RESUMO
Deficiency of the cysteine protease inhibitor cystatin M/E (Cst6) in mice leads to disturbed epidermal cornification, impaired barrier function, and neonatal lethality. We report the rescue of the lethal skin phenotype of ichq (Cst6-deficient; Cst6-/-) mice by transgenic, epidermis-specific, reexpression of Cst6 under control of the human involucrin (INV) promoter. Rescued Tg(INV-Cst6)Cst6ichq/ichq mice survive the neonatal phase, but display severe eye pathology and alopecia after 4 mo. We observed keratitis and squamous metaplasia of the corneal epithelium, comparable to Cst6-/-Ctsl+/- mice, as we have reported in other studies. We found the INV promoter to be active in the hair follicle infundibulum; however, we did not observe Cst6 protein expression in the lower regions of the hair follicle in Tg(INV-Cst6)Cst6ichq/ichq mice. This result suggests that unrestricted activity of proteases is involved in disturbance of hair follicle biology, eventually leading to baldness. Using quenched activity-based probes, we identified mouse cathepsin B (CtsB), which is expressed in the lower regions of the hair follicle, as an additional target of mouse Cst6. These data suggest that Cst6 is necessary to control CtsB activity in hair follicle morphogenesis and highlight Cst6-controlled proteolytic pathways as targets for preventing hair loss.-Oortveld, M. A. W., van Vlijmen-Willems, I. M. J. J., Kersten, F. F. J., Cheng, T., Verdoes, M., van Erp, P. E. J., Verbeek, S., Reinheckel, T., Hendriks, W. J. A. J., Schalkwijk, J., Zeeuwen, P. L. J. M. Cathepsin B as a potential cystatin M/E target in the mouse hair follicle.
Assuntos
Catepsina B/metabolismo , Diferenciação Celular/fisiologia , Cistatina M/metabolismo , Epiderme/metabolismo , Folículo Piloso/metabolismo , Alopecia/metabolismo , Animais , Catepsina L/metabolismo , Células Cultivadas , Cistatina M/deficiência , Humanos , Camundongos , Pele/metabolismoRESUMO
Leber congenital amaurosis (LCA) causes blindness or severe visual impairment at or within a few months of birth. Here we show, using homozygosity mapping, that the LCA5 gene on chromosome 6q14, which encodes the previously unknown ciliary protein lebercilin, is associated with this disease. We detected homozygous nonsense and frameshift mutations in LCA5 in five families affected with LCA. In a sixth family, the LCA5 transcript was completely absent. LCA5 is expressed widely throughout development, although the phenotype in affected individuals is limited to the eye. Lebercilin localizes to the connecting cilia of photoreceptors and to the microtubules, centrioles and primary cilia of cultured mammalian cells. Using tandem affinity purification, we identified 24 proteins that link lebercilin to centrosomal and ciliary functions. Members of this interactome represent candidate genes for LCA and other ciliopathies. Our findings emphasize the emerging role of disrupted ciliary processes in the molecular pathogenesis of LCA.
Assuntos
Proteínas do Olho/genética , Proteínas Associadas aos Microtúbulos/genética , Atrofia Óptica Hereditária de Leber/genética , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Cílios/genética , Códon sem Sentido , Proteínas do Olho/metabolismo , Feminino , Mutação da Fase de Leitura , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Linhagem , Ratos , Ratos WistarRESUMO
Late cornified envelope (LCE) proteins are small cationic epidermal proteins with antimicrobial properties, and the combined deletion of LCE3B and LCE3C genes is a risk factor for psoriasis that affects skin microbiome composition. In a yeast two-hybrid screen, we identified CYSRT1 as an interacting partner of members of all LCE groups except LCE6. These interactions were confirmed in a mammalian cell system by coimmunoprecipitation. CYSRT1 is a protein of unknown function that is specifically expressed in cutaneous and oral epithelia and spatially colocalizes with LCE proteins in the upper layers of the suprabasal epidermis. Constitutive CYSRT1 expression is present in fully differentiated epidermis and can be further induced in vivo by disruption of the skin barrier upon stratum corneum removal. Transcriptional regulation correlates to keratinocyte terminal differentiation but not to skin bacteria exposure. Similar to LCEs, CYSRT1 was found to have antibacterial activity against Pseudomonas aeruginosa. Comparative gene sequence analysis and protein amino acid alignment indicate that CYSRT1 is highly conserved among vertebrates and has putative antimicrobial activity. To summarize, we identified CYSRT1 in the outer skin layer, where it colocalizes with LCE proteins and contributes to the constitutive epidermal antimicrobial host defense repertoire.
Assuntos
Anti-Infecciosos , Psoríase , Anti-Infecciosos/metabolismo , Proteínas Ricas em Prolina do Estrato Córneo/genética , Proteínas Ricas em Prolina do Estrato Córneo/metabolismo , Epiderme/metabolismo , Queratinócitos/metabolismo , Proteínas/metabolismo , Psoríase/genética , Psoríase/metabolismo , Pele/metabolismo , HumanosRESUMO
The protease inhibitor cystatin M/E (CST6) regulates a biochemical pathway involved in stratum corneum homeostasis, and its deficiency in mice causes ichthyosis and neonatal lethality. Cystatin M/E deficiency has not been described in humans so far, and we did not detect disease-causing mutations in the CST6 gene in a large number of patients with autosomal recessive congenital ichthyosis, who were negative for mutations in known ichthyosis-associated genes. To investigate the phenotype of CST6 deficiency in human epidermis, we used lentiviral delivery of short hairpin RNAs that target CST6 in a 3D reconstructed skin model. Surprisingly, CST6 deficiency did not cause an ichthyosis-like phenotype, but prevented the development of a multilayered epidermis. From this study, we conclude that CST6 deficiency may be incompatible with normal human foetal development.
Assuntos
Cistatina M/genética , Epiderme/crescimento & desenvolvimento , Lentivirus/genética , Morfogênese/genética , RNA Interferente Pequeno/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cistatina M/fisiologia , Células Epidérmicas , Epiderme/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Ictiose/patologia , Modelos Biológicos , Morfogênese/efeitos dos fármacos , Fenótipo , RNA Interferente Pequeno/farmacologia , Pele Artificial , Alicerces TeciduaisRESUMO
Usher syndrome (USH) and Leber congenital amaurosis (LCA) are autosomal recessive disorders resulting in syndromic and non-syndromic forms of blindness. In order to gain insight into the pathogenic mechanisms underlying retinal degeneration, we searched for interacting proteins of USH2A isoform B (USH2A(isoB)) and the LCA5-encoded protein lebercilin. We identified a novel isoform of the centrosomal ninein-like protein, hereby named Nlp isoform B (Nlp(isoB)), as a common interactor. Although we identified the capacity of this protein to bind calcium with one of its three EF-hand domains, the interacton with USH2A(isoB) did not depend on this. Upon expression in ARPE-19 cells, recombinant Nlp(isoB), lebercilin and USH2A(isoB) were all found to co-localize at the centrosomes. Staining of retinal sections with specific antibodies against all three proteins revealed their co-localization at the basal bodies of the photoreceptor-connecting cilia. Based on this subcellular localization and the nature of their previously identified binding partners, we hypothesize that the pathogenic mechanisms for LCA and USH show significant overlap and involve defects in ciliogenesis, cilia maintenance and intraflagellar and/or microtubule-based transport. The direct association of Nlp(isoB) with USH2A(isoB) and lebercilin indicates that Nlp can be considered as a novel candidate gene for USH, LCA and allied retinal ciliopathies.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Atrofia Óptica Hereditária de Leber/metabolismo , Síndromes de Usher/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Atrofia Óptica Hereditária de Leber/genética , Células Fotorreceptoras/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Wistar , Retina/metabolismo , Alinhamento de Sequência , Técnicas do Sistema de Duplo-Híbrido , Síndromes de Usher/genéticaRESUMO
OBJECTIVE: To describe the mutations in the CLRN1 gene in patients from 2 consanguineous Pakistani families diagnosed with autosomal recessive retinitis pigmentosa (arRP). DESIGN: Case-series study. PARTICIPANTS: Affected and unaffected individuals of 2 consanguineous Pakistani families and 90 unaffected controls from the same population. Informed consent was obtained from participants and the protocol was approved by a local institutional review board. METHODS: Patients of 2 consanguineous families were genotyped with single-nucleotide polymorphism microarrays for genome-wide linkage analysis. The search for potential candidate genes within the 8-Mb overlapping homozygous region in these families revealed the presence of CLRN1, a gene previously known to cause Usher's syndrome type III (USH3), which was analyzed by direct sequence analysis. The clinical diagnosis was based on the presence of night blindness, fundoscopic findings, and electroretinography (ERG) results. Additionally, pure tone audiometry was performed to rule out Usher's syndrome. MAIN OUTCOME MEASURES: Fundoscopy, single-nucleotide polymorphism microarray, DNA sequence analysis, ERG, and audiometry. RESULTS: Sequencing of CLRN1 revealed novel missense mutations (p.Pro31Leu and p.Leu154Trp) segregating in 2 families. Analysis of fundus photographs indicated attenuation of the retinal vessels, and bone spicule pigmentation in the periphery of the retina. The ERG responses were indicative of a rod-cone pattern of the disease. Audiometric assessment revealed no hearing impairment, thereby excluding Usher's syndrome. Subcellular localization studies demonstrated the retention of the mutant proteins in the endoplasmic reticulum, whereas the wild-type protein was mainly present at the cell membrane. CONCLUSIONS: The RP-associated mutations p.Pro31Leu and p.Leu154Trp may represent hypomorphic mutations, because the substituted amino acids located in the transmembrane domains remain polar, whereas more severe changes have been detected in patients with USH3. These data indicate that mutations in CLRN1 are associated not only with USH3, but also with nonsyndromic arRP.
Assuntos
Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Retinose Pigmentar/genética , Adulto , Audiometria de Tons Puros , Consanguinidade , DNA/genética , Eletrorretinografia , Fundo de Olho , Genes Recessivos , Ligação Genética , Humanos , Espaço Intracelular/metabolismo , Leucina , Análise em Microsséries , Mutação de Sentido Incorreto/genética , Cegueira Noturna/etiologia , Polimorfismo de Nucleotídeo Único , Prolina , Retinose Pigmentar/complicações , Retinose Pigmentar/diagnóstico , Retinose Pigmentar/fisiopatologia , Distribuição Tecidual , Triptofano , Adulto JovemRESUMO
Ca2+ ions play a fundamental role in many cellular processes, and the extracellular concentration of Ca2+ is kept under strict control to allow the proper physiological functions to take place. The kidney, small intestine, and bone determine the Ca2+ flux to the extracellular Ca2+ pool in a concerted fashion. Transient receptor potential (TRP) cation channel subfamily V, members 5 and 6 (TRPV5 and TRPV6) have recently been postulated to be the molecular gatekeepers facilitating Ca2+ influx in these tissues and are members of the TRP family, which mediates diverse biological effects ranging from pain perception to male aggression. Genetic ablation of TRPV5 in the mouse allowed us to investigate the function of this novel Ca2+ channel in maintaining the Ca2+ balance. Here, we demonstrate that mice lacking TRPV5 display diminished active Ca2+ reabsorption despite enhanced vitamin D levels, causing severe hypercalciuria. In vivo micropuncture experiments demonstrated that Ca2+ reabsorption was malfunctioning within the early part of the distal convolution, exactly where TRPV5 is localized. In addition, compensatory hyperabsorption of dietary Ca2+ was measured in TRPV5 knockout mice. Furthermore, the knockout mice exhibited significant disturbances in bone structure, including reduced trabecular and cortical bone thickness. These data demonstrate the key function of TRPV5 in active Ca2+ reabsorption and its essential role in the Ca2+ homeostasis.
Assuntos
Osso e Ossos/fisiologia , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Canais de Cálcio/fisiologia , Cálcio/metabolismo , Rim/metabolismo , Absorção , Animais , Sinalização do Cálcio , Cátions , Feminino , Fêmur/metabolismo , Biblioteca Gênica , Genótipo , Imuno-Histoquímica , Íons , Nefropatias/metabolismo , Masculino , Camundongos , Camundongos Knockout , Modelos Genéticos , Osteoporose , Fenótipo , Potássio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sódio/metabolismo , Canais de Cátion TRPV , Fatores de TempoRESUMO
In two large Turkish consanguineous families, a locus for autosomal recessive nonsyndromic hearing loss (ARNSHL) was mapped to chromosome 6p21.3 by genome-wide linkage analysis in an interval overlapping with the loci DFNB53 (COL11A2), DFNB66, and DFNB67. Fine mapping excluded DFNB53 and subsequently homozygous mutations were identified in the lipoma HMGIC fusion partner-like 5 (LHFPL5) gene, also named tetraspan membrane protein of hair cell stereocilia (TMHS) gene, which was recently shown to be mutated in the "hurry scurry" mouse and in two DFNB67-linked families from Pakistan. In one family, we found a homozygous one-base pair deletion, c.649delG (p.Glu216ArgfsX26) and in the other family we identified a homozygous transition c.494C>T (p.Thr165Met). Further screening of index patients from 96 Turkish ARNSHL families and 90 Dutch ARNSHL patients identified one additional Turkish family carrying the c.649delG mutation. Haplotype analysis revealed that the c.649delG mutation was located on a common haplotype in both families. Mutation screening of the LHFPL5 homologs LHFPL3 and LHFPL4 did not reveal any disease causing mutation. Our findings indicate that LHFPL5 is essential for normal function of the human cochlea.
Assuntos
Mutação da Fase de Leitura , Perda Auditiva Bilateral/genética , Perda Auditiva Neurossensorial/genética , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Mapeamento Cromossômico , Cromossomos Humanos Par 5 , Consanguinidade , Análise Mutacional de DNA , Feminino , Ligação Genética , Haplótipos , Perda Auditiva Bilateral/diagnóstico , Perda Auditiva Neurossensorial/diagnóstico , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Alinhamento de SequênciaRESUMO
PURPOSE: Usher syndrome is the most common form of hereditary deaf-blindness. It is both clinically and genetically heterogeneous. The USH2D protein whirlin interacts via its PDZ domains with other Usher-associated proteins containing a C-terminal type I PDZ-binding motif. These proteins co-localize with whirlin at the region of the connecting cilium and at the synapse of photoreceptor cells. This study was undertaken to identify novel, Usher syndrome-associated, interacting partners of whirlin and thereby obtain more insights into the function of whirlin. METHODS: The database of ciliary proteins was searched for proteins that are present in both the retina and inner ear and contain a PDZ-binding motif. Interactions with whirlin were evaluated by yeast two-hybrid analyses and validated by glutathione S-transferase pull-down assays, co-immunoprecipitation, and co-localization in the retina with immunofluorescence and immunoelectron microscopy. RESULTS: The L-type calcium channel subunit Ca(v)1.3 (alpha(1D)) specifically interacts with whirlin. In adult photoreceptors, Ca(v)1.3 (alpha(1D)) and whirlin co-localize in the region of the connecting cilium and at the synapse. During murine embryonic development, the expression patterns of the Whrn and Cacna1d genes show significant overlap and include expression in the eye, the inner ear, and the central nervous system. CONCLUSIONS: The findings indicate that Ca(v)1.3 (alpha(1D)) is connected to the Usher protein network. This conclusion leads to the hypothesis that, in the retina, whirlin scaffolds Ca(v)1.3 (alpha(1D)) and therefore contributes to the organization of calcium channels in the photoreceptor cells, where both proteins may be involved in membrane fusions.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteínas de Membrana/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Animais , Western Blotting , Células COS , Canais de Cálcio Tipo L/genética , Chlorocebus aethiops , Biologia Computacional , Bases de Dados de Proteínas , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Imunoeletrônica , Cílio Conector dos Fotorreceptores/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Retina/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The human Usher syndrome (USH) is the most frequent cause of combined deaf-blindness. USH is genetically heterogeneous with at least 12 chromosomal loci assigned to three clinical types, USH1-3. Although these USH types exhibit similar phenotypes in human, the corresponding gene products belong to very different protein classes and families. The scaffold protein harmonin (USH1C) was shown to integrate all identified USH1 and USH2 molecules into protein networks. Here, we analyzed a protein network organized in the absence of harmonin by the scaffold proteins SANS (USH1G) and whirlin (USH2D). Immunoelectron microscopic analyses disclosed the colocalization of all network components in the apical inner segment collar and the ciliary apparatus of mammalian photoreceptor cells. In this complex, whirlin and SANS directly interact. Furthermore, SANS provides a linkage to the microtubule transport machinery, whereas whirlin may anchor USH2A isoform b and VLGR1b (very large G-protein coupled receptor 1b) via binding to their cytodomains at specific membrane domains. The long ectodomains of both transmembrane proteins extend into the gap between the adjacent membranes of the connecting cilium and the apical inner segment. Analyses of Vlgr1/del7TM mice revealed the ectodomain of VLGR1b as a component of fibrous links present in this gap. Comparative analyses of mouse and Xenopus photoreceptors demonstrated that this USH protein network is also part of the periciliary ridge complex in Xenopus. Since this structural specialization in amphibian photoreceptor cells defines a specialized membrane domain for docking and fusion of transport vesicles, we suggest a prominent role of the USH proteins in cargo shipment.
Assuntos
Células Fotorreceptoras de Vertebrados/metabolismo , Síndromes de Usher/genética , Síndromes de Usher/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células COS , Proteínas de Ciclo Celular , Chlorocebus aethiops , Proteínas do Citoesqueleto , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Humanos , Técnicas In Vitro , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Imunoeletrônica , Modelos Biológicos , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células Fotorreceptoras de Vertebrados/ultraestrutura , Mapeamento de Interação de Proteínas , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Frações Subcelulares/metabolismo , Transfecção , Síndromes de Usher/classificação , Xenopus/genética , Xenopus/metabolismoRESUMO
The highly ordered distribution of neurons is an essential feature of a functional mammalian retina. Disruptions in the apico-basal polarity complexes at the outer limiting membrane (OLM) of the retina are associated with retinal patterning defects in vertebrates. We have analyzed the binding repertoire of MPP5/Pals1, a key member of the apico-basal Crumbs polarity complex, that has functionally conserved counterparts in zebrafish (nagie oko) and Drosophila (Stardust). We show that MPP5 interacts with its MAGUK family member MPP1/p55 at the OLM. Mechanistically, this interaction involves heterodimerization of both MAGUK modules in a directional fashion. MPP1 expression in the retina throughout development resembles the expression of whirlin, a multi-PDZ scaffold protein and an important organizer in the Usher protein network. We demonstrate that both proteins interact strongly by both a classical PDZ domain-to-PDZ binding motif (PBM) mechanism, and a mechanism involving internal epitopes. MPP1 and whirlin colocalize in the retina at the OLM, at the outer synaptic layer and at the basal bodies and the ciliary axoneme. In view of the known roles of the Crumbs and Usher protein networks, our findings suggest a novel link of the core developmental processes of actin polymerization and establishment/maintenance of apico-basal cell polarity through MPP1. These processes, essential in neural development and patterning of the retina, may be disrupted in eye disorders that are associated with defects in these protein networks.
Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Núcleosídeo-Fosfato Quinase/metabolismo , Retina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas Sanguíneas/genética , Membrana Celular/metabolismo , Embrião de Mamíferos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Olho/genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Modelos Genéticos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Núcleosídeo-Fosfato Quinase/química , Núcleosídeo-Fosfato Quinase/genética , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Técnicas do Sistema de Duplo-HíbridoRESUMO
Mutations in the DFNB31 gene encoding the PDZ scaffold protein whirlin are causative for hearing loss in man and mouse. Whirlin is known to be essential for the elongation process of the stereocilia of sensory hair cells in the inner ear, though its complete spatial and temporal expression patterns remained elusive. Here, we demonstrate that, in embryonic development, the gene is not only expressed in the inner ear, but also in the developing brain and the retina. Various isoforms of whirlin are widely and differentially expressed, and we provide evidence that whirlin directly associates with USH2A isoform b and VLGR1b, two proteins that we previously reported to be part of the Usher protein interactome. These proteins co-localize with whirlin at the synaptic regions of both photoreceptor cells and outer hair cells in the cochlea. These findings indicate that whirlin is part of a macromolecular PDZ protein scaffold that functions in the organization of the pre- and/or postsynaptic side of photoreceptor and hair cell synapses. Whirlin might be involved in synaptic adhesion through interaction with USH2A and VLGR1b as well as in synaptic development as suggested by its spatial and temporal expression patterns. In addition, we demonstrate that whirlin, USH2A and Vlgr1b co-localize at the connecting cilium and the outer limiting membrane of photoreceptor cells and in spiral ganglion neurons of the inner ear. Our data show that whirlin is connected to the dynamic Usher protein interactome and indicate that whirlin has a pleiotropic function in both the retina and the inner ear.
Assuntos
Cóclea/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas de Membrana/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Retina/metabolismo , Animais , Células COS , Chlorocebus aethiops , Proteínas da Matriz Extracelular/genética , Cobaias , Imuno-Histoquímica , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Receptores Acoplados a Proteínas G/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
The primary mediator of NaCl reabsorption in the renal distal tubule is the human bumetanide-sensitive Na(+)-K(+)-2Cl(-) co-transporter (hNKCC2), located at the apical membrane of the thick ascending limb of Henle's loop. The physiologic importance of this transporter is emphasized by the tubular disorder Bartter syndrome type I, which arises from the functional impairment of hNKCC2 as a result of mutations in the SLC12A1 gene. The aim of the present study was to investigate the oligomeric state of hNKCC2 to understand further its operational mechanism. To this end, hNKCC2 was heterologously expressed in Xenopus laevis oocytes. Chemical cross-linking with dimethyl-3,3-dithio-bis-propionamidate indicated that hNKCC2 subunits can reversibly form high molecular weight complexes. Co-immunoprecipitation of tagged hNKCC2 subunits further substantiated a physical interaction between individual hNKCC2 subunits. The size of the hNKCC2 multimers was determined by sucrose gradient centrifugation, and a preference for dimeric complexes (approximately 320 kD) was demonstrated. Finally, concatemeric constructs consisting of two wild-type subunits or a wild-type and a functionally impaired hNKCC2 subunit (G319R) were expressed in oocytes. Subsequently, the concatemers were functionally characterized, resulting in a significant bumetanide-sensitive (22)Na(+) uptake of 2.5 +/- 0.2 nmol/oocyte per 30 min for the wild-type-wild-type concatemer, which was reduced to 1.3 +/- 0.1 nmol/oocyte per 30 min for the wild-type-G319R concatemer. In conclusion, this study suggests that hNKCC2 forms at least functional dimers when expressed in Xenopus laevis oocytes of which the individual subunits transport Na(+) independently.
Assuntos
Acidose Tubular Renal/metabolismo , Simportadores de Cloreto de Sódio-Potássio/química , Precipitação Química , Humanos , Membro 2 da Família 12 de Carreador de SolutoRESUMO
Bartter syndrome (BS) is a heterogeneous renal tubular disorder affecting Na-K-Cl reabsorption in the thick ascending limb of Henle's loop. BS type I patients typically present with profound hypokalemia and metabolic alkalosis. The main goal of the present study was to elucidate the functional implications of six homozygous mutations (G193R, A267S, G319R, A508T, del526N, and Y998X) in the bumetanide-sensitive Na-K-2Cl cotransporter (hNKCC2) identified in patients diagnosed with BS type I. To this end, capped RNA (cRNA) of FLAG-tagged hNKCC2 and the corresponding mutants was injected in Xenopus laevis oocytes and transporter activity was measured after 72 h by means of a bumetanide-sensitive (22)Na(+) uptake assay at 30 degrees C. Injection of 25 ng of hNKCC2 cRNA resulted in bumetanide-sensitive (22)Na(+) uptake of 2.5 +/- 0.5 nmol/oocyte per 30 min. Injection of 25 ng of mutant cRNA yielded no significant bumetanide-sensitive (22)Na(+) uptake. Expression of wild-type and mutant transporters was confirmed by immunoblotting, showing significantly less mutant protein compared with wild-type at the same cRNA injection levels. However, when the wild-type cRNA injection level was reduced to obtain a protein expression level equal to that of the mutants, the wild-type still exhibited a significant bumetanide-sensitive (22)Na(+) uptake. Immunocytochemical analysis showed immunopositive staining of hNKCC2 at the plasma membrane for wild-type and all studied mutants. In conclusion, mutations in hNKCC2 identified in type I BS patients, when expressed in Xenopus oocytes, result in a low expression of normally routed but functionally impaired transporters. These results are in line with the hypothesis that the mutations in hNKCC2 are the underlying cause of the clinical abnormalities seen in patients with type I BS.