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1.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199759

RESUMO

The TWIK-related spinal cord potassium channel (TRESK) is encoded by KCNK18, and variants in this gene have previously been associated with susceptibility to familial migraine with aura (MIM #613656). A single amino acid substitution in the same protein, p.Trp101Arg, has also been associated with intellectual disability (ID), opening the possibility that variants in this gene might be involved in different disorders. Here, we report the identification of KCNK18 biallelic missense variants (p.Tyr163Asp and p.Ser252Leu) in a family characterized by three siblings affected by mild-to-moderate ID, autism spectrum disorder (ASD) and other neurodevelopment-related features. Functional characterization of the variants alone or in combination showed impaired channel activity. Interestingly, Ser252 is an important regulatory site of TRESK, suggesting that alteration of this residue could lead to additive downstream effects. The functional relevance of these mutations and the observed co-segregation in all the affected members of the family expand the clinical variability associated with altered TRESK function and provide further insight into the relationship between altered function of this ion channel and human disease.


Assuntos
Alelos , Deficiência Intelectual/genética , Mutação/genética , Transtornos do Neurodesenvolvimento/genética , Canais de Potássio/genética , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Calcineurina/metabolismo , Feminino , Genoma Humano , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ionomicina/farmacologia , Masculino , Linhagem , Canais de Potássio/química , Irmãos , Xenopus laevis/metabolismo , Adulto Jovem
2.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205849

RESUMO

The ability of spermatozoa to swim towards an oocyte and fertilize it depends on precise K+ permeability changes. Kir5.1 is an inwardly-rectifying potassium (Kir) channel with high sensitivity to intracellular H+ (pHi) and extracellular K+ concentration [K+]o, and hence provides a link between pHi and [K+]o changes and membrane potential. The intrinsic pHi sensitivity of Kir5.1 suggests a possible role for this channel in the pHi-dependent processes that take place during fertilization. However, despite the localization of Kir5.1 in murine spermatozoa, and its increased expression with age and sexual maturity, the role of the channel in sperm morphology, maturity, motility, and fertility is unknown. Here, we confirmed the presence of Kir5.1 in spermatozoa and showed strong expression of Kir4.1 channels in smooth muscle and epithelial cells lining the epididymal ducts. In contrast, Kir4.2 expression was not detected in testes. To examine the possible role of Kir5.1 in sperm physiology, we bred mice with a deletion of the Kcnj16 (Kir5.1) gene and observed that 20% of Kir5.1 knock-out male mice were infertile. Furthermore, 50% of knock-out mice older than 3 months were unable to breed. By contrast, 100% of wild-type (WT) mice were fertile. The genetic inactivation of Kcnj16 also resulted in smaller testes and a greater percentage of sperm with folded flagellum compared to WT littermates. Nevertheless, the abnormal sperm from mutant animals displayed increased progressive motility. Thus, ablation of the Kcnj16 gene identifies Kir5.1 channel as an important element contributing to testis development, sperm flagellar morphology, motility, and fertility. These findings are potentially relevant to the understanding of the complex pHi- and [K+]o-dependent interplay between different sperm ion channels, and provide insight into their role in fertilization and infertility.


Assuntos
Infertilidade Masculina/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Espermatozoides/metabolismo , Animais , Fertilidade/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Infertilidade Masculina/patologia , Masculino , Potenciais da Membrana/genética , Camundongos , Camundongos Knockout , Músculo Liso/metabolismo , Oócitos/crescimento & desenvolvimento , Potássio/metabolismo , Motilidade Espermática/genética , Espermatozoides/crescimento & desenvolvimento , Testículo/crescimento & desenvolvimento , Testículo/metabolismo
4.
Pflugers Arch ; 472(7): 899-909, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32577860

RESUMO

Investigating the Shaker-related K+ channel Kv1.1, the dysfunction of which is responsible for episodic ataxia 1 (EA1), at the functional and molecular level provides valuable understandings on normal channel dynamics, structural correlates underlying voltage-gating, and disease-causing mechanisms. Most studies focused on apparently functional amino acid residues composing voltage-gated K+ channels, neglecting the simplest ones. Glycine at position 311 of Kv1.1 is highly conserved both evolutionarily and within the Kv channel superfamily, is located in a region functionally relevant (the S4-S5 linker), and results in overt disease when mutated (p.G311D). By mutating the G311 residue to aspartate, we show here that the channel voltage-gating, activation, deactivation, inactivation, and window currents are markedly affected. In silico, modeling shows this glycine residue is strategically placed at one end of the linker helix which must be free to both bend and move past other portions of the protein during the channel's opening and closing. This is befitting of a glycine residue as its small neutral side chain allows for movement unhindered by interaction with any other amino acid. Results presented reveal the crucial importance of a distinct glycine residue, within the S4-S5 linker, in the voltage-dependent electromechanical coupling that control channel gating.


Assuntos
Aminoácidos/metabolismo , Ativação do Canal Iônico/fisiologia , Canal de Potássio Kv1.1/genética , Sequência de Aminoácidos , Animais , Ataxia/metabolismo , Ataxia/patologia , Xenopus laevis/metabolismo
5.
Int J Mol Sci ; 21(11)2020 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-32471306

RESUMO

Episodic ataxia type 2 (EA2) is characterized by paroxysmal attacks of ataxia with typical onset in childhood or early adolescence. The disease is associated with mutations in the voltage-gated calcium channel alpha 1A subunit (Cav2.1) that is encoded by the CACNA1A gene. However, previously unrecognized atypical symptoms and the genetic overlap existing between EA2, spinocerebellar ataxia type 6, familial hemiplegic migraine type 1, and other neurological diseases blur the genotype/phenotype correlations, making a differential diagnosis difficult to formulate correctly and delaying early therapeutic intervention. Here we report a new clinical phenotype of a CACNA1A-associated disease characterized by absence epilepsy occurring during childhood. However, much later in life the patient displayed non-episodic, slowly progressive gait ataxia. Gene panel sequencing for hereditary ataxias led to the identification of a novel heterozygous CACNA1A mutation (c.1913 + 2T > G), altering the donor splice site of intron 14. This genetic defect was predicted to result in an in-frame deletion removing 44 amino acids from the voltage-gated calcium channel Cav2.1. An RT-PCR analysis of cDNA derived from patient skin fibroblasts confirmed the skipping of the entire exon 14. Furthermore, two-electrode voltage-clamp recordings performed from Xenopus laevis oocytes expressing a wild-type versus mutant channel showed that the genetic defect caused a complete loss of channel function. This represents the first description of distinct clinical manifestations that remarkably expand the genetic and phenotypic spectrum of CACNA1A-related diseases and should be considered for an early diagnosis and effective therapeutic intervention.


Assuntos
Canais de Cálcio/genética , Ataxia Cerebelar/genética , Epilepsia/genética , Mutação com Perda de Função , Animais , Canais de Cálcio/metabolismo , Células Cultivadas , Ataxia Cerebelar/complicações , Ataxia Cerebelar/patologia , Epilepsia/complicações , Epilepsia/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Splicing de RNA , Xenopus
6.
Neuroscience ; 440: 337-359, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32473276

RESUMO

Inherited and sporadic mutations in genes encoding for brain ion channels, affecting membrane expression or biophysical properties, have been associated with neurodevelopmental disorders characterized by epilepsy, cognitive and behavioral deficits with significant phenotypic and genetic heterogeneity. Over the years, the screening of a growing number of patients and the functional characterization of newly identified mutations in ion channels genes allowed to recognize new phenotypes and to widen the clinical spectrum of known diseases. Furthermore, advancements in understanding disease pathogenesis at atomic level or using patient-derived iPSCs and animal models have been pivotal to orient therapeutic intervention and to put the basis for the development of novel pharmacological options for drug-resistant disorders. In this review we will discuss major improvements and critical issues concerning neurodevelopmental disorders caused by dysfunctions in brain sodium, potassium, calcium, chloride and ligand-gated ion channels.


Assuntos
Epilepsia , Transtornos do Neurodesenvolvimento , Animais , Encéfalo/metabolismo , Epilepsia/genética , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Mutação , Transtornos do Neurodesenvolvimento/genética
7.
Pflugers Arch ; 472(7): 923-930, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32394190

RESUMO

Mutations in the KCNK18 gene that encodes the TRESK K2P potassium channel have previously been linked with typical familial migraine with aura. Recently, an atypical clinical case has been reported in which a male individual carrying the p.Trp101Arg (W101R) missense mutation in the KCNK18 gene was diagnosed with intellectual disability and migraine with brainstem aura. Here we report the functional characterization of this new missense variant. This mutation is located in a highly conserved residue close to the selectivity filter, and our results show although these mutant channels retain their K+ selectivity and calcineurin-dependent regulation, the variant causes an overall dramatic loss of TRESK channel function as well as an initial dominant-negative effect when co-expressed with wild-type channels in Xenopus laevis oocytes. The dramatic functional consequences of this mutation thereby support a potentially pathogenic role for this variant and provide further insight into the relationship between the structure and function of this ion channel.


Assuntos
Deficiência Intelectual/genética , Transtornos de Enxaqueca/genética , Mutação de Sentido Incorreto/genética , Canais de Potássio/genética , Animais , Calcineurina/genética , Humanos , Deficiência Intelectual/metabolismo , Potenciais da Membrana/genética , Transtornos de Enxaqueca/metabolismo , Oócitos/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo
8.
Int J Mol Sci ; 21(8)2020 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-32331416

RESUMO

Kv1.1 belongs to the Shaker subfamily of voltage-gated potassium channels and acts as a critical regulator of neuronal excitability in the central and peripheral nervous systems. KCNA1 is the only gene that has been associated with episodic ataxia type 1 (EA1), an autosomal dominant disorder characterized by ataxia and myokymia and for which different and variable phenotypes have now been reported. The iterative characterization of channel defects at the molecular, network, and organismal levels contributed to elucidating the functional consequences of KCNA1 mutations and to demonstrate that ataxic attacks and neuromyotonia result from cerebellum and motor nerve alterations. Dysfunctions of the Kv1.1 channel have been also associated with epilepsy and kcna1 knock-out mouse is considered a model of sudden unexpected death in epilepsy. The tissue-specific association of Kv1.1 with other Kv1 members, auxiliary and interacting subunits amplifies Kv1.1 physiological roles and expands the pathogenesis of Kv1.1-associated diseases. In line with the current knowledge, Kv1.1 has been proposed as a novel and promising target for the treatment of brain disorders characterized by hyperexcitability, in the attempt to overcome limited response and side effects of available therapies. This review recounts past and current studies clarifying the roles of Kv1.1 in and beyond the nervous system and its contribution to EA1 and seizure susceptibility as well as its wide pharmacological potential.


Assuntos
Canalopatias/etiologia , Canalopatias/terapia , Predisposição Genética para Doença , Canal de Potássio Kv1.1/genética , Mutação , Alelos , Animais , Canalopatias/diagnóstico , Canalopatias/metabolismo , Gerenciamento Clínico , Regulação da Expressão Gênica , Estudos de Associação Genética , Genótipo , Humanos , Ativação do Canal Iônico , Canal de Potássio Kv1.1/química , Canal de Potássio Kv1.1/metabolismo , Terapia de Alvo Molecular , Fenótipo , Relação Estrutura-Atividade
9.
Front Mol Neurosci ; 12: 65, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30983966

RESUMO

Glioblastoma multiforme (GBM) is the most common and malignant of the glial tumors. The world-wide estimates of new cases and deaths annually are remarkable, making GBM a crucial public health issue. Despite the combination of radical surgery, radio and chemotherapy prognosis is extremely poor (median survival is approximately 1 year). Thus, current therapeutic interventions are highly unsatisfactory. For many years, GBM-induced brain oedema and inflammation have been widely treated with dexamethasone (DEX), a synthetic glucocorticoid (GC). A number of studies have reported that DEX also inhibits GBM cell proliferation and migration. Nevertheless, recent controversial results provided by different laboratories have challenged the widely accepted dogma concerning DEX therapy for GBM. Here, we have reviewed the main clinical features and genetic and epigenetic abnormalities underlying GBM. Finally, we analyzed current notions and concerns related to DEX effects on cerebral oedema, cancer cell proliferation and migration and clinical outcome.

10.
Front Neurol ; 9: 587, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30140249

RESUMO

Episodic ataxia type 1 (EA1), a Shaker-like K+channelopathy, is a consequence of genetic anomalies in the KCNA1 gene that lead to dysfunctions in the voltage-gated K+ channel Kv1. 1. Generally, KCNA1 mutations are inherited in an autosomal dominant manner. Here we report the clinical phenotype of an EA1 patient characterized by ataxia attacks that decrease in frequency with age, and eventually leading to therapy discontinuation. A new de novo mutation (c.932G>A) that changed a highly conserved glycine residue into an aspartate (p.G311D) was identified by using targeted next-generation sequencing. The conserved glycine is located in the S4-S5 linker, a crucial domain controlling Kv1.1 channel gating. In silico analyses predicted the mutation deleterious. Heterologous expression of the mutant (Kv1.1-G311D) channels resulted in remarkably decreased amplitudes of measured current, confirming the identified variant is pathogenic. Collectively, these findings corroborate the notion that EA1 also results from de novo variants and point out that regardless of the mutation-induced deleterious loss of Kv1.1 channel function the ataxia phenotype may improve spontaneously.

13.
Nat Med ; 24(9): 1481, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29934534

RESUMO

In the version of this article originally published, some labels in Fig. 1f are incorrect. The "ß-actin" labels on the second and fourth rows of blots should instead be "ß-tubulin". The error has been corrected in the HTML and PDF versions of this article.

14.
Nat Med ; 24(9): 1482, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29934535

RESUMO

In the version of this article originally published, the amino acid sequence for Tα1 described in the Online Methods is incorrect. The sequence is described as "Ac-SDAAVDTSSEITTJDLKEKKEVVEEAEN-OH". It should be "Ac-SDAAVDTSSEITTKDLKEKKEVVEEAEN-OH". The error has been corrected in the HTML and PDF versions of this article.

15.
Curr Neuropharmacol ; 16(5): 608-617, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-28875832

RESUMO

BACKGROUND: The KCa3.1 channel is the intermediate-conductance member of the Ca2+- activated K channel superfamily. It is widely expressed in excitable and non-excitable cells, where it plays a major role in a number of cell functions. This paper aims at illustrating the main structural, biophysical and modulatory properties of the KCa3.1 channel, and providing an account of experimental data on its role in volume regulation and Ca2+ signals. METHODS: Research and online content related to the structure, structure/function relationship, and physiological role of the KCa3.1 channel are reviewed. RESULTS: Expressed in excitable and non-excitable cells, the KCa3.1 channel is voltage independent, its opening being exclusively gated by the binding of intracellular Ca2+ to calmodulin, a Ca2+- binding protein constitutively associated with the C-terminus of each KCa3.1 channel α subunit. The KCa3.1 channel activates upon high affinity Ca2+ binding, and in highly coordinated fashion giving steep Hill functions and relatively low EC50 values (100-350 nM). This high Ca2+ sensitivity is physiologically modulated by closely associated kinases and phosphatases. The KCa3.1 channel is normally activated by global Ca2+ signals as resulting from Ca2+ released from intracellular stores, or by the refilling influx through store operated Ca2+ channels, but cases of strict functional coupling with Ca2+-selective channels are also found. KCa3.1 channels are highly expressed in many types of cells, where they play major roles in cell migration and death. The control of these complex cellular processes is achieved by KCa3.1 channel regulation of the driving force for Ca2+ entry from the extracellular medium, and by mediating the K+ efflux required for cell volume control. CONCLUSION: Much work remains to be done to fully understand the structure/function relationship of the KCa3.1 channels. Hopefully, this effort will provide the basis for a beneficial modulation of channel activity under pathological conditions.


Assuntos
Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Canais de Potássio Cálcio-Ativados/fisiologia , Animais , Calmodulina/metabolismo , Simulação de Dinâmica Molecular , Canais de Potássio Cálcio-Ativados/química , Ligação Proteica , Estrutura Terciária de Proteína
16.
Mol Cell Neurosci ; 83: 6-12, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28666963

RESUMO

Episodic ataxia type 1 (EA1) is a human dominant neurological syndrome characterized by continuous myokymia, episodic attacks of ataxic gait and spastic contractions of skeletal muscles that can be triggered by emotional stress and fatigue. This rare disease is caused by missense mutations in the KCNA1 gene coding for the neuronal voltage gated potassium channel Kv1.1, which contributes to nerve cell excitability in the cerebellum, hippocampus, cortex and peripheral nervous system. We identified a novel KCNA1 mutation, E283K, in an Italian proband presenting with paroxysmal ataxia and myokymia aggravated by painful contractures and metabolic dysfunctions. The E283K mutation is located in the S3-S4 extracellular linker belonging to the voltage sensor domain of Kv channels. In order to test whether the E283K mutation affects Kv1.1 biophysical properties we transfected HEK293 cells with WT or mutant cDNAs alone or in a 1:1 combination, and recorded relative potassium currents in the whole-cell configuration of patch-clamp. Mutant E283K channels display voltage-dependent activation shifted by 10mV toward positive potentials and kinetics of activation slowed by ~2 fold compared to WT channels. Potassium currents resulting from heteromeric WT/E283K channels show voltage-dependent gating and kinetics of activation intermediate between WT and mutant homomeric channels. Based on homology modeling studies of the mutant E283K, we propose a molecular explanation for the reduced voltage sensitivity and slow channel opening. Overall, our results suggest that the replacement of a negatively charged residue with a positively charged lysine at position 283 in Kv1.1 causes a drop of potassium current that likely accounts for EA-1 symptoms in the heterozygous carrier.


Assuntos
Ataxia/genética , Canal de Potássio Kv1.1/metabolismo , Mutação de Sentido Incorreto , Mioquimia/genética , Ataxia/metabolismo , Ataxia/patologia , Feminino , Células HEK293 , Humanos , Ativação do Canal Iônico , Canal de Potássio Kv1.1/química , Canal de Potássio Kv1.1/genética , Pessoa de Meia-Idade , Mioquimia/metabolismo , Mioquimia/patologia , Linhagem
17.
Sci Rep ; 7(1): 4583, 2017 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-28676720

RESUMO

Channelopathy mutations prove informative on disease causing mechanisms and channel gating dynamics. We have identified a novel heterozygous mutation in the KCNA1 gene of a young proband displaying typical signs and symptoms of Episodic Ataxia type 1 (EA1). This mutation is in the S4 helix of the voltage-sensing domain and results in the substitution of the highly conserved phenylalanine 303 by valine (p.F303V). The contributions of F303 towards K+ channel voltage gating are unclear and here have been assessed biophysically and by performing structural analysis using rat Kv1.2 coordinates. We observed significant positive shifts of voltage-dependence, changes in the activation, deactivation and slow inactivation kinetics, reduced window currents, and decreased current amplitudes of both Kv1.1 and Kv1.1/1.2 channels. Structural analysis revealed altered interactions between F303V and L339 and I335 of the S5 helix of a neighboring subunit. The substitution of an aromatic phenylalanine with an aliphatic valine within the voltage-sensor destabilizes the open state of the channel. Thus, F303 fine-tunes the Kv1.1 gating properties and contributes to the interactions between the S4 segment and neighboring alpha helices. The resulting channel's loss of function validates the clinical relevance of the mutation for EA1 pathogenesis.


Assuntos
Ataxia/genética , Ataxia/metabolismo , Canalopatias/genética , Canalopatias/metabolismo , Ativação do Canal Iônico/genética , Canal de Potássio Kv1.1/genética , Canal de Potássio Kv1.1/metabolismo , Mutação , Alelos , Sequência de Aminoácidos , Ataxia/diagnóstico , Canalopatias/diagnóstico , Sequência Conservada , Feminino , Genótipo , Humanos , Canal de Potássio Kv1.1/química , Masculino , Modelos Moleculares , Linhagem , Fenilalanina/genética , Conformação Proteica , Avaliação de Sintomas
18.
J Neurophysiol ; 118(4): 2402-2411, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28747464

RESUMO

A 2-yr-old boy presented profound developmental delay, failure to thrive, ataxia, hypotonia, and tonic-clonic seizures that caused the death of the patient. Targeted and whole exome sequencing revealed two heterozygous missense variants: a novel mutation in the KCNJ10 gene that encodes for the inward-rectifying K+ channel Kir4.1 and another previously characterized mutation in KCNT1 that encodes for the Na+-activated K+ channel known as Slo2.2 or SLACK. The objectives of this study were to perform the clinical and genetic characterization of the proband and his family and to examine the functional consequence of the Kir4.1 mutation. The mutant and wild-type KCNJ10 constructs were generated and heterologously expressed in Xenopus laevis oocytes, and whole cell K+ currents were measured using the two-electrode voltage-clamp technique. The KCNJ10 mutation c.652C>T resulted in a p.L218F substitution at a highly conserved residue site. Wild-type KCNJ10 expression yielded robust Kir current, whereas currents from oocytes expressing the mutation were reduced, remarkably. Western Blot analysis revealed reduced protein expression by the mutation. Kir5.1 subunits display selective heteromultimerization with Kir4.1 constituting channels with unique kinetics. The effect of the mutation on Kir4.1/5.1 channel activity was twofold: a reduction in current amplitudes and an increase in the pH-dependent inhibition. We thus report a novel loss-of-function mutation in Kir4.1 found in a patient with a coexisting mutation in SLACK channels that results in a fatal disease.NEW & NOTEWORTHY We present and characterize a novel mutation in KCNJ10 Unlike previously reported EAST/SeSAME patients, our patient was heterozygous, and contrary to previous studies, mimicking the heterozygous state by coexpression resulted in loss of channel function. We report in the same patient co-occurrence of a KCNT1 mutation resulting in a more severe phenotype. This study provides new insights into the phenotypic spectrum and to the genotype-phenotype correlations associated with EAST/SeSAME and MMFSI.


Assuntos
Deficiências do Desenvolvimento/genética , Mutação com Perda de Função , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio/genética , Convulsões/genética , Animais , Deficiências do Desenvolvimento/patologia , Heterozigoto , Humanos , Lactente , Masculino , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio Ativados por Sódio , Convulsões/patologia , Síndrome , Xenopus
19.
Nat Med ; 23(5): 590-600, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28394330

RESUMO

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that compromise its chloride channel activity. The most common mutation, p.Phe508del, results in the production of a misfolded CFTR protein, which has residual channel activity but is prematurely degraded. Because of the inherent complexity of the pathogenetic mechanisms involved in CF, which include impaired chloride permeability and persistent lung inflammation, a multidrug approach is required for efficacious CF therapy. To date, no individual drug with pleiotropic beneficial effects is available for CF. Here we report on the ability of thymosin alpha 1 (Tα1)-a naturally occurring polypeptide with an excellent safety profile in the clinic when used as an adjuvant or an immunotherapeutic agent-to rectify the multiple tissue defects in mice with CF as well as in cells from subjects with the p.Phe508del mutation. Tα1 displayed two combined properties that favorably opposed CF symptomatology: it reduced inflammation and increased CFTR maturation, stability and activity. By virtue of this two-pronged action, Tα1 has strong potential to be an efficacious single-molecule-based therapeutic agent for CF.


Assuntos
Adjuvantes Imunológicos/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Fibrose Cística/genética , Citocinas/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Timosina/análogos & derivados , Animais , Autofagia/efeitos dos fármacos , Western Blotting , Linhagem Celular , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/metabolismo , Fibrose Cística/imunologia , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Citocinas/imunologia , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Imunofluorescência , Humanos , Imuno-Histoquímica , Imunoprecipitação , Indolamina-Pirrol 2,3,-Dioxigenase/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Inflamação , Camundongos , Camundongos Endogâmicos CFTR , Técnicas de Patch-Clamp , Estabilidade Proteica/efeitos dos fármacos , Células RAW 264.7 , Mucosa Respiratória/citologia , Timalfasina , Timosina/farmacologia , Ubiquitina Tiolesterase/efeitos dos fármacos , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação/efeitos dos fármacos
20.
J Cell Physiol ; 232(1): 91-100, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27028592

RESUMO

The malignancy of glioblastoma multiforme (GBM), the most common human brain tumor, correlates with the presence of hypoxic areas, but the underlying mechanisms are unclear. GBM cells express abundant Cl channels whose activity supports cell volume and membrane potential changes, ultimately leading to cell proliferation, migration, and escaping death. In non-tumor tissues Cl channels are modulated by hypoxia, which prompted us to verify whether hypoxia would also modulate Cl channels in GBM cells. Our results show that in GBM cell lines, acute application of a hypoxic solution activates a Cl current displaying the biophysical and pharmacological features of the swelling-activated Cl current (ICl,swell ). We also found that acute hypoxia increased the cell volume by about 20%, and a 30% hypertonic solution partially inhibited the hypoxia-activated Cl current, suggesting that cell swelling and the activation of the Cl current are sequential events. Notably, the hypoxia-induced cell swelling was followed by a regulatory volume decrease (RVD) mediated mainly by ICl,swell . Since, a hypoxia-induced prolonged cell swelling is usually regarded as a death insult, we hypothesized that the hypoxia-activated Cl current could limit cell swelling and prevent necrotic death of GBM cells under hypoxic conditions. In accordance, we found that the ICl,swell inhibitor DCPIB hampered the RVD process, and more importantly it sensibly increased the hypoxia-induced necrotic death in these cells. Taken together, these results suggest that Cl channels are strongly involved in the survival of GBM cells in a hypoxic environment, and may thus represent a new therapeutic target for this malignant tumor. J. Cell. Physiol. 232: 91-100, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Tamanho Celular , Canais de Cloreto/metabolismo , Glioblastoma/metabolismo , Potenciais da Membrana/fisiologia , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Glioblastoma/patologia , Humanos , Pressão Osmótica/fisiologia , Técnicas de Patch-Clamp/métodos
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