Assuntos
Síndrome de Brugada , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5 , Fenótipo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Síndrome de Brugada/genética , Síndrome de Brugada/fisiopatologia , Síndrome de Brugada/metabolismo , Humanos , Potenciais de Ação , Potenciais da Membrana , AnimaisRESUMO
AIMS: Caveolinopathies are a family of genetic disorders arising from alterations of the caveolin-3 (cav-3) gene. The T78M cav-3 variant has been associated with both skeletal and cardiac muscle pathologies but its functional contribution, especially to cardiac diseases, is still controversial. Here, we evaluated the effect of the T78M cav-3 variant on cardiac ion channel function and membrane excitability. METHODS AND RESULTS: We transfected either the wild type (WT) or T78M cav-3 in caveolin-1 knock-out mouse embryonic fibroblasts and found by immunofluorescence and electron microscopy that both are expressed at the plasma membrane and form caveolae. Two ion channels known to interact and co-immunoprecipitate with the cav-3, hKv1.5 and hHCN4, interact also with T78M cav-3 and reside in lipid rafts. Electrophysiological analysis showed that the T78M cav-3 causes hKv1.5 channels to activate and inactivate at more hyperpolarized potentials and the hHCN4 channels to activate at more depolarized potentials, in a dominant way. In spontaneously beating neonatal cardiomyocytes, the expression of the T78M cav-3 significantly increased action potential peak-to-peak variability without altering neither the mean rate nor the maximum diastolic potential. We also found that in a small cohort of patients with supraventricular arrhythmias, the T78M cav-3 variant is more frequent than in the general population. Finally, in silico analysis of both sinoatrial and atrial cell models confirmed that the T78M-dependent changes are compatible with a pro-arrhythmic effect. CONCLUSION: This study demonstrates that the T78M cav-3 induces complex modifications in ion channel function that ultimately alter membrane excitability. The presence of the T78M cav-3 can thus generate a susceptible substrate that, in concert with other structural alterations and/or genetic mutations, may become arrhythmogenic.
Assuntos
Potenciais de Ação , Caveolina 3/genética , Caveolina 3/metabolismo , Fibroblastos/metabolismo , Mutação , Miócitos Cardíacos/metabolismo , Células 3T3 , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Cavéolas/metabolismo , Caveolina 1/deficiência , Caveolina 1/genética , Simulação por Computador , Fibroblastos/ultraestrutura , Frequência Cardíaca , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico , Cinética , Canal de Potássio Kv1.5/genética , Canal de Potássio Kv1.5/metabolismo , Camundongos , Camundongos Knockout , Modelos Cardiovasculares , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Contração Miocárdica , Miócitos Cardíacos/ultraestrutura , Canais de Potássio/genética , Canais de Potássio/metabolismo , Ratos Sprague-Dawley , TransfecçãoRESUMO
To optimise the efficiency of cell machinery, cells can use the same protein (often called a hub protein) to participate in different cell functions by simply changing its target molecules. There are large data sets describing protein-protein interactions ("interactome") but they frequently fail to consider the functional significance of the interactions themselves. We studied the interaction between two potential hub proteins, ICln and 4.1R (in the form of its two splicing variants 4.1R80 and 4.1R135), which are involved in such crucial cell functions as proliferation, RNA processing, cytoskeleton organisation and volume regulation. The sub-cellular localisation and role of native and chimeric 4.1R over-expressed proteins in human embryonic kidney (HEK) 293 cells were examined. ICln interacts with both 4.1R80 and 4.1R135 and its over-expression displaces 4.1R from the membrane regions, thus affecting 4.1R interaction with ß-actin. It was found that 4.1R80 and 4.1R135 are differently involved in regulating the swelling activated anion current (ICl,swell) upon hypotonic shock, a condition under which both isoforms are dislocated from the membrane region and thus contribute to ICl,swell current regulation. Both 4.1R isoforms are also differently involved in regulating cell morphology, and ICln counteracts their effects. The findings of this study confirm that 4.1R plays a role in cell volume regulation and cell morphology and indicate that ICln is a new negative regulator of 4.1R functions.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas ELAV/metabolismo , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/metabolismo , Linhagem Celular , Citoesqueleto/metabolismo , Proteína Semelhante a ELAV 2 , Células HEK293 , Humanos , Ligação ProteicaRESUMO
BACKGROUND: Cigarette smoke extract (CSE), a model for studying the effects of tobacco smoke in vivo and in vitro, induces cell oxidative stress and affects the antioxidative glutathione system. We evaluated the impact of CSE on airway epithelial cells and the possible cytoprotective effect of the mucolitic drug S-carboximethilcysteine lysine salt (S-CMC-Lys). METHODS: Reduced glutathione (GSH) and reactive oxygen species (ROS) intracellular levels were evaluated by fluorimetry in human bronchial epithelial cells (16-HBE) and the expression and activity of enzymes of the GSH metabolic pathway were investigated by RT-PCR, Western blot and colorimetric assays. RESULTS: CSE significantly increased cell mortality in a time and dose dependent manner, via an apoptosis-independent pathway. Short-term (3 hours) CSE exposure induced an increase in ROS levels and a GSH intracellular concentration drop. In parallel, the expression of glutathione peroxidases 2 and 3, glutathione reductase and glutamate-cysteine-ligase was increased. S-CMC-Lys was effective in counteracting these effects. CONCLUSION: CSE affects ROS levels, GSH concentration and GSH enzymes pathway. These effects can be to some extent reversed by S-CMC-Lys, that could represent a therapeutic tool to counteract CSE induced oxidative cellular injuries.
Assuntos
Brônquios/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Glutationa/metabolismo , Fumar/efeitos adversos , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Homeostase/efeitos dos fármacos , Humanos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Patients with PS or non-syndromic deafness were submitted to genetic/functional analyzes of SLC26A4, of its binding domain for FOXI1 (FOXI1-DBD), of the transcription activator FOXI1, and of the potassium channel KCNJ10. SLC26A4 was the most frequently mutated gene. An altered intracellular localization with immunocytochemistry, and a hampered maturation process were demonstrated for two novel SLC26A4 variants. Biochemical and immunocytochemical analyzes led to the development of a more sensitive fluorometric functional assay able to reveal the partial loss-of-function of SLC26A4 mutations. A novel missense variant was found in FOXI1 gene, though functional analysis showed no significant impairment in the transcriptional activation of SLC26A4. Finally, 3 patients were found to harbor a variant in KCNJ10, which was classified as polymorphism. The novelty of the study resides in the analysis of all the 4 candidate genetic loci linked to PS/non-syndromic deafness, and in the precise definition of the thyroid phenotype. PS was invariably associated with biallelic mutations of SLC26A4, whereas the genetic origin of non-syndromic deafness remained largely undetermined, since monoallelic SLC26A4 variants accounted for one fourth of the cases and FOXI1 and KCNJ10 were not involved in this series.
Assuntos
Fatores de Transcrição Forkhead/genética , Bócio Nodular/genética , Perda Auditiva Neurossensorial/genética , Proteínas de Membrana Transportadoras/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Adolescente , Adulto , Alelos , Animais , Células COS , Linhagem Celular , Criança , Pré-Escolar , Chlorocebus aethiops , Feminino , Fatores de Transcrição Forkhead/metabolismo , Variação Genética , Genótipo , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mutação , Transportadores de Sulfato , Ativação Transcricional , Adulto JovemRESUMO
The CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) activity and localization are influenced by the cytoskeleton, in particular by actin and its polymerization state. In this study we investigated whether the expression of the hypertensive mutations of α-adducin (G460W-S586C in humans, F316Y in rats), an actin capping protein, led to a functional modification of CFTR activity and surface expression. The experiments were performed on HEK293 T cells cotransfected with CFTR and the human wild type (WT) or G460W mutated α-adducin. In whole-cell patch-clamp experiments, both the CFTR chloride current and the slope of current activation after forskolin addition were significantly higher in HEK cells overexpressing the G460W adducin. A higher plasma membrane density of active CFTR channels was confirmed by cell-attached patch-clamp experiments, both in HEK cells and in cultured primary DCT cells, isolated from MHS (Milan Hypertensive Strain, a Wistar rat (Rattus norvegicus) hypertensive model carrying the F316Y adducin mutation), compared to MNS (Milan Normotensive Strain) rats. Western blot experiments demonstrated an increase of the plasma membrane CFTR protein expression, with a modification of the channel glycosylation state, in the presence of the mutated adducin. A higher retention of CFTR protein in the plasma membrane was confirmed both by FRAP (Fluorescence Recovery After Photobleaching) and photoactivation experiments. The present data indicate that in HEK cells and in isolated DCT cells the presence of the G460W-S586C hypertensive variant of adducin increases CFTR channel activity, possibly by altering its membrane turnover and inducing a retention of the channel in the plasmamembrane. Since CFTR is known to modulate the activity of many others transport systems, the increased surface expression of the channel could have consequences on the whole network of transport in kidney cells.
Assuntos
Proteínas de Ligação a Calmodulina/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Hipertensão/genética , Hipertensão/metabolismo , Túbulos Renais Distais/metabolismo , Mutação , Animais , Membrana Celular/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Expressão Gênica , Células HEK293 , Humanos , Masculino , Técnicas de Patch-Clamp , Ligação Proteica , Ratos , Transdução de SinaisRESUMO
The pendrin (SLC26A4 or PDS) gene is responsible, when mutated, for the Pendred syndrome, a recessive disorder characterized by sensorineural hearing loss often accompanied by thyroid dysfunctions. Pendrin protein is an anion exchanger and we focused on a still unexplored function that it might play in view of its importance in the inner ear: Cl(-) fluxes regulation during cellular volume control. We challenged HEK-293 Phoenix cells over-expressing wild type pendrin (PDS HEK cells) together with the EYFP (Enhanced Yellow Fluorescent Protein) or over-expressing the EYFP alone (control HEK cells) with hypo-osmolar solutions. Taking advantage of the confocal optical sectioning we measured the cell volume. In addition, we determined the intracellular pH and chloride concentration with fluorescent probes (EYFP and seminaphthorhodafluor-5F, SNARF-5F). Consequently, we could estimate simultaneously Cl(-) fluxes, cellular volume and intracellular pH variations. Cl(-) movements markedly differed between PDS and control HEK cells upon hypotonic shock and are accompanied by an attenuation of the swelling induced pH drop in PDS HEK cells. The contemporary measurements of the three variables not yet reported in living cells, allowed to assess a possible influence of pendrin upregulation in volume homeostasis and evidenced its participation to Cl(-) fluxes.
Assuntos
Cloretos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Tamanho Celular/efeitos dos fármacos , Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Soluções Hipotônicas/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Transportadores de Sulfato , TransfecçãoRESUMO
The mucoactive drug S-carbocysteine lysine salt monohydrate (S-CMC-Lys) stimulates glutathione (GSH) efflux from respiratory cells. Since GSH is one of the most important redox regulatory mechanisms, the aim of this study was to evaluate the S-CMC-Lys effects on GSH efflux and intracellular concentration during an oxidative stress induced by the hydroxyl radical (xOH). Experiments were performed on cultured human respiratory WI-26VA4 cells by means of patch-clamp experiments in whole-cell configuration and of fluorimetric analyses at confocal microscope. xOH exposure induced an irreversible inhibition of the GSH and chloride currents that was prevented if the cells were incubated with S-CMC-Lys. In this instance, the currents were inhibited by the specific blocker CFTR(inh)-172. CFT1-C2 cells, which lack a functional CFTR channel, were not responsive to S-CMC-Lys, but the stimulatory effect of the drug was restored in LCFSN-infected CFT1 cells, functionally corrected to express CFTR. Fluorimetric measurements performed on the S-CMC-Lys-incubated cells revealed a significant increase of the GSH concentration that was completely hindered after oxidative stress and abolished by CFTR(inh)-172. The cellular content of reactive oxygen species was significantly lower in the S-CMC-Lys-treated cells either before or after xOH exposure. As a conclusion, S-CMC-Lys could exert a protective function during oxidative stress, therefore preventing or reducing the ROS-mediated inflammatory response.
Assuntos
Carbocisteína/análogos & derivados , Citoproteção/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Sistema Respiratório/citologia , Carbocisteína/farmacologia , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fluorometria , Glutationa/metabolismo , Humanos , Radical Hidroxila/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Ativação do Canal Iônico/efeitos dos fármacosRESUMO
Fluorescence resonance energy transfer (FRET) is a technique used for the study of functional interactions between molecules. The intimate vicinity between two fluorescent molecules (FRET-pair; donor and acceptor) allows for an energy transfer, which can be directly calculated as the so called FRET efficiency. This technique is used in fixed as well as living cells. Here we show first, measured by the FRET technique, that the ICln ion channel is transposed from the cytosol towards the cellular membrane in HEK cells after swelling, and second, that the calculation of the FRET efficiency by de-quenching the donor cyan-fluorescent-protein (CFP) emission due to acceptor-photobleaching leads to erroneous estimate of the FRET efficiency in fixed, mounted and sealed specimens. The acceptor photobleaching leads to a modification of the donor cyan-fluorescent-protein, which shows then a strong emission, thus mimicking functional interaction between CFP (donor) and yellow-fluorescent-protein (YFP; acceptor). Moreover, the procedure of acceptor photobleaching masks physiological (non random) interaction between molecules within the fixed, mounted and sealed cell. We show that no artifactual CFP modifications arise when using the acceptor photobleaching technique under in vivo conditions, and we offer strategies to minimize erroneous FRET efficiency calculations if cells need to be fixed.
Assuntos
Artefatos , Cosméticos/química , Fixadores/química , Transferência Ressonante de Energia de Fluorescência , Técnicas de Preparação Histocitológica/métodos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Forma Celular , Genes Reporter/genética , Humanos , Fotodegradação , Fixação de TecidosRESUMO
Malfunction of the SLC26A4 protein leads to Pendred syndrome, characterized by sensorineural hearing loss, often associated with mild thyroid dysfunction and goiter. It is generally assumed that SLC26A4 acts as a chloride/anion exchanger, which in the thyroid gland transports iodide, and in the inner ear contributes to the conditioning of the endolymphatic fluid. Here we describe a fast fluorometric method able to be used to functionally scrutinize SLC26A4 and its mutants described in Pendred syndrome. The validation of the method was done by functionally characterizing the chloride/iodide transport of SLC26A4, and a mutant, i.e. SLC26A4(S28R), which we previously described in a patient with sensorineural hearing loss, hypothyroidism and goiter. Using the fluorometric method we describe here we can continuously monitor and quantify the iodide or chloride amounts transported by the cells, and we found that the transport capability of the SLC26A4(S28R) mutant protein is markedly reduced if compared to wild-type SLC26A4.
Assuntos
Cloretos/metabolismo , Fluorometria/métodos , Iodetos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Fluoresceínas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana Transportadoras/genética , Mutagênese Sítio-Dirigida/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transportadores de Sulfato , TransfecçãoRESUMO
BACKGROUND: Malfunction of the SLC26A4 protein leads to prelingual deafness often associated with mild thyroid dysfunction and goiter. It is assumed that SLC26A4 acts as a chloride/anion exchanger responsible for the iodide organification in the thyroid gland, and conditioning of the endolymphatic fluid in the inner ear. METHODS: Chloride uptake studies were made using HEK293-Phoenix cells expressing human wild type SLC26A4 (pendrin) and a mutant (SLC26A4(S28R)) we recently described in a patient with hypothyroidism, goiter and sensorineural hearing loss. RESULTS: Experiments are summarized showing the functional characterization of wild type SLC26A4 and a mutant (S28R), which we described recently. This mutant protein is transposed towards the cell membrane, however, its transport capability is markedly reduced if compared to wild-type SLC26A4. Furthermore, we show that the SLC26A4 induced chloride uptake in HEK293-Phoenix cells competes with iodide, and, in addition, that the chloride uptake can be blocked by NPPB and niflumic acid, whereas DIDS is ineffective. CONCLUSIONS: The functional characteristics of SLC26A4(S28R) we describe here, are consistent with the clinical phenotype observed in the patient from which the mutant was derived.
Assuntos
Bócio/genética , Perda Auditiva Neurossensorial/genética , Hipotireoidismo/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Cloretos/metabolismo , Citoplasma/metabolismo , Humanos , Iodetos/metabolismo , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Ácido Niflúmico/farmacologia , Nitrobenzoatos/farmacologia , Transportadores de Sulfato , SíndromeRESUMO
OBJECTIVE: The SLC26A4 protein (pendrin) seems to be involved in the exchange of chloride with other anions, therefore being responsible for iodide organification in the thyroid gland and the conditioning of the endolymphatic fluid in the inner ear. Malfunction of SLC26A4 leads to Pendred syndrome, characterized by mild thyroid dysfunction often associated with goiter and/or prelingual deafness. The precise function of the SLC26A4 protein, however, is still elusive. An open question is still whether the SLC26A4-induced ion exchange mechanism is electrogenic or electroneutral. Recently, it has been shown that human pendrin expressed in monkey cells leads to chloride currents. METHODS: We overexpressed the human SLC26A4 isoform in HEK293 Phoenix cells and measured cationic and anionic currents by the patch-clamp technique in whole cell configuration. RESULTS: Here we show that human pendrin expressed in human cells does not lead to the activation of chloride currents, but, in contrast, leads to an increase of cationic currents. CONCLUSION: Our experiments suggest that the SLC26A4-induced chloride transport is electroneutral when expressed in human cellular systems.
Assuntos
Cátions/metabolismo , Canais Iônicos/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Linhagem Celular , Canais de Cloreto/fisiologia , Condutividade Elétrica , Humanos , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Transportadores de SulfatoRESUMO
ICln is a multifunctional protein involved in regulatory mechanisms as different as membrane ion transport and RNA splicing. The protein is water-soluble, and during regulatory volume decrease after cell swelling, it is able to migrate from the cytosol to the cell membrane. Purified, water-soluble ICln is able to insert into lipid bilayers to form ion channels. Here, we show that ICln159, a truncated ICln mutant, which is also able to form ion channels in lipid bilayers, belongs to the pleckstrin homology (PH) domain superfold family of proteins. The ICln PH domain shows unusual properties as it lacks the electrostatic surface polarization seen in classical PH domains. However, similar to many classical PH domain-containing proteins, ICln interacts with protein kinase C, and in addition, interacts with cAMP-dependent protein kinase and cGMP-dependent protein kinase type II but not cGMP-dependent protein kinase type Ibeta. A major phosphorylation site for all three kinases is Ser-45 within the ICln PH domain. Furthermore, ICln159 interacts with LSm4, a protein involved in splicing and mRNA degradation, suggesting that the ICln159 PH domain may serve as a protein-protein interaction platform.
Assuntos
Proteínas Sanguíneas/química , Canais Iônicos/química , Canais Iônicos/metabolismo , Fosfoproteínas/química , Dobramento de Proteína , Estrutura Terciária de Proteína , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Sequência de Aminoácidos , Animais , Cães , Humanos , Canais Iônicos/genética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Células NIH 3T3 , Ressonância Magnética Nuclear Biomolecular , Técnicas de Patch-Clamp , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/genéticaRESUMO
BACKGROUND INFORMATION: Transepithelial transport of water is one of the most distinctive functions by which the gall-bladder rearranges its bile content. Water is reabsorbed from the gall-bladder lumen during fasting, whereas it is secreted into the lumen following meal ingestion. Nevertheless, the molecular mechanism by which water is transported across the gall-bladder epithelium remains mostly unclear. RESULTS: In the present study, we investigate the presence and subcellular localization of AQP (aquaporin) water channels in the mouse gall-bladder epithelium. Considerable AQP8 mRNA was detected in the gall-bladder epithelium of mouse, calf, rabbit, guinea pig and man. Studies of subcellular localization were then addressed to the mouse gall-bladder where the transcript of a second AQP, AQP1, was also detected. Immunoblotting experiments confirmed the presence of AQP8 and AQP1 at a protein level. Immunohistochemistry showed intense expression of AQP8 and AQP1 in the gall-bladder epithelial cells where AQP8 was localized in the apical membrane, whereas AQP1 was seen both in the apical and basolateral membranes, and in vesicles located in the subapical cytoplasm. CONCLUSIONS: The pattern of subcellular distribution of AQP8 and AQP1 strongly corroborates the hypothesis of a transcellular route for the movement of water across the gall-bladder epithelium. Osmotic water would cross the apical membrane through AQP8 and AQP1, although AQP1 would be the facilitated pathway for the movement of water across the basolateral membrane. The presence of two distinct AQPs in the apical membrane is an unusual finding and may relate to the membrane's ability both to absorb and secrete fluid. It is tempting to hypothesize that AQP1 is hormonally translocated to the gall-bladder apical membrane to secrete water as in the bile duct epithelium, a functional homologue of the gall-bladder epithelium, whereas apical AQP8 may account for the absorption of water from gall-bladder bile.
Assuntos
Aquaporinas/biossíntese , Epitélio/metabolismo , Vesícula Biliar/metabolismo , Regulação da Expressão Gênica , Canais Iônicos/biossíntese , Sequência de Aminoácidos , Animais , Aquaporina 1 , Aquaporinas/química , Transporte Biológico , Membrana Celular/metabolismo , Citoplasma/metabolismo , Cobaias , Hormônios/metabolismo , Immunoblotting , Imuno-Histoquímica , Hibridização In Situ , Canais Iônicos/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Água/metabolismoRESUMO
Thiazides, such as hydrochlorothiazide (HCTZ), are used to control blood pressure and to reduce renal calcium excretion. These effects are a result of interactions with the NaCl-cotransporter (NCC). This is demonstrated by the fact that mutations within the NCC protein lead to salt-resistant hypotension and hypocalciuria, paralleled by an increase in bone mineral density. These symptoms are also known as Gitelman syndrome. It has become increasingly evident that the effect of HCTZ on blood pressure and calcium homeostasis cannot be attributed exclusively to kidney functions, where the primary action of HCTZ on NCC is postulated to occur. We demonstrated the presence of the NCC transporter in the rat small intestine (ileum and jejunum) and human HT-29 cells, by using reverse transcription-PCR, Northern blot, Western blot, and immunofluorescence. Furthermore, we show that HCTZ modulates Ca(2+) uptake by intestinal cells, while affecting the electrical parameters of the cellular membrane, thus suggesting a functional interaction between NCC and the epithelial voltage-dependent calcium channel. The experiments presented here support the hypothesis of a direct involvement of the intestinal cells in the interaction between HCTZ and NaCl, as well as calcium homeostasis.
Assuntos
Hidroclorotiazida/farmacologia , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Receptores de Droga/metabolismo , Simportadores/metabolismo , Animais , Sequência de Bases , Cálcio/metabolismo , DNA Complementar/genética , Células HT29 , Homeostase , Humanos , Transporte de Íons/efeitos dos fármacos , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de Droga/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Simportadores de Cloreto de Sódio , Membro 3 da Família 12 de Carreador de Soluto , Simportadores/genéticaRESUMO
The plasma membrane is a highly dynamic cell-barrier if the nature and distribution of its constituents are considered. Ion channels are embedded in these double lipid bilayers, which modulate their 3D-structures. The structure modulations by the lipid bilayer can assume such a degree that channel activation depends on them, as was shown for the KcsA potassium channel. Here we show that the cation-over-anion selectivity of reconstituted ICln channels can be varied by the thickness of a bilayer build of phosphatidylcholines. The shorter the acyl-chains and therefore the thinner the bilayers of the membrane are, the more potassium selective the channels are. In contrast, the longer the acyl-chains and therefore the thicker the membranes are, the more chloride selective the channels become.
Assuntos
Membrana Celular/química , Membrana Celular/fisiologia , Canais Iônicos/fisiologia , Bicamadas Lipídicas/química , Animais , Membrana Celular/ultraestrutura , Cães , Humanos , Canais Iônicos/genética , Transporte de Íons , Potenciais da Membrana , Microscopia de Força AtômicaRESUMO
Anion channels in human mesothelial and mesothelioma cell lines were characterized by patch-clamp and biomolecular approaches. We found an outwardly rectifying anionic current which was inactivated at positive voltages and inhibited by extracellular adenosine 5'-triphosphate (ATP). Mesothelial and mesothelioma cells behaved differently concerning current inactivation properties. Inactivation is more pronounced and has a steeper onset in mesothelial cells. Different reversal potentials, in asymmetrical Cl(-) solutions, that could be attributed to a different selectivity of the channel, have been observed in the two cell lines. Mesothelioma cell single-channel analysis indicates that the number of the same active anion channel (3-4 pS) increased under hypoosmotic conditions. Immunocytochemistry experiments showed the presence of ICln protein in the cytosol and in the plasma membrane. Western blot analysis revealed an increase of ICln in the membrane under hypotonic conditions, an event possibly related to the activation of Cl(-) channels.
Assuntos
Canais de Cloreto/fisiologia , Canais Iônicos/análise , Mesotelioma/patologia , Trifosfato de Adenosina/farmacologia , Linhagem Celular Tumoral , Membrana Celular/química , Canais de Cloreto/metabolismo , Citosol/química , Células Epiteliais/metabolismo , Humanos , Imuno-Histoquímica , Canais Iônicos/fisiologia , Mesotelioma/metabolismo , Pressão Osmótica , Técnicas de Patch-Clamp , Derrame Pleural/patologia , Células Tumorais CultivadasRESUMO
How can a large number of different phenotypes be generated by a limited number of genotypes? Promiscuity between different, structurally related and/or unrelated proteins seems to provide a plausible explanation to this pertinent question. Strategies able to predict such functional interrelations between different proteins are important to restrict the number of putative candidate proteins, which can then be subjected to time-consuming functional tests. Here we describe the use of the operon structure of the nematode genome to identify partner proteins in human cells. In this work we focus on ion channels proteins, which build an interface between the cell and the outside world and are responsible for a growing number of diseases in humans. However, the proposed strategy for the partner protein quest is not restricted to this scientific area but can be adopted in virtually every field of human biology where protein-protein interactions are assumed.
Assuntos
Caenorhabditis elegans/genética , Genoma , Canais Iônicos/genética , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Íons/química , Íons/metabolismo , Luz , Modelos Genéticos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Óperon , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Cell volume alterations are involved in numerous cellular events like epithelial transport, metabolic processes, hormone secretion, cell migration, proliferation and apoptosis. Above all it is a need for every cell to counteract osmotic cell swelling in order to avoid cell damage. The defence against excess cell swelling is accomplished by a reduction of the intracellular osmolarity by release of organic- or inorganic osmolytes from the cell or by synthesis of osmotically less active macromolecules from their specific subunits. De-spite the large amount of experimental data that has accumulated, the intracellular mechanisms underlying the sensing of cell volume perturbations and the activation of volume compensatory processes, commonly summarized as regulatory volume decrease (RVD), are still only partly revealed. Moving into this field opens a complex scenario of molecular rearrangements and interactions involving intracellular messengers such as calcium, phosphoinositides and inositolphosphates as well as phosphoryla-tion/dephosphorylation processes and cytoskeletal reorganization with marked cell type- and tissue specific variations. Even in one and the same cell type significant differences regarding the activated pathways during RVD may be evident. This makes it virtually im-possible to unambigously define common sensing- and sinaling pathways used by differ-ent cells to readjust their celll volume, even if all these pathways converge to the activa-tion of comparatively few sets of effectors serving for osmolyte extrusion, including ion channels and transporters. This review is aimed at providing an insight into the manifold cellular mechanisms and alterations occuring during cell swelling and RVD.
Assuntos
Tamanho Celular/fisiologia , Transdução de Sinais/fisiologia , Animais , Ácido Araquidônico/farmacologia , Comunicação Autócrina/fisiologia , Cálcio/metabolismo , Membrana Celular/fisiologia , Citoesqueleto/metabolismo , Eicosanoides/farmacologia , Humanos , Fosfatos de Inositol/metabolismo , Fosfatos de Inositol/farmacologia , Canais Iônicos/metabolismo , Osmose/fisiologia , Fosfatidilinositóis/metabolismo , Fosfatidilinositóis/farmacologia , Fosforilação , Receptores Purinérgicos P2/metabolismoRESUMO
We examined whether protein kinase C (PKC) modulates the transport systems involved in bicarbonate movements across the plasma membranes of rat jejunum. Results of enzymatic assays provide evidence that under basal conditions conventional PKC (cPKC) is present in both basolateral membranes (BLMs) and apical (brush border) membranes (BBMs) of the enterocyte. In BLMs the basal expression of the kinase is low compared to expression in BBMs; however, treatment with Ca(2+) and phorbol 12-myristate 13-acetate (PMA) causes a significant increase, thus suggesting an asymmetrical kinase translocation. To explore the effect of PKC activation on membrane-bound transport mechanisms, 'in vitro' phosphorylated membrane vesicles were used to perform uptake studies. Results suggest that PKC activation exerts an inhibitory effect on the basolateral Cl(-)-HCO(3)(-) antiporter, whereas the basolateral HCO(3)(-) conductive pathway seems to be stimulated and Cl(-) conductance unaffected. The apical, but not basolateral, Na(+)-H(+) exchanger is inhibited by PKC activation. The specificity of the response to PKC was confirmed by using the kinase inhibitor staurosporine or the inactive phorbol ester 4-alpha-PMA. The inhibition of both apical Na(+)-H(+) and basolateral Cl(-)-HCO(3)(-) exchange activities suggests that the overall action of PKC causes a reduction of transepithelial bicarbonate transport.