RESUMO
Many human connexin50 (Cx50) mutants have been linked to cataracts including two carboxyl terminus serine mutants that are known phosphorylation sites in the lens (Cx50S258F and Cx50S259Y). To examine the behavior of these mutants and the role of phosphorylation at these positions, we stably transfected HeLa cells with cataract-linked and phosphorylation-mimicking (Cx50S258D and Cx50S259D) Cx50 mutants. We observed that gap junctional plaques were rarely detected in Cx50S258F-expressing and Cx50S259Y-expressing cells compared with wild-type cells. In contrast, gap junction abundance and size were greatly increased for Cx50S258D and Cx50S259D mutants. Cx50S258F and Cx50S259Y supported very low levels of gap junctional coupling, whereas Cx50S258D and Cx50S259D supported extensive intercellular communication. Furthermore, Cx50 levels as detected by immunoblotting were lower in Cx50S258F and Cx50S259Y mutants than in the wild-type or the aspartate substitution mutants, and chloroquine or ammonium chloride treatment significantly increased Cx50S258F and Cx50S259Y protein levels, implying participation of the lysosome in their increased degradation. Alanine substitution of amino acids within a predicted tyrosine-based sorting signal in Cx50S258F and Cx50S259Y increased levels of gap junctional plaques and intercellular transfer of neurobiotin. These results suggest that the absence of phosphorylatable serines at these positions exposes a sorting signal leading to lysosomal degradation of Cx50, whereas phosphorylation at these sites conceals this signal and allows targeting of Cx50 to the plasma membrane and stabilization of gap junction plaques. We propose that in the lens, degradation of Cx50S258F and Cx50S259Y decreases Cx50 levels at the plasma membrane and consequently Cx50 function, leading to cataracts.
Assuntos
Catarata , Conexinas , Cristalino , Mutação , Catarata/genética , Catarata/metabolismo , Conexinas/genética , Conexinas/metabolismo , Proteínas do Olho/metabolismo , Junções Comunicantes/genética , Junções Comunicantes/metabolismo , Células HeLa , Humanos , Cristalino/metabolismo , Lisossomos/genética , Lisossomos/metabolismo , Serina/genética , Serina/metabolismoRESUMO
Connexin-50 (Cx50) is among the most frequently mutated genes associated with congenital cataracts. Although most of these disease-linked variants cause loss of function because of misfolding or aberrant trafficking, others directly alter channel properties. The mechanistic bases for such functional defects are mostly unknown. We investigated the functional and structural properties of a cataract-linked mutant, Cx50T39R (T39R), in the Xenopus oocyte system. T39R exhibited greatly enhanced hemichannel currents with altered voltage-gating properties compared to Cx50 and induced cell death. Coexpression of mutant T39R with wild-type Cx50 (to mimic the heterozygous state) resulted in hemichannel currents whose properties were indistinguishable from those induced by T39R alone, suggesting that the mutant had a dominant effect. Furthermore, when T39R was coexpressed with Cx46, it produced hemichannels with increased activity, particularly at negative potentials, which could potentially contribute to its pathogenicity in the lens. In contrast, coexpression of wild-type Cx50 with Cx46 was associated with a marked reduction in hemichannel activity, indicating that it may have a protective effect. All-atom molecular dynamics simulations indicate that the R39 substitution can form multiple electrostatic salt-bridge interactions between neighboring subunits that could stabilize the open-state conformation of the N-terminal (NT) domain while also neutralizing the voltage-sensing residue D3 as well as residue E42, which participates in loop gating. Together, these results suggest T39R acts as a dominant gain-of-function mutation that produces leaky hemichannels that may cause cytotoxicity in the lens and lead to development of cataracts.
Assuntos
Catarata , Cristalino , Animais , Catarata/congênito , Catarata/genética , Catarata/metabolismo , Conexinas/genética , Conexinas/metabolismo , Proteínas do Olho/metabolismo , Junções Comunicantes/metabolismo , Humanos , Cristalino/metabolismo , Mutação de Sentido Incorreto , XenopusRESUMO
An N-terminal mutant of connexin46 (T19M) alters a highly conserved threonine and has been linked to autosomal dominant cataracts. To study the cellular and functional consequences of substitution of this amino acid, T19M was expressed in Xenopus oocytes and in HeLa cells. Unlike wild-type Cx46, T19M did not induce intercellular conductances in Xenopus oocytes. In transfected HeLa cells, T19M was largely localized within the cytoplasm, with drastically reduced formation of gap junction plaques. Expression of rat T19M was cytotoxic, as evidenced by an almost complete loss of viable cells expressing the mutant protein by 48-72 h following transfection. When incubated in medium containing physiological concentrations of divalent cations, T19M-expressing cells showed increased uptake of DAPI as compared with cells expressing wild-type Cx46, suggesting aberrant connexin hemi-channel activity. Time-lapse and dye uptake studies suggested that T19M hemi-channels had reduced sensitivity to Ca(2+). Whole cell patch clamp studies of single transfected HeLa cells demonstrated that rat T19M formed functional hemi-channels with altered voltage-dependent gating. These data suggest that T19M causes cataracts by loss of gap junctional channel function and abnormally increased hemi-channel activity. Furthermore, they implicate this conserved threonine in both gap junction plaque formation and channel/hemi-channel gating in Cx46.
Assuntos
Conexinas/metabolismo , Junções Comunicantes/metabolismo , Ativação do Canal Iônico/fisiologia , Animais , Catarata/genética , Catarata/metabolismo , Conexinas/genética , Junções Comunicantes/fisiologia , Células HeLa , Humanos , Ativação do Canal Iônico/genética , Mutação , Ratos , Xenopus laevisRESUMO
The lens is proposed to have an internal microcirculation system consisting of continuously circulating ionic fluxes that play an essential role in maintaining lens transparency. One of the key components of this system is the sodium leak conductance. Here we investigate the contribution of Cx46 hemichannels to the basal membrane permeability of peripheral fiber cells isolated from transgenic mouse lenses lacking Cx50 or both Cx50 and Cx46 (dKO) using the whole cell patch-clamp technique. Our results show that Cx46 hemichannels were largely closed at a resting voltage of -60 mV in the presence of millimolar divalent cation concentrations. However, even though the vast majority of these channels were closed at -60 mV, a small, persistent, inward current could still be detected. This current could be mostly blocked by exposure to 1 mM La(3+) and was not observed in fiber cells isolated from dKO mouse lenses suggesting that it was due to Cx46 hemichannels. In addition, Cx50(-/-) fiber cells showed increased open channel noise and a depolarized resting potential compared with dKO fiber cells. Exposure of Cx50(-/-) fiber cells to La(3+) hyperpolarized the resting potential to -58 mV, which is similar to the value of resting potential measured in dKO fiber and significantly reduced the open channel noise. In conclusion, these results suggest that Cx46 hemichannels may contribute to the sodium leak conductance in lens fiber cells.
Assuntos
Permeabilidade da Membrana Celular , Membrana Celular/metabolismo , Conexinas/metabolismo , Cristalino/metabolismo , Sódio/metabolismo , Animais , Conexinas/deficiência , Conexinas/genética , Proteínas do Olho/genética , Ativação do Canal Iônico , Transporte de Íons , Cristalino/citologia , Potenciais da Membrana , Camundongos , Camundongos KnockoutRESUMO
Mutations in connexin 46 are associated with congenital cataracts. The purpose of this project was to characterize cellular and functional properties of two congenital cataract-associated mutations located in the NH2 terminus of connexin 46: Cx46D3Y and Cx46L11S, which we found localized to gap junctional plaques like wild-type Cx46 in transfected HeLa cells. Dual two-microelectrode-voltage-clamp studies of Xenopus oocyte pairs injected with wild-type or mutant rat Cx46 showed that oocyte pairs injected with D3Y or L11S cRNA failed to induce gap junctional coupling, whereas oocyte pairs injected with Cx46 showed high levels of coupling. D3Y, but not L11S, functionally paired with wild-type Cx46. To determine whether coexpression of D3Y or L11S affected the junctional conductance produced by wild-type lens connexins, we studied pairs of oocytes coinjected with equal amounts of mutant and wild-type connexin cRNA. Expression of D3Y or L11S almost completely abolished gap junctional coupling induced by Cx46. In contrast, expression of D3Y or L11S failed to inhibit junctional conductance induced by Cx50. To examine effects of the D3Y and L11S mutations on hemichannel activity, hemichannel currents were measured in connexin cRNA-injected oocytes. Oocytes expressing D3Y exhibited reduced hemichannel activity as well as alterations in voltage gating and charge selectivity while oocytes expressing L11S showed no hemichannel activity. Moreover, coexpression of mutant with wild-type Cx50 or Cx46 gave rise to hemichannels with distinct electrophysiological properties, suggesting that the mutant connexins were forming heteromeric channels with wild-type connexins. These data suggest D3Y and L11S cause cataracts by similar but not identical mechanisms.
Assuntos
Catarata/genética , Conexinas/genética , Mutação de Sentido Incorreto , Animais , Catarata/congênito , Catarata/patologia , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Junções Comunicantes/fisiologia , Células HeLa , Humanos , Ativação do Canal Iônico , Potenciais da Membrana , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Ratos , Homologia Estrutural de Proteína , Xenopus laevisRESUMO
During accommodation, the lens changes focus by altering its shape following contraction and relaxation of the ciliary muscle. At the cellular level, these changes in shape may be accompanied by fluid flow in and out of individual lens cells. We tested the hypothesis that some of this flow might be directly modulated by pressure-activated channels. In particular, we used the whole cell patch clamp technique to test whether calcium-activated-chloride channels (CaCCs) expressed in differentiating lens cells are activated by mechanical stimulation. Our results show that mechanical stress, produced by focally perfusing the lens cell at a constant rate, caused a significant increase in a chloride current that could be fully reversed by stopping perfusion. The time course of activation and recovery from activation of the flow-induced current occurred rapidly over a time frame similar to that of accommodation. The flow-induced current could be inhibited by the TMEM16A specific CaCC blocker, Ani9, suggesting that the affected current was predominantly due to TMEM16A chloride channels. The mechanism of action of mechanical stress did not appear to involve calcium influx through other mechanosensitive ion channels since removal of calcium from the bath solution failed to block the flow-induced chloride current. In conclusion, our results suggest that CaCCs in the lens can be rapidly and reversibly modulated by mechanical stress, consistent with their participation in regulation of volume in this organ.
RESUMO
Gap junction channels, which are made of connexins, are critical for intercellular communication, a function that may be disrupted in a variety of diseases. We studied the consequences of two cataract-associated mutations at adjacent positions at the first extracellular boundary in human connexin50 (Cx50), W45S and G46V. Both of these mutants formed gap junctional plaques when they were expressed in HeLa cells, suggesting that they trafficked to the plasma membrane properly. However, their functional properties differed. Dual two-microelectrode voltage-clamp studies showed that W45S did not form functional intercellular channels in paired Xenopus oocytes or hemichannel currents in single oocytes. When W45S was coexpressed with wild-type Cx50, the mutant acted as a dominant negative inhibitor of wild-type function. In contrast, G46V formed both functional gap junctional channels and hemichannels. G46V exhibited greatly enhanced currents compared with wild-type Cx50 in the presence of physiological calcium concentrations. This increase in hemichannel activity persisted when G46V was coexpressed with wild-type lens connexins, consistent with a dominant gain of hemichannel function for G46V. These data suggest that although these two mutations are in adjacent amino acids, they have very different effects on connexin function and cause disease by different mechanisms: W45S inhibits gap junctional channel function; G46V reduces cell viability by forming open hemichannels.
Assuntos
Catarata/genética , Conexinas/genética , Proteínas do Olho/genética , Mutação , Animais , Sequência de Bases , Cálcio/fisiologia , Catarata/fisiopatologia , Conexinas/fisiologia , Fenômenos Eletrofisiológicos/genética , Proteínas do Olho/fisiologia , Feminino , Junções Comunicantes/genética , Junções Comunicantes/fisiologia , Células HeLa , Humanos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Xenopus laevisRESUMO
BACKGROUND: Atrial fibrillation is the most common type of cardiac arrhythmia and a leading cause of cardiovascular morbidity, particularly stroke. The cardiac gap-junction protein connexin 40 is expressed selectively in atrial myocytes and mediates the coordinated electrical activation of the atria. We hypothesized that idiopathic atrial fibrillation has a genetic basis and that tissue-specific mutations in GJA5, the gene encoding connexin 40, may predispose the atria to fibrillation. METHODS: We sequenced GJA5 from genomic DNA isolated from resected cardiac tissue and peripheral lymphocytes from 15 patients with idiopathic atrial fibrillation. Identified GJA5 mutations were transfected into a gap-junction-deficient cell line to assess their functional effects on protein transport and intercellular electrical coupling. RESULTS: Four novel heterozygous missense mutations were identified in 4 of the 15 patients. In three patients, the mutations were found in the cardiac-tissue specimens but not in the lymphocytes, indicating a somatic source of the genetic defects. In the fourth patient, the sequence variant was detected in both cardiac tissue and lymphocytes, suggesting a germ-line origin. Analysis of the expression of mutant proteins revealed impaired intracellular transport or reduced intercellular electrical coupling. CONCLUSIONS: Mutations in GJA5 may predispose patients to idiopathic atrial fibrillation by impairing gap-junction assembly or electrical coupling. Our data suggest that common diseases traditionally considered to be idiopathic may have a genetic basis, with mutations confined to the diseased tissue.
Assuntos
Fibrilação Atrial/genética , Conexinas/genética , Mutação de Sentido Incorreto , Adulto , Idade de Início , Sequência de Aminoácidos , Fibrilação Atrial/metabolismo , Fibrilação Atrial/patologia , Sequência de Bases , Linhagem Celular Tumoral , Conexinas/metabolismo , Análise Mutacional de DNA , Feminino , Junções Comunicantes/metabolismo , Átrios do Coração/patologia , Humanos , Linfócitos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neuroblastoma/genética , Neuroblastoma/metabolismo , Homologia de Sequência , Transfecção , Proteína alfa-5 de Junções ComunicantesRESUMO
PURPOSE: The aim of this study was the genetic, cellular, and physiological characterization of a connexin50 (CX50) variant identified in a child with congenital cataracts. METHODS: Lens material from surgery was collected and used for cDNA production. Genomic DNA was prepared from blood obtained from the proband and her parents. PCR amplified DNA fragments were sequenced and characterized by restriction digestion. Connexin protein distribution was studied by immunofluorescence in transiently transfected HeLa cells. Formation of functional channels was assessed by two-microelectrode voltage-clamp in cRNA-injected Xenopus oocytes. RESULTS: Ophthalmologic examination showed that the proband suffered from bilateral white, diffuse cataracts, but the parents were free of lens opacities. Direct sequencing of the PCR product produced from lens cDNA showed that the proband was heterozygous for a G>T transition at position 741 of the GJA8 gene, encoding the exchange of methionine for isoleucine at position 247 of CX50 (CX50I247M). The mutation was confirmed in the genomic DNA, but it was also present in the unaffected mother. When expressed in HeLa cells, both wild type CX50 and CX50I247M formed gap junction plaques. Both CX50 and CX50I247M induced gap junctional currents in pairs of Xenopus oocytes. CONCLUSIONS: Although the CX50I247M substitution has previously been suggested to cause cataracts, our genetic, cellular, and electrophysiological data suggest that this allele more likely represents a rare silent, polymorphic variant.
Assuntos
Alelos , Substituição de Aminoácidos/genética , Catarata/genética , Conexinas/genética , Proteínas do Olho/genética , Mutação/genética , Polimorfismo Genético , Adulto , Idoso , Sequência de Aminoácidos , Sequência de Bases , Conexinas/química , Proteínas do Olho/química , Família , Feminino , Junções Comunicantes/metabolismo , Células HeLa , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Mutantes , Linhagem , TransfecçãoRESUMO
Purpose: Chloride channels have been proposed to play an important role in the regulation of lens volume. Unfortunately, little information is available about the molecular identity of these channels or how they are regulated in the lens due to the difficulties in isolating mouse fiber cells. Recently, our laboratory has developed a new technique for isolating these cells by using transgenic mouse lenses that lack both Cx50 and Cx46. The purpose of this study was to test the hypothesis that newly differentiating mouse fiber cells express calcium-activated chloride channels (CaCCs) by using this technique. Methods: Differentiating fiber cells were isolated from lenses of double knockout mice that lack both Cx50 and Cx46 by using collagenase. Membrane currents were studied using the whole-cell patch clamp technique. The molecular identity and distribution of CaCCs were investigated using RT-PCR and immunofluorescence. Results: Our electrophysiologic experiments suggest that peripheral fiber cells express a calcium-activated chloride current. The voltage gating properties, calcium sensitivity, and pharmacologic properties of this current resembled those of TMEM16 CaCCs. RT-PCR analysis demonstrated the presence of TMEM16A and TMEM16B transcripts in wild-type and double knockout mouse lenses. Both TMEM16A and TMEM16B proteins were detected in the differentiating epithelial cells and newly elongating fiber cells near the equator of the lens by immunohistochemistry. Conclusions: Our results demonstrate that membrane conductance of peripheral fiber cells contain CaCCs that can be attributed to TMEM16A and TMEM16B. Given their critical role in volume regulation in other tissues, we speculate that these channels play a similar role in the lens.
Assuntos
Canais de Cloreto/metabolismo , Cristalino/metabolismo , Animais , Anoctaminas/metabolismo , Diferenciação Celular/fisiologia , Tamanho Celular , Canais de Cloreto/genética , Conexinas/genética , Células Epiteliais/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Técnicas de Inativação de Genes , Cristalino/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase em Tempo RealRESUMO
A mutant human connexin50 (hCx50), hCx50P88S, has been linked to cataracts inherited as an autosomal dominant trait. The functional, biochemical and cellular behavior of wild-type and mutant hCx50 were examined in transfected cells. hCx50P88S was unable to induce gap junctional currents by itself, and it abolished gap junctional currents when co-expressed with wild-type (wt) hCx50. Cells transfected with hCx50P88S showed cytoplasmic accumulations of Cx50 immunoreactivity in addition to staining at appositional membranes; these accumulations did not significantly co-localize with markers for the endoplasmic reticulum, Golgi apparatus, lysosomes, endosomes or vimentin filaments. Immunoelectron microscopy studies localized hCx50P88S to cytoplasmic membrane stacks in close vicinity to the endoplasmic reticulum. In contrast, aggresome-like accumulations were induced by treatment of wt hCx50-transfected cells with proteasomal inhibitors. The formation of hCx50P88S accumulations in transiently transfected cells was not blocked by treatment with Brefeldin A suggesting that they form before Cx50 transits through the Golgi apparatus to the plasma membrane. Treatment of HeLa-hCx50P88S cells with cycloheximide demonstrated the presence of a very stable pool of hCx50P88S. Taken together, these results suggest that the P to S mutation at amino acid residue 88 causes a defect that leads to decreased degradation and subsequent accumulation of hCx50P88S in a cellular structure different from aggresomes.
Assuntos
Catarata/genética , Proteínas do Olho/genética , Animais , Linhagem Celular , Conexinas , Eletrofisiologia , Proteínas do Olho/metabolismo , Células HeLa/ultraestrutura , Humanos , Immunoblotting , Microscopia Eletrônica , Mutação , Oligopeptídeos/farmacologia , Fenilbutiratos/farmacologia , Transporte Proteico/efeitos dos fármacos , TransfecçãoRESUMO
The lens is a multicellular, but avascular tissue that must stay transparent to allow normal transmission of light and focusing of it on the retina. Damage to lens cells and/or proteins can cause cataracts, opacities that disrupt these processes. The normal survival of the lens is facilitated by an extensive network of gap junctions formed predominantly of connexin46 and connexin50. Mutations of the genes that encode these connexins (GJA3 and GJA8) have been identified and linked to inheritance of cataracts in human families and mouse lines. In vitro expression studies of several of these mutants have shown that they exhibit abnormalities that may lead to disease. Many of the mutants reduce or modify intercellular communication due to channel alterations (including loss of function or altered gating) or due to impaired cellular trafficking which reduces the number of gap junction channels within the plasma membrane. However, the abnormalities detected in studies of other mutants suggest that they cause cataracts through other mechanisms including gain of hemichannel function (leading to cell injury and death) and formation of cytoplasmic accumulations (that may act as light scattering particles). These observations and the anticipated results of ongoing studies should elucidate the mechanisms of cataract development due to mutations of lens connexins and abnormalities of other lens proteins. They may also contribute to our understanding of the mechanisms of disease due to connexin mutations in other tissues.
RESUMO
PURPOSE: To characterize the properties of connexin 46 hemichannels in differentiating fiber cells isolated from mouse lenses. METHODS: Differentiating fiber cells were isolated from mouse lenses using collagenase. Cellular localization of connexin 50 (Cx50) and connexin 46 (Cx46) was assessed by immunofluorescence. Membrane currents were recorded using whole cell patch clamping. Dye uptake was measured using time-lapse imaging. RESULTS: In freshly dissociated fiber cells isolated from knockout Cx50 (KOCx50) mouse lenses, removal of external divalent cations induced a macroscopic current composed of large conductance channels. This current was reduced at a holding potential of -60 mV, activated on depolarization, and had a reversal potential near 0 mV. These properties were very similar to those of Cx46 hemichannel currents recorded in single Xenopus oocytes. If the currents observed in divalent cation-free Ringer's solution were due to Cx46 hemichannel opening, then dye influx by gap junctional/hemichannel permeable dyes should be measurable in the fiber cells. To measure dye influx, the authors used the positively charged dyes, propidium iodide (PrI) and 4'-6-diamidino-2-phenylindole (DAPI). In the absence of external calcium, fiber cells took up both dyes. Furthermore, dye influx could be inhibited by hemichannel blockers. To confirm that this current was due to Cx46 hemichannels, the authors studied fiber cells isolated from the lenses of double knockout (Cx46(-/-); Cx50(-/-)) mice and demonstrated that both the calcium-sensitive conductance and dye influx were absent. CONCLUSIONS: These results show that Cx46 can form functional hemichannels in the nonjunctional membrane of fiber cells.
Assuntos
Diferenciação Celular/fisiologia , Conexinas/metabolismo , Canais Iônicos/metabolismo , Cristalino/citologia , Animais , Proteínas do Olho/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Corantes Fluorescentes/metabolismo , Junções Comunicantes/metabolismo , Indóis/metabolismo , Cristalino/metabolismo , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Propídio/metabolismo , Imagem com Lapso de TempoRESUMO
PURPOSE: To determine the consequences of expression of a novel connexin50 (CX50) mutant identified in a child with congenital total cataracts. METHODS: The GJA8 gene was directly sequenced. Formation of functional channels was assessed by the two-microelectrode voltage-clamp METHOD: Connexin protein levels and distribution were assessed by immunoblot analysis and immunofluorescence. The proportion of apoptotic cells was determined by flow cytometry. RESULTS: Direct sequencing of the GJA8 gene identified a 137 G>T transition that resulted in the replacement of glycine by valine at position 46 of the coding region of CX50 (CX50G46V). Both CX50 and CX50G46V induced gap junctional currents in pairs of Xenopus oocytes. In single Xenopus oocytes, CX50G46V induced connexin hemichannel currents that were activated by removal of external calcium; their magnitudes were much higher than those in oocytes injected with similar amounts of CX50 cRNA. When expressed in HeLa cells under the control of an inducible promoter, both CX50 and CX50G46V formed gap junctional plaques. Induction of CX50G46V expression led to a decrease in the number of cells and an increase in the proportion of apoptotic cells. CX50G46V-induced cell death was prevented by high concentrations of extracellular calcium ions. CONCLUSIONS: Unlike previously characterized CX50 mutants that exhibit impaired trafficking and/or lack of function, CX50G46V traffics properly to the plasma membrane and forms functional hemichannels and gap junction channels; however, it causes cell death even when expressed at minute levels. The biochemical results indirectly suggest a potential novel mechanism by which connexin mutants could lead to cataracts: cytotoxicity due to enhanced hemichannel function.
Assuntos
Apoptose/genética , Catarata/genética , Conexinas/genética , Proteínas do Olho/genética , Junções Comunicantes/fisiologia , Regulação da Expressão Gênica/fisiologia , Mutação Puntual , Animais , Catarata/congênito , Catarata/patologia , Técnicas de Cultura de Células , Pré-Escolar , Ecdisterona/análogos & derivados , Ecdisterona/farmacologia , Feminino , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Células HeLa , Humanos , Immunoblotting , Oócitos/fisiologia , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , Transfecção , Xenopus laevisRESUMO
To examine the effects of increased expression of Cx50 in the mouse lens, transgenic mice were generated using a DNA construct containing the human Cx50 coding region and a C-terminal FLAG epitope driven by the chicken betaB1-crystallin promoter. Expression of this protein in paired Xenopus oocytes induced gap junctional currents of similar magnitude to wild type human Cx50. Three lines of transgenic mice expressing the transgenic protein were analyzed. Lenses from transgenic mice were smaller than those from non-transgenic littermates, and had cataracts that were already visible at postnatal day 1. Expression of the transgene resulted in a 3- to 13-fold increase in Cx50 protein levels above those of non-transgenic animals. Light microscopy revealed alterations in epithelial cell differentiation, fiber cell structure, interactions between fiber cells and areas of liquefaction. Scanning electron microscopy showed fiber cells of varying widths with bulging areas along single fibers. Anti-Cx50 and anti-FLAG immunoreactivities were detected at appositional membranes and in intracellular vesicles in transgenic lenses. N-cadherin, Cx46, ZO-1 and aquaporin 0 localized mainly at the plasma membrane, although some N-cadherin and aquaporin 0 was associated with the intracellular vesicles. The abundance and solubility/integrity of alphaA-, alphaB-, beta- and gamma-crystallin were unaffected. These results demonstrate that transgenic expression of Cx50 in mice leads to cataracts associated with formation of cytoplasmic vesicles containing Cx50 and decreased or slowed epithelial differentiation without major alterations in the distribution of other integral membrane or membrane-associated proteins or the integrity/solubility of crystallins.
Assuntos
Catarata/etiologia , Conexinas/metabolismo , Proteínas do Olho/metabolismo , Animais , Aquaporinas/análise , Aquaporinas/metabolismo , Catarata/metabolismo , Catarata/patologia , Células Cultivadas , Conexinas/análise , Conexinas/genética , Cristalinas/metabolismo , Eletrofisiologia , Proteínas do Olho/análise , Proteínas do Olho/genética , Expressão Gênica , Engenharia Genética , Humanos , Immunoblotting/métodos , Cristalino/patologia , Cristalino/ultraestrutura , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica , Oócitos/fisiologia , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Solubilidade , Xenopus , Proteínas de Xenopus , Proteína da Zônula de Oclusão-1RESUMO
The voltage- and calcium-dependent gating properties of two lens gap-junctional hemichannels were compared at the macroscopic and single channel level. In solutions containing zero added calcium and 1 mM Mg, chicken Cx56 hemichannels were mostly closed at negative potentials and application of depolarizing voltage clamp steps elicited a slowly activating outward current. In contrast, chicken Cx45.6 hemichannels were predominantly open at negative potentials and rapidly closed in response to application of large depolarizing potentials. Another difference was that macroscopic Cx45.6 currents were much smaller in size than the hemichannel currents induced by oocytes with similar amounts of cRNA for Cx56. The aim of this study was to identify which regions of the connexins were responsible for the differences in voltage-dependent gating and macroscopic current amplitude by constructing a series of chimeric Cx45.6-Cx56 channels. Our results show that two charged amino acids that are specific for the alpha3-group connexins (R9 in the N-terminus and E43 in the first extracellular loop) are important determinants for the difference in voltage-dependent gating between Cx45.6 and Cx56 hemichannels; the first transmembrane-spanning domain, M1, is an important determinant of macroscopic current magnitude; R9 and E43 are also determinants of single channel conductance and rectification.
Assuntos
Conexinas/fisiologia , Proteínas do Olho/fisiologia , Junções Comunicantes/fisiologia , Ativação do Canal Iônico , Sequência de Aminoácidos , Animais , Membrana Celular/fisiologia , Galinhas , Feminino , Técnicas In Vitro , Cristalino/fisiologia , Potenciais da Membrana , Dados de Sequência Molecular , Oócitos/fisiologia , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
Previous studies have shown that gap junctional channels formed from the lens connexins Cx50 (or its chicken orthologue, Cx45.6) and Cx43 exhibit marked differences in transjunctional voltage gating and unitary conductance. In the present study, we used the negatively charged dye, Lucifer Yellow (LY), to examine and compare quantitative differences in dye transfer between pairs of HeLa cells stably transfected with Cx45.6 or Cx43. Our results show that Cx45.6 gap junctional channels are three times less permeable to LY than Cx43 channels. Replacement of the N-terminus of Cx45.6 with the corresponding domain of Cx43 increased LY permeability, reduced the transjunctional voltage (V(j)) gating sensitivity, and reduced the unitary conductance of Cx45.6-43N gap junctional channels. Further experiments, using a series of Alexa probes that had similar net charge but varied in size showed that the Cx45.6-43N had a significantly higher permeability for the two largest Alexa dyes than Cx45.6. These data suggest that the N-terminus plays a critical role in determining many of biophysical properties of Cx45.6 gap junctional channels, including molecular permeability and voltage gating.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Conexinas/análise , Conexinas/fisiologia , Ativação do Canal Iônico/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/genética , Galinhas , Quimera/genética , Conexina 43/análise , Conexina 43/química , Conexina 43/genética , Conexina 43/fisiologia , Conexinas/química , Conexinas/genética , Células HeLa , Humanos , Isoquinolinas , Matemática , Camundongos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Ratos , Fatores de Tempo , TransfecçãoRESUMO
An increasing number of diseases have been mapped to genes coding for ion channel proteins, including the gap junction proteins, connexins. Here, we report on the identification of an amino acid sequence underlying the behavior of a non-functional mutant connexin46 (CX46) associated with congenital cataracts. The mutant protein, CX46fs380, is 31 amino acids longer than CX46 and contains 87 aberrant amino acids in its C terminus. When expressed in mammalian cells, the mutant CX46 was not found at gap junctional plaques, but it showed extensive co-localization with markers for ERGIC and Golgi. The severe reductions in function and formation of gap junctional plaques were transferred to other connexins by creating chimeras containing the last third (or more) of the aberrant C terminus of the CX46 mutant. This sequence also impaired trafficking of a CD8 chimera. Site-directed mutagenesis of a diphenylalanine restored appositional membrane localization and function. These results suggest a novel mechanism in which a mutation causes disease by generating a motif that leads to retention within the synthetic/secretory pathway.
Assuntos
Catarata/congênito , Catarata/genética , Conexinas/química , Conexinas/genética , Mutação , Sequência de Aminoácidos , Animais , Brefeldina A/farmacologia , Membrana Celular/química , Conexinas/fisiologia , Cicloeximida/farmacologia , Condutividade Elétrica , Citometria de Fluxo , Imunofluorescência , Junções Comunicantes/fisiologia , Expressão Gênica , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Oócitos/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Recombinantes de Fusão/análise , Relação Estrutura-Atividade , Transfecção , XenopusRESUMO
Connexons or gap junction hemichannels are large, nonselective ion channels that reside in the nonjunctional plasma membrane before their assembly into gap junction channels. Increasing evidence suggests that these channels can open under certain conditions and may participate in a number of cellular processes, including the release of small metabolites such as ATP and NAD(+), which are involved in paracrine signaling.
Assuntos
Junções Comunicantes/metabolismo , Canais Iônicos/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Membrana Celular/metabolismo , Conexinas/metabolismo , HumanosRESUMO
One of the most striking features of hemi-gap-junctional channels is that they are dramatically modulated by extracellular divalent cations. In this study, we characterized the effects of external divalent cations and voltage on macroscopic human connexin46 (hCx46) hemi-gap-junctional currents using the two-electrode voltage-clamp technique. Increasing extracellular magnesium resulted in a shift of the voltage dependence of activation to more positive potentials, a decrease in the maximum conductance, an acceleration of deactivation, and a slowing of activation. Hyperpolarizing the membrane potential could mimic the effect of raising external magnesium on the activation kinetics and maximum conductance. These results could be interpreted in terms of a sequential model of channel activation with two independent divalent cation binding sites. This model could also explain the effects of external calcium on hCx46 hemichannels. However, the apparent binding affinities for calcium were significantly higher than for magnesium. In addition, we identified a mutation in the first extracellular domain of hCx46 (hCx46*N63S) that resulted in hemichannels that showed increased sensitivity to magnesium blockade.