RESUMEN
Red cell volume is a major determinant of HbS concentration in sickle cell disease. Cellular deoxy-HbS concentration determines the delay time, the interval between HbS deoxygenation and deoxy-HbS polymerization. Major membrane transporter protein determinants of sickle red cell volume include the SLC12/KCC K-Cl cotransporters KCC3/SLC12A6 and KCC1/SLC12A4, and the KCNN4/KCa3.1 Ca2+-activated K+ channel (Gardos channel). Among standard inhibitors of KCC-mediated K-Cl cotransport, only [(dihydroindenyl)oxy]acetic acid (DIOA) has been reported to lack inhibitory activity against the related bumetanide-sensitive erythroid Na-K-2Cl cotransporter NKCC1/SLC12A2. DIOA has been often used to inhibit K-Cl cotransport when studying the expression and regulation of other K+ transporters and K+ channels. We report here that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also abrogate activity of the KCNN4/KCa3.1 Gardos channel in human and mouse red cells and in human sickle red cells. DIOA inhibition of A23187-stimulated erythroid K+ uptake (Gardos channel activity) was chloride-independent and persisted in mouse red cells genetically devoid of the principal K-Cl cotransporters KCC3 and KCC1. DIOA also inhibited YODA1-stimulated, chloride-independent erythroid K+ uptake. In contrast, DIOA exhibited no inhibitory effect on K+ influx into A23187-treated red cells of Kcnn4-/- mice. DIOA inhibition of human KCa3.1 was validated (IC50 42 µM) by whole cell patch clamp in HEK-293 cells. RosettaLigand docking experiments identified a potential binding site for DIOA in the fenestration region of human KCa3.1. We conclude that DIOA at concentrations routinely used to inhibit K-Cl cotransport can also block the KCNN4/KCa3.1 Gardos channel in normal and sickle red cells.
Asunto(s)
Anemia de Células Falciformes , Simportadores , Ácido Acético , Anemia de Células Falciformes/tratamiento farmacológico , Animales , Calcimicina , Cloruros/metabolismo , Células HEK293 , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Ratones , Potasio/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12 , Simportadores/metabolismoRESUMEN
The molecular identity of Psickle, the deoxygenation-activated cation conductance of the human sickle erythrocyte, remains unknown. We observed in human sickle red cells that inhibitors of TRPA1 and TRPV1 inhibited Psickle, whereas a TRPV1 agonist activated a Psickle-like cation current. These observations prompted us to test the roles of TRPV1 and TRPA1 in Psickle in red cells of the SAD mouse model of sickle cell disease. We generated SAD mice genetically deficient in either TRPV1 or TRPA1. SAD;Trpv1-/- and SAD;Trpa1-/- mice were indistinguishable in appearance, hematological indices, and osmotic fragility from SAD mice. We found that deoxygenation-activated cation currents remained robust in SAD;Trpa1-/- and SAD;Trpv1-/- mice. In addition, 45Ca2+ influx into SAD mouse red cells during prolonged deoxygenation was not reduced in red cells from SAD;Trpa1-/- and SAD;Trpv1-/- mice. We conclude that the nonspecific cation channels TRPA1 and TRPV1 are not required for deoxygenation to stimulate Psickle-like activity in red cells of the SAD mouse model of sickle cell disease. (159).
Asunto(s)
Anemia de Células Falciformes/metabolismo , Eritrocitos/patología , Canal Catiónico TRPA1/metabolismo , Canales Catiónicos TRPV/metabolismo , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/patología , Animales , Cationes/metabolismo , Modelos Animales de Enfermedad , Eritrocitos/metabolismo , Eliminación de Gen , Humanos , Ratones , Ratones Noqueados , Canal Catiónico TRPA1/genética , Canales Catiónicos TRPV/genéticaRESUMEN
Distal renal tubular acidosis is a rare renal tubular disorder characterized by hyperchloremic metabolic acidosis and impaired urinary acidification. Mutations in three genes (ATP6V0A4, ATP6V1B1 and SLC4A1) constitute a monogenic causation in 58-70% of familial cases of distal renal tubular acidosis. Recently, mutations in FOXI1 have been identified as an additional cause. Therefore, we hypothesized that further monogenic causes of distal renal tubular acidosis remain to be discovered. Panel sequencing and/or whole exome sequencing was performed in a cohort of 17 families with 19 affected individuals with pediatric onset distal renal tubular acidosis. A causative mutation was detected in one of the three "classical" known distal renal tubular acidosis genes in 10 of 17 families. The seven unsolved families were then subjected to candidate whole exome sequencing analysis. Potential disease causing mutations in three genes were detected: ATP6V1C2, which encodes another kidney specific subunit of the V-type proton ATPase (1 family); WDR72 (2 families), previously implicated in V-ATPase trafficking in cells; and SLC4A2 (1 family), a paralog of the known distal renal tubular acidosis gene SLC4A1. Two of these mutations were assessed for deleteriousness through functional studies. Yeast growth assays for ATP6V1C2 revealed loss-of-function for the patient mutation, strongly supporting ATP6V1C2 as a novel distal renal tubular acidosis gene. Thus, we provided a molecular diagnosis in a known distal renal tubular acidosis gene in 10 of 17 families (59%) with this disease, identified mutations in ATP6V1C2 as a novel human candidate gene, and provided further evidence for phenotypic expansion in WDR72 mutations from amelogenesis imperfecta to distal renal tubular acidosis.
Asunto(s)
Acidosis Tubular Renal , ATPasas de Translocación de Protón Vacuolares , Acidosis Tubular Renal/genética , Proteína 1 de Intercambio de Anión de Eritrocito , Niño , Antiportadores de Cloruro-Bicarbonato , Análisis Mutacional de ADN , Factores de Transcripción Forkhead , Humanos , Mutación , ATPasas de Translocación de Protón Vacuolares/genética , Secuenciación del ExomaRESUMEN
ß-thalassemia (ß-Thal) is caused by defective ß-globin production leading to globin chain imbalance, aggregation of free alpha chain in developing erythroblasts, reticulocytes, and mature circulating red blood cells. The hypochromic thalassemic red cells exhibit increased cell dehydration in association with elevated K+ leak and increased K-Cl cotransport activity, each of which has been linked to globin chain imbalance and related oxidative stress. We therefore tested the effect of genetic inactivation of K-Cl cotransporters KCC1 and KCC3 in a mouse model of ß-thalassemia intermedia. In the absence of these transporters, the anemia of ß-Thal mice was ameliorated, in association with increased MCV and reductions in CHCM and hyperdense cells, as well as in spleen size. The resting K+ content of ß-Thal red cells was greatly increased, and Thal-associated splenomegaly slightly decreased. Lack of KCC1 and KCC3 activity in Thal red cells reduced red cell density and improved ß-Thal-associated osmotic fragility. We conclude that genetic inactivation of K-Cl cotransport can reverse red cell dehydration and partially attenuate the hematologic phenotype in a mouse model of ß-thalassemia.
Asunto(s)
Simportadores/genética , Talasemia beta/genética , Anemia/prevención & control , Animales , Deshidratación , Modelos Animales de Enfermedad , Eritrocitos/química , Eritrocitos/patología , Ratones , Fragilidad Osmótica , Fenotipo , Esplenomegalia , Simportadores/metabolismo , Talasemia beta/patología , Cotransportadores de K ClRESUMEN
Hereditary xerocytosis (HX) is caused by missense mutations in either the mechanosensitive cation channel PIEZO1 or the Ca2+-activated K+ channel KCNN4. All HX-associated KCNN4 mutants studied to date have revealed increased current magnitude and red cell dehydration. Baseline KCNN4 activity was increased in HX red cells heterozygous for KCNN4 mutant V282M. However, HX red cells maximally stimulated by Ca2+ ionophore A23187 or by PMCA Ca2+-ATPase inhibitor orthovanadate displayed paradoxically reduced KCNN4 activity. This reduced Ca2+-stimulated mutant KCNN4 activity in HX red cells was associated with unchanged sensitivity to KCNN4 inhibitor senicapoc and KCNN4 activator Ca2+, with slightly elevated Ca2+ uptake and reduced PMCA activity, and with decreased KCNN4 activation by calpain inhibitor PD150606. The altered intracellular monovalent cation content of HX red cells prompted experimental nystatin manipulation of red cell Na and K contents. Nystatin-mediated reduction of intracellular K+ with corresponding increase in intracellular Na+ in wild-type cells to mimic conditions of HX greatly suppressed vanadate-stimulated and A23187-stimulated KCNN4 activity in those wild-type cells. However, conferral of wild-type cation contents on HX red cells failed to restore wild-type-stimulated KCNN4 activity to those HX cells. The phenotype of reduced, maximally stimulated KCNN4 activity was shared by HX erythrocytes expressing heterozygous PIEZO1 mutants R2488Q and V598M, but not by HX erythrocytes expressing heterozygous KCNN4 mutant R352H or PIEZO1 mutant R2456H. Our data suggest that chronic KCNN4-driven red cell dehydration and intracellular cation imbalance can lead to reduced KCNN4 activity in HX and wild-type red cells.
Asunto(s)
Anemia Hemolítica Congénita/sangre , Eritrocitos/metabolismo , Hidropesía Fetal/sangre , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/sangre , Potasio/sangre , Anemia Hemolítica Congénita/diagnóstico , Anemia Hemolítica Congénita/genética , Señalización del Calcio , Estudios de Casos y Controles , Índices de Eritrocitos , Predisposición Genética a la Enfermedad , Humanos , Hidropesía Fetal/diagnóstico , Hidropesía Fetal/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Canales Iónicos/sangre , Canales Iónicos/genética , Potenciales de la Membrana , Mutación Missense , Fragilidad Osmótica , FenotipoRESUMEN
Excessive red cell dehydration contributes to the pathophysiology of sickle cell disease (SCD). The densest fraction of sickle red cells (with the highest corpuscular hemoglobin concentration) undergoes the most rapid polymerization of deoxy-hemoglobin S, leading to accelerated cell sickling and increased susceptibility to endothelial activation, red cell adhesion, and vaso-occlusion. Increasing red cell volume in order to decrease red cell density can thus serve as an adjunct therapeutic goal in SCD. Regulation of circulating mouse red cell volume and density is mediated largely by the Gardos channel, KCNN4, and the K-Cl cotransporters, KCC3 and KCC1. Whereas inhibition of the Gardos channel in subjects with sickle cell disease increased red cell volume, decreased red cell density, and improved other hematological indices in subjects with SCD, specific KCC inhibitors have not been available for testing. We therefore investigated the effect of genetic inactivation of KCC3 and KCC1 in the SAD mouse model of sickle red cell dehydration, finding decreased red cell density and improved hematological indices. We describe here generation of mice genetically deficient in the three major red cell volume regulatory gene products, KCNN4, KCC3, and KCC1 in C57BL6 non-sickle and SAD sickle backgrounds. We show that combined loss-of-function of all three gene products in SAD mice leads to incrementally increased MCV, decreased CHCM and % hyperchromic cells, decreased red cell density (phthalate method), increased resistance to hypo-osmotic lysis, and increased cell K content. The data show that combined genetic deletion of the Gardos channel and K-Cl cotransporters in a mouse SCD model decreases red cell density and improves several hematological parameters, supporting the strategy of combined pharmacological inhibition of these ion transport pathways in the adjunct treatment of human SCD.
Asunto(s)
Anemia de Células Falciformes/sangre , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Animales , Tamaño de la Célula/efectos de los fármacos , Deshidratación/tratamiento farmacológico , Modelos Animales de Enfermedad , Eritrocitos/patología , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/deficiencia , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Ratones , Simportadores/deficiencia , Simportadores/genética , Cotransportadores de K ClRESUMEN
Senicapoc, a Gardos channel inhibitor, prevented erythrocyte dehydration in clinical trials of patients with sickle cell disease. We tested the hypothesis that senicapoc-induced blockade of the Gardos channel inhibits Plasmodium growth. Senicapoc inhibited in vitro growth of human and primate plasmodia during the clinical blood stage. Senicapoc treatment suppressed P. yoelii parasitemia in vivo in C57BL/6 mice. The reassuring safety and biochemical profile of senicapoc encourage its use in antimalarial development.
Asunto(s)
Acetamidas/farmacología , Antimaláricos/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium knowlesi/efectos de los fármacos , Plasmodium yoelii/efectos de los fármacos , Compuestos de Tritilo/farmacología , Trofozoítos/efectos de los fármacos , Animales , Transporte Biológico/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Eritrocitos/parasitología , Interacciones Huésped-Parásitos , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Macaca mulatta , Ratones , Ratones Endogámicos C57BL , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Plasmodium knowlesi/crecimiento & desarrollo , Plasmodium knowlesi/metabolismo , Plasmodium yoelii/crecimiento & desarrollo , Plasmodium yoelii/metabolismo , Trofozoítos/crecimiento & desarrollo , Trofozoítos/metabolismo , Agua/metabolismoRESUMEN
BACKGROUND AND PURPOSE: WNK kinases, including WNK3, and the associated downstream Ste20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress responsive 1 (OSR1) kinases, comprise an important signaling cascade that regulates the cation-chloride cotransporters. Ischemia-induced stimulation of the bumetanide-sensitive Na(+)-K(+)-Cl(-) cotransporter (NKCC1) plays an important role in the pathophysiology of experimental stroke, but the mechanism of its regulation in this context is unknown. Here, we investigated the WNK3-SPAK/OSR1 pathway as a regulator of NKCC1 stimulation and their collective role in ischemic brain damage. METHOD: Wild-type WNK3 and WNK3 knockout mice were subjected to ischemic stroke via transient middle cerebral artery occlusion. Infarct volume, brain edema, blood brain barrier damage, white matter demyelination, and neurological deficits were assessed. Total and phosphorylated forms of WNK3 and SPAK/OSR1 were assayed by immunoblotting and immunostaining. In vitro ischemia studies in cultured neurons and immature oligodendrocytes were conducted using the oxygen-glucose deprivation/reoxygenation method. RESULTS: WNK3 knockout mice exhibited significantly decreased infarct volume and axonal demyelination, less cerebral edema, and accelerated neurobehavioral recovery compared with WNK3 wild-type mice subjected to middle cerebral artery occlusion. The neuroprotective phenotypes conferred by WNK3 knockout were associated with a decrease in stimulatory hyperphosphorylations of the SPAK/OSR1 catalytic T-loop and of NKCC1 stimulatory sites Thr(203)/Thr(207)/Thr(212), as well as with decreased cell surface expression of NKCC1. Genetic inhibition of WNK3 or small interfering RNA knockdown of SPAK/OSR1 increased the tolerance of cultured primary neurons and oligodendrocytes to in vitro ischemia. CONCLUSIONS: These data identify a novel role for the WNK3-SPAK/OSR1-NKCC1 signaling pathway in ischemic neuroglial injury and suggest the WNK3-SPAK/OSR1 kinase pathway as a therapeutic target for neuroprotection after ischemic stroke.
Asunto(s)
Lesiones Encefálicas/enzimología , Enfermedades del Sistema Nervioso/enzimología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/biosíntesis , Recuperación de la Función , Accidente Cerebrovascular/enzimología , Animales , Lesiones Encefálicas/patología , Lesiones Encefálicas/fisiopatología , Células Cultivadas , Femenino , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Ratones Transgénicos , Enfermedades del Sistema Nervioso/patología , Enfermedades del Sistema Nervioso/fisiopatología , Embarazo , Proteínas Serina-Treonina Quinasas/deficiencia , Recuperación de la Función/fisiología , Transducción de Señal/fisiología , Accidente Cerebrovascular/patología , Accidente Cerebrovascular/fisiopatologíaRESUMEN
Autosomal dominant dehydrated hereditary stomatocytosis (DHSt) usually presents as a compensated hemolytic anemia with macrocytosis and abnormally shaped red blood cells (RBCs). DHSt is part of a pleiotropic syndrome that may also exhibit pseudohyperkalemia and perinatal edema. We identified PIEZO1 as the disease gene for pleiotropic DHSt in a large kindred by exome sequencing analysis within the previously mapped 16q23-q24 interval. In 26 affected individuals among 7 multigenerational DHSt families with the pleiotropic syndrome, 11 heterozygous PIEZO1 missense mutations cosegregated with disease. PIEZO1 is expressed in the plasma membranes of RBCs and its messenger RNA, and protein levels increase during in vitro erythroid differentiation of CD34(+) cells. PIEZO1 is also expressed in liver and bone marrow during human and mouse development. We suggest for the first time a correlation between a PIEZO1 mutation and perinatal edema. DHSt patient red cells with the R2456H mutation exhibit increased ion-channel activity. Functional studies of PIEZO1 mutant R2488Q expressed in Xenopus oocytes demonstrated changes in ion-channel activity consistent with the altered cation content of DHSt patient red cells. Our findings provide direct evidence that R2456H and R2488Q mutations in PIEZO1 alter mechanosensitive channel regulation, leading to increased cation transport in erythroid cells.
Asunto(s)
Anemia Hemolítica Congénita/genética , Hidropesía Fetal/genética , Canales Iónicos/genética , Mutación , Adulto , Secuencia de Aminoácidos , Anemia Hemolítica Congénita/clasificación , Anemia Hemolítica Congénita/diagnóstico , Animales , Embrión de Mamíferos , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Hidropesía Fetal/clasificación , Hidropesía Fetal/diagnóstico , Ratones , Ratones Transgénicos , Modelos Biológicos , Datos de Secuencia Molecular , Mutación/fisiología , Linaje , Homología de Secuencia de Aminoácido , Transfección , Xenopus laevisRESUMEN
Dehydrated hereditary stomatocytosis (DHSt) is an autosomal dominant congenital hemolytic anemia with moderate splenomegaly and often compensated hemolysis. Affected red cells are characterized by a nonspecific cation leak of the red cell membrane, reflected in elevated sodium content, decreased potassium content, elevated MCHC and MCV, and decreased osmotic fragility. The majority of symptomatic DHSt cases reported to date have been associated with gain-of-function mutations in the mechanosensitive cation channel gene, PIEZO1. A recent study has identified two families with DHSt associated with a single mutation in the KCNN4 gene encoding the Gardos channel (KCa3.1), the erythroid Ca(2+) -sensitive K(+) channel of intermediate conductance, also expressed in many other cell types. We present here, in the second report of DHSt associated with KCNN4 mutations, two previously undiagnosed DHSt families. Family NA exhibited the same de novo missense mutation as that recently described, suggesting a hot spot codon for DHSt mutations. Family WO carried a novel, inherited missense mutation in the ion transport domain of the channel. The patients' mild hemolytic anemia did not improve post-splenectomy, but splenectomy led to no serious thromboembolic events. We further characterized the expression of KCNN4 in the mutated patients and during erythroid differentiation of CD34+ cells and K562 cells. We also analyzed KCNN4 expression during mouse embryonic development.
Asunto(s)
Anemia Hemolítica Congénita/genética , Hidropesía Fetal/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Mutación Missense , Adolescente , Adulto , Anemia Hemolítica Congénita/cirugía , Animales , Femenino , Humanos , Hidropesía Fetal/cirugía , Células K562 , Masculino , Ratones , Esplenectomía/efectos adversos , Tromboembolia/etiología , Tromboembolia/genéticaRESUMEN
The zebrafish genome encodes two slc4a1 genes, one expressed in erythroid tissues and the other in the HR (H(+)-ATPase-rich) type of embryonic skin ionocytes, and two slc4a2 genes, one in proximal pronephric duct and the other in several extrarenal tissues of the embryo. We now report cDNA cloning and functional characterization of zebrafish slc4a3/ae3 gene products. The single ae3 gene on chromosome 9 generates at least two low-abundance ae3 transcripts differing only in their 5'-untranslated regions and encoding a single definitive Ae3 polypeptide of 1170 amino acids. The 7 kb upstream of the apparent initiator Met in ae3 exon 3 comprises multiple diverse, mobile repeat elements which disrupt and appear to truncate the Ae3 N-terminal amino acid sequence that would otherwise align with brain Ae3 of other species. Embryonic ae3 mRNA expression was detected by whole mount in situ hybridization only in fin buds at 24-72 hpf, but was detectable by RT-PCR across a range of embryonic and adult tissues. Epitope-tagged Ae3 polypeptide was expressed at or near the surface of Xenopus oocytes, and mediated low rates of DIDS-sensitive (36)Cl(-)/Cl(-) exchange in influx and efflux assays. As previously reported for Ae2 polypeptides, (36)Cl(-) transport by Ae3 was inhibited by both extracellular and intracellular acidic pH, and stimulated by alkaline pH. However, zebrafish Ae3 differed from Ae2 polypeptides in its insensitivity to NH4Cl and to hypertonicity. We conclude that multiple repeat elements have disrupted the 5'-end of the zebrafish ae3 gene, associated with N-terminal truncation of the protein and reduced anion transport activity.
Asunto(s)
Antiportadores de Cloruro-Bicarbonato/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Antiportadores de Cloruro-Bicarbonato/metabolismo , Clonación Molecular , Humanos , Hibridación in Situ , Microscopía Confocal , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
AE2/SLC4A2 is the most widely expressed of the Na(+)-independent SLC4 Cl(-)/HCO3 (-) exchangers and is essential for postnatal survival, but its structure remains unknown. We have generated and expressed a mouse AE2 construct devoid of transmembrane domain cysteine (Cys) residues, mAE2Cys-less, to enhance the utility of Cys-substitution mutagenesis for structural and structure-function studies of mAE2. mAE2Cys-less expressed in Xenopus oocytes exhibited partial reduction of stilbene disulfonate-sensitive anion exchange activity. This activity was independent of the mAE2 N-terminal cytosolic domain and was accompanied by near-normal surface expression, without change in K 1/2 for extracellular Cl(-). mAE2Cys-less exhibited wildtype activation of anion exchange by hypertonicity and by NH4Cl, and wildtype inhibition of anion exchange by acidic intracellular pH (pHi) in the absence of NH4 (+). However, inhibition of anion exchange by extracellular pH (pHo) exhibited an alkaline shifted pHo(50) value of at least 0.6-0.7 pH units. Although SO4 (2-) transport by mAE2Cys-less resembled wildtype mAE2 in its stimulation by acidic pHo, the absence of transmembrane domain Cys residues abrogated activation of oxalate transport by acidic pHo. The contrasting enhancement of SO4 (2-) transport by alkaline pHo in the mAE1 anion translocation pathway mutant E699Q (Am J Physiol Cell Physiol 295: C302) was phenocopied by the corresponding mutant E1007Q in both AE2 and AE2Cys-less. However, the absence of transmembrane domain Cys residues exacerbated the reduced basal anion transport function exhibited by this and other missense substitutions at AE2 residue E1007. AE2Cys-less will be a valuable experimental tool for structure-function studies of the SLC4 gene family, but its utility for studies of AE2 regulation by extracellular pH must be evaluated in the context of its alkaline-shifted pHo sensitivity, resembling that of AE2 gastric parietal cell variant AE2c1.
Asunto(s)
Sustitución de Aminoácidos , Cloruros/metabolismo , Cisteína/genética , Sulfatos/metabolismo , Animales , Antiportadores de Cloruro-Bicarbonato/química , Antiportadores de Cloruro-Bicarbonato/genética , Antiportadores de Cloruro-Bicarbonato/metabolismo , Concentración de Iones de Hidrógeno , Transporte Iónico , Ratones , Ratones Noqueados , Oxalatos/metabolismo , Estructura Terciaria de Proteína , XenopusRESUMEN
The K-Cl cotransporters (KCCs) of mouse erythrocytes exhibit higher basal activity than those of human erythrocytes, but are similarly activated by cell swelling, by hypertonic urea, and by staurosporine. However, the dramatic stimulation of human erythroid KCCs by N-ethylmaleimide (NEM) is obscured in mouse erythrocytes by a prominent NEM-stimulated K(+) efflux that lacks Cl(-)-dependence. The NEM-sensitivity of Cl(-)-independent K(+) efflux of mouse erythrocytes is lower than that of KCC. The genetically engineered absence of the K-Cl cotransporters KCC3 and KCC1 from mouse erythrocytes does not modify Cl(-)-independent K(+) efflux. Mouse erythrocytes genetically devoid of the Gardos channel KCNN4 show increased NEM-sensitivity of both Cl(-)-independent K(+) efflux and K-Cl cotransport. The increased NEM-sensitivity and stimulation magnitude of Cl(-)-independent K(+) efflux in mouse erythrocytes expressing transgenic hypersickling human hemoglobin SAD (HbSAD) are independent of the presence of KCC3 and KCC1, but absence of KCNN4 reduces the stimulatory effect of HbSAD. NEM-stimulated Cl(-)-independent K(+) efflux of mouse red cells is insensitive to ouabain and bumetanide, but partially inhibited by chloroquine, barium, and amiloride. The NEM-stimulated activity is modestly reduced at pH6.0 but not significantly altered at pH8.0, and is abolished at 0°C. Although the molecular identity of this little-studied K(+) efflux pathway of mouse erythrocytes remains unknown, its potential role in the pathophysiology of sickle red cell dehydration will be important for the extrapolation of studies in mouse models of sickle cell disease to our understanding of humans with sickle cell anemia.
Asunto(s)
Cloruros/metabolismo , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Etilmaleimida/farmacología , Potasio/metabolismo , Simportadores/agonistas , Simportadores/metabolismo , Amilorida/farmacología , Animales , Cloroquina/farmacología , Resistencia a Medicamentos/genética , Hemoglobina Falciforme/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Ratones , Ratones Noqueados , Simportadores/antagonistas & inhibidores , Temperatura , Cotransportadores de K ClRESUMEN
Familial Pseudohyperkalemia (FP) is a dominant red cell trait characterized by increased serum [K(+)] in whole blood stored at or below room temperature, without additional hematological abnormalities. Functional gene mapping and sequencing analysis of the candidate genes within the 2q35-q36 critical interval identified-in 20 affected individuals among three multigenerational FP families-two novel heterozygous missense mutations in the ABCB6 gene that cosegregated with disease phenotype. The two genomic substitutions altered two adjacent nucleotides within codon 375 of ABCB6, a porphyrin transporter that, in erythrocyte membranes, bears the Langereis blood group antigen system. The ABCB6 R375Q mutation did not alter the levels of mRNA or protein, or protein localization in mature erythrocytes or erythroid precursor cells, but it is predicted to modestly alter protein structure. ABCB6 mRNA and protein levels increase during in vitro erythroid differentiation of CD34(+) erythroid precursors and the erythroleukemia cell lines HEL and K562. These data suggest that the two missense mutations in residue 375 of the ABCB6 polypeptide found in affected individuals of families with chromosome 2-linked FP could contribute to the red cell K(+) leak characteristic of this condition.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Cromosomas Humanos Par 2/genética , Hiperpotasemia/congénito , Errores Innatos del Metabolismo/genética , Mutación Missense , Transportadoras de Casetes de Unión a ATP/metabolismo , Adulto , Sustitución de Aminoácidos , Codón , Células Precursoras Eritroides , Femenino , Humanos , Hiperpotasemia/sangre , Hiperpotasemia/genética , Células K562 , Errores Innatos del Metabolismo/sangre , Potasio/sangreRESUMEN
Autosomal dominant overhydrated cation-leak stomatocytosis in humans has been associated with missense mutations in the erythroid membrane transport genes AE1, RhAG, and GLUT1. Syndromic stomatocytosis has been reported in three dog breeds, but stomatocytosis in Standard Schnauzers is usually asymptomatic, and is accompanied by minimal if any anemia. We have extended the evaluation of a cohort of schnauzers. We found that low-level stomatocytosis was accompanied by increased MCV and increased red cell Na content, and minimal or no reticulocytosis. Red cells from two affected dogs exhibited increased currents in on-cell patches measured in symmetrical NaCl solutions, but Na,K-ATPase and NKCC-mediated cation flux was minimal. Three novel coding polymorphisms found in canine RhAG cDNA and three novel polymorphisms found in canine SLC4A1 cDNA did not cosegregate with MCV or Na content. The GLUT1 cDNA sequence was normal. We conclude that unlike human overhydrated cation-leak stomatocytosis, stomatocytosis in this cohort of Standard Schnauzers is not caused by mutations in the genes encoding RhAG, SLC4A1, or GLUT1.
Asunto(s)
Anemia Hemolítica Congénita/genética , Proteína 1 de Intercambio de Anión de Eritrocito/genética , Transportador de Glucosa de Tipo 1/genética , Glicoproteínas de Membrana/genética , Mutación , Anemia Hemolítica Congénita/metabolismo , Animales , Transporte Biológico , Cationes/metabolismo , Perros , Índices de Eritrocitos , Eritrocitos/metabolismo , Femenino , Humanos , Iones/sangre , Masculino , Sistemas de Lectura Abierta , Linaje , Polimorfismo Genético , Análisis de Secuencia de ADNAsunto(s)
Acetamidas/uso terapéutico , Anemia Hemolítica Congénita/sangre , Eritrocitos/metabolismo , Mutación con Ganancia de Función , Hidropesía Fetal/sangre , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Transporte Iónico/efectos de los fármacos , Mutación Missense , Bloqueadores de los Canales de Potasio/uso terapéutico , Potasio/sangre , Compuestos de Tritilo/uso terapéutico , Acetamidas/farmacología , Anemia Hemolítica Congénita/genética , Recuento de Células , Evaluación Preclínica de Medicamentos , Deformación Eritrocítica , Eritrocitos/efectos de los fármacos , Heterocigoto , Humanos , Hidropesía Fetal/genética , Técnicas In Vitro , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Fragilidad Osmótica , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Compuestos de Tritilo/farmacologíaRESUMEN
Investigation of erythrocytes from spontaneous or engineered germ-line mutant mice has been instrumental in characterizing the physiological functions of components of the red cell cytoskeleton and membrane. However, the red blood cell expresses some proteins whose germline loss-of-function is embryonic-lethal, perinatal-lethal, or confers reduced post-weaning viability. Promoter regions of erythroid-specific genes have been used to engineer erythroid-specific expression of Cre recombinase. Through breeding with mice carrying appropriately spaced insertions of loxP sequences, generation of erythroid-specific knockouts has been carried out for signaling enzymes, transcription factors, peptide hormones, and single transmembrane span signaling receptors. We report here the use of Cre recombinase expression driven by the erythropoietin receptor (EpoR) promoter to generate EpoR-Cre;Kcc3f/f mice, designed to express erythroid-specific knockout of the KCC3 K-Cl cotransporter encoded by Kcc3/Slc12A6. We confirm KCC3 as the predominant K-Cl cotransporter of adult mouse red cells in mice with better viability than previously exhibited by Kcc3-/- germline knockouts. We demonstrate roughly proportionate preservation of K-Cl stimulation by hypotonicity, staurosporine, and urea in the context of reduced, but not abrogated, K-Cl function in EpoR-Cre;Kcc3f/f mice. We also report functional evidence suggesting incomplete recombinase-mediated excision of the Kcc3 gene in adult erythroid tissues.
Asunto(s)
Eritrocitos , Integrasas , Receptores de Eritropoyetina , Simportadores , Animales , Eritrocitos/metabolismo , Integrasas/biosíntesis , Integrasas/sangre , Integrasas/genética , Ratones , Regiones Promotoras Genéticas , Receptores de Eritropoyetina/sangre , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismo , Simportadores/sangre , Simportadores/genética , Simportadores/metabolismoRESUMEN
We report the novel, heterozygous AE1 mutation R730C associated with dominant, overhydrated, cation leak stomatocytosis and well-compensated anemia. Parallel elevations of red blood cell cation leak and ouabain-sensitive Na(+) efflux (pump activity) were apparently unaccompanied by increased erythroid cation channel-like activity, and defined ouabain-insensitive Na(+) efflux pathways of nystatin-treated cells were reduced. Epitope-tagged AE1 R730C at the Xenopus laevis oocyte surface exhibited severely reduced Cl(-) transport insensitive to rescue by glycophorin A (GPA) coexpression or by methanethiosulfonate (MTS) treatment. AE1 mutant R730K preserved Cl(-) transport activity, but R730 substitution with I, E, or H inactivated Cl(-) transport. AE1 R730C expression substantially increased endogenous oocyte Na(+)-K(+)-ATPase-mediated (86)Rb(+) influx, but ouabain-insensitive flux was minimally increased and GPA-insensitive. The reduced AE1 R730C-mediated sulfate influx did not exhibit the wild-type pattern of stimulation by acidic extracellular pH (pH(o)) and, unexpectedly, was partially rescued by exposure to sodium 2-sulfonatoethyl methanethiosulfonate (MTSES) but not to 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) or 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET). AE1 R730E correspondingly exhibited acid pH(o)-stimulated sulfate uptake at rates exceeding those of wild-type AE1 and AE1 R730K, whereas mutants R730I and R730H were inactive and pH(o) insensitive. MTSES-treated oocytes expressing AE1 R730C and untreated oocytes expressing AE1 R730E also exhibited unprecedented stimulation of Cl(-) influx by acid pH(o). Thus recombinant cation-leak stomatocytosis mutant AE1 R730C exhibits severely reduced anion transport unaccompanied by increased Rb(+) and Li(+) influxes. Selective rescue of acid pH(o)-stimulated sulfate uptake and conferral of acid pH(o)-stimulated Cl(-) influx, by AE1 R730E and MTSES-treated R730C, define residue R730 as critical to selectivity and regulation of anion transport by AE1.