RESUMEN
Mitochondria contain a specific translation machinery for the synthesis of mitochondria-encoded respiratory chain components. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial DNA and, similar to their cytoplasmic counterparts, are post-transcriptionally modified. Here, we find that the RNA methyltransferase METTL8 is a mitochondrial protein that facilitates 3-methyl-cytidine (m3C) methylation at position C32 of the mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knockout cells show a reduction in respiratory chain activity, whereas overexpression increases activity. In pancreatic cancer, METTL8 levels are high, which correlates with lower patient survival and an enhanced respiratory chain activity. Mitochondrial ribosome profiling uncovered mitoribosome stalling on mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons. Further analysis of the respiratory chain complexes using mass spectrometry revealed reduced incorporation of the mitochondrially encoded proteins ND6 and ND1 into complex I. The well-balanced translation of mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons through METTL8-mediated m3C32 methylation might, therefore, facilitate the optimal composition and function of the mitochondrial respiratory chain.
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Metiltransferasas/metabolismo , ARN Mitocondrial/química , ARN de Transferencia/química , Animales , Anticodón , Proliferación Celular , Codón , Citoplasma , ADN Mitocondrial/metabolismo , Transporte de Electrón , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Mitocondrias/metabolismo , Membranas Mitocondriales , Proteínas Mitocondriales/química , Consumo de Oxígeno , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidad , Ribosomas/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: The endocytic reabsorption of proteins in the proximal tubule requires a complex machinery and defects can lead to tubular proteinuria. The precise mechanisms of endocytosis and processing of receptors and cargo are incompletely understood. EHD1 belongs to a family of proteins presumably involved in the scission of intracellular vesicles and in ciliogenesis. However, the relevance of EHD1 in human tissues, in particular in the kidney, was unknown. METHODS: Genetic techniques were used in patients with tubular proteinuria and deafness to identify the disease-causing gene. Diagnostic and functional studies were performed in patients and disease models to investigate the pathophysiology. RESULTS: We identified six individuals (5-33 years) with proteinuria and a high-frequency hearing deficit associated with the homozygous missense variant c.1192C>T (p.R398W) in EHD1. Proteinuria (0.7-2.1 g/d) consisted predominantly of low molecular weight proteins, reflecting impaired renal proximal tubular endocytosis of filtered proteins. Ehd1 knockout and Ehd1R398W/R398W knockin mice also showed a high-frequency hearing deficit and impaired receptor-mediated endocytosis in proximal tubules, and a zebrafish model showed impaired ability to reabsorb low molecular weight dextran. Interestingly, ciliogenesis appeared unaffected in patients and mouse models. In silico structural analysis predicted a destabilizing effect of the R398W variant and possible inference with nucleotide binding leading to impaired EHD1 oligomerization and membrane remodeling ability. CONCLUSIONS: A homozygous missense variant of EHD1 causes a previously unrecognized autosomal recessive disorder characterized by sensorineural deafness and tubular proteinuria. Recessive EHD1 variants should be considered in individuals with hearing impairment, especially if tubular proteinuria is noted.
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Sordera , Pez Cebra , Adolescente , Adulto , Animales , Niño , Preescolar , Sordera/genética , Endocitosis , Humanos , Túbulos Renales Proximales/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Mutación , Proteinuria/metabolismo , Proteínas de Transporte Vesicular/genética , Adulto Joven , Pez Cebra/metabolismoRESUMEN
Background For many patients with kidney failure, the cause and underlying defect remain unknown. Here, we describe a novel mechanism of a genetic order characterized by renal Fanconi syndrome and kidney failure.Methods We clinically and genetically characterized members of five families with autosomal dominant renal Fanconi syndrome and kidney failure. We performed genome-wide linkage analysis, sequencing, and expression studies in kidney biopsy specimens and renal cells along with knockout mouse studies and evaluations of mitochondrial morphology and function. Structural studies examined the effects of recognized mutations.Results The renal disease in these patients resulted from monoallelic mutations in the gene encoding glycine amidinotransferase (GATM), a renal proximal tubular enzyme in the creatine biosynthetic pathway that is otherwise associated with a recessive disorder of creatine deficiency. In silico analysis showed that the particular GATM mutations, identified in 28 members of the five families, create an additional interaction interface within the GATM protein and likely cause the linear aggregation of GATM observed in patient biopsy specimens and cultured proximal tubule cells. GATM aggregates-containing mitochondria were elongated and associated with increased ROS production, activation of the NLRP3 inflammasome, enhanced expression of the profibrotic cytokine IL-18, and increased cell death.Conclusions In this novel genetic disorder, fully penetrant heterozygous missense mutations in GATM trigger intramitochondrial fibrillary deposition of GATM and lead to elongated and abnormal mitochondria. We speculate that this renal proximal tubular mitochondrial pathology initiates a response from the inflammasome, with subsequent development of kidney fibrosis.
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Amidinotransferasas/genética , Síndrome de Fanconi/genética , Fallo Renal Crónico/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Anciano , Amidinotransferasas/metabolismo , Animales , Simulación por Computador , Síndrome de Fanconi/complicaciones , Síndrome de Fanconi/metabolismo , Síndrome de Fanconi/patología , Femenino , Heterocigoto , Humanos , Lactante , Inflamasomas/metabolismo , Fallo Renal Crónico/etiología , Fallo Renal Crónico/metabolismo , Fallo Renal Crónico/patología , Masculino , Ratones , Ratones Noqueados , Conformación Molecular , Mutación , Mutación Missense , Linaje , Especies Reactivas de Oxígeno/metabolismo , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
BACKGROUND: In renal Fanconi's syndrome, dysfunction in proximal tubular cells leads to renal losses of water, electrolytes, and low-molecular-weight nutrients. For most types of isolated Fanconi's syndrome, the genetic cause and underlying defect remain unknown. METHODS: We clinically and genetically characterized members of a five-generation black family with isolated autosomal dominant Fanconi's syndrome. We performed genomewide linkage analysis, gene sequencing, biochemical and cell-biologic investigations of renal proximal tubular cells, studies in knockout mice, and functional evaluations of mitochondria. Urine was studied with the use of proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. RESULTS: We linked the phenotype of this family's Fanconi's syndrome to a single locus on chromosome 3q27, where a heterozygous missense mutation in EHHADH segregated with the disease. The p.E3K mutation created a new mitochondrial targeting motif in the N-terminal portion of EHHADH, an enzyme that is involved in peroxisomal oxidation of fatty acids and is expressed in the proximal tubule. Immunocytofluorescence studies showed mistargeting of the mutant EHHADH to mitochondria. Studies of proximal tubular cells revealed impaired mitochondrial oxidative phosphorylation and defects in the transport of fluids and a glucose analogue across the epithelium. (1)H-NMR spectroscopy showed elevated levels of mitochondrial metabolites in urine from affected family members. Ehhadh knockout mice showed no abnormalities in renal tubular cells, a finding that indicates a dominant negative nature of the mutation rather than haploinsufficiency. CONCLUSIONS: Mistargeting of peroxisomal EHHADH disrupts mitochondrial metabolism and leads to renal Fanconi's syndrome; this indicates a central role of mitochondria in proximal tubular function. The dominant negative effect of the mistargeted protein adds to the spectrum of monogenic mechanisms of Fanconi's syndrome. (Funded by the European Commission Seventh Framework Programme and others.).
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Síndrome de Fanconi/genética , Túbulos Renales Proximales/metabolismo , Mitocondrias/metabolismo , Mutación Missense , Enzima Bifuncional Peroxisomal/genética , Secuencia de Aminoácidos , Animales , Población Negra , Cromosomas Humanos Par 3 , Modelos Animales de Enfermedad , Síndrome de Fanconi/etnología , Femenino , Ligamiento Genético , Humanos , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Linaje , Enzima Bifuncional Peroxisomal/química , Enzima Bifuncional Peroxisomal/metabolismo , Fenotipo , Análisis de Secuencia de ADNRESUMEN
Renal Fanconi syndrome (RFS) refers to the generalized dysfunction of the proximal tubule (PT) (Kleta R. Fanconi or not Fanconi? Lowe syndrome revisited. Clin J Am Soc Nephrol 2008; 3: 1244-1245). In its isolated form, RFS only affects the PT, but not the other nephron segments. The study of isolated RFS can thus provide specific insights into the function of the PT. In a recent paper, Klootwijk et al. investigated one such form of isolated RFS and revealed the underlying molecular basis (Klootwijk ED, Reichold M, Helip-Wooley A et al. Mistargeting of peroxisomal EHHADH and inherited renal Fanconi's syndrome. N Engl J Med 2014; 370: 129-138). The affected family had been described previously, demonstrating the typical features of RFS, such as low-molecular weight proteinuria, aminoaciduria, glycosuria and phosphaturia with consequent rickets; yet, importantly, patients had no evidence of impaired glomerular filtration (Tolaymat A, Sakarcan A, Neiberger R. Idiopathic Fanconi syndrome in a family. Part I. Clinical aspects. J Am Soc Nephrol 1992; 2: 1310-1317). Inheritance was consistent with an autosomal dominant mode. Klootwijk et al. discovered a surprising explanation: a heterozygous missense mutation causing partial mistargeting of the peroxisomal enzyme EHHADH to the mitochondria. Notably, disease causing was not the absence of the enzyme in the peroxisome, but its interference with mitochondrial function. The discovery of this novel disease mechanism not only confirmed the importance of mitochondrial function for PT transport, but also demonstrated the critical dependence of PT on fatty acid metabolism for energy generation.
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Síndrome de Fanconi/patología , Túbulos Renales Proximales/patología , ADN Mitocondrial/genética , Síndrome de Fanconi/genética , Heterocigoto , Humanos , Mutación Missense/genética , Enzima Bifuncional Peroxisomal/genéticaRESUMEN
TWIK1 belongs to the family of background K(+) channels with two pore domains. In native and transfected cells, TWIK1 is detected mainly in recycling endosomes. In principal cells in the kidney, TWIK1 gene inactivation leads to the loss of a nonselective cationic conductance, an unexpected effect that was attributed to adaptive regulation of other channels. Here, we show that TWIK1 ion selectivity is modulated by extracellular pH. Although TWIK1 is K(+) selective at neutral pH, it becomes permeable to Na(+) at the acidic pH found in endosomes. Selectivity recovery is slow after restoration of a neutral pH. Such hysteresis makes plausible a role of TWIK1 as a background channel in which selectivity and resulting inhibitory or excitatory influences on cell excitability rely on its recycling rate between internal acidic stores and the plasma membrane. TWIK1(-/-) pancreatic ß cells are more polarized than control cells, confirming a depolarizing role of TWIK1 in kidney and pancreatic cells.
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Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Concentración de Iones de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Canales de Potasio/química , Homología de Secuencia de Aminoácido , XenopusRESUMEN
Genetically modified mice represent important models for elucidating renal pathophysiology, but gene deletions frequently cause severe failure to thrive. In such cases, the analysis of the phenotype is often limited to the first weeks of life when renal excretory function undergoes dramatic physiological changes. Here, we investigated the postnatal dynamics of urinary ion excretion in mice. The profiles of urinary electrolyte excretion of mice were examined from birth until after weaning using an automated ion chromatography system. Postnatally, mice grew about 0.4 g/day, except during two phases with slower weight gain: (i) directly after birth during adaptation to extrauterine conditions (P0-P2) and (ii) during the weaning period (P15-P21), when nutrition changed from mother's milk to solid chow and water. During the first 3 days after birth, remarkable changes in urinary Na(+), Ca(2+), Mg(2+), and phosphate concentrations occurred, whereas K(+) and Cl(-) concentrations hardly changed. From days 4-14 after birth, Na(+), Ca(2+), Mg(2+), K(+), and Cl(-) concentrations remained relatively stable at low levels. Urinary concentrations of creatinine, NH4(+), phosphate, and sulfate constantly increased from birth until after weaning. Profiles of salt excretion in KCNJ10(-/-) mice exemplified the relevance of age-dependent analysis of urinary excretion. In conclusion, the most critical phases for analysis of renal ion excretion during the first weeks of life are directly after birth and during the weaning period. The age dependence of urinary excretion varies for the different ions. This should be taken into consideration when the renal phenotype of mice is investigated during the first weeks of life.
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Animales Recién Nacidos/crecimiento & desarrollo , Creatinina/orina , Iones/orina , Riñón/fisiología , Factores de Edad , Animales , Cromatografía por Intercambio Iónico , Masculino , Ratones , Ratones Endogámicos C57BL , DesteteRESUMEN
Task2 K(+) channel expression in the central nervous system is surprisingly restricted to a few brainstem nuclei, including the retrotrapezoid (RTN) region. All Task2-positive RTN neurons were lost in mice bearing a Phox2b mutation that causes the human congenital central hypoventilation syndrome. In plethysmography, Task2(-/-) mice showed disturbed chemosensory function with hypersensitivity to low CO(2) concentrations, leading to hyperventilation. Task2 probably is needed to stabilize the membrane potential of chemoreceptive cells. In addition, Task2(-/-) mice lost the long-term hypoxia-induced respiratory decrease whereas the acute carotid-body-mediated increase was maintained. The lack of anoxia-induced respiratory depression in the isolated brainstem-spinal cord preparation suggested a central origin of the phenotype. Task2 activation by reactive oxygen species generated during hypoxia could silence RTN neurons, thus contributing to respiratory depression. These data identify Task2 as a determinant of central O(2) chemoreception and demonstrate that this phenomenon is due to the activity of a small number of neurons located at the ventral medullary surface.
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Dióxido de Carbono/fisiología , Oxígeno/fisiología , Canales de Potasio de Dominio Poro en Tándem/fisiología , Centro Respiratorio/fisiología , Animales , Animales Recién Nacidos , Tronco Encefálico/patología , Tronco Encefálico/fisiología , Tronco Encefálico/fisiopatología , Células Quimiorreceptoras/patología , Células Quimiorreceptoras/fisiología , Modelos Animales de Enfermedad , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Humanos , Hipercapnia/fisiopatología , Hipoxia/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Pletismografía Total , Canales de Potasio de Dominio Poro en Tándem/deficiencia , Canales de Potasio de Dominio Poro en Tándem/genética , Embarazo , Fenómenos Fisiológicos Respiratorios , Apnea Central del Sueño/etiología , Apnea Central del Sueño/genética , Apnea Central del Sueño/fisiopatología , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
Mutations of the KCNJ10 (Kir4.1) K(+) channel underlie autosomal recessive epilepsy, ataxia, sensorineural deafness, and (a salt-wasting) renal tubulopathy (EAST) syndrome. We investigated the localization of KCNJ10 and the homologous KCNJ16 in kidney and the functional consequences of KCNJ10 mutations found in our patients with EAST syndrome. Kcnj10 and Kcnj16 were found in the basolateral membrane of mouse distal convoluted tubules, connecting tubules, and cortical collecting ducts. In the human kidney, KCNJ10 staining was additionally observed in the basolateral membrane of the cortical thick ascending limb of Henle's loop. EM of distal tubular cells of a patient with EAST syndrome showed reduced basal infoldings in this nephron segment, which likely reflects the morphological consequences of the impaired salt reabsorption capacity. When expressed in CHO and HEK293 cells, the KCNJ10 mutations R65P, G77R, and R175Q caused a marked impairment of channel function. R199X showed complete loss of function. Single-channel analysis revealed a strongly reduced mean open time. Qualitatively similar results were obtained with coexpression of KCNJ10/KCNJ16, suggesting a dominance of KCNJ10 function in native renal KCNJ10/KCNJ16 heteromers. The decrease in the current of R65P and R175Q was mainly caused by a remarkable shift of pH sensitivity to the alkaline range. In summary, EAST mutations of KCNJ10 lead to impaired channel function and structural changes in distal convoluted tubules. Intriguingly, the metabolic alkalosis present in patients carrying the R65P mutation possibly improves residual function of KCNJ10, which shows higher activity at alkaline pH.
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Anomalías Múltiples/genética , Mutación Missense , Canales de Potasio de Rectificación Interna/genética , Animales , Ataxia , Línea Celular , Epilepsia , Pérdida Auditiva Sensorineural , Humanos , Enfermedades Renales , Túbulos Renales Distales/patología , Ratones , Ratones Endogámicos C57BL , Canales de Potasio de Rectificación Interna/análisis , Síndrome , TransfecciónRESUMEN
TASK1 (KCNK3) and TASK3 (KCNK9) are two-pore domain potassium channels highly expressed in adrenal glands. TASK1/TASK3 heterodimers are believed to contribute to the background conductance whose inhibition by angiotensin II stimulates aldosterone secretion. We used task1-/- mice to analyze the role of this channel in adrenal gland function. Task1-/- exhibited severe hyperaldosteronism independent of salt intake, hypokalemia, and arterial 'low-renin' hypertension. The hyperaldosteronism was fully remediable by glucocorticoids. The aldosterone phenotype was caused by an adrenocortical zonation defect. Aldosterone synthase was absent in the outer cortex normally corresponding to the zona glomerulosa, but abundant in the reticulo-fasciculata zone. The impaired mineralocorticoid homeostasis and zonation were independent of the sex in young mice, but were restricted to females in adults. Patch-clamp experiments on adrenal cells suggest that task3 and other K+ channels compensate for the task1 absence. Adrenal zonation appears as a dynamic process that even can take place in adulthood. The striking changes in the adrenocortical architecture in task1-/- mice are the first demonstration of the causative role of a potassium channel in development/differentiation.
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Glándulas Suprarrenales/metabolismo , Homeostasis/genética , Mineralocorticoides/antagonistas & inhibidores , Mineralocorticoides/metabolismo , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Canales de Potasio de Dominio Poro en Tándem/deficiencia , Canales de Potasio de Dominio Poro en Tándem/genética , Glándulas Suprarrenales/patología , Aldosterona/sangre , Aldosterona/metabolismo , Animales , Femenino , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Potasio/sangre , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Renina/sangreRESUMEN
BACKGROUND: Five children from two consanguineous families presented with epilepsy beginning in infancy and severe ataxia, moderate sensorineural deafness, and a renal salt-losing tubulopathy with normotensive hypokalemic metabolic alkalosis. We investigated the genetic basis of this autosomal recessive disease, which we call the EAST syndrome (the presence of epilepsy, ataxia, sensorineural deafness, and tubulopathy). METHODS: Whole-genome linkage analysis was performed in the four affected children in one of the families. Newly identified mutations in a potassium-channel gene were evaluated with the use of a heterologous expression system. Protein expression and function were further investigated in genetically modified mice. RESULTS: Linkage analysis identified a single significant locus on chromosome 1q23.2 with a lod score of 4.98. This region contained the KCNJ10 gene, which encodes a potassium channel expressed in the brain, inner ear, and kidney. Sequencing of this candidate gene revealed homozygous missense mutations in affected persons in both families. These mutations, when expressed heterologously in xenopus oocytes, caused significant and specific decreases in potassium currents. Mice with Kcnj10 deletions became dehydrated, with definitive evidence of renal salt wasting. CONCLUSIONS: Mutations in KCNJ10 cause a specific disorder, consisting of epilepsy, ataxia, sensorineural deafness, and tubulopathy. Our findings indicate that KCNJ10 plays a major role in renal salt handling and, hence, possibly also in blood-pressure maintenance and its regulation.
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Ataxia/genética , Epilepsia/genética , Pérdida Auditiva Sensorineural/genética , Mutación Missense , Canales de Potasio de Rectificación Interna/genética , Defectos Congénitos del Transporte Tubular Renal/genética , Secuencia de Aminoácidos , Animales , Preescolar , Cromosomas Humanos Par 1 , Femenino , Genes Recesivos , Humanos , Escala de Lod , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Linaje , Fenotipo , Potasio/metabolismo , Análisis de Secuencia de ADN , Sodio/metabolismo , SíndromeRESUMEN
The SARS-CoV-2 virus has triggered a worldwide pandemic. According to the BioGrid database, CLN7 (MFSD8) is thought to interact with several viral proteins. The aim of this work was to investigate a possible involvement of CLN7 in the infection process. Experiments on a CLN7-deficient HEK293T cell line exhibited a 90% reduced viral load compared to wild-type cells. This observation may be linked to the finding that CLN7 ko cells have a significantly reduced GM1 content in their cell membrane. GM1 is found highly enriched in lipid rafts, which are thought to play an important role in SARS-CoV-2 infection. In contrast, overexpression of CLN7 led to an increase in viral load. This study provides evidence that CLN7 is involved in SARS-CoV-2 infection. This makes it a potential pharmacological target for drug development against COVID-19. Furthermore, it provides insights into the physiological function of CLN7 where still only little is known about.
RESUMEN
Mutations in the K+ channel gene KCNJ10 (Kir4.1) cause the autosomal recessive EAST syndrome which is characterized by epilepsy, ataxia, sensorineural deafness, and a salt-wasting tubulopathy. The renal salt-wasting pathology of EAST syndrome is caused by transport defects in the distal convoluted tubule where KCNJ10 plays a pivotal role as a basolateral K+ channel. This review on EAST syndrome outlines the molecular aspects of the physiology and pathophysiology of KCNJ10 in the distal convoluted tubule.
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Enfermedades Renales/genética , Canales de Potasio de Rectificación Interna/genética , Sales (Química)/metabolismo , Ataxia/genética , Epilepsia/genética , Humanos , Enfermedades Renales/fisiopatología , Túbulos Renales Distales/fisiopatología , Mutación/genética , Canales de Potasio de Rectificación Interna/fisiología , SíndromeRESUMEN
BACKGROUND/AIMS: Mutations in the inwardly-rectifying K+ channel KCNJ10/Kir4.1 cause an autosomal recessive disorder characterized by epilepsy, ataxia, sensorineural deafness and tubulopathy (EAST syndrome). KCNJ10 is expressed in the kidney distal convoluted tubule, cochlear stria vascularis and brain glial cells. Patients clinically diagnosed with EAST syndrome were genotyped to identify and study mutations in KCNJ10. METHODS: Patient DNA was sequenced and new mutations identified. Mutant and wild-type KCNJ10 constructs were cloned and heterologously expressed in Xenopus oocytes. Whole-cell K+ currents were measured by two-electrode voltage clamping. RESULTS: Three new mutations in KCNJ10 (p.R65C, p.F75L and p.V259fs259X) were identified, and mutation p.R297C, previously only seen in a compound heterozygous patient, was found in a homozygous state. Wild-type human KCNJ10-expressing oocytes showed strongly inwardly-rectified currents, which by comparison were significantly reduced in all the mutants (p < 0.001). Specific inhibition of KCNJ10 currents by Ba2+ demonstrated residual function in all mutant channels (p < 0.05) but V259X. CONCLUSION: This study confirms that EAST syndrome can be caused by many different mutations in KCNJ10 that significantly reduce K+ conductance. EAST syndrome should be considered in any patient with a renal Gitelman-like phenotype with additional neurological signs and symptoms like ataxia, epilepsy or sensorineural deafness.
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Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/fisiopatología , Discapacidad Intelectual/genética , Discapacidad Intelectual/fisiopatología , Canales de Potasio de Rectificación Interna/fisiología , Convulsiones/genética , Convulsiones/fisiopatología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Femenino , Genotipo , Pérdida Auditiva Sensorineural/metabolismo , Humanos , Discapacidad Intelectual/metabolismo , Túbulos Renales Distales/metabolismo , Masculino , Datos de Secuencia Molecular , Mutación , Oocitos/fisiología , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Convulsiones/metabolismo , Análisis de Secuencia de ADN , Xenopus laevis/genética , Xenopus laevis/fisiologíaRESUMEN
The mitochondria of the proximal tubule are essential for providing energy in this nephron segment, whose ATP generation is almost exclusively oxygen dependent. In addition, mitochondria are involved in a variety of metabolic processes and complex signaling networks. Proximal tubular mitochondrial dysfunction can therefore affect renal function in very different ways. Two autosomal dominantly inherited forms of renal Fanconi syndrome illustrate how multifaceted mitochondrial pathology can be: Mutation of EHHADH, an enzyme in fatty acid metabolism, results in decreased ATP synthesis and a consecutive transport defect. In contrast, mutations of GATM, an enzyme in the creatine biosynthetic pathway, leave ATP synthesis unaffected but do lead to mitochondrial protein aggregates, inflammasome activation, and renal fibrosis with progressive renal failure. In this review article, the distinct pathophysiological mechanisms of these two diseases are presented, which are examples of the spectrum of proximal tubular mitochondrial diseases.
RESUMEN
We recently reported an autosomal dominant form of renal Fanconi syndrome caused by a missense mutation in the third codon of the peroxisomal protein EHHADH. The mutation mistargets EHHADH to mitochondria, thereby impairing mitochondrial energy production and, consequently, reabsorption of electrolytes and low-molecular-weight nutrients in the proximal tubule. Here, we further elucidate the molecular mechanism underlying this pathology. We find that mutated EHHADH is incorporated into mitochondrial trifunctional protein (MTP), thereby disturbing ß-oxidation of long-chain fatty acids. The resulting MTP deficiency leads to a characteristic accumulation of hydroxyacyl- and acylcarnitines. Mutated EHHADH also limits respiratory complex I and corresponding supercomplex formation, leading to decreases in oxidative phosphorylation capacity, mitochondrial membrane potential maintenance, and ATP generation. Activity of the Na(+)/K(+)-ATPase is thereby diminished, ultimately decreasing the transport activity of the proximal tubule cells.
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Síndrome de Fanconi/metabolismo , Riñón/metabolismo , Riñón/patología , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Animales , Transporte Biológico , Extractos Celulares , Metabolismo Energético , Síndrome de Fanconi/complicaciones , Síndrome de Fanconi/patología , Ácidos Grasos/metabolismo , Células LLC-PK1 , Microscopía Confocal , Enfermedades Mitocondriales/complicaciones , Enfermedades Mitocondriales/patología , Mutación/genética , Oxidación-Reducción , Enzima Bifuncional Peroxisomal/metabolismo , Proteómica , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Fracciones Subcelulares/metabolismo , PorcinosRESUMEN
Task1 and Task3 potassium channels (Task: tandem of P domains in a weak inward rectifying K(+) channel-related acid-sensitive K(+) channel) are believed to control the membrane voltage of aldosterone-producing adrenal glomerulosa cells. This study aimed at understanding the role of Task3 for the control of aldosterone secretion. The adrenal phenotype of Task3(-/-) mice was investigated using electrophysiology, adrenal slices, and blood pressure measurements. Primary adrenocortical cells of Task3(-/-) mice were strongly depolarized compared with wild-type (-52 vs. -79 mV), and in fresh adrenal slices Ca(2+) signaling of Task3(-/-) glomerulosa cells was abnormal. In living Task3(-/-) mice, the regulation of aldosterone secretion showed specific deficits: Under low Na(+) and high K(+) diets, protocols known to increase aldosterone, and under standard diet, Task3 inactivation was compensated and aldosterone was normal. However, high Na(+) and low K(+) diets, two protocols known to lower aldosterone, failed to lower aldosterone in Task3(-/-) mice. The physiological regulation of aldosterone was disturbed: aldosterone-renin ratio, an indicator of autonomous aldosterone secretion, was 3-fold elevated at standard and high Na(+) diets. Isolated adrenal glands of Task3(-/-) produced 2-fold more aldosterone. As a consequence, Task3(-/-) mice showed salt-sensitive arterial hypertension (plus 10 mm Hg). In conclusion, Task3 plays an important role in the adaptation of aldosterone secretion to dietary salt intake.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Presión Sanguínea/genética , Hipertensión/genética , Canales de Potasio/genética , Renina/sangre , Cloruro de Sodio Dietético , Adaptación Fisiológica/genética , Glándulas Suprarrenales/citología , Aldosterona/sangre , Animales , Calcio/metabolismo , Células Cultivadas , Hipertensión/metabolismo , Ratones , Ratones Noqueados , Fenotipo , Canales de Potasio/metabolismo , Zona Glomerular/citología , Zona Glomerular/metabolismoRESUMEN
The positively charged fluorescent dyes ethidium (Et(+)) and propidium (Pr(2+)) are widely used as DNA and necrosis markers. Et(+) is cytotoxic and mutagenic. The polyspecific organic cation transporters OCT1 (SLC22A1), OCT2 (SLC22A2), and OCT3 (SLC22A3) mediate electrogenic facilitated diffusion of small (< or =500 Da) organic cations with broad specificities. In humans, OCT2 mediates basolateral uptake by kidney proximal tubules (PT), whereas in rodents OCT1/2 are involved. In mouse kidney, perfused Et(+) accumulated predominantly in the S2/S3 segments of the PT, but not Pr(2+). In cells stably overexpressing human OCTs (hOCTs), Et(+) uptake was observed with K(m) values of 0.8 +/- 0.2 microM (hOCT1), 1.7 +/- 0.5 microM (hOCT2), and 2.0 +/- 0.5 microM (hOCT3), whereas Pr(2+) was not transported. Accumulation of Et(+) was inhibited by OCT substrates quinine, 3-methyl-4-phenylpyridinium (MPP(+)), cimetidine, and tetraethylammonium (TEA(+)). For hOCT1 and hOCT2, the IC(50) values for MPP(+), TEA(+), and cimetidine were higher than for inhibition of previously tested transported substrates. For hOCT2, the inhibition of Et(+) uptake by MPP(+) and cimetidine was shown to be competitive. Et(+) also inhibited transport of 0.1 microM [(3)H]MPP(+) by all hOCT isoforms with IC(50) values between 0.4 and 1.3 microM, and the inhibition of hOCT1-mediated uptake of MPP(+) by Et(+) was competitive. In Oct1/2(-/-) mice, Et(+) uptake in the PT was almost abolished. The data demonstrate that Et(+) is taken up avidly by the PT, which is mediated by OCT1 and/or OCT2. Considering the high affinity of OCTs for Et(+) and their strong expression in various organs, strict safety guidelines for Et(+) handling should be reinforced.
Asunto(s)
Colorantes/farmacocinética , Etidio/farmacocinética , Túbulos Renales Proximales/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , Animales , Transporte Biológico Activo/fisiología , Células CHO , Cricetinae , Cricetulus , Regulación de la Expresión Génica/fisiología , Humanos , Concentración 50 Inhibidora , Túbulos Renales Proximales/citología , Ratones , Ratones Noqueados , Paraquat/química , Paraquat/farmacología , Propidio/química , Propidio/farmacología , Isoformas de Proteínas , Especificidad por SustratoRESUMEN
BACKGROUND/AIMS: Heteromeric KCNEx/KCNQ1 (=KvLQT1, Kv7.1) K(+) channels are important for repolarization of cardiac myocytes, endolymph secretion in the inner ear, gastric acid secretion, and transport across epithelia. They are modulated by pH in a complex way: homomeric KCNQ1 is inhibited by external acidification (low pH(e)); KCNE2/KCNQ1 is activated; and for KCNE1/KCNQ1, variable effects have been reported. METHODS: The role of KCNE subunits for the effect of pH(e) on KCNQ1 was analyzed in transfected COS cells and cardiac myocytes by the patch-clamp technique. RESULTS: In outside-out patches of transfected cells, hKCNE2/hKCNQ1 current was increased by acidification down to pH 4.5. Chimeras with the acid-insensitive hKCNE3 revealed that the extracellular N-terminus and at least part of the transmembrane domain of hKCNE2 are needed for activation by low pH(e). hKCNE1/hKCNQ1 heteromeric channels exhibited marked changes of biophysical properties at low pH(e): The slowly activating hKCNE1/hKCNQ1 channels were converted into constitutively open, non-deactivating channels. Experiments on guinea pig and mouse cardiac myocytes pointed to an important role of KCNQ1 during acidosis implicating a significant contribution to cardiac repolarization under acidic conditions. CONCLUSION: External pH can modify current amplitude and biophysical properties of KCNQ1. KCNE subunits work as molecular switches by modulating the pH sensitivity of human KCNQ1.
Asunto(s)
Canal de Potasio KCNQ1/química , Miocitos Cardíacos/efectos de los fármacos , Canales de Potasio con Entrada de Voltaje/química , Ácidos/farmacología , Animales , Conductividad Eléctrica , Cobayas , Humanos , Concentración de Iones de Hidrógeno , Canal de Potasio KCNQ1/fisiología , Miocitos Cardíacos/fisiología , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/fisiología , Estructura Terciaria de Proteína , RatasRESUMEN
A novel model for the regulation of cell excitability has recently been proposed. It originates from the observation that the background K(+) channel K2P1 (TWIK1) may be silenced by sumoylation in Xenopus oocytes and that inactivation of the putative sumoylation site (mutation K274E) gives rise to robust current expression in transfected COS-7 cells. Here, we show that only the mutation K274E, and not K274R, is associated with an increase of K2P1 current density, suggesting a charge effect of K274E. Furthermore, we failed to observe any band shift by western blot analysis that would confirm an eventual sumoylation of K2P1 in COS-7 cells and oocytes.