RESUMEN
Mutations in PIEZO1 are the primary cause of hereditary xerocytosis, a clinically heterogeneous, dominantly inherited disorder of erythrocyte dehydration. We used next-generation sequencing-based techniques to identify PIEZO1 mutations in individuals from 9 kindreds referred with suspected hereditary xerocytosis (HX) and/or undiagnosed congenital hemolytic anemia. Mutations were primarily found in the highly conserved, COOH-terminal pore-region domain. Several mutations were novel and demonstrated ethnic specificity. We characterized these mutations using genomic-, bioinformatic-, cell biology-, and physiology-based functional assays. For these studies, we created a novel, cell-based in vivo system for study of wild-type and variant PIEZO1 membrane protein expression, trafficking, and electrophysiology in a rigorous manner. Previous reports have indicated HX-associated PIEZO1 variants exhibit a partial gain-of-function phenotype with generation of mechanically activated currents that inactivate more slowly than wild type, indicating that increased cation permeability may lead to dehydration of PIEZO1-mutant HX erythrocytes. In addition to delayed channel inactivation, we found additional alterations in mutant PIEZO1 channel kinetics, differences in response to osmotic stress, and altered membrane protein trafficking, predicting variant alleles that worsen or ameliorate erythrocyte hydration. These results extend the genetic heterogeneity observed in HX and indicate that various pathophysiologic mechanisms contribute to the HX phenotype.
Asunto(s)
Anemia Hemolítica Congénita/genética , Hidropesía Fetal/genética , Canales Iónicos/genética , Adulto , Anemia Hemolítica Congénita/metabolismo , Niño , Estudios de Cohortes , Análisis Mutacional de ADN , Deshidratación/genética , Deshidratación/metabolismo , Eritrocitos/metabolismo , Familia , Femenino , Células HEK293 , Humanos , Hidropesía Fetal/metabolismo , Mutación INDEL , Recién Nacido , Canales Iónicos/metabolismo , Cinética , Masculino , Mutación Missense , Presión Osmótica/fisiologíaRESUMEN
Hereditary xerocytosis (HX; MIM 194380) is an autosomal-dominant hemolytic anemia characterized by primary erythrocyte dehydration. In many patients, heterozygous mutations associated with delayed channel inactivation have been identified in PIEZO1. This report describes patients from 2 well-phenotyped HX kindreds, including from one of the first HX kindreds described, who lack predicted heterozygous PIEZO1-linked variants. Whole-exome sequencing identified novel, heterozygous mutations affecting the Gardos channel, encoded by the KCNN4 gene, in both kindreds. Segregation analyses confirmed transmission of the Gardos channel mutations with disease phenotype in affected individuals. The KCNN4 variants were different mutations in the same residue, which is highly conserved across species and within members of the small-intermediate family of calcium-activated potassium channel proteins. Both mutations were predicted to be deleterious by mutation effect algorithms. In sickle erythrocytes, the Gardos channel is activated under deoxy conditions, leading to cellular dehydration due to salt and water loss. The identification of KCNN4 mutations in HX patients supports recent studies that indicate it plays a critical role in normal erythrocyte deformation in the microcirculation and participates in maintenance of erythrocyte volume homeostasis.
Asunto(s)
Anemia Hemolítica Congénita/genética , Hidropesía Fetal/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Mutación Missense , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Análisis Mutacional de ADN , Exoma/genética , Femenino , Genes Dominantes , Estudios de Asociación Genética , Heterocigoto , Humanos , Canales Iónicos/genética , Masculino , Datos de Secuencia Molecular , Linaje , Homología de Secuencia de AminoácidoRESUMEN
Plasmodium falciparum relies on anion channels activated in the erythrocyte membrane to ensure the transport of nutrients and waste products necessary for its replication and survival after invasion. The molecular identity of these anion channels, termed "new permeability pathways" is unknown, but their currents correspond to up-regulation of endogenous channels displaying complex gating and kinetics similar to those of ligand-gated channels. This report demonstrates that a peripheral-type benzodiazepine receptor, including the voltage dependent anion channel, is present in the human erythrocyte membrane. This receptor mediates the maxi-anion currents previously described in the erythrocyte membrane. Ligands that block this peripheral-type benzodiazepine receptor reduce membrane transport and conductance in P falciparum-infected erythrocytes. These ligands also inhibit in vitro intraerythrocytic growth of P falciparum. These data support the hypothesis that dormant peripheral-type benzodiazepine receptors become the "new permeability pathways" in infected erythrocytes after up-regulation by P falciparum. These channels are obvious targets for selective inhibition in anti-malarial therapies, as well as potential routes for drug delivery in pharmacologic applications.
Asunto(s)
Eritrocitos/metabolismo , Eritrocitos/parasitología , Plasmodium falciparum/metabolismo , Receptores de GABA-A/sangre , Canales Aniónicos Dependientes del Voltaje/sangre , Antimaláricos/farmacología , Benzodiazepinonas/farmacología , Diazepam/farmacología , Eritrocitos/efectos de los fármacos , Humanos , Técnicas In Vitro , Activación del Canal Iónico , Isoquinolinas/farmacología , Ligandos , Malaria Falciparum/sangre , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/genética , Malaria Falciparum/parasitología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/patogenicidad , ARN Mensajero/sangre , ARN Mensajero/genética , Receptores de GABA-A/efectos de los fármacos , Regulación hacia Arriba , Canales Aniónicos Dependientes del Voltaje/genéticaAsunto(s)
Sustitución de Aminoácidos , Anemia Hemolítica Congénita/diagnóstico , Hidropesía Fetal/diagnóstico , Canales Iónicos/genética , Mutación Missense , Anemia Hemolítica Congénita/sangre , Anemia Hemolítica Congénita/complicaciones , Anemia Hemolítica Congénita/genética , Colelitiasis/complicaciones , Secuencia Conservada , Deformación Eritrocítica , Índices de Eritrocitos , Femenino , Hemoglobinas/análisis , Humanos , Hidropesía Fetal/sangre , Hidropesía Fetal/etiología , Hidropesía Fetal/genética , Canales Iónicos/química , Canales Iónicos/fisiología , Ictericia Neonatal/etiología , Masculino , Linaje , Fenotipo , Recuento de ReticulocitosRESUMEN
During the past three decades, electrophysiological studies revealed that human red blood cell membrane is endowed with a large variety of ion channels. The physiological role of these channels, if any, remains unclear; they do not participate in red cell homeostasis which is rather based on the almost total absence of cationic permeability and minute anionic conductance. They seem to be inactive in the "resting cell." However, when activated experimentally, ion channels can lead to a very high single cell conductance and potentially induce disorders, with the major risks of fast dehydration and dissipation of gradients. Could there be physiological conditions under which the red cell needs to activate these high conductances, or are ion channels relics of a function lost in anucleated cells? It has been demonstrated that they play a key role in diseases such as sickle cell anemia or malaria. This short overview of ion channels identified to-date in the human red cell membrane is an attempt to propose a dynamic role for these channels in circulating cells in health and disease.
Asunto(s)
Membrana Eritrocítica/metabolismo , Eritrocitos/química , Canales Iónicos/fisiología , Eritrocitos/fisiología , HumanosRESUMEN
Mechanoelectrical transduction is mediated by the opening of different types of force-sensitive ion channels, including Piezo1/2 and the TREK/TRAAK K2P channels. Piezo1 curves the membrane locally into an inverted dome that reversibly flattens in response to force application. Moreover, Piezo1 forms numerous preferential interactions with various membrane lipids, including cholesterol. Whether this structural architecture influences the functionality of neighboring membrane proteins is unknown. Here, we show that Piezo1/2 increase TREK/TRAAK current amplitude, slow down activation/deactivation, and remove inactivation upon mechanical stimulation. These findings are consistent with a mechanism whereby Piezo1/2 cause a local depletion of membrane cholesterol associated with a prestress of TREK/TRAAK channels. This regulation occurs in mouse fibroblasts between endogenous Piezo1 and TREK-1/2, both channel types acting in concert to delay wound healing. In conclusion, we demonstrate a community effect between different structural and functional classes of mechanosensitive ion channels.
Asunto(s)
Activación del Canal Iónico , Canales Iónicos/fisiología , Mecanotransducción Celular , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Colesterol/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Encía/citología , Encía/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Canales de Potasio de Dominio Poro en Tándem/genéticaRESUMEN
Historically, the anion transport through the human red cell membrane has been perceived to be mediated by Band 3, in the two-component concept with the large electroneutral anion exchange accompanied by the conductance proper, which dominated the total membrane conductance. The status of anion channels proper has never been clarified, and the informations obtained by different groups of electrophysiologists are rather badly matched. This study, using the cell-attached configuration of the patch-clamp technique, rationalizes and explains earlier confusing results by demonstrating that the diversity of anionic channel activities recorded in human erythrocytes corresponds to different kinetic modalities of a unique type of maxi-anion channel with multiple conductance levels and probably multiple gating properties and pharmacology, depending on conditions. It demonstrates the role of activator played by serum in the recruitment of multiple new conductance levels showing very complex kinetics and gating properties upon serum addition. These channels, which seem to be dormant under normal physiological conditions, are potentially activable and could confer a far higher anion conductance to the red cell than the ground leak mediated by Band 3.