RESUMEN
Inherited disorders of erythrocyte volume homeostasis are a heterogeneous group of rare disorders with phenotypes ranging from dehydrated to overhydrated erythrocytes. Clinical, laboratory, physiologic, and genetic heterogeneities characterize this group of disorders. A series of recent reports have provided novel insights into our understanding of the genetic bases underlying some of these disorders of red cell volume regulation. This report reviews this progress in understanding determinants that influence erythrocyte hydration and how they have yielded a better understanding of the pathways that influence cellular water and solute homeostasis.
Asunto(s)
Volumen de Eritrocitos , Eritrocitos/metabolismo , Enfermedades Hematológicas/fisiopatología , Homeostasis , Proteína 1 de Intercambio de Anión de Eritrocito/genética , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Enfermedades Hematológicas/genética , Enfermedades Hematológicas/metabolismo , Humanos , Mutación , Agua/metabolismo , Desequilibrio Hidroelectrolítico/genéticaRESUMEN
The REDUCE MRSA Trial (Randomized Evaluation of Decolonization vs Universal Clearance to Eliminate Methicillin-Resistant Staphylococcus aureus), a large multicenter, randomized controlled trial in adult intensive care units (ICUs), found universal decolonization to be more effective than surveillance and isolation procedures with or without targeted decolonization for reducing rates of MRSA-positive clinical cultures. The Agency for Healthcare Research and Quality and the Centers for Disease Control and Prevention subsequently published protocols for implementing universal decolonization in ICUs based on the trial's methods. Caution should be exercised before widely adopting these procedures in neonatal intensive care units (NICUs), particularly strategies that involve bathing with chlorhexidine and mupirocin application due to the potential for adverse events in their unique patient population, especially preterm infants. Large multicenter trials in the NICUs are needed to evaluate the efficacy, short- and long-term safety, and cost effectiveness of these strategies prior to their widespread implementation.
Asunto(s)
Unidades de Cuidado Intensivo Neonatal , Unidades de Cuidados Intensivos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/prevención & control , Adulto , Clorhexidina/efectos adversos , Humanos , Recién Nacido , Enfermedades del Prematuro , Mupirocina/efectos adversosAsunto(s)
Cromosomas Humanos Par 1 , Cromosomas Humanos Par 22 , Exoma , Leucemia Megacarioblástica Aguda/genética , Metaloproteinasa 8 de la Matriz/genética , Mutación , Translocación Genética , Niño , Proteínas de Unión al ADN/genética , Humanos , Leucemia Megacarioblástica Aguda/tratamiento farmacológico , Masculino , Proteínas de Fusión Oncogénica/genética , Polimorfismo de Nucleótido Simple , Proteínas de Unión al ARN/genética , TransactivadoresRESUMEN
The epidemiology, etiology and outcome of neonatal sepsis are changing over time. While monitoring longitudinal trends in neonatal sepsis in our institution, we encountered a case of late-onset neonatal sepsis due to Leclercia adecarboxylata. A Gram-negative rod previously not encountered in the clinical setting, L. adecarboxylata has recently emerged as a human pathogen, primarily in immunosuppressed patients. This report describes the clinical and laboratory features of this case of late-onset L. adecarboxylata sepsis, and reviews significant features of infection associated with this emerging pathogen.
Asunto(s)
Infecciones por Enterobacteriaceae/diagnóstico , Enterobacteriaceae , Insuficiencia Multiorgánica/etiología , Síndrome de Dificultad Respiratoria del Recién Nacido/terapia , Choque Séptico/diagnóstico , Rotura Gástrica/complicaciones , Infecciones por Enterobacteriaceae/terapia , Resultado Fatal , Femenino , Humanos , Recién Nacido , Recien Nacido Prematuro , Insuficiencia Multiorgánica/diagnóstico , Insuficiencia Multiorgánica/terapia , Síndrome de Dificultad Respiratoria del Recién Nacido/complicaciones , Choque Séptico/terapia , Rotura Gástrica/diagnóstico , Rotura Gástrica/terapiaRESUMEN
The hereditary stomatocytoses are a group of heterogeneous conditions associated with chronic red cell hemolysis for which the causative genetic mutations are not known. We investigated 137 members of a large Canadian kindred with phenotypic findings consistent with hereditary xerocytosis, one of the most common stomatocytosis syndromes. The objectives of this study were to characterize the clinical hallmarks of the hemolytic process, and to define the chromosomal region carrying the disease locus. The mode of inheritance was autosomal dominant. Affected family members had a well-compensated hemolysis, associated with an elevated MCHC, decreased osmotic fragility, decreased haptoglobin, and indirect hyperbilirubinemia. Cholelithiasis and progressive iron loading were common, despite normal hemoglobin levels. Quantitative erythrocyte morphologic evaluation revealed increased schistocytes, target cells, reticulocytes, and eccentrocytes in affected individuals; stomatocytes were not increased. Genetic linkage analysis confirmed the localization of the disease phenotype to chromosome 16q, and refined the candidate region to 16q24.2-16qter, a 2.4 million base pair interval containing 51 known or predicted genes.
Asunto(s)
Anemia Hemolítica Congénita/genética , Cromosomas Humanos Par 16 , Sitios Genéticos , Hidropesía Fetal/genética , Adolescente , Adulto , Anemia Hemolítica Congénita/patología , Canadá , Niño , Mapeo Cromosómico , Femenino , Ligamiento Genético , Haplotipos , Humanos , Hidropesía Fetal/patología , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Adulto JovenRESUMEN
Disruption of spectrin self-association underlies many inherited hemolytic disorders. Using dynamic modeling and energy minimization, the 3-dimensional structure of the self-association domain has been estimated in human erythrocyte spectrin and the structural consequences of 17 elliptogenic mutations determined. The predicted structure of the normal self-association domain was remarkably similar to the crystal structure of the Drosophila alpha-spectrin 14th repeat unit, despite replacement in the human sequence of over 70% of the amino acids relative to fly spectrin, including 2 prolines in the human sequence that appear in helical regions of the fly structure. The predicted structure placed all hydrophilic residues at the surface and identified 4 salt bridges, 9 hydrophobic interactions, and 4 H-bonds that stabilize the native self-association unit. Remarkably, every pathologic point mutation, including seemingly conservative substitutions such as G for A, A for V, or K for R (single-letter amino acid codes), led to conformational rearrangements in the predicted structure. The degree of structural disruption, as measured by root-mean-square deviation of the predicted backbone structure from the Drosophila structure, correlated strongly with the severity of clinical disease associated with each mutation. This approach thus enables an accurate prediction, from the primary sequence, of the clinical consequences of specific point mutations in spectrin. The 3-dimensional structure of the self-association domain derived here is likely to be accurate. It provides a powerful heuristic model for understanding how point mutations disrupt cytoskeletal function in a variety of hemolytic disorders.
Asunto(s)
Anemia Hemolítica Congénita/genética , Eliptocitosis Hereditaria/genética , Modelos Moleculares , Mutación Puntual , Espectrina/química , Espectrina/genética , Secuencia de Aminoácidos , Anemia Hemolítica Congénita/diagnóstico , Animales , Drosophila/genética , Eliptocitosis Hereditaria/diagnóstico , Humanos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Ankyrin defects are the most common cause of hereditary spherocytosis (HS). In several kindreds with recessive, ankyrin-deficient HS, mutations have been identified in the ankyrin promoter that have been proposed to decrease ankyrin synthesis. We analyzed the effects of two mutations, -108T to C and -108T to C in cis with -153G to A, on ankyrin expression. No difference between wild type and mutant promoters was demonstrated in transfection or gel shift assays in vitro. Transgenic mice with a wild type ankyrin promoter linked to a human (A)gamma-globin gene expressed gamma-globin in 100% of erythrocytes in a copy number-dependent, position-independent manner. Transgenic mice with the mutant -108 promoter demonstrated variegated gamma-globin expression, but showed copy number-dependent and position-independent expression similar to wild type. Severe effects in ankyrin expression were seen in mice with the linked -108/-153 mutations. Three transgenic lines had undetectable levels of (A)gamma-globin mRNA, indicating position-dependent expression, and four lines expressed significantly lower levels of (A)gamma-globin mRNA than wild type. Two of four expressing lines showed variegated gamma-globin expression, and there was no correlation between transgene copy number and RNA level, indicating copy number-independent expression. These data are the first demonstration of functional defects caused by HS-related, ankyrin gene promoter mutations.
Asunto(s)
Ancirinas/genética , Mutación , Regiones Promotoras Genéticas , Esferocitosis Hereditaria/genética , Animales , ADN/metabolismo , Dosificación de Gen , Globinas/análisis , Globinas/genética , Humanos , Células K562 , Ratones , Ratones Transgénicos , ARN Mensajero/análisis , TransfecciónRESUMEN
Gene therapy for patients with hemoglobin disorders such has been hampered by the inability of retrovirus vectors to transfer globin genes and the locus control region (LCR) into hematopoietic stem cells without rearrangement. In addition, the expression from intact globin gene vectors has been variable in red blood cells as a result of position effects and retrovirus silencing. We hypothesized that by substituting the globin gene promoter for the promoter of another gene expressed in red blood cells, we could generate stable retrovirus vectors that would express globin at sufficient levels to treat hemoglobinopathies. Transgenic mice containing the human ankyrin (Ank) gene promoter fused to the human gamma-globin gene showed position-independent, copy number-dependent expression of a linked gamma-globin mRNA. We generated a "double-copy" Ank/A gamma-globin retrovirus vector that transferred two copies of the Ank/A gamma-globin gene into target cells. Stable gene transfer was observed in primary primary mouse progenitor cells and long-term repopulating hematopoietic stem cells. Expression of Ank/A gamma-globin mRNA in mature red blood cells was approximately 8% of the level of mouse alpha-globin mRNA. We conclude that this novel retrovirus vector may be valuable for treating a variety of hemoglobinopathies by gene therapy if the level of expression can be further increased.
Asunto(s)
Eritrocitos/metabolismo , Vectores Genéticos/genética , ARN Mensajero/biosíntesis , Retroviridae/genética , gammaglobulinas/genética , Células 3T3 , Anemia/genética , Anemia/terapia , Animales , Ancirinas/genética , Citometría de Flujo , Expresión Génica , Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
In the erythrocyte, ankyrin is the major adapter protein linking tetramers of band 3 to the spectrin-actin cytoskeleton. This linkage involves a direct interaction between ankyrin and the 14th-15th repeat unit of beta-spectrin. The spectrin cytoskeleton itself is stabilized by the self-association of spectrin heterodimers into tetramers and larger oligomers, a process mediated by the 17th repeat unit of beta-spectrin and a short NH(2) -terminal sequence in alpha-spectrin. The self-association of spectrin and its ankyrin-mediated membrane binding have generally been considered independent events. We now demonstrate that spectrin self-association, the binding of spectrin to ankyrin, and the binding of ankyrin to the 43-kDa cytoplasmic domain of band 3 (cdb3) are coupled in a positively cooperative way. In solution, [(125)I]-labeled ankyrin was found by ND-PAGE3 to enhance the affinity of spectrin self-association by 10-fold. The reciprocal process was also true, in that spectrin tetramers and oligomers bound ankyrin with enhanced affinity relative to dimer spectrin. Saturation of the beta-spectrin self-association site by an NH(2) -terminal 80-kDa alpha-spectrin peptide enhanced the affinity of spectrin dimer for ankyrin, indicating a direct relationship between ankyrin binding and the occupancy of the beta-spectrin self-association site. cdb3 accentuated these cooperative interactions. Several inherited spectrin mutations that cause hemolytic disease but that do not directly destabilize the self-association or ankyrin-binding sites can be explained by these results. Three classes of mutations appear to disrupt cooperative coupling between self-association and ankyrin binding: (i) mutation of the linker sequences that join helices C and A in repeat units that intervene between the two functional sites, mutations that presumably block repeat-to-repeat transfer of conformational information; (ii) mutations in alpha-spectrin repeats 4 to 6 that disrupt the ability of this region to trans-regulate ankyrin binding by the adjacent beta-spectrin repeats 14-15; and (iii) exon-skipping mutations that shorten alpha-spectrin and force repeats 4 to 6 to fall out-of-register with the ankyrin-binding motif in beta-spectrin. Collectively, these results demonstrate a molecular mechanism whereby a membrane receptor can directly promote cytoskeletal assembly.
Asunto(s)
Anemia Hemolítica/genética , Ancirinas/química , Ancirinas/metabolismo , Membrana Eritrocítica/fisiología , Espectrina/química , Anemia Hemolítica/sangre , Citoplasma/fisiología , Dimerización , Eliptocitosis Hereditaria/sangre , Eliptocitosis Hereditaria/genética , Membrana Eritrocítica/ultraestructura , Eritrocitos/fisiología , Humanos , Sustancias Macromoleculares , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de Proteína , Subunidades de Proteína , Espectrina/genética , Espectrina/ultraestructura , Esferocitosis Hereditaria/sangre , Esferocitosis Hereditaria/genéticaRESUMEN
OBJECTIVE: To record weight changes, fluid intake and changes in serum sodium concentration in ultradistance triathletes. DESIGN: Descriptive research. SETTING: Ironman triathlon (3.8 km swim, 180 km cycle, 42.2 km run). Air temperature at 1200 h was 21 degrees C, (relative humidity 91%). Water temperature was 20.7 degrees C. PARTICIPANTS: 18 triathletes. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Subjects were weighed and had blood drawn for serum sodium concentration [Na], hemoglobin, and hematocrit, pre-race, post-race, and at 0800 h on the morning following the race ("recovery"); subjects were also weighed at transitions. Fluid intake during the race was estimated by athlete recall. RESULTS: Median weight change during the race = -2.5 kg (p < 0.0006). Subjects lost weight during recovery (median = -1.0 kg) (p < 0.03). Median hourly fluid intake = 716 ml/h (range 421-970). Fluid intakes were higher on the bike than on the run (median 889 versus 632 ml/h, p = 0.03). Median calculated fluid losses cycling were 808 ml/h and running were 1,021 ml/h. No significant difference existed between pre-race and post-race [Na] (median 140 versus 138 mmol/L) or between post-race and recovery [Na] (median 138 versus 137 mmol/L). Plasma volume increased during the race, median + 10.8% (p = 0.0005). There was an inverse relationship between change in [Na] pre-race to post-race and relative weight change (r = -0.68, p = 0.0029). Five subjects developed hyponatremia ([Na] 128-133 mmol/L). CONCLUSIONS: Athletes lose 2.5 kg of weight during an ultradistance triathlon. most likely from sources other than fluid loss. Fluid intakes during this event are more modest than that recommended for shorter duration exercise. Plasma volume increases during the ultradistance triathlon. Subjects who developed hyponatremia had evidence of fluid overload despite modest fluid intakes.
Asunto(s)
Ciclismo/fisiología , Hiponatremia/fisiopatología , Carrera/fisiología , Natación/fisiología , Equilibrio Hidroelectrolítico , Adulto , Deshidratación , Conducta de Ingestión de Líquido , Femenino , Humanos , Hiponatremia/etiología , Masculino , Persona de Mediana Edad , Resistencia Física , Pérdida de PesoAsunto(s)
Movimiento Fetal , Feto/anomalías , Imagenología Tridimensional , Ultrasonografía Prenatal , Adulto , Femenino , Humanos , Recién Nacido , Masculino , EmbarazoRESUMEN
Gene therapy for patients with hemoglobin disorders has been hampered by the inability of retrovirus vectors to transfer globin genes and their cis-acting regulatory sequences into hematopoietic stem cells without rearrangement. In addition, the expression from intact globin gene vectors has been variable in red blood cells due to position effects and retrovirus silencing. We hypothesized that by substituting the globin gene promoter for the promoter of another gene expressed in red blood cells, we could generate stable retrovirus vectors that would express globin at sufficient levels to treat hemoglobinopathies. Recently, we have shown that the human ankyrin (Ank) gene promoter directs position-independent, copy number-dependent expression of a linked gamma-globin gene in transgenic mice. We inserted the Ank/(A)gamma-globin gene into retrovirus vectors that could transfer one or two copies of the Ank/(A)gamma-globin gene to target cells. Both vectors were stable, transferring only intact proviral sequences into primary mouse hematopoietic stem cells. Expression of Ank/(A)gamma-globin mRNA in mature red blood cells was 3% (single copy) and 8% (double copy) of the level of mouse alpha-globin mRNA. We conclude that these novel retrovirus vectors may be valuable for treating a variety of red cell disorders by gene replacement therapy including severe beta-thalassemia if the level of expression can be further increased.
Asunto(s)
Ancirinas/genética , Eritrocitos/metabolismo , Células Precursoras Eritroides/metabolismo , Globinas/genética , Transcripción Genética , Animales , Ensayo de Unidades Formadoras de Colonias , Células Precursoras Eritroides/citología , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos , Humanos , Ratones , Regiones Promotoras Genéticas , ARN Mensajero/sangre , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/sangre , RetroviridaeRESUMEN
To begin to study the sequence variations identified in the 5' flanking genomic DNA of the ankyrin gene in ankyrin-deficient hereditary spherocytosis patients and to provide additional insight into our understanding of the regulation of genes encoding erythrocyte membrane proteins, we have identified and characterized the erythroid promoter of the human ankyrin-1 gene. This compact promoter has characteristics of a housekeeping gene promoter, including very high G+C content and enzyme restriction sites characteristic of an HTF-island, no TATA, InR, or CCAAT consensus sequences, and multiple transcription initiation sites. In vitro DNAseI footprinting analyses revealed binding sites for GATA-1, CACCC-binding, and CGCCC-binding proteins. Transfection of ankyrin promoter/reporter plasmids into tissue culture cell lines yielded expression in erythroid, but not muscle, neural, or HeLa cells. Electrophoretic mobility shift assays, including competition and antibody supershift experiments, demonstrated binding of GATA-1, BKLF, and Sp1 to core ankyrin promoter sequences. In transfection assays, mutation of the Sp1 site had no effect on reporter gene expression, mutation of the CACCC site decreased expression by half, and mutation of the GATA-1 site completely abolished activity. The ankyrin gene erythroid promoter was transactivated in heterologous cells by forced expression of GATA-1 and to a lesser degree BKLF.
Asunto(s)
Ancirinas/genética , Eritrocitos/fisiología , Regulación de la Expresión Génica , Transcripción Genética , Secuencia de Bases , Línea Celular , Humanos , Datos de Secuencia Molecular , Regiones Promotoras GenéticasRESUMEN
In red blood cells ankyrin (ANK-1) provides the primary linkage between the erythrocyte membrane skeleton and the plasma membrane. We have previously demonstrated that a 271-bp 5'-flanking region of the ANK-1 gene has promoter activity in erythroid, but not non-erythroid, cell lines. To determine whether the ankyrin promoter could direct erythroid-specific expression in vivo, we analyzed transgenic mice containing the ankyrin promoter fused to the human (A)gamma-globin gene. Sixteen of 17 lines expressed the transgene in erythroid cells indicating nearly position-independent expression. We also observed a significant correlation between the level of Ank/(A)gamma-globin mRNA and transgene copy number. The level of Ank/(A)gamma mRNA averaged 11% of mouse alpha-globin mRNA per gene copy at all developmental stages. The addition of the HS2 enhancer from the beta-globin locus control region to the Ank/(A)gamma-globin transgene resulted in Ank/(A)gamma-globin mRNA expression in embryonic and fetal erythroid cells in six of eight lines but resulted in absent or dramatically reduced levels of Ank/(A)gamma-globin mRNA in adult erythroid cells in eight of eight transgenic lines. These data indicate that the minimal ankyrin promoter contains all sequences necessary and sufficient for erythroid-specific, copy number-dependent, position-independent expression of the human (A)gamma-globin gene.
Asunto(s)
Ancirinas/genética , Elementos de Facilitación Genéticos , Dosificación de Gen , Globinas/genética , Regiones Promotoras Genéticas , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , ARN Mensajero/análisisRESUMEN
OBJECTIVE: The goal of this study was to transfer by retroviral vector the cDNA for ankyrin to progenitors from normal bone marrow and from the nb/nb spherocytosis mutant deficient in expression of full-length ankyrin to achieve erythroid expression of functional ankyrin protein. MATERIALS AND METHODS: A minigene composed of the human ankyrin promoter, murine ankyrin cDNA, and the 3' human domain corresponding to the ankyrin 2.2 isoform was assembled in the retroviral vector, pG1. Murine erythroleukemia (MEL) cells, normal murine bone marrow cells, 3T3 fibroblasts, and nb/nb mutant bone marrow and spleen cells were transduced with the retroviral supernatant. Transduced mutant cells were induced to differentiate in liquid culture. Gene transfer was assessed by colony polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR, immunofluorescence, and Southern, Northern, and Western blot analysis. RESULTS: MEL cells, normal bone marrow progenitors, and nb/nb cells were all successfully transduced and expressed ankyrin by RT-PCR and Western blot. Transduced murine 3T3 fibroblasts and MEL cells exhibited cell membrane staining by immunofluorescence. Colony RT-PCR demonstrated dependence of expression on erythropoietin. In vitro, the transduced nb/nb cells matured to polychromatophils, whereas nontransduced nb/nb cells matured to microspherocytes. CONCLUSION: Retroviral transfer of ankyrin corrected the defect leading to formation of microspherocytes in erythroid differentiation cultures from the nb/nb mutant. The human ankyrin promoter conferred erythropoietin-dependent expression in normal and mutant erythroid progenitors, which could have implications for the gene therapy of human hemolytic anemias.
Asunto(s)
Ancirinas/deficiencia , Ancirinas/genética , Médula Ósea , Técnicas de Transferencia de Gen , Terapia Genética , Esferocitosis Hereditaria/terapia , Animales , Western Blotting , Línea Celular , Electroforesis en Gel de Poliacrilamida , Eritropoyetina/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Retroviridae , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A case of acquired diaphragmatic paralysis in an extremely low birth weight infant complicated by respiratory failure, recurrent atelectasis, and pneumonia is described. Diaphragmatic plication led to a rapid improvement in pulmonary function and allowed for discontinuation of mechanical ventilation in less than 1 week. Therapeutic options for acquired diaphragmatic paralysis, including the rationale for early operative intervention, in this patient population are discussed.
Asunto(s)
Enfermedades del Prematuro/terapia , Recién Nacido de muy Bajo Peso , Respiración con Presión Positiva , Respiración Artificial , Parálisis Respiratoria/terapia , Humanos , Recién Nacido , Recien Nacido Prematuro , MasculinoRESUMEN
Potassium leak conductances were recently revealed to exist as independent molecular entities. Here, the genomic structure, cardiac localization, and biophysical properties of a murine example are considered. Kcnk3 subunits have two pore-forming P domains and unique functional attributes. At steady state, Kcnk3 channels behave like open, potassium-selective, transmembrane holes that are inhibited by physiological levels of proton. With voltage steps, Kcnk3 channels open and close in two phases, one appears to be immediate and one is time-dependent (tau = approximately 5 ms). Both proton block and gating are potassium-sensitive; this produces an anomalous increase in outward flux as external potassium levels rise because of decreased proton block. Single Kcnk3 channels open across the physiological voltage range; hence they are "leak" conductances; however, they open only briefly and rarely even after exposure to agents that activate other potassium channels.
Asunto(s)
Activación del Canal Iónico , Canales de Potasio/metabolismo , Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario , Cinética , Potenciales de la Membrana , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso , Canales de Potasio/fisiología , Canales de Potasio de Dominio Poro en Tándem , Protones , Xenopus laevisRESUMEN
A 2 P domain potassium channel expressed in eye, lung, and stomach, Kcnk8, has recently been identified. To initiate further biochemical and genetic studies of this channel, we assembled the murine Kcnk8 cDNA sequence, characterized the genomic structure of the Kcnk8 gene, determined its chromosomal localization, and analyzed its activity in a Xenopus laevis oocyte expression system. The composite cDNA has an open reading frame of 1029 bp and encodes a protein of 343 amino acids with a predicted molecular mass of 36 kDa. Structure analyses predict 2 P domains and four potential transmembrane helices with a potential single EF-hand motif and four potential SH3-binding motifs in the COOH-terminus. Cloning of the Kcnk8 chromosomal gene revealed that it is composed of three exons distributed over 4 kb of genomic DNA. Genome database searching revealed that one of the intron/exon boundaries identified in Kcnk8 is present in other mammalian 2 P domain potassium channels genes and many C. elegans 2P domain potassium channel genes, revealing evolutionary conservation of gene structure. Using fluorescence in situ hybridization, the murine Kcnk8 gene was mapped to chromosome 19, 2B, the locus of the murine dancer phenotype, and syntenic to 11q11-11q13, the location of the human homologue. No significant currents were generated in a Xenopus laevis oocyte expression system using the composite Kcnk8 cDNA sequence, suggesting, like many potassium channels, additional channel subunits, modulator substances, or cellular chaperones are required for channel function.