/ajpcell.00465.2019.
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New milestones have been reached in the field of cation-Cl− cotransporters with the recently released cryo-electron microscopy (EM) structures of the Danio rerio (zebrafish) Na+-K+-2Cl− cotransporter ( DrNKCC1) and the human K+-Cl− cotransporter (hKCC1). In this review we provide a brief timeline that identifies the multiple breakthroughs in the field of solute carrier 12 transporters that led to the structure resolution of two of its key members. While cation-Cl− cotransporters share the overall architecture of carriers belonging to the amino acid-polyamine-organocation (APC) superfamily and some of their substrate binding sites, several new insights are gained from the two individual structures. A first major feature relates to the largest extracellular domain between transmembrane domain (TMD) 5 and TMD6 of KCC1, which stabilizes the dimer and forms a cap that likely participates in extracellular gating. A second feature is the conservation of the K+ and Cl− binding sites in both structures and evidence of an unexpected second Cl− coordination site in the KCC1 structure. Structural data are discussed in the context of previously published studies that examined the basic and kinetics properties of these cotransport mechanisms. A third characteristic is the evidence of an extracellular gate formed by conserved salt bridges between charged residues located toward the end of TMD3 and TMD4 in both transporters and the existence of an additional neighboring bridge in the hKCC1 structure. A fourth feature of these newly solved structures relates to the multiple points of contacts between the monomer forming the cotransporter homodimer units. These involve the TMDs, the COOH-terminal domains, and the large extracellular loop for hKCC1.
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Joiner, Clinton H., Richard Papes, Scott Crable, Dao Pan, and David B. Mount. "Functional Comparison of Red Cell KCl Cotransporter Isoforms, KCC1, KCC3, and KCC4." Blood 108, no. 11 (November 16, 2006): 1245. http://dx.doi.org/10.1182/blood.v108.11.1245.1245.
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Abstract The KCl cotransporter mediates volume reduction in normal (AA) reticulocytes, and its abnormal regulation in sickle (SS) reticulocytes contributes to cellular dehydration that facilitates Hb S sickling. mRNA for three KCC genes - KCC1, KCC3, and KCC4 - as well as a splicing isoform, KCC1ex1b, is present in reticulocytes (Exp. Hem.2005; 33:624). Western blotting has demonstrated KCC1 in human RBC membranes (Su et al, AJPhysiol.1999;277:C899) and KCC3 in sheep (Lauf et al, CompBiochemPhysiol2001;130:499); KCC4 protein was found in hRBC membranes by proteomic analysis (Pasini et al, Blood2006;108:791). We confirm here the presence of KCC3 protein in hRBC via western blotting using an antibody to an exon 3 epitope distal to known N-terminal splicing sites. We sought to characterize and compare human KCC isoforms expressed in human cells (HEK 293) and assess their similarity to KCC activity in RBC. cDNAs for human KCC1, KCC1ex1b, KCC3a, and KCC4, with N-terminal c-myc epitope tags were expressed in HEK 293 cells. Stable cell lines were selected by growth in neomycin, and expression monitored by quantitative PCR analysis of the expressed construct and other endogenous isoforms and by western blotting (anti-myc) of plasma membranes. KCC activity was measured as N-ethylmaleimide(NEM)-stimulated, Cl-dependent Rb uptake in cells grown to 75–90 % confluency. Cells were incubated at 37°C in isotonic saline media with various concentrations of Rb (Na replacement), plus 0.1 mM ouabain and 0.01 mM bumetanide. At 2 and 4 min cells were washed with iced Rb-free media, then lysed and assayed for Rb by flame emission, normalized to sample protein. Flux rates were calculated from Rb uptake at 2 and 4 min. The flux rate in Cl-free sulfamate media was subtracted from that in Cl-media to yield the Cl-dependent flux rate. Wild-type HEK cells showed no increase in Cl-dependent Rb influx when exposed to 1 mM NEM, but Cl-dependent, NEM-stimulated Rb uptake was apparent in cells expressing KCC isoforms. All isoforms were stimulated by hypotonic conditions (75 mOsm), but relative to NEM-stimulated activity, KCC3 was most responsive. Kinetic characteristics of Cl-dependent, NEM-stimulated Rb influx in HEK cell expressing human KCC1, KCC3a, and KCC4 isoforms isoforms are given below, compared to RBC KCC fluxes. A splice variant, KCC1ex1b, coding for a protein with a truncated N-terminus, exhibited 48 ± 7% of the activity of the full-length KCC1 isoform, but did not alter transport activity of coexpressed KCC3a. Thus, KCC isoforms expressed in HEK cells share some, but not all, characteristics with RBC KCC fluxes. The isoform kinetics also differ from those previously published in xenopus oocyte expression systems, suggesting that the membrane milieu in which KCC proteins are expressed may influence their functional characteristics. Likewise, KCC activity in RBC may represent an average of several transporters operating in parallel. The truncated KCC1ex1b isoform, expressed at higher levels in normal than in sickle reticulocytes, has lower activity than KCC1. KCC3 and KCC4 exhibit more robust transport activity than KCC1 and may mediate a substantial part of KCC activity in RBC. KCC Kinetic Characteristics VMax (μmol/mgprot./min) Km (ext. Rb, mM) Anion Selectivity hKCC1 28.9 ± 5.6 16.7 ± 5.4 Cl > Br > I > SCN hKCC3a 107 ± 45 11.0 ± 7.1 Cl = Br > I > SCN hKCC4 206 ± 16 15.6 ± 8.2 Cl > Br >> I = SCN RBC NA 12 - 20 Cl = Br >> I = SCN
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Risinger, Mary, Jesse Rinehart, Scott Crable, Anna Ottlinger, Richard Winkelmann, Dao Pan, Christian Huebner, Patrick G. Gallagher, and Clinton H. Joiner. "Structural and Functional Interactions of KCl Cotransport Proteins KCC1 and KCC3 in Sickle and Normal Erythrocyte Membranes." Blood 112, no. 11 (November 16, 2008): 2474. http://dx.doi.org/10.1182/blood.v112.11.2474.2474.
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Abstract The KCl cotransporter (KCC) mediates volume reduction in normal reticulocytes and exaggerated KCC activity in sickle red blood cells (SS RBC) (Joiner et al, Blood109:1728, 2007) contributes to pathological dehydration that potentiates sickling. Three separate genes (KCC1, KCC3, KCC4) are expressed in RBC (Crable et al, Exp. Hem.33:624, 2005). KCC1 and KCC3 proteins have been shown to interact in ex vivo expression systems (Simard et al, JBC282(25):18083, 2007), and co-expression of an N-terminal truncation of KCC1 reduces KCC activity mediated by full-length KCC1 or KCC3 (Casula et al. JBC276:41870, 2001), suggesting functional interaction. We show here via western blot analysis that SS RBC membranes contain more KCC1 protein (relative to KCC3) than AA RBC, independent of the reticulocytosis of sickle blood. Immunoprecipitation of solubilized SS RBC membranes with KCC3-specific antibody yielded a band at 125 kD on SDS PAGE which contained KCC1, as identified by western blotting with KCC1-specific antibody and by TOF mass spectroscopy. The effect of co-expression of KCC1 and KCC3 on KCC activity was assessed by measuring NEM-stimulated, Cl-dependent, (ouabain + bumetanide)-insensitive Rb uptake in HEK 293 cells. The Flip-In T-rex HEK 293 cell line (Invitrogen) containing a tetracycline-response promoter was transfected with a pcDNA5a plasmid containing KCC3a cDNA. Recombination of the plasmid with the integrated tet-promoter construct inserts the KCC3a gene under control of a tetracycline-responsive promoter. These cells were subsequently transduced with a retroviral vector (SF-91. Hildinger et at, Gene Ther. 5:1575, 1998) containing KCC1 cDNA linked to a GFP cassette. Control cells contained SF-91 vector lacking KCC1. Cells were selected for GFP expression and grown in the absence (un-induced, no KCC3a expression) or presence of tetracycline (induced, KCC3a expression). From this binary matrix, four types of cells were obtained: Cells with no additional KCC expression, representing endogenous KCC activity; cells with only KCC1 or KCC3a expression; cells with both KCC1 and KCC3a expression. Western blots indicated similar KCC1 expression in cells with KCC1 only and [KCC1 + KCC3] and similar KCC3 expression in cells with KCC3 only and [KCC1 + KCC3]. Thus, the expression of neither isoform was affected by the presence of the other. KCC activity in cells overexpressing KCC1 only was similar to endogenous activity in HEK 293 cells; i.e., transport activity of KCC1 alone was minimal. Cells overexpressing KCC3 only had a 5-fold increase in KCC activity over endogenous levels. When KCC1 was co-expressed with KCC3 in [KCC1 + KCC3] cells, an additional 50% increase in KCC activity was observed (p < 0.05 by paired t-test, N=4), despite similar levels of KCC3 expression by western blot analysis. This synergistic effect was dependent on the cytoplasmic N-terminus of KCC1, as it was not seen when the first 39 amino acids of KCC1 were removed. Interestingly, removal of the entire cytoplasmic N-terminal domain (117 aa) produced an inhibitory effect when co-expressed with KCC3a in HEK cells, as previously reported in Xenopus oocytes (Casula et al.). These data indicate that KCC1 and KCC3 interact structurally and functionally in RBC membranes, and provide another potential mechanism for regulation of KCC activity via multimeric associations between KCC isoforms. Thus, KCC activity could be modulated not only by transcriptional mechanisms and post-translational modification (phosphorylation), but also by altering the ratios of KCC isoforms or the kinetics of their association. We speculate that higher levels of KCC1 protein relative to KCC3 in SS RBC membranes could account for higher KCC activity in these cells relative to AA RBC.
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Mercado, Adriana, Paola de los Heros, Zesergio Melo, María Chávez-Canales, Adrián R. Murillo-de-Ozores, Erika Moreno, Silvana Bazúa-Valenti, Norma Vázquez, Juliette Hadchouel, and Gerardo Gamba. "With no lysine L-WNK1 isoforms are negative regulators of the K+-Cl− cotransporters." American Journal of Physiology-Cell Physiology 311, no. 1 (July 1, 2016): C54—C66. http://dx.doi.org/10.1152/ajpcell.00193.2015.
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The K+-Cl− cotransporters (KCC1-KCC4) encompass a branch of the SLC12 family of electroneutral cation-coupled chloride cotransporters that translocate ions out of the cell to regulate various factors, including cell volume and intracellular chloride concentration, among others. L-WNK1 is an ubiquitously expressed kinase that is activated in response to osmotic stress and intracellular chloride depletion, and it is implicated in two distinct hereditary syndromes: the renal disease pseudohypoaldosteronism type II (PHAII) and the neurological disease hereditary sensory neuropathy 2 (HSN2). The effect of L-WNK1 on KCC activity is unknown. Using Xenopus laevis oocytes and HEK-293 cells, we show that the activation of KCCs by cell swelling was prevented by L-WNK1 coexpression. In contrast, the activity of the Na+-K+-2Cl− cotransporter NKCC1 was remarkably increased with L-WNK1 coexpression. The negative effect of L-WNK1 on the KCCs is kinase dependent. Elimination of the STE20 proline-alanine rich kinase (SPAK)/oxidative stress-responsive kinase (OSR1) binding site or the HQ motif required for the WNK-WNK interaction prevented the effect of L-WNK1 on KCCs, suggesting a required interaction between L-WNK1 molecules and SPAK. Together, our data support that NKCC1 and KCCs are coordinately regulated by L-WNK1 isoforms.
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Su, Wanfang, Boris E. Shmukler, Marina N. Chernova, Alan K. Stuart-Tilley, Lucia de Franceschi, Carlo Brugnara, and Seth L. Alper. "Mouse K-Cl cotransporter KCC1: cloning, mapping, pathological expression, and functional regulation." American Journal of Physiology-Cell Physiology 277, no. 5 (November 1, 1999): C899—C912. http://dx.doi.org/10.1152/ajpcell.1999.277.5.c899.
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Although K-Cl cotransporter (KCC1) mRNA is expressed in many tissues, K-Cl cotransport activity has been measured in few cell types, and detection of endogenous KCC1 polypeptide has not yet been reported. We have cloned the mouse erythroid KCC1 (mKCC1) cDNA and its flanking genomic regions and mapped the mKCC1 gene to chromosome 8. Three anti-peptide antibodies raised against recombinant mKCC1 function as immunoblot and immunoprecipitation reagents. The tissue distributions of mKCC1 mRNA and protein are widespread, and mKCC1 RNA is constitutively expressed during erythroid differentiation of ES cells. KCC1 polypeptide or related antigen is present in erythrocytes of multiple species in which K-Cl cotransport activity has been documented. Erythroid KCC1 polypeptide abundance is elevated in proportion to reticulocyte counts in density-fractionated cells, in bleeding-induced reticulocytosis, in mouse models of sickle cell disease and thalassemia, and in the corresponding human disorders. mKCC1-mediated uptake of86Rb into Xenopus oocytes requires extracellular Cl−, is blocked by the diuretic R(+)-[2- n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1 H-indenyl-5-yl-)oxy]acetic acid, and exhibits an erythroid pattern of acute regulation, with activation by hypotonic swelling, N-ethylmaleimide, and staurosporine and inhibition by calyculin and okadaic acid. These reagents and findings will expedite studies of KCC1 structure-function relationships and of the pathobiology of KCC1-mediated K-Cl cotransport.
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Liu, Si, Shenghai Chang, Binming Han, Lingyi Xu, Mingfeng Zhang, Cheng Zhao, Wei Yang, et al. "Cryo-EM structures of the human cation-chloride cotransporter KCC1." Science 366, no. 6464 (October 24, 2019): 505–8. http://dx.doi.org/10.1126/science.aay3129.
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Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations with chloride across the plasma membrane and are vital for cell volume regulation, salt reabsorption in the kidney, and γ-aminobutyric acid (GABA)–mediated modulation in neurons. Here we present cryo–electron microscopy (cryo-EM) structures of human potassium-chloride cotransporter KCC1 in potassium chloride or sodium chloride at 2.9- to 3.5-angstrom resolution. KCC1 exists as a dimer, with both extracellular and transmembrane domains involved in dimerization. The structural and functional analyses, along with computational studies, reveal one potassium site and two chloride sites in KCC1, which are all required for the ion transport activity. KCC1 adopts an inward-facing conformation, with the extracellular gate occluded. The KCC1 structures allow us to model a potential ion transport mechanism in KCCs and provide a blueprint for drug design.
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De Franceschi, Lucia, Luisa Ronzoni, Achille Iolascon, Francesca Cimmino, Seth L. Alper, Valeria Servedio, Franco Turrini, and Maria D. Cappellini. "Pharmacological Inhibition of K-Cl Cotransport Alters In Vitro Maturation of Normal and β-Thalassemic Human Erythroid Progenitors." Blood 106, no. 11 (November 16, 2005): 3819. http://dx.doi.org/10.1182/blood.v106.11.3819.3819.
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Abstract The K-Cl cotransporter family (KCC) plays a crucial role in cell volume regulation, and KCC1 and KCC3 have been reported to participate in cell growth events (Shen MR, PNAS98, 2001; Shen MR JBC278, 2003). Expression of KCC1, KCC3 and KCC4 has been reported in erythroid cells. In β-thalassemic red blood cells (RBCs), K-Cl cotransport activity is abnormally activated and contributes to red cell loss of water and K. This study evaluated the gene expression of two KCC gene products and the effects of the KCC inhibitor [(dihydroindoenyl)oxy]alkanoic acid (DIOA) on in vitro liquid-culture expansion of human normal and β thalassemic (β thal) erythroid precursors from peripheral blood CD34+ cells. Cells from normal subjects and from β thalassemia major patients (cod39cod39) were cultured for 7 days (to the pro-normobast stage) and 14 days (to the eythroblast stage) in the presence or absence of 10 mM DIOA, At each time point the following parameters were evaluated; cells counts; cytospins stained with Wright-Giemsa to assess differential cell counts and morphology, cell cycle stage by fluorescence-activated cell sorting after propidium iodide staining; KCC protein expression by Western-blot analysis with antibody to the shared KCC carboxy-terminus; mRNA by real time-PCR analysis. KCC protein expression increased during erythropoiesis in both normal and β thal cells, and was higher in β thal cells than in normal controls. KCC1 mRNA level was increased only in β thal cells at day 14, whereas KCC3 mRNA level was increased at day 14 in both normal and β thal cells. DIOA significantly reduced the number of both normal and β thal cells, parallelled by increases in the percentage of polychromatophilic normoblasts among normal progenitors and of basophilic normoblasts among b thal progenitors. We further investigated the inhibitory effects of DIOA on cell growth by FACS evaluation of cell cycle distributions and by determination ofCycD, p21, Casp3 and Casp8 gene expression. At day 14 DIOA exposure was associated with: significant reduction in the percentage of β thal cells in S-phase compared to either untreated cells or DIOA-treated normal controls; up-regulation of CycD gene expression in both normal and β thal cells; down-regulation of p21 in β thal cells; up-regulation of casp3 and casp8 in both normal and β thal cells. These data suggest that KCC is involved in the late phase of erythropoiesis mainly in β thal cells, and support a novel role of KCC in erythroid cell growth.
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Joiner, C. H., M. Jiang, H. Fathallah, F. Giraud, and R. S. Franco. "Deoxygenation of sickle red blood cells stimulates KCl cotransport without affecting Na+/H+exchange." American Journal of Physiology-Cell Physiology 274, no. 6 (June 1, 1998): C1466—C1475. http://dx.doi.org/10.1152/ajpcell.1998.274.6.c1466.
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KCl cotransport activated by swelling of sickle red blood cells (SS RBC) is inhibited by deoxygenation. Yet recent studies found a Cl−-dependent increase in sickle reticulocyte density with cyclic deoxygenation. This study sought to demonstrate cotransporter stimulation by deoxygenation of SS RBC in isotonic media with normal pH. Low-density SS RBC exhibited a Cl−-dependent component of the deoxygenation-induced net K+efflux, which was blocked by two inhibitors of KCl cotransport, [(dihydroindenyl)oxy]alkanoic acid and okadaic acid. Cl−-dependent K+efflux stimulated by deoxygenation was enhanced 2.5-fold by clamping of cellular Mg2+at the level in oxygenated cells using ionophore A-23187. Incubating cells in high external K+or Rb+minimized inhibition of KCl cotransport by internal Mg2+, and under these conditions deoxygenation markedly stimulated KCl cotransport in the absence of ionophore. Activation of KCl cotransport by deoxygenation of SS RBC in isotonic media at normal pH is consistent with the generalized dephosphorylation of membrane proteins induced by deoxygenation and activation of the cotransporter by a dephosphorylation mechanism. Na+/H+exchange activity, known to be modulated by cytosolic Ca2+elevation and cell shrinkage, remained silent under deoxygenation conditions.
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Zhou, Guo-Ping, Clara Wong, Robert Su, Scott C. Crable, Kathleen P. Anderson, and Patrick G. Gallagher. "Human potassium chloride cotransporter 1 (SLC12A4) promoter is regulated by AP-2 and contains a functional downstream promoter element." Blood 103, no. 11 (June 1, 2004): 4302–9. http://dx.doi.org/10.1182/blood-2003-01-0107.
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Abstract Most K-Cl cotransport in the erythrocyte is attributed to potassium chloride cotransporter 1 (KCC1). K-Cl cotransport is elevated in sickle erythrocytes, and the KCC1 gene has been proposed as a modifier gene in sickle cell disease. To provide insight into our understanding of the regulation of the human KCC1 gene, we mapped the 5′ end of the KCC1 cDNA, cloned the corresponding genomic DNA, and identified the KCC1 gene promoter. The core promoter lacks a TATA box and is composed of an initiator element (InR) and a downstream promoter element (DPE), a combination found primarily in Drosophila gene promoters and rarely observed in mammalian gene promoters. Mutational analyses demonstrated that both the InR and DPE sites were critical for full promoter activity. In vitro DNase I footprinting, electrophoretic mobility shift assays, and reporter gene assays identified functional AP-2 and Sp1 sites in this region. The KCC1 promoter was transactivated by forced expression of AP-2 in heterologous cells. Sequences encoding the InR, DPE, AP-2, and Sp1 sites were 100% conserved between human and murine KCC1 genes. In vivo studies using chromatin immunoprecipitation assays with antihistone H3 and antihistone H4 antibodies demonstrated hyperacetylation of this core promoter region. (Blood. 2004;103:4302-4309)
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Mercado, Adriana, Paola de los Heros, Norma Vázquez, Patricia Meade, David B. Mount, and Gerardo Gamba. "Functional and molecular characterization of the K-Cl cotransporter of Xenopus laevis oocytes." American Journal of Physiology-Cell Physiology 281, no. 2 (August 1, 2001): C670—C680. http://dx.doi.org/10.1152/ajpcell.2001.281.2.c670.
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The K-Cl cotransporters (KCCs) have a broad range of physiological roles, in a number of cells and species. We report here that Xenopus laevis oocytes express a K-Cl cotransporter with significant functional and molecular similarity to mammalian KCCs. Under isotonic conditions, defolliculated oocytes exhibit a Cl−-dependent86Rb+ uptake mechanism after activation by the cysteine-reactive compounds N-ethylmaleimide (NEM) and mercuric chloride (HgCl2). The activation of this K-Cl cotransporter by cell swelling is prevented by inhibition of protein phosphatase-1 with calyculin A; NEM activation of the transporter was not blocked by phosphatase inhibition. Kinetic characterization reveals apparent values for the Michaelis-Menten constant of 27.7 ± 3.0 and 15.4 ± 4.7 mM for Rb+ and Cl−, respectively, with an anion selectivity for K+ transport of Cl− = PO[Formula: see text] = Br−> I− > SCN− > gluconate. The oocyte K-Cl cotransporter was sensitive to several inhibitors, including loop diuretics, with apparent half-maximal inhibition values of 200 and 500 μM for furosemide and bumetanide, respectively. A partial cDNA encoding the Xenopus K-Cl cotransporter was cloned from oocyte RNA; the corresponding transcript is widely expressed in Xenopus tissues. The predicted COOH-terminal protein fragment exhibited particular homology to the KCC1/KCC3 subgroup of the mammalian KCCs, and the functional characteristics are the most similar to those of KCC1 (Mercado A, Song L, Vazquez N, Mount DB, and Gamba G. J Biol Chem 275: 30326–30334, 2000).
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DMITRIEV, ANDREY V., NINA A. DMITRIEVA, KENT T. KEYSER, and STUART C. MANGEL. "Multiple functions of cation-chloride cotransporters in the fish retina." Visual Neuroscience 24, no. 4 (July 2007): 635–45. http://dx.doi.org/10.1017/s0952523807070629.
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A GABA- or glycine-induced increase in Cl− permeability can produce either a depolarization or hyperpolarization, depending on the Cl− equilibrium potential. It has been shown that retinal neurons express the chloride cotransporters, Na-K-2Cl (NKCC) and K-Cl (KCC), the primary molecular mechanisms that control the intracellular Cl− concentration. We thus studied (1) the localization of these cotransporters in the fish retina, and (2) how suppression of cotransporter activity in the fish retina affects function. Specific antibodies against NKCC and KCC2 revealed that both cotransporters were expressed in the outer and inner plexiform layers, and colocalized in many putative amacrine cells and in cells of the ganglion cell layer. However, the somata of putative horizontal cells displayed only NKCC immunoreactivity and many bipolar cells were only immunopositive for KCC2. In the outer retina, application of bumetanide, a specific inhibitor of NKCC activity, (1) increased the steady-state extracellular concentration of K+ ([K+]o) and enhanced the light-induced decrease in the [K+]o, (2) increased the sPIII photoreceptor-dependent component of the ERG, and (3) reduced the extracellular space volume. In contrast, in the outer retina, application of furosemide, a specific inhibitor of KCC activity, decreased sPIII and the light-induced reduction in [K+]o, but had little effect on steady-state [K+]o. In the inner retina, bumetanide increased the sustained component of the light-induced increase in [K+]o. These findings thus indicate that NKCC and KCC2 control the [K+]o and extracellular space volume in the retina in addition to regulating GABA- and glycine-mediated synaptic transmission. In addition, the anatomical and electrophysiological results together suggest that all of the major neuronal types in the fish retina are influenced by chloride cotransporter activity.
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Anderson, Kathleen, Scott C. Crable, Suzan M. Hammond, Clinton H. Joiner, and Patrick G. Gallagher. "A Second Promoter for the Human KCl Cotransporter 1 (KCC1) Gene Drives Differential Expression of a Variant Isoform in Sickle Versus Normal Reticulocytes." Blood 104, no. 11 (November 16, 2004): 3592. http://dx.doi.org/10.1182/blood.v104.11.3592.3592.
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Abstract The K+Cl- cotransporter plays a significant role in the maintenance of red cell volume. During cellular maturation, this cotransporter actively moves K+ and Cl- out of the cell. The accompanying movement of water results in dehydration and shrinking of the red cell. Because KCl cotransporter activity is higher in sickle compared to normal reticulocytes, it has been considered a potential modifier gene for sickle cell disease. We have evidence for expression of three KCC genes in human reticulocytes and have investigated the promoter for KCC1. While the expression of the principal KCC1 transcript did not differ in SS compared to normal reticulocytes, we now describe an alternative transcript of the KCC1 gene emanating from a second promoter and exhibiting a restricted tissue distribution. Investigation of the EST databases revealed spliced ESTs corresponding to the use of four distinct N-terminal exons in the KCC1 gene, each reported multiple times in the dataset. Primers were developed for these 5′ regions (exon1, 1a, 1b, and 1c) and used in an RT-PCR reaction with human reticulocyte RNA. The exon1 form and the exon1b variant were expressed. When the relative levels of these forms were compared, expression of the exon1 transcript was unchanged, while significantly higher levels of the exon1b variant was evident in the AA reticulocyte RNA compared to numerous SS samples. In an analysis of seven other human tissue samples, the exon1b isoform was highly expressed in kidney, lung, and heart, while the KCC1ex1 transcript was expressed at a constant level in all tissues. Although the transcript for this variant could arise from the KCC1 promoter we have previously characterized, the pattern of expression suggested control from a second promoter. A 915bp region corresponding to −787 to +128 was isolated and cloned into a reporter construct to test for promoter activity. This clone was compared with KCC1 promoter constructs in transient transfection assays. The exon1b construct not only exhibited promoter activity by directing high levels of luciferase expression in K562 cells, it also demonstrated tissue-specificity with a low level of activity in Jurkat cells. This recapitulates the endogenous levels detected by RT-PCR analysis of these cell lines. The −787/+128 exon1b construct is also 3-fold more active than the ubiquitously expressed exon1 promoter. To identify the control elements for this promoter, we produced a series of deletion constructs; the smallest construct contained 181bp. No reduction in reporter gene activity was evident, indicating the major regulatory elements lie very close to this promoter. Since exon1 encodes 39aa and exon1b only 7aa, the use of this smaller first exon effectively produces an N-terminal truncation in the protein. Studies with the mouse KCC1 cDNA have demonstrated that proteins produced by an N-terminal truncation are not only inactive for K+Cl- cotransport, but also function as dominant negative regulators of a full-length KCC1 protein. High level expression of this variant in AA cells compared to SS cells would therefore be consistent with the low reported activity in the AA reticulocytes. Induction or modulation of the expression of the KCC1ex1b variant may be an key factor in the control of red cell hydration.
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Anderson, Kathleen P., Scott C. Crable, Suzan M. Hammond, Patrick G. Gallagher, and Clinton H. Joiner. "Expression of a KCC1 Splice Variant Is Suppressed by NF-ΚB in Erythroid Cells." Blood 106, no. 11 (November 16, 2005): 1669. http://dx.doi.org/10.1182/blood.v106.11.1669.1669.
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Abstract The K+Cl- cotransporter (KCC) plays a significant role in the maintenance of red cell volume. Activity of the cotransporter is higher in sickle (SS) compared to normal (AA) reticulocytes, and contributes to SS dehydration. Thus, KCC is considered a potential modifier gene for sickle cell disease (SCD). We have demonstrated the presence of transcripts for KCC1, KCC3, and KCC4 in human reticulocytes (Exp.Hem.2005;33:624–31) and shown that one splice variant of KCC1 (KCC1ex1b), which codes for a protein with a small (7aa) alternative N-terminal exon, is detected in AA, but not SS reticulocytes. Studies with murine KCC1 have demonstrated that proteins produced by N-terminal truncation are inactive for K+Cl- cotransport, and function as dominant negative regulators of full-length KCC1 and KCC3 proteins. Since high level expression of this variant in AA cells compared to SS cells might explain the relatively low KCC activity in AA reticulocytes, we have identified the promoter for the KCC1ex1b transcript and investigated the regulatory elements that control its expression. Here we report the involvement of TNFα and NF-ΚB in the transcriptional regulation of the KCC1ex1b variant. Although KCC1ex1b is not expressed in reticulocytes isolated from sickle cell patients, we found that SS erythroid precursor cells cultured in vitro express this variant. SS and AA peripheral blood mononuclear cells were cultured in semi-liquid media with stem cell factor and erythropoietin, and collected after 5, 10, and 14 days in culture. Cells harvested at 14 days and isolated by binding to micromagnetic beads coated with transferrin receptor antibody were 95–98% positive for glycophorin A. RNA was extracted and analyzed by semi-quantitative RT-PCR, using primers for KCC1ex1 and KCC1ex1b. In both AA and SS cells, the transcript level for KCC1ex1b rose over the time in culture, while the KCC1ex1 transcript was constant. This difference between the in vitro and in vivo expression patterns for the KCC1ex1b variant could be explained by regulation via an external factor, such as a cytokine present in the blood of sickle cell patients, but absent in the in vitro culture system. The levels of numerous cytokines, including TNF, VEGF, and various interleukins, are elevated in SCD. We therefore assayed the effect of TNFα on endogenous KCC1ex1b expression in K562 cells by RT-PCR analysis at 24 and 48 hours after the addition of TNFα to the tissue culture medium. The steady-state mRNA levels of the KCC1ex1b variant decreased approximately 40% in response to TNF treatment. The transcription factor NF-ΚB is activated by TNF signaling, and an NF-ΚB consensus site is present in the KCC1ex1b promoter region. We assayed the effect of co-expressing NF-ΚB and our KCC1ex1b promoter constructs in K562 cells. NF-ΚB expression produced an 8-fold decrease in luciferase activity from these promoter constructs indicating NF-ΚB transcriptionally represses this promoter, either directly or indirectly. Our current model proposes that induction or modulation of the expression of the KCC1ex1bvariant could be an important factor in the control of red cell hydration.
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Zhang, Jing, Peter K. Lauf, and Norma C. Adragna. "Platelet-derived growth factor regulates K-Cl cotransport in vascular smooth muscle cells." American Journal of Physiology-Cell Physiology 284, no. 3 (March 1, 2003): C674—C680. http://dx.doi.org/10.1152/ajpcell.00312.2002.
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Platelet-derived growth factor (PDGF), a potent serum mitogen for vascular smooth muscle cells (VSMCs), plays an important role in membrane transport regulation and in atherosclerosis. K-Cl cotransport (K-Cl COT/KCC), the coupled-movement of K and Cl, is involved in ion homeostasis. VSMCs possess K-Cl COT activity and the KCC1 and KCC3 isoforms. Here, we report on the effect of PDGF on K-Cl COT activity and mRNA expression in primary cultures of rat VSMCs. K-Cl COT was determined as the Cl-dependent Rb influx and mRNA expression by semiquantitative RT-PCR. Twenty four-hour serum deprivation inhibited basal K-Cl COT activity. Addition of PDGF increased total protein content and K-Cl COT activity in a time-dependent manner. PDGF activated K-Cl COT in a dose-dependent manner, both acutely (10 min) and chronically (12 h). AG-1296, a selective inhibitor of the PDGF receptor tyrosine kinase, abolished these effects. Actinomycin D and cycloheximide had no effect on the acute PDGF activation of K-Cl COT, suggesting posttranslational regulation by the drug. Furthermore, PDGF increased KCC1 and decreased KCC3 mRNA expression in a time-dependent manner. These results indicate that chronic activation of K-Cl COT activity by PDGF may involve regulation of the two KCC mRNA isoforms, with KCC1 playing a dominant role in the mechanism of PDGF-mediated activation.
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Di Fulvio, Mauricio, Peter K. Lauf, Shalin Shah, and Norma C. Adragna. "NONOates regulate KCl cotransporter-1 and -3 mRNA expression in vascular smooth muscle cells." American Journal of Physiology-Heart and Circulatory Physiology 284, no. 5 (May 1, 2003): H1686—H1692. http://dx.doi.org/10.1152/ajpheart.00710.2002.
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Nitric oxide (NO) donors regulate KCl cotransport (KCC) activity and cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in sheep erythrocytes and in primary cultures of rat vascular smooth muscle cells (VSMCs), respectively. In this study, we used NONOates as rapid and slow NO releasers to provide direct evidence implicating NO as a regulator of KCC3 gene expression at the mRNA level. In addition, we used the expression of KCC3 mRNA to further investigate the mechanism of action of these NO donors at the cellular level. Treatment of VSMCs with rapid NO releasers, like NOC-5 and NOC-9, as well as with the direct NO-independent soluble guanylyl cyclase (sGC) stimulator YC-1, acutely increased KCC3 mRNA expression in a concentration- and time-dependent manner. The slow NO releaser NOC-18 had no effect on KCC3 gene expression. A specific NO scavenger completely prevented the NONOate-induced KCC3 mRNA expression. Inhibition of sGC with LY-83583 blocked the NONOate- and YC-1-induced KCC3 mRNA expression. This study shows that in primary cultures of rat VSMCs, the fast NO releasers NOC-9 and NOC-5, but not the slow NO releaser NOC-18, acutely upregulate KCC3 mRNA expression in a NO/sGC-dependent manner.
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Bergeron, Marc J., Édith Gagnon, Bernadette Wallendorff, Jean-Yves Lapointe, and Paul Isenring. "Ammonium transport and pH regulation by K+-Cl- cotransporters." American Journal of Physiology-Renal Physiology 285, no. 1 (July 2003): F68—F78. http://dx.doi.org/10.1152/ajprenal.00032.2003.
Full textAbstract:
The Na+-K+-Cl- cotransporters (NKCCs), which belong to the cation-Cl- cotransporter (CCC) family, are able to translocate [Formula: see text] across cell membranes. In this study, we have used the oocyte expression system to determine whether the K+-Cl- cotransporters (KCCs) can also transport [Formula: see text] and whether they play a role in pH regulation. Our results demonstrate that all of the CCCs examined (NKCC1, NKCC2, KCC1, KCC3, and KCC4) can promote [Formula: see text] translocation, presumably through binding of the ion at the K+ site. Moreover, kinetic studies for both NKCCs and KCCs suggest that [Formula: see text] is an excellent surrogate of Rb+ or K+ and that [Formula: see text] transport and cellular acidification resulting from CCC activity are relevant physiologically. In this study, we have also found that CCCs are strongly and differentially affected by changes in intracellular pH (independently of intracellular [[Formula: see text]]). Indeed, NKCC2, KCC1, KCC2, and KCC3 are inhibited at intracellular pH <7.5, whereas KCC4 is activated. These results indicate that certain CCC isoforms may be specialized to operate in acidic environments. CCC-mediated [Formula: see text] transport could bear great physiological implication given the ubiquitous distribution of these carriers.
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Bazúa-Valenti, Silvana, María Castañeda-Bueno, and Gerardo Gamba. "Physiological role of SLC12 family members in the kidney." American Journal of Physiology-Renal Physiology 311, no. 1 (July 1, 2016): F131—F144. http://dx.doi.org/10.1152/ajprenal.00071.2016.
Full textAbstract:
The solute carrier family 12, as numbered according to Human Genome Organisation (HUGO) nomenclature, encodes the electroneutral cation-coupled chloride cotransporters that are expressed in many cells and tissues; they play key roles in important physiological events, such as cell volume regulation, modulation of the intracellular chloride concentration, and transepithelial ion transport. Most of these family members are expressed in specific regions of the nephron. The Na-K-2Cl cotransporter NKCC2, which is located in the thick ascending limb, and the Na-Cl cotransporter, which is located in the distal convoluted tubule, play important roles in salt reabsorption and serve as the receptors for loop and thiazide diuretics, respectively (Thiazide diuretics are among the most commonly prescribed drugs in the world.). The activity of these transporters correlates with blood pressure levels; thus, their regulation has been a subject of intense research for more than a decade. The K-Cl cotransporters KCC1, KCC3, and KCC4 are expressed in several nephron segments, and their role in renal physiology is less understood but nevertheless important. Evidence suggests that they are involved in modulating proximal tubule glucose reabsorption, thick ascending limb salt reabsorption and collecting duct proton secretion. In this work, we present an overview of the physiological roles of these transporters in the kidney, with particular emphasis on the knowledge gained in the past few years.
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Liapis, Helen, M. Nag, and Deepak M. Kaji. "K-Cl cotransporter expression in the human kidney." American Journal of Physiology-Cell Physiology 275, no. 6 (December 1, 1998): C1432—C1437. http://dx.doi.org/10.1152/ajpcell.1998.275.6.c1432.
Full textAbstract:
The K-Cl cotransporter protein KCC1 is a membrane transport protein that mediates the coupled, electroneutral transport of K and Cl across plasma membranes. The precise cell type(s) in the kidney that express the K-Cl cotransporter have remained unknown. The aim of the present investigation was to define the distribution of KCC1 mRNA in the human kidney. We used in situ hybridization with a nonradioactive digoxigenin-labeled riboprobe. We identified abundant KCC1 mRNA expression in the epithelial cells throughout the distal and proximal renal tubular epithelium. The transporter was also expressed in glomerular mesangial cells and endothelial cells of the renal vessels. These findings suggest that the K-Cl cotransporter may have an important role in transepithelial K and Cl reabsorption.
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Pacheco-Alvarez, Diana, Diego Luis Carrillo-Pérez, Adriana Mercado, Karla Leyva-Ríos, Erika Moreno, Elisa Hernández-Mercado, María Castañeda-Bueno, Norma Vázquez, and Gerardo Gamba. "WNK3 and WNK4 exhibit opposite sensitivity with respect to cell volume and intracellular chloride concentration." American Journal of Physiology-Cell Physiology 319, no. 2 (August 1, 2020): C371—C380. http://dx.doi.org/10.1152/ajpcell.00488.2019.
Full textAbstract:
Cation-coupled chloride cotransporters (CCC) play a role in modulating intracellular chloride concentration ([Cl−]i) and cell volume. Cell shrinkage and cell swelling are accompanied by an increase or decrease in [Cl−]i, respectively. Cell shrinkage and a decrease in [Cl−]i increase the activity of NKCCs (Na-K-Cl cotransporters: NKCC1, NKCC2, and Na-Cl) and inhibit the activity of KCCs (K-Cl cotransporters: KCC1 to KCC4), wheras cell swelling and an increase in [Cl−]i activate KCCs and inhibit NKCCs; thus, it is unlikely that the same kinase is responsible for both effects. WNK1 and WNK4 are chloride-sensitive kinases that modulate the activity of CCC in response to changes in [Cl−]i. Here, we showed that WNK3, another member of the serine-threonine kinase WNK family with known effects on CCC, is not sensitive to [Cl−]i but can be regulated by changes in extracellular tonicity. In contrast, WNK4 is highly sensitive to [Cl−]i but is not regulated by changes in cell volume. The activity of WNK3 toward NaCl cotransporter is not affected by eliminating the chloride-binding site of WNK3, further confirming that the kinase is not sensitive to chloride. Chimeric WNK3/WNK4 proteins were produced, and analysis of the chimeras suggests that sequences within the WNK’s carboxy-terminal end may modulate the chloride affinity. We propose that WNK3 is a cell volume-sensitive kinase that translates changes in cell volume into phosphorylation of CCC.
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Brown, Fiona C., Ashlee J. Conway, Loretta Cerruti, Janelle E. Collinge, Catriona McLean, James S. Wiley, Ben T. Kile, Stephen M. Jane, and David J. Curtis. "Activation of the erythroid K-Cl cotransporter Kcc1 enhances sickle cell disease pathology in a humanized mouse model." Blood 126, no. 26 (December 24, 2015): 2863–70. http://dx.doi.org/10.1182/blood-2014-10-609362.
Full textAbstract:
Key Points A missense mutation in the cytoplasmic tail of Kcc1 activates K-Cl cotransporter activity by impairing phosphorylation of nearby threonines. In vivo evidence shows that activation of Kcc1 directly contributes to the pathogenesis of sickle cell disease.
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Garneau, A. P., A. A. Marcoux, R. Frenette-Cotton, F. Mac-Way, J. L. Lavoie, and P. Isenring. "Molecular insights into the normal operation, regulation, and multisystemic roles of K+-Cl− cotransporter 3 (KCC3)." American Journal of Physiology-Cell Physiology 313, no. 5 (November 1, 2017): C516—C532. http://dx.doi.org/10.1152/ajpcell.00106.2017.
Full textAbstract:
Long before the molecular identity of the Na+-dependent K+-Cl− cotransporters was uncovered in the mid-nineties, a Na+-independent K+-Cl− cotransport system was also known to exist. It was initially observed in sheep and goat red blood cells where it was shown to be ouabain-insensitive and to increase in the presence of N-ethylmaleimide (NEM). After it was established between the early and mid-nineties, the expressed sequence tag (EST) databank was found to include a sequence that was highly homologous to those of the Na+-dependent K+-Cl− cotransporters. This sequence was eventually found to code for the Na+-independent K+-Cl− cotransport function that was described in red blood cells several years before. It was termed KCC1 and led to the discovery of three isoforms called KCC2, KCC3, and KCC4. Since then, it has become obvious that each one of these isoforms exhibits unique patterns of distribution and fulfills distinct physiological roles. Among them, KCC3 has been the subject of great attention in view of its important role in the nervous system and its association with a rare hereditary sensorimotor neuropathy (called Andermann syndrome) that affects many individuals in Quebec province (Canada). It was also found to play important roles in the cardiovascular system, the organ of Corti, and circulating blood cells. As will be seen in this review, however, there are still a number of uncertainties regarding the transport properties, structural organization, and regulation of KCC3. The same is true regarding the mechanisms by which KCC3 accomplishes its numerous functions in animal cells.
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Garzón-Muvdi, Tomas, Diana Pacheco-Alvarez, Kenneth B. E. Gagnon, Norma Vázquez, José Ponce-Coria, Erika Moreno, Eric Delpire, and Gerardo Gamba. "WNK4 kinase is a negative regulator of K+-Cl− cotransporters." American Journal of Physiology-Renal Physiology 292, no. 4 (April 2007): F1197—F1207. http://dx.doi.org/10.1152/ajprenal.00335.2006.
Full textAbstract:
WNK kinases [with no lysine (K) kinase] are emerging as regulators of several membrane transport proteins in which WNKs act as molecular switches that coordinate the activity of several players. Members of the cation-coupled chloride cotransporters family (solute carrier family number 12) are one of the main targets. WNK3 activates the Na+-driven cotransporters NCC, NKCC1, and NKCC2 and inhibits the K+-driven cotransporters KCC1 to KCC4. WNK4 inhibits the activity of NCC and NKCC1, while in the presence of the STE20-related proline-alanine-rich kinase SPAK activates NKCC1. Nothing is known, however, regarding the effect of WNK4 on the K+-Cl− cotransporters. Using the heterologous expression system of Xenopus laevis oocytes, here we show that WNK4 inhibits the activity of the K+-Cl− cotransporters KCC1, KCC3, and KCC4 under cell swelling, a condition in which these cotransporters are maximally active. The effect of WNK4 requires its catalytic activity because it was lost by the substitution of aspartate 318 for alanine (WNK4-D318A) that renders WNK4 catalytically inactive. In contrast, three different WNK4 missense mutations that cause pseudohypoaldosteronism type II do not affect the WNK4-induced inhibition of KCC4. Finally, we observed that catalytically inactive WNK4-D318A is able to bypass the tonicity requirements for KCC2 and KCC3 activation in isotonic conditions. This effect is enhanced by the presence of catalytically inactive SPAK, was prevented by the presence of protein phosphatase inhibitors, and was not present in KCC1 and KCC4. Our results reveal that WNK4 regulates the activity of the K+-Cl− cotransporters expressed in the kidney.
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Zhang, Di, Sujatha M. Gopalakrishnan, Gail Freiberg, and Carol S. Surowy. "A Thallium Transport FLIPR-Based Assay for the Identification of KCC2-Positive Modulators." Journal of Biomolecular Screening 15, no. 2 (January 19, 2010): 177–84. http://dx.doi.org/10.1177/1087057109355708.
Full textAbstract:
KCC2, potassium chloride cotransporter 2, is expressed exclusively in the CNS (on inhibitory neurons) and plays a major role in maintaining appropriately low intracellular chloride levels that ensure inhibitory actions of GABAA and glycine receptors. As such, it plays a pivotal role in inhibitory mechanisms that control neuronal excitation in the CNS. KCC2 downregulation has been implicated in various excitatory disorders, such as epilepsy and neuropathic pain. Positive modulators of KCC2 expression or activity may thus provide effective therapy for these disorders. However, the identification of such agents is hindered by the lack of a high-throughput screening method. Here the authors report the development of a fluorescence-based thallium (Tl+) transport assay using a Fluorometric Imaging Plate Reader (FLIPR), in which KCC2 activity is assessed by measuring the initial rate of KCC2-mediated Tl+ transport/influx. The authors demonstrate Tl+/Cl− cotransport by KCC2, which exhibits a high apparent affinity for Tl+ and dependency on the presence of the Cl− ion. Pharmacological studies revealed anticipated effects and potencies of known KCC-positive (NEM, staurosporine) and KCC-negative (DIOA, furosemide) modulators. The authors demonstrate that the assay is robust and reproducible and can be employed in high-throughput screening for positive modulators of KCC2 as potential therapeutic agents.
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Gonsalves, Caryn S., Scott C. Crable, Sharat Chandra, and Clinton H. Joiner. "VEGF Mediated Regulation of KCl Cotransporters Gene Expression in Erythroid Cells,." Blood 118, no. 21 (November 18, 2011): 3163. http://dx.doi.org/10.1182/blood.v118.21.3163.3163.
Full textAbstract:
Abstract Abstract 3163 The KCl co-transporter (KCC) family of proteins catalyzes the electroneutral, coupled movement of K+ and Cl− ions across the plasma membrane, thereby mediating transepithelial ion transport and regulating cell volume. These proteins play an important role in disease states such as cancer, numerous neurological conditions as well as sickle cell disease (SCD). KCC activity is increased in sickle red blood cells and contributes to their dehydration, which potentiates sickling. The mechanisms of increased KCC activity and its abnormal regulation are not understood. Of the four mammalian KCC isoforms, KCC 1, 3 and 4 are expressed in erythroid cells (Crable et al. Exp Hematol. 2005; 33:624). Hiki et. al. showed that the angiogenic factor vascular endothelial growth factor (VEGF) increased KCC 3a expression in HUVEC cells (J.B.C. 274, 10661–10667, 1999). As levels of VEGF and related family member, placenta growth factor (PlGF) are elevated in sickle cell patients, we hypothesized that VEGF and PlGF may influence KCC expression in erythroid cells. RT-PCR revealed that erythroid K562 cells expressed the VEGF receptor-1 (VEGF-R1, or Flt-1) but not VEGF receptor-2, (VEGF-R2 or Flk-1). Additionally, flow cytometric analysis of WT C57Bl6 mouse bone marrow showed the presence of the Flt-1 receptor, but not Flk-1 or Flk-3 in erythroid progenitors and expression decreased with maturation. VEGF treatment (50 ng/ml) of K562 cells increased KCC 1, 3a, 3b and 4 mRNA levels; PlGF treatment increased KCC 1, 3a and 4 mRNA levels but not KCC 3b. The VEGF receptor inhibitor, SU5416, ablated the effect of VEGF. VEGF-stimulated KCC 4 expression was blocked by pharmacological inhibitors that implicated PI3 kinase, p38 MAP kinase, mTOR, JNK kinase and the transcription factor hypoxia inducible factor-1α (HIF-1α), as with other VEGF effects. Analysis of the KCC 4 promoter showed that the −875 and −90 bp promoter luciferase constructs exhibited similar levels of activity as the −1200 bp promoter construct, when compared to the promoterless reporter plasmid. Deleted constructs corresponding to −65 bp from transcription start site showed ∼90% reduced promoter activity. In silico analysis of the −90 bp region of the KCC 4 promoter showed potential binding sites for transcription factor SP-1 and HIF-1α. Binding sites for transcription factor SP-1 at positions −35 to −44 bp and −56 to −64 bp were shown to be active by site directed mutagenesis. Mutation of the HIF-1α binding site at −73 to −76 bp significantly inhibited promoter activity, whereas mutation of the HIF-1α binding site at position −21 bp to −18 bp did not have any effect on activity. Similar analysis of the KCC 3a promoter indicate potential binding sites for SP-1 at positions −8 to −4 bp and a HIF-1α binding site at position −23 to −20 bp, and the KCC 3b promoter has binding sites for HIF-1α at –9 to −6 bp and −49 to −46 bp and an AP-1 binding site at position −13 to −10 bp. Luciferase assays with KCC 3b promoter constructs indicated that the −190 bp promoter region containing HIF-1α sites at –9 to −6 bp and −49 to −46 bp and an AP-1 binding site at −13 to −10 bp contained minimal promoter required for transcription activity. Mutations within both HIF-1α binding sites attenuated promoter activity indicating a role for HIF-1α in regulating KCC 3b activity, as well. EMSA and ChIP assays with the KCC 4 promoter demonstrated that VEGF treatment of K562 cells increased HIF-1α binding to the HIF-1α sites, which was abrogated by mutating these sites. Similar results were obtained for the KCC 3a and 3b promoters.These results suggest that activation of VEGF-R1 by VEGF, and presumably its other ligand, PlGF, leads to non-hypoxic activation of HIF-1α and SP-1-mediated up-regulation of KCC3a, 3b and 4 expressions in erythroid K562 cells via its canonical signaling pathways. Variation in KCC gene expression and its modulation by cytokines and growth factors may be a source of inter-individual variation in SS RBC volume regulation and thus of phenotypic variability of SCD. Identifying the factors that modulate transcriptional control of KCC expression is important to understanding volume regulation in reticulocytes and its dysregulation in SS RBC. Disclosures: No relevant conflicts of interest to declare.
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Lytle, Christian, and Thomas McManus. "Coordinate modulation of Na-K-2Cl cotransport and K-Cl cotransport by cell volume and chloride." American Journal of Physiology-Cell Physiology 283, no. 5 (November 1, 2002): C1422—C1431. http://dx.doi.org/10.1152/ajpcell.00130.2002.
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Na-K-2Cl cotransporter (NKCC) and K-Cl cotransporter (KCC) play key roles in cell volume regulation and epithelial Cl− transport. Reductions in either cell volume or cytosolic Cl− concentration ([Cl−]i) stimulate a corrective uptake of KCl and water via NKCC, whereas cell swelling triggers KCl loss via KCC. The dependence of these transporters on volume and [Cl−]i was evaluated in model duck red blood cells. Replacement of [Cl−]i with methanesulfonate elevated the volume set point at which NKCC activates and KCC inactivates. The set point was insensitive to cytosolic ionic strength. Reducing [Cl−]i at a constant driving force for inward NKCC and outward KCC caused the cells to adopt the new set point volume. Phosphopeptide maps of NKCC indicated that activation by cell shrinkage or low [Cl−]iis associated with phosphorylation of a similar constellation of Ser/Thr sites. Like shrinkage, reduction of [Cl−]i accelerated NKCC phosphorylation after abrupt inhibition of the deactivating phosphatase with calyculin A in vivo, whereas [Cl−] had no specific effect on dephosphorylation in vitro. Our results indicate that NKCC and KCC are reciprocally regulated by a negative feedback system dually modulated by cell volume and [Cl−]. The major effect of Cl− on NKCC is exerted through the volume-sensitive kinase that phosphorylates the transport protein.
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Franco, RS, M. Palascak, H. Thompson, DL Rucknagel, and CH Joiner. "Dehydration of transferrin receptor-positive sickle reticulocytes during continuous or cyclic deoxygenation: role of KCl cotransport and extracellular calcium." Blood 88, no. 11 (December 1, 1996): 4359–65. http://dx.doi.org/10.1182/blood.v88.11.4359.4359.
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Abstract The K+ efflux that mediates sickle-cell dehydration may occur through several pathways, including two with a high capacity for mediating rapid K+ loss, KCl cotransport and the Ca(2+)-dependent K+ channel [K(Ca2+)]. The rate and pathway of red blood cell (RBC) dehydration most likely depends on cell age and hemoglobin (Hb) composition, with the presence of HbF playing an important role. Oxygenated sickle RBCs have relatively stable cell volume during incubation in vitro, whereas deoxygenated cells become dehydrated, and therefore more dense, due to activation of one or more K+ efflux pathways. In this investigation, sickle RBCs were deoxygenated either continuously or in 15-minute cycles for 4 hours, and the density increases of very young, transferrin receptor-positive (TfR+) cells and the remaining TfR-cells were determined. The contribution of KCl cotransport was estimated by replacing Cl-with NO3-.K(Ca2+) was inhibited by removal of Ca2+ or addition of charybdotoxin (ChTX). For both continuous and cyclic deoxygenation, TfR+ cells had a greater density increase when compared with TfR-cells. The lower percentage of HbF found in the TfR+ population may contribute to this difference. With continuous deoxygenation, the density shift was decreased by inhibition of K(Ca2+), but not by inhibition of KCl cotransport. With cyclic deoxygenation, the density shift was decreased in an independent, additive manner by inhibition of both pathways. Thus, cyclic deoxygenation of sickle cells under these conditions appears to activate both K(Ca2+) and the KCl cotransporter.
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Franco, RS, M. Palascak, H. Thompson, DL Rucknagel, and CH Joiner. "Dehydration of transferrin receptor-positive sickle reticulocytes during continuous or cyclic deoxygenation: role of KCl cotransport and extracellular calcium." Blood 88, no. 11 (December 1, 1996): 4359–65. http://dx.doi.org/10.1182/blood.v88.11.4359.bloodjournal88114359.
Full textAbstract:
The K+ efflux that mediates sickle-cell dehydration may occur through several pathways, including two with a high capacity for mediating rapid K+ loss, KCl cotransport and the Ca(2+)-dependent K+ channel [K(Ca2+)]. The rate and pathway of red blood cell (RBC) dehydration most likely depends on cell age and hemoglobin (Hb) composition, with the presence of HbF playing an important role. Oxygenated sickle RBCs have relatively stable cell volume during incubation in vitro, whereas deoxygenated cells become dehydrated, and therefore more dense, due to activation of one or more K+ efflux pathways. In this investigation, sickle RBCs were deoxygenated either continuously or in 15-minute cycles for 4 hours, and the density increases of very young, transferrin receptor-positive (TfR+) cells and the remaining TfR-cells were determined. The contribution of KCl cotransport was estimated by replacing Cl-with NO3-.K(Ca2+) was inhibited by removal of Ca2+ or addition of charybdotoxin (ChTX). For both continuous and cyclic deoxygenation, TfR+ cells had a greater density increase when compared with TfR-cells. The lower percentage of HbF found in the TfR+ population may contribute to this difference. With continuous deoxygenation, the density shift was decreased by inhibition of K(Ca2+), but not by inhibition of KCl cotransport. With cyclic deoxygenation, the density shift was decreased in an independent, additive manner by inhibition of both pathways. Thus, cyclic deoxygenation of sickle cells under these conditions appears to activate both K(Ca2+) and the KCl cotransporter.
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Joiner, Clinton H., R. Kirk Rettig, Maorong Jiang, and Robert S. Franco. "KCl cotransport mediates abnormal sulfhydryl-dependent volume regulation in sickle reticulocytes." Blood 104, no. 9 (November 1, 2004): 2954–60. http://dx.doi.org/10.1182/blood-2004-01-0112.
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Abstract KCl cotransport (KCC) activation by cell swelling and pH was compared in sickle (SS) and normal (AA) red blood cells (RBCs). KCC fluxes had the same relationship to mean corpuscular hemoglobin concentration (MCHC) in SS and AA RBCs when normalized to the maximal volume-stimulated (VSmax) flux (MCHC < 270 g/L [27 g/dL]). Acid-stimulated (pH 6.9) KCC flux in SS RBCs was 60% to 70% of VSmax KCC versus 20% in AA RBCs. Density gradients were used to track changes in reticulocyte MCHC during KCC-mediated regulatory volume decrease (RVD). Swelling to MCHC of 260 g/L (26 g/dL) produced Cl-dependent RVD that resulted in higher MCHC in SS than AA reticulocytes. In acid pH, RVD was also greater in SS than AA reticulocytes. Sulfhydryl reduction by dithiothreitol (DTT) lowered VSmax KCC flux in AA and SS RBCs by one third but did not alter swelling-induced RVD. DTT lowered acid-activated KCC in SS RBCs by 50% and diminished acid-induced RVD in SS reticulocytes. Thus, swelling activation of KCC is normal in SS RBCs but KCC-mediated RVD produces higher MCHC in SS than AA reticulocytes. Acid activation of KCC is exaggerated in SS RBCs and causes dehydration in SS reticulocytes. KCC response to acid stimulation was mitigated by DTT, suggesting that it arises from sulfhydryl oxidation.
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Joiner, Clinton H., R. Kirk Rettig, Maorong Jiang, Mary Risinger, and Robert S. Franco. "Urea stimulation of KCl cotransport induces abnormal volume reduction in sickle reticulocytes." Blood 109, no. 4 (October 5, 2006): 1728–35. http://dx.doi.org/10.1182/blood-2006-04-018630.
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Abstract KCl cotransport (KCC) activity contributes to pathologic dehydration in sickle (SS) red blood cells (RBCs). KCC activation by urea was measured in SS and normal (AA) RBCs as Cl-dependent Rb influx. KCC-mediated volume reduction was assessed by measuring reticulocyte cellular hemoglobin concentration (CHC) cytometrically. Urea activated KCC fluxes in fresh RBCs to levels seen in swollen cells, although SS RBCs required lower urea concentrations than did normal (AA) RBCs. Little additional KCC stimulation by urea occurred in swollen AA or SS RBCs. The pH dependence of KCC in “euvolemic” SS RBCs treated with urea was similar to that in swollen cells. Urea triggered volume reduction in SS and AA reticulocytes, establishing a higher CHC. Volume reduction was Cl dependent and was limited by the KCC inhibitor, dihydro-indenyl-oxyalkanoic acid. Final CHC depended on urea concentration, but not on initial CHC. Under all activation conditions, volume reduction was exaggerated in SS reticulocytes and produced higher CHCs than in AA reticulocytes. The sulfhydryl-reducing agent, dithiothreitol, normalized the sensitivity of KCC activation to urea in SS RBCs and mitigated the urea-stimulated volume decrease in SS reticulocytes, suggesting that the dysfunctional activity of KCC in SS RBCs was due in part to reversible sulfhydryl oxidation.
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Jennings, Michael L. "Volume-Sensitive K+/Cl− Cotransport in Rabbit Erythrocytes." Journal of General Physiology 114, no. 6 (November 15, 1999): 743–58. http://dx.doi.org/10.1085/jgp.114.6.743.
Full textAbstract:
The kinetics of activation and inactivation of K+/Cl− cotransport (KCC) have been measured in rabbit red blood cells for the purpose of determining the individual rate constants for the rate-limiting activation and inactivation events. Four different interventions (cell swelling, N-ethylmaleimide [NEM], low intracellular pH, and low intracellular Mg2+) all activate KCC with a single exponential time course; the kinetics are consistent with the idea that there is a single rate-limiting event in the activation of transport by all four interventions. In contrast to LK sheep red cells, the KCC flux in Mg2+-depleted rabbit red cells is not affected by cell volume. KCC activation kinetics were examined in cells pretreated with NEM at 0°C, washed, and then incubated at higher temperatures. The forward rate constant for activation has a very high temperature dependence (Ea ∼ 32 kCal/mol), but is not affected measurably by cell volume. Inactivation kinetics were examined by swelling cells at 37°C to activate KCC, and then resuspending at various osmolalities and temperatures to inactivate most of the transporters. The rate of transport inactivation increases steeply as cell volume decreases, even in a range of volumes where nearly all the transporters are inactive in the steady state. This finding indicates that the rate-limiting inactivation event is strongly affected by cell volume over the entire range of cell volumes studied, including normal cell volume. The rate-limiting inactivation event may be mediated by a protein kinase that is inhibited, either directly or indirectly, by cell swelling, low Mg2+, acid pH, and NEM.
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Bräuer, Margot, Eva Frei, Lutz Claes, Stephan Grissmer, and Heike Jäger. "Influence of K-Cl cotransporter activity on activation of volume-sensitive Cl– channels in human osteoblasts." American Journal of Physiology-Cell Physiology 285, no. 1 (July 2003): C22—C30. http://dx.doi.org/10.1152/ajpcell.00289.2002.
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The whole cell recording mode of the patch-clamp technique was used to study the effect of hypotonic NaCl or isotonic high-KCl solution on membrane currents in a human osteoblast-like cell line, C1. Both hypotonic NaCl or isotonic high-KCl solution activated Cl– channels expressed in these cells as described previously. The reversal potential of the induced Cl– current is more negative when activated through hypotonic NaCl solution (–47 ± 5 mV; n = 6) compared with activation through isotonic high-KCl solution (–35 ± 3 mV; n = 8). This difference can be explained by an increase in intracellular [Cl–] through the activity of a K-Cl cotransporter. Potassium aspartate was unable to activate the current, and furosemide or DIOA suppressed the increase in Cl– current induced by isotonic high-KCl solution. In addition, we used the polymerase chain reaction to demonstrate the presence of KCC1–KCC4 mRNA in the osteoblast-like cell line. From these results, we conclude that human osteoblasts express functional K-Cl cotransporters in their cell membrane that seem to be able to induce the indirect activation of volume-sensitive Cl– channels by KCl through an increase in the intracellular ion concentration followed by water influx and cell swelling.
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Glover, Mark, Annie Mercier Zuber, Nikki Figg, and Kevin M. O’Shaughnessy. "The activity of the thiazide-sensitive Na+–Cl– cotransporter is regulated by protein phosphatase PP4." Canadian Journal of Physiology and Pharmacology 88, no. 10 (October 2010): 986–95. http://dx.doi.org/10.1139/y10-080.
Full textAbstract:
Cation transport in the distal mammalian nephron relies on the SLC12 family of membrane cotransporters that include the thiazide-sensitive Na+–Cl– cotransporter (NCC). NCC is regulated through a scaffold of interacting proteins, including the WNK kinases, WNK 1 and WNK 4, which are mutated in the hypertensive Gordon’s syndrome. Dynamic regulation of NCC function by kinases must involve dephosphorylation by phosphatases, as illustrated by the role of PP1 and PP2B in the regulation of KCC members of the SLC12 family. There are 2 phosphorylation-controlled regulatory pathways for NCC: type 1, mediated by WNK4 and affecting trafficking to the surface membrane, and type 2, affecting intrinsic transporter kinetics by phosphorylation of conserved N-terminal S/T amino acids. Using the Xenopus oocyte expression system, we show that PP4 inhibits NCC activity — but not trafficking to the surface membrane — by a mechanism that requires phosphatase activity and a conserved N-terminal amino acid of NCC, threonine 58. This action is distinct from WNK4 regulation of membrane trafficking. In the mouse kidney, PP4 is selectively expressed in the distal nephron, including cells of the distal convoluted tubule cells, suggesting that PP4 may have a physiological role in regulating NCC and hence NaCl reabsorption in vivo.
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Su, Xiao-Tong, Nathan J. Klett, Avika Sharma, Charles N. Allen, Wen-Hui Wang, Chao-Ling Yang, and David H. Ellison. "Distal convoluted tubule Cl− concentration is modulated via K+ channels and transporters." American Journal of Physiology-Renal Physiology 319, no. 3 (September 1, 2020): F534—F540. http://dx.doi.org/10.1152/ajprenal.00284.2020.
Full textAbstract:
Cl−-sensitive with-no-lysine kinase (WNK) plays a key role in regulating the thiazide-sensitive Na+-Cl− cotransporter (NCC) in the distal convoluted tubule (DCT). Cl− enters DCT cells through NCC and leaves the cell across the basolateral membrane via the Cl− channel ClC-K2 or K+-Cl− cotransporter (KCC). While KCC is electroneutral, Cl− exit via ClC-K2 is electrogenic. Therefore, an alteration in DCT basolateral K+ channel activity is expected to influence Cl− movement across the basolateral membrane. Although a role for intracellular Cl− in the regulation of WNK and NCC has been established, intracellular Cl− concentrations ([Cl−]i) have not been directly measured in the mammalian DCT. Therefore, to measure [Cl−]i in DCT cells, we generated a transgenic mouse model expressing an optogenetic kidney-specific Cl-Sensor and measured Cl− fluorescent imaging in the isolated DCT. Basal measurements indicated that the mean [Cl−]i was ~7 mM. Stimulation of Cl− exit with low-Cl− hypotonic solutions decreased [Cl−]i, whereas inhibition of KCC by DIOA or inhibition of ClC-K2 by NPPB increased [Cl−]i, suggesting roles for both KCC and ClC-K2 in the modulation of [Cl−]i . Blockade of basolateral K+ channels (Kir4.1/5.1) with barium significantly increased [Cl−]i. Finally, a decrease in extracellular K+ concentration transiently decreased [Cl−]i, whereas raising extracellular K+ transiently increased [Cl−]i, further suggesting a role for Kir4.1/5.1 in the regulation of [Cl−]i. We conclude that the alteration in ClC-K2, KCC, and Kir4.1/5.1 activity influences [Cl−]i in the DCT.
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Muzyamba, M. C., P. F. Speake, and J. S. Gibson. "Oxidants and regulation of K+-Cl−cotransport in equine red blood cells." American Journal of Physiology-Cell Physiology 279, no. 4 (October 1, 2000): C981—C989. http://dx.doi.org/10.1152/ajpcell.2000.279.4.c981.
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The effect of oxidants on K+-Cl−cotransport (KCC) was investigated in equine red blood cells. Carbon monoxide mimicked O2. The substituted benzaldehyde, 12C79 (5 mM), markedly increased O2affinity. In N2, however, O2saturation was low (<10%) but KCC remained active. Nitrite (NO2−) oxidized heme to methemoglobin (metHb). High concentrations of NO2−(1 and 5 mM vs. 0.5 mM) increased KCC activity above control levels; it became O2independent but remained sensitive to other stimuli. 1-Chloro-2,4-dinitrobenzene (1–3 mM) depleted reduced glutathione (GSH). Prolonged exposure (60–120 min, 1 mM) or high concentrations (3 mM) stimulated an O2-independent KCC activity; short exposures and low concentrations (30 min, 0.5 or 1 mM) did not. The effect of these manipulations was correlated with changes in GSH and metHb concentrations. An oxy conformation of Hb was necessary for KCC activation. An increase in its activity over the level found in oxygenated control cells required both accumulation of metHb and depletion of GSH. Findings are relevant to understanding the physiology and pathology of regulation of KCC.
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Lauf, Peter K., Neelima Sharma, and Norma C. Adragna. "Kinetic studies of K-Cl cotransport in cultured rat vascular smooth muscle cells." American Journal of Physiology-Cell Physiology 316, no. 2 (February 1, 2019): C274—C284. http://dx.doi.org/10.1152/ajpcell.00002.2017.
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During aging, and development of atherosclerosis and cardiovascular disease (CVD), aortic vascular smooth muscle cells (VSMCs) transition from healthy contractile to diseased synthetic phenotypes. K-Cl cotransport (KCC) maintains cell volume and ion homeostasis in growth and differentiation, and hence is important for VSMC proliferation and migration. Therefore, KCC activity may play a role in the contractile-to-synthetic VSMC phenotypic transition. Early, medium, and late synthetic passage VSMCs were tested for specific cytoskeletal protein expression. KCC-mediated ouabain- and bumetanide-insensitive Rb+ (a K+ congener) influx was determined as Cl−-dependent Rb+ influx at different external Rb+ and Cl− ion concentrations, [Rb+]o and [Cl−]o. Expressions of the cytoskeletal proteins α-actin, vimentin, and desmin fell from early through late synthetic VSMCs. KCC kinetic parameters, such as maximum velocity ( Vm), and apparent Cl− and Rb+ affinities ( Km), were calculated with Lineweaver-Burk, Hanes-Woolf, and Hill approximations. Vm values of both Rb+- and Cl−-dependent influxes were of equal magnitude, commensurate with a KCC stoichiometry of unity, and rose threefold from early to late synthetic VSMCs. Hill coefficients for Rb+ and Cl− correlated with cell passage number, suggesting increased KCC ligand cooperativity. However, Km values for [Cl−]o were strikingly bimodal with 60–80 mM in early, ~20–30 mM in medium, and 60 mM in late passage cells. In contrast, Km values for [Rb+]o remained steady at ~17 mM. Since total KCC isoform expression was similar with cell passage, structure/function changes of the KCC signalosome may accompany the transition of aortic VSMCs from a healthy to a diseased phenotype.
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Bize, Isabel, Samara Taher, and Carlo Brugnara. "Regulation of K-Cl cotransport during reticulocyte maturation and erythrocyte aging in normal and sickle erythrocytes." American Journal of Physiology-Cell Physiology 285, no. 1 (July 2003): C31—C38. http://dx.doi.org/10.1152/ajpcell.00447.2002.
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The age/density-dependent decrease in K-Cl cotransport (KCC), PP1 and PP2A activities in normal and sickle human erythrocytes, and the effect of urea, a known KCC activator, were studied using discontinuous, isotonic gradients. In normal erythrocytes, the densest fraction (d ∼33.4 g/dl) has only about ∼5% of the KCC and 4% of the membrane (mb)-PP1 activities of the least-dense fraction (d ∼24.7 g/dl). In sickle and normal erythrocytes, density-dependent decreases for mb-PP1 activity were similar (d50% 28.1 ± 0.4 vs. 27.2 ± 0.2 g/dl, respectively), whereas those for KCC activity were not (d50% 31.4 ± 0.9 vs. 26.8 ± 0.3 g/dl, respectively, P = 0.004). Excluding the 10% least-dense cells, a very tight correlation exists between KCC and mb-PP1 activities in normal ( r2 = 0.995) and sickle erythrocytes ( r2 = 0.93), but at comparable mb-PP1 activities, KCC activity is higher in sickle erythrocytes, suggesting a defective, mb-PP1-independent KCC regulation. In normal, least-dense but not in densest cells, urea stimulates KCC (two- to fourfold) and moderately increases mb-PP1 (20–40%). Thus mb-PP1 appears to mediate part of urea-stimulated KCC activity.
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Velázquez, Heino, and Teresa Silva. "Cloning and localization of KCC4 in rabbit kidney: expression in distal convoluted tubule." American Journal of Physiology-Renal Physiology 285, no. 1 (July 2003): F49—F58. http://dx.doi.org/10.1152/ajprenal.00389.2002.
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Cl-dependent K secretion is a feature of renal distal tubules and collecting ducts. Recent cloning and identification of K-Cl cotransporter proteins led us to search for additional novel KCC isoforms expressed in the renal distal nephron. A human expressed sequence tag (EST) with high homology to KCC1 was identified. The rabbit isoform was cloned by homology using degenerate primers and rapid amplification of cDNA ends (RACE). Our isoform is the rabbit homologue of mouse and human KCC4 published previously. The 4.35-kb rabbit KCC4 cDNA encodes a protein of 1,106 amino acids. Antibodies were generated to both NH2-terminal and COOH-terminal fusion proteins. Northern and Western blot analyses showed widespread mRNA and protein expression in many rabbit organs, in renal cortex, outer medulla, and inner medulla but not in skeletal muscle. Immunohistochemical localization of KCC4 showed expression exclusively along the basolateral membrane in many nephron segments. The distal convoluted tubule and connecting tubule exhibited the highest level of KCC4 immunoreactivity, followed by the medullary thick ascending limb. A low level of immunoreactivity was detected in the proximal tubule and collecting ducts. We postulate that KCC4 mediates potassium and chloride exit from the cell and may play an important role in salt absorption by the distal convoluted tubule.
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Lauf, Peter K., Sandeep Misri, Ameet A. Chimote, and Norma C. Adragna. "Apparent intermediate K conductance channel hyposmotic activation in human lens epithelial cells." American Journal of Physiology-Cell Physiology 294, no. 3 (March 2008): C820—C832. http://dx.doi.org/10.1152/ajpcell.00375.2007.
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This study explores the nature of K fluxes in human lens epithelial cells (LECs) in hyposmotic solutions. Total ion fluxes, Na-K pump, Cl-dependent Na-K-2Cl (NKCC), K-Cl (KCC) cotransport, and K channels were determined by 85Rb uptake and cell K (Kc) by atomic absorption spectrophotometry, and cell water gravimetrically after exposure to ouabain ± bumetanide (Na-K pump and NKCC inhibitors), and ion channel inhibitors in varying osmolalities with Na, K, or methyl-d-glucamine and Cl, sulfamate, or nitrate. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blot analyses, and immunochemistry were also performed. In isosmotic (300 mosM) media ∼90% of the total Rb influx occurred through the Na-K pump and NKCC and ∼10% through KCC and a residual leak. Hyposmotic media (150 mosM) decreased Kc by a 16-fold higher K permeability and cell water, but failed to inactivate NKCC and activate KCC. Sucrose replacement or extracellular K to >57 mM, but not Rb or Cs, in hyposmotic media prevented Kc and water loss. Rb influx equaled Kc loss, both blocked by clotrimazole (IC50 ∼25 μM) and partially by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) inhibitors of the IK channel KCa3.1 but not by other K channel or connexin hemichannel blockers. Of several anion channel blockers (dihydro-indenyl)oxy]alkanoic acid (DIOA), 4-2(butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)oxybutyric acid (DCPIB), and phloretin totally or partially inhibited Kc loss and Rb influx, respectively. RT-PCR and immunochemistry confirmed the presence of KCa3.1 channels, aside of the KCC1, KCC2, KCC3 and KCC4 isoforms. Apparently, IK channels, possibly in parallel with volume-sensitive outwardly rectifying Cl channels, effect regulatory volume decrease in LECs.
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Lauf, P. K., N. C. Adragna, N. Dupre, J. P. Bouchard, and G. A. Rouleau. "K–Cl cotransport in red blood cells from patients with KCC3 isoform mutantsThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease." Biochemistry and Cell Biology 84, no. 6 (December 2006): 1034–44. http://dx.doi.org/10.1139/o06-203.
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Red blood cells (RBCs) possess the K–Cl cotransport (KCC) isoforms 1, 3, and 4. Mutations within a given isoform may affect overall KCC activity. In a double-blind study, we analyzed, with Rb as a K congener, K fluxes (total flux, ouabain-sensitive Na+/K+ pump, and bumetanide-sensitive Na–K–2Cl cotransport, Cl-dependent, and ouabain- and bumetanide-insensitive KCC with or without stimulation by N-ethylmaleimide (NEM) and staurosporine or Mg removal, and basal channel-mediated fluxes, osmotic fragility, and ions and water in the RBCs of 8 controls, and of 8 patients with hereditary motor and sensory neuropathy with agenesis of corpus callosum (HMSN–ACC) with defined KCC3 mutations (813FsX813 and Phe529FsX532) involving the truncations of 338 and 619 C-terminal amino acids, respectively. Water and ion content and, with one exception, mean osmotic fragility, as well as K fluxes without stimulating agents, were similar in controls and HMSN–ACC RBCs. However, the NEM-stimulated KCC was reduced 5-fold (p < 0.0005) in HMSN–ACC vs control RBCs, as a result of a lower Vmax (p < 0.05) rather than a lower Km (p = 0.109), accompanied by corresponding differences in Cl activation. Low intracellular Mg activated KCC in 6 out of 7 controls vs 1 out of 6 HMSN–ACC RBCs, suggesting that regulation is compromised. The lack of differences in staurosporine-activated KCC indicates different action mechanisms. Thus, in HMSN–ACC patients with KCC3 mutants, RBC KCC activity, although indistinguishable from that of the control group, responded differently to biochemical stressors, such as thiol alkylation or Mg removal, thereby indirectly indicating an important contribution of KCC3 to overall KCC function and regulation.
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Turner, R. J., and J. N. George. "Bumetanide binding to the parotid NaCl/KCl cotransporter." Comparative Biochemistry and Physiology Part A: Physiology 90, no. 4 (January 1988): 836. http://dx.doi.org/10.1016/0300-9629(88)90804-3.
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Joiner, Clinton H., R. Kirk Rettig, Mary Palascak, Amher Sheriff, Robert M. Cohen, and Robert S. Franco. "Mature Sickle and Normal Red Cells Exhibit Regulatory Volume Decrease Activated by Urea and Mediated by KCl Cotransport." Blood 106, no. 11 (November 16, 2005): 2321. http://dx.doi.org/10.1182/blood.v106.11.2321.2321.
Full textAbstract:
Abstract KCl Cotransport (KCC) is active in normal (AA) reticulocytes and overly active in sickle (SS) reticulocytes. Cell swelling activates KCC and induces a powerful regulatory volume decrease (RVD) in reticulocytes, which increases cellular hemoglobin concentration (CHC) to new steady state values that are higher in SS than AA cells (Blood2004;104(9):2954–60). We recently showed that urea (300–900 mM), which strongly activates KCC, also induces an intense RVD with even higher final CHC values (SS>AA) (Blood2004; 104 (11): 976a). Because KCC activity is high in reticulocyte-rich samples in both SS and AA blood, KCC activity has been assumed to be minimal in mature cells. We now report that mature RBC exhibit RVD stimulated by urea and mediated by KCC. AA and SS RBC were washed in HBS and treated with nystatin to increase cation content and decrease CHC to 22–24 gm/dl. During incubation at 37o in HBS (145 mM NaCl, 5 KCl, 1 MgCl2, 10 glucose, 20 HEPES, pH 7.4) ± 600 mM urea, timed samples were taken into iced HBS, washed, and kept on ice until analyzed later that day on an Advia 120 automated cell counter, which reports frequency distributions for CHC of both mature RBC and reticulocytes. As previously reported, within 30 min reticulocytes achieved a new steady state CHC which was higher for SS than AA cells, though the speed of RVD was similar. Surprisingly, mean CHC of mature (non-reticulocyte) RBC in both AA and SS blood also increased upon incubation with urea. RVD in mature cells was slower than in reticulocytes and was apparently incomplete after 2 hours. RVD in mature RBC was completely abrogated (CHC was stable) in the absence of Cl- (sulfamate substitution) or in the presence of 100 uM DIOA (dihydro-indenyl-oxy-alkanoic acid), both of which inhibit KCC activity. Whereas reticulocyte CHC frequency distributions after urea-stimulated KCC-mediated RVD showed a single population, CHC distributions for mature RBC revealed two distinct sub-populations: One in which CHC changed little during incubation and a second which achieved a CHC similar to that achieved by reticulocytes after RVD. The relative size of the volume regulating (high CHC) sub-population increased steadily throughout the incubation, which was responsible for the progressive increase in mean CHC values. The high CHC sub-population was not apparent when cells were incubated in Cl- free media or with DIOA, indicating that RVD was mediated by KCC. After 2 hours incubation, 67 ± 8 % of SS RBC had shifted to higher CHC, compared to 37 ± 11 % of mature AA RBC (p<<0.001 by t-test). The progressive change in CHC histograms during incubation was consistent with cells achieving the same final CHC values at various rates. In preliminary studies with biotin-labeled AA cells ageing in vivo, urea-stimulated RVD in mature cells diminished with time, but persisted through most of RBC lifespan. These data indicate that the KCl cotransporter remains in the membrane of mature AA RBC, and is capable of producing RVD under the strong stimulation of urea. In SS RBC, which have shorter lifespan, a majority of non-reticulocytes retain urea-stimulated KCC activity.
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Joiner, Clinton H., R. Kirk Rettig, Maorong Jiang, and Robert S. Franco. "Activation of KCI Cotransport by Urea Induces Dehydration in Both Sickle and Normal Reticulocytes." Blood 104, no. 11 (November 16, 2004): 3591. http://dx.doi.org/10.1182/blood.v104.11.3591.3591.
Full textAbstract:
Abstract KCl Cotransport (KCC) is highly expressed in sickle red blood cells (SS RBC) and recent data have demonstrated its abnormal response to cell swelling and acid pH. We showed that the final MCHC achieved by SS reticulocytes upon activation of KCC by these stimuli was higher than that of normal (AA) reticulocytes (Joiner et al, Blood, in Press). Here we report studies examining the sensitivity of KCC to activation by urea at concentrations present in the kidney and the effect of urea stimulation of KCC on reticulocyte MCHC. KCC fluxes were assayed as Rb uptake over 20 min in isotonic saline solutions buffered with HEPES to pH 7.4 (37°C) containing 0.1 mM ouabain, and 0.01 mM bumetanide, with 27 mM RbCl replacing equimolar NaCl. Under these conditions > 95 % of Rb uptake was Cl-dependent (assessed by sulfamate replacement of Cl). The maximal volume-stimulated KCC flux (VSmax KCC) was measured for each sample in cells swollen isotonically to MCHC < 27 gm/dl (nystatin method). Urea (100 to 1000 mM) increased osmolality of buffers, but did not alter initial MCHC. MCHC of reticulocytes (detected by flow cytometry) was tracked by measuring density changes on calibrated OPTIprep® gradients. Cl-dependent, ouabain- and bumetanide-insensitive Rb influx in both AA and SS RBC was increased by urea, reaching a plateau at 1000 mM urea that was similar to VSmax KCC. SS RBC were more sensitive to urea stimulation than AA RBC: 50% VSSmax KCC was achieved at 330 mM urea in SS RBC vs 550 mM in AA RBC. This effect was sulfhydryl dependent: exposure to the reducing agent dithiothreitol (preincubation for 30 min at 37°C with 10 mM DTT, then 1 mM in flux media) normalized the response to urea in SS RBC, with no effect in AA RBC. When swollen to MCHC 30 gm/dl, SS and AA retics exhibited Regulatory Volume Decrease (RVD) which increased MCHC. RVD in both AA and SS retics was abolished by incubation in sulfamate media, indicating mediation by KCC. As previously reported, final MCHC achieved after two hours incubation by SS retics was greater than AA retics (see Table, Control). Final Reticulocyte MCHC, gm/dl [mean (SD), n = 3] AA SS p (AA vs SS) Control 31.9 (0.7) 34.7 (1.2 0.03 Urea 35.3 (0.5) 37.8 (0.3) 0.002 p (Control vs Urea) 0.005 0.03 Urea (600 mM) enhanced RVD in both AA retics and SS retics. Sulfhydryl reduction with DTT had no effect on urea-stimulated RVD in SS reticulocytes. RVD stimulated by urea was complete within 60 minutes, and was irreversible: additional incubation without urea did not lower MCHC. The partially dehydrating effect of brief (10 min) exposure to urea was also irreversible, and was cumulative: cells exposed to two 10 min exposures to high urea (600 mM), separated by 10 min at a low, non-stimulating concentration of urea (100 mM), yielded the same MCHC as a continuous 20 min exposure. These data demonstrate that urea, at concentrations found in the renal medulla, is a powerful stimulant of KCC and intiates a striking RVD in reticulocytes. To the extent that intermittent stimulation of KCC by urea in the kidney occurs in vivo, this could contribute an exaggerated RVD resulting in dehydration of SS reticulocytes.
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Sun, Hui, Omkar Paudel, and James S. K. Sham. "Increased intracellular Cl− concentration in pulmonary arterial myocytes is associated with chronic hypoxic pulmonary hypertension." American Journal of Physiology-Cell Physiology 321, no. 2 (August 1, 2021): C297—C307. http://dx.doi.org/10.1152/ajpcell.00172.2021.
Full textAbstract:
Chloride channels play an important role in regulating smooth muscle contraction and proliferation, and contribute to the enhanced constriction of pulmonary arteries (PAs) in pulmonary hypertension (PH). The intracellular Cl− concentration ([Cl−]i), tightly regulated by various Cl− transporters, determines the driving force for Cl− conductance, thereby the functional outcome of Cl− channel activation. This study characterizes for the first time the expression profile of Cl− transporters/exchangers in PA smooth muscle and provides the first evidence that the intracellular Cl− homeostasis is altered in PA smooth muscle cells (PASMCs) associated with chronic hypoxic PH (CHPH). Quantitative RT-PCR revealed that the endothelium-denuded intralobar PA of rats expressed Slc12a gene family-encoded Na-K-2Cl cotransporter 1 (NKCC1), K-Cl cotransporters (KCC) 1, 3, and 4, and Slc4a gene family-encoded Na+-independent and Na+-dependent Cl−/HCO3− exchangers. Exposure of rats to chronic hypoxia (10% O2, 3 wk) caused CHPH and selectively increased the expression of Cl−-accumulating NKCC1 and reduced the Cl−-extruding KCC4. The intracellular Cl− concentration ([Cl−]i) averaged at 45 mM and 47 mM in normoxic PASMCs as determined by fluorescent indicator MEQ and by gramicidin-perforated patch-clamp technique, respectively. The ([Cl−]i was increased by ∼10 mM in PASMCs of rats with CHPH. Future studies are warranted to further establish the hypothesis that the altered intracellular Cl− homeostasis contributes to the pathogenesis of CHPH.
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Jennings, Michael L., and Mark F. Adame. "Direct estimate of 1:1 stoichiometry of K+-Cl−cotransport in rabbit erythrocytes." American Journal of Physiology-Cell Physiology 281, no. 3 (September 1, 2001): C825—C832. http://dx.doi.org/10.1152/ajpcell.2001.281.3.c825.
Full textAbstract:
This work was undertaken to obtain a direct measure of the stoichiometry of Na+-independent K+-Cl−cotransport (KCC), with rabbit red blood cells as a model system. To determine whether86Rb+can be used quantitatively as a tracer for KCC,86Rb+and K+effluxes were measured in parallel after activation of KCC with N-ethylmaleimide (NEM). The rate constant for NEM-stimulated K+efflux into isosmotic NaCl was smaller than that for86Rb+by a factor of 0.68 ± 0.11 (SD, n = 5). This correction factor was used in all other experiments to calculate the K+efflux from the measured86Rb+efflux. To minimize interference from the anion exchanger, extracellular Cl−was replaced with SO[Formula: see text], and 4,4′-diisothiocyanothiocyanatodihydrostilbene-2,2′-disulfonic acid was present in the flux media. The membrane potential was clamped near 0 mV with the protonophore 2,4-dinitrophenol. The Cl−efflux at 25°C under these conditions is ∼100,000-fold smaller than the uninhibited Cl−/Cl−exchange flux and is stimulated ∼2-fold by NEM. The NEM-stimulated36Cl−flux is inhibited by okadaic acid and calyculin A, as expected for KCC. The ratio of the NEM-stimulated K+to Cl−efflux is 1.12 ± 0.26 (SD, n = 5). We conclude that K+-Cl−cotransport in rabbit red blood cells has a stoichiometry of 1:1.