RESUMEN

Functional biological synthetic composite (BSC) membranes were made using phospholipids, biological membrane proteins and permeable synthetic supports or membranes. Lipid bilayers were formed on porous polycarbonate (PC), polyethylene terephthalate (PETE) and poly (l-lactic acid) (PLLA) membranes and in 10-100 microm laser-drilled pores in a 96-well plastic plate as measured by increased resistance or decreased currents. Bilayers in 50 microm and smaller pores were stable for up to 4 h as measured by resistance changes or a current after gramicidin D reconstitution. Biological membrane transport reconstitution was then carried out. Using vesicles containing Kv1.5 K(+) channels, K(+) currents and decreased resistance were measured across bilayers in 50 microm pores in the plastic plate and PLLA membranes, respectively, which were inhibited by compound B, a Kv1.5 K(+) channel inhibitor. Functional reconstitution of Kv1.5 K(+) channels was successful. Incorporation of membrane proteins in functional form in stable permeable membrane-supported lipid bilayers is a simple technology to create BSC membranes that mimic biological function which is readily adaptable for high throughput screening.

Asunto(s)

Biofisica/métodos , Iones , Membrana Dobles de Lípidos/química , Membranas Artificiales , Membrana Celular/metabolismo , Gramicidina/química , Concentración 50 Inhibidora , Canal de Potasio Kv1.5/antagonistas & inhibidores , Canal de Potasio Kv1.5/química , Ácido Láctico/química , Potenciales de la Membrana , Microscopía Electrónica de Rastreo , Cemento de Policarboxilato/química , Poliésteres , Tereftalatos Polietilenos/química , Polímeros/química , Potasio/química , Canales de Potasio/química , Espectrometría por Rayos X

RESUMEN

ClC-2 Cl- channels represent a potential target for therapy in cystic fibrosis. Key questions regarding the feasibility of using ClC-2 as a therapeutic target are addressed in the present studies, including whether the channels are present in human lung epithelia and whether activators of the channel can be identified. Two new mechanisms of activation of human recombinant ClC-2 Cl- channels expressed in HEK-293 cells were identified: amidation with glycine methyl ester catalyzed by 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) and treatment with acid-activated omeprazole. ClC-2 mRNA was detected by RT-PCR. Channel function was assessed by measuring Cl- currents by patch clamp in the presence of a cAMP-dependent protein kinase (PKA) inhibitor, myristoylated protein kinase inhibitor, to prevent PKA-activated Cl- currents. Calu-3, A549, and BEAS-2B cell lines derived from different human lung epithelia contained ClC-2 mRNA, and Cl- currents were increased by amidation, acid-activated omeprazole, and arachidonic acid. Similar results were obtained with buccal cells from healthy individuals and cystic fibrosis patients. The ClC-2 Cl- channel is thus a potential target for therapy in cystic fibrosis.

Asunto(s)

Ácidos Araquidónicos/farmacología , Canales de Cloruro/metabolismo , Omeprazol/farmacología , Mucosa Respiratoria/efectos de los fármacos , 1-Metil-3-Isobutilxantina/farmacología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Amidas/metabolismo , Canales de Cloruro CLC-2 , Línea Celular , Células Cultivadas , Canales de Cloruro/genética , Cloruros/metabolismo , Colforsina/farmacología , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Inhibidores Enzimáticos/farmacología , Etildimetilaminopropil Carbodiimida/farmacología , Humanos , Magnesio/metabolismo , Mucosa Bucal/citología , Técnicas de Placa-Clamp , Inhibidores de Fosfodiesterasa/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Mucosa Respiratoria/metabolismo

RESUMEN

HCl secretion across the parietal cell apical secretory membrane involves the H+-K+-ATPase, the ClC-2 Cl- channel, and a K+ channel. In the present study, the cellular and subcellular distribution of ClC-2 mRNA and protein was determined in the rabbit gastric mucosa and in isolated gastric glands. ClC-2 mRNA was localized to parietal cells by in situ hybridization and by direct in situ RT-PCR. By immunoperoxidase microscopy, ClC-2 protein was concentrated in parietal cells. Immunofluorescent confocal microscopy suggested that the ClC-2 was localized to the secretory canalicular membrane of stimulated parietal cells and to intracellular structures of resting parietal cells. Immunogold electron microscopy confirmed that ClC-2 is in the secretory canalicular membrane of stimulated cells and in tubulovesicles of resting parietal cells. These findings, together with previous functional characterization of the native and recombinant channel, strongly indicate that ClC-2 is the Cl- channel, which together with the H+-K+-ATPase and a K+ channel, results in HCl secretion across the parietal cell secretory membrane.

Asunto(s)

Canales de Cloruro/análisis , Canales de Cloruro/genética , Mucosa Gástrica/química , Animales , Feto/química , Ácido Gástrico/metabolismo , Mucosa Gástrica/metabolismo , Mucosa Gástrica/ultraestructura , Inmunohistoquímica , Hibridación in Situ , Microscopía Inmunoelectrónica , ARN Mensajero/análisis , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

1. The CIC-2 Cl- channels are present in the adult human lung epithelia and, therefore, are a potential target for therapy in cystic fibrosis. 2. Activators of CIC-2 Cl- channels that may have physiological relevance include activation by reduced external pH, protein kinase A and arachidonic acid. 3. Activators of CIC-2 Cl- channels that have therapeutic potential include amidation and omeprazole and, perhaps, effectors of arachidonic acid metabolism.

Asunto(s)

Canales de Cloruro/metabolismo , Fibrosis Quística/metabolismo , Secuencia de Aminoácidos , Animales , Biotransformación , Agonistas de los Canales de Cloruro , Canales de Cloruro/química , Fibrosis Quística/genética , Humanos , Datos de Secuencia Molecular

RESUMEN

An HEK-293 cell line stably expressing the human recombinant ClC-2 Cl(-) channel was used in patch-clamp studies to study its regulation. The relative permeability P(x)/P(Cl) calculated from reversal potentials was I(-) > Cl(-) = NO(3)(-) = SCN(-)>/=Br(-). The absolute permeability calculated from conductance ratios was Cl(-) = Br(-) = NO(3)(-) >/= SCN(-) > I(-). The channel was activated by cAMP-dependent protein kinase (PKA), reduced extracellular pH, oleic acid (C:18 cisDelta9), elaidic acid (C:18 transDelta9), arachidonic acid (AA; C:20 cisDelta5,8,11,14), and by inhibitors of AA metabolism, 5,8,11,14-eicosatetraynoic acid (ETYA; C:20 transDelta5,8,11,14), alpha-methyl-4-(2-methylpropyl)benzeneacetic acid (ibuprofen), and 2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2 Cl(-) channels were activated by a combination of forskolin plus IBMX and were inhibited by the cell-permeant myristoylated PKA inhibitor (mPKI). Channel activation by reduction of bath pH was increased by PKA and prevented by mPKI. AA activation of the ClC-2 Cl(-) channel was not inhibited by mPKI or staurosporine and was therefore independent of PKA or protein kinase C activation.

Asunto(s)

Ácido Araquidónico/fisiología , Canales de Cloruro/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Ácido 5,8,11,14-Eicosatetrainoico/farmacología , Antiinflamatorios no Esteroideos/farmacología , Ácido Araquidónico/farmacología , Azoles/farmacología , Canales de Cloruro CLC-2 , Línea Celular , Canales de Cloruro/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Conductividad Eléctrica , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Ácidos Grasos no Esterificados/farmacología , Humanos , Concentración de Iones de Hidrógeno , Ibuprofeno/farmacología , Iones , Isoindoles , Nitroprusiato/farmacología , Compuestos de Organoselenio/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo

Asunto(s)

Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cloruros/metabolismo , Sordera/congénito , Sordera/metabolismo , Formiatos/metabolismo , Proteínas de Transporte de Membrana , Animales , Expresión Génica , Humanos , Oocitos/fisiología , Transportadores de Sulfato , Xenopus laevis

Asunto(s)

Mama/metabolismo , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Clonación Molecular , Pulmón/metabolismo , Tráquea/metabolismo , Canales de Cloruro/química , Femenino , Humanos , Isoformas de Proteínas/metabolismo

RESUMEN

Rabbit and human ClC-2G Cl- channels are voltage sensitive and activated by protein kinase A and low extracellular pH. The objective of the present study was to investigate the mechanism involved in acid activation of the ClC-2G Cl- channel and to determine which amino acid residues play a role in this acid activation. Channel open probability (Po) at +/-80 mV holding potentials increased fourfold in a concentration-dependent manner with extracellular H+ concentration (that is, extracellular pH, pHtrans), with an apparent acidic dissociation constant of pH 4.95 +/- 0.27. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide-catalyzed amidation of the channel with glycine methyl ester increased Po threefold at pHtrans 7.4, at which the channel normally exhibits low Po. With extracellular pH reduction (protonation) or amidation, increased Po was due to a significant increase in open time constants and a significant decrease in closed time constants of the channel gating, and this effect was insensitive to applied voltage. With the use of site-directed mutagenesis, the extracellular region EELE (amino acids 416-419) was identified as the pH sensor and amino acid Glu-419 was found to play the key or predominant role in activation of the ClC-2G Cl- channel by extracellular acid.

Asunto(s)

Canales de Cloruro/fisiología , Concentración de Iones de Hidrógeno , Animales , Canales de Cloruro CLC-2 , Carbodiimidas/farmacología , Canales de Cloruro/efectos de los fármacos , Canales de Cloruro/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ácido Egtácico/farmacología , Femenino , Humanos , Activación del Canal Iónico/fisiología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Oocitos/fisiología , Técnicas de Placa-Clamp , ARN Complementario , Conejos , Xenopus laevis

RESUMEN

A ClC-2G(2 alpha) Cl- channel was identified to be present in human lung and stomach, and a partial cDNA for this Cl- channel was cloned from a human fetal lung library. A full-length expressible human ClC-2G(2 alpha) cDNA was constructed by ligation of mutagenized expressible rabbit ClC-2G(2 alpha) cDNA with the human lung ClC-2G(2 alpha) cDNA, expressed in oocytes, and characterized at the single-channel level. Adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) treatment increased the probability of opening of the channel (Po). After PKA activation, the channel exhibited a linear (r = 0.99) current-voltage curve with a slope conductance of 22.1 +/- 0.8 pS in symmetric 800 mM tetraethylammonium chloride (TEACl; pH 7.4). Under fivefold gradient conditions of TEACl, a reversal potential of +21.5 +/- 2.8 mV was measured demonstrating anion-to-cation discrimination. As previously demonstrated for the rabbit ClC-2G(2 alpha) Cl- channel, the human analog, hClC-2G(2 alpha), was active at pH 7.4 as well as when the pH of the extracellular face of the channel (trans side of the bilayer; pHtrans) was asymmetrically reduced to pH 3.0. The extent of PKA activation was dependent on pHtrans. With PKA treatment, Po increased fourfold with a pHtrans of 7.4 and eightfold with a pHtrans of 3.0. Effects of sequential PKA addition followed by pHtrans reduction on the same channel suggested that the PKA- and pH-dependent increases in channel Po were separable and cumulative. Northern analysis showed ClC-2G(2 alpha) mRNA to be present in human adult and fetal lung and adult stomach, and quantitative reverse transcriptase-polymerase chain reaction showed this channel to be present in the adult human lung and stomach at about one-half the level found in fetal lung. The findings of the present study suggest that the ClC-2G(2 alpha) Cl- channel may play an important role in Cl- transport in the fetal and adult human lung.

Asunto(s)

Quimera , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Hidrógeno/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Línea Celular , ADN Complementario/genética , Femenino , Feto , Mucosa Gástrica/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Pulmón/embriología , Pulmón/metabolismo , Datos de Secuencia Molecular , Oocitos/metabolismo , Conejos , Estómago/citología , Xenopus laevis

RESUMEN

Recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) has been produced in a Saccharomyces cerevisiae expression system used previously to produce transport ATPases with high yields. The arrangement of the bases in the region immediately upstream from the ATG start codon of the CFTR is extremely important for high expression levels. The maximal CFTR expression level is about 5-10% of that in Sf9 insect cells as judged by comparison of immunoblots. Upon sucrose gradient centrifugation, the majority of the CFTR is found in a light vesicle fraction separated from the yeast plasma membrane in a heavier fraction. It thus appears that most of expressed CFTR is not directed to the plasma membrane in this system. CFTR expressed in yeast has the same mobility (ca. 140 kDa) as recombinant CFTR produced in Sf9 cells in a high resolution SDS-PAGE gel before and after N-glycosidase F treatment, suggesting that it is not glycosylated. The channel function of the expressed CFTR was measured by an isotope flux assay in isolated yeast membrane vesicles and single channel recording following reconstitution into planar lipid bilayers. In the isotope flux assay, protein kinase A (PKA) increased the rate of 125I- uptake by about 30% in membrane vesicles containing the CFTR, but not in control membranes. The single channel recordings showed that a PKA-activated small conductance anion channel (8 pS) with a linear I-V relationship was present in the CFTR membranes, but not in control membranes. These results show that the human CFTR has been expressed in functional form in yeast. With the reasonably high yield and the ability to grow massive quantities of yeast at low cost, this CFTR expression system may provide a valuable new source of starting material for purification of large quantities of the CFTR for biochemical studies.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Expresión Génica , Saccharomyces cerevisiae/genética , Animales , Membrana Celular/metabolismo , Centrifugación por Gradiente de Densidad , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Humanos , Mutagénesis Sitio-Dirigida , Plásmidos , Proteínas Recombinantes , Saccharomyces cerevisiae/ultraestructura , Spodoptera/metabolismo

RESUMEN

5,10,15,20-Tetraphenyl-21H,23H-porphine manganese (III) chloride [TPPMn(III)] is a positively charged lipophilic anion carrier that is widely used as a Cl- sensor. TPPMn(III) increased anion permeability of cultured mouse lung epithelial (MLE) cells as measured by short-circuit current (ISC) to a level similar to that induced by forskolin analogues. Anion permeability was also studied in cultured human lung epithelial (A549) cells by measurement of the rates of change of fluorescence of the anion sensitive fluorescent dye, 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). In these studies, cells were incubated with SPQ in SO2-4- medium, washed free of extracellular SPQ, and then perfused with medium containing anions that are known to quench SPQ fluorescence. The effect of TPPMn(III) on anion transport was then determined either microscopically in single cell studies or using cell monolayers mounted in a front face fluorimeter. TPPMn(III) in the range from 1 to 100 micrograms/ml induced a dose-dependent increase in Br- transport. The half-maximal quenching effect was estimated to be approximate 5 micrograms/ml. TPPMn(III) increased the rates of fluorescence quench of anions by up to fourfold. TPPMn(III) was without effect on -Ca2+-i level in A549 cells as measured with fura 2-AM. This indicates that TPPMn(III) effects were not mediated through effects on Ca+2 -activated Cl- channels, or by compromise of energy metabolism or membrane integrity of the cells. This study suggests that TPPMn(III) and, by extension, other lipophilic Mn(III) or Co(III) derivatives wherein the selectivity of lipophilicity is altered, could increase the anion permeability of biological membranes, and suggests a new approach for treatment of diseases such as cystic fibrosis, where transport of Cl- is defective.

Asunto(s)

Permeabilidad de la Membrana Celular/efectos de los fármacos , Ionóforos/farmacología , Pulmón/fisiología , Metaloporfirinas/farmacología , Animales , Bumetanida/farmacología , Línea Celular , Colforsina/análogos & derivados , Colforsina/farmacología , Relación Dosis-Respuesta a Droga , Electrofisiología , Epitelio/efectos de los fármacos , Epitelio/fisiología , Colorantes Fluorescentes , Humanos , Ionomicina/farmacología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Ratones , Valinomicina/farmacología

RESUMEN

In cardiomyocytes glucose transport is activated not only by insulin but also by contractile activity that causes translocation of the glucose transporter, GLUT-4, from intracellular vesicles to the plasma membrane. The latter effect may possibly be mediated by intracellular Ca2+, as suggested by previous studies. To investigate the role of Ca2+, we permeabilized neonatal rat myocytes with alpha-toxin and incubated them for 1 h either at a pCa (i.e.--log10 [Ca2+]) of 8 (control) or at a pCa of 5 in the presence of adenosine 5'-triphosphate (ATP). Translocation of GLUT-4 was then monitored by a novel immunoprecipitation method using a peptide antibody directed against an exofacial (extracellular) loop of GLUT-4 (residues 58-80). Incorporation of GLUT-4 into the plasmalemma was stimulated 1.8-fold by 10 microM Ca2+ and 1.7-fold by insulin (as in the case of intact cells). The insulin effect was Ca2+ independent, i.e. it was identical in the absence and presence of Ca2+ (10 microM). Guanosine 5'-O-(3-thio-triphosphate) (GTP[gamma S]), which was inactive in intact cells, also caused translocation of GLUT-4 in permeabilized cardiomyocytes. Thus, incorporation of GLUT-4 into the plasma membrane was enhanced 2.5-fold by 200 microM GTP[gamma S] in the virtual absence of Ca2+ (pCa 8) and even 3.5-fold at 10 microM free Ca2+. We conclude that an increase in intracellular Ca2+ concentration increases GLUT-4 translocation of (permeabilized) cardiomyocytes to a similar extent as do insulin and GTP[gamma S] in the absence of Ca2+, but that the effects of Ca2+ and GTP[gamma S] may be additive.

Asunto(s)

Calcio/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Miocardio/metabolismo , Animales , Western Blotting , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Transportador de Glucosa de Tipo 4 , Corazón/efectos de los fármacos , Miocardio/citología , Pruebas de Precipitina , Ratas , Transducción de Señal/efectos de los fármacos

RESUMEN

cDNA encoding a Cl- channel was isolated from a rabbit gastric library, sequenced, and expressed in Xenopus oocytes. The predicted protein (898 amino acids, relative molecular mass 98,433 Da) was overall 93% similar to the rat brain ClC-2 Cl- channel. However, a 151-amino acid stretch toward the COOH-terminus was 74% similar to ClC-2 with six amino acids deleted. Two new potential protein kinase A (PKA) phosphorylation sites (also protein kinase C phosphorylation sites) were introduced. cRNA-injected Xenopus oocytes expressed a Cl- channel that was active at pHtrans 3 and had a linear current-voltage (I-V) curve and a slope conductance of 29 +/- 1 pS at 800 mM CsCl. A fivefold Cl- gradient caused a rightward shift in the I-V curve with a reversal potential of +30 +/- 3 mV, indicating anion selectivity. The selectivity was I- > Cl- > NO3-. The native and recombinant Cl- channel were both activated in vitro by PKA catalytic subunit and ATP. The electrophysiological and regulatory properties of the cloned and the native channel were similar. The cloned protein may be the Cl- channel involved in gastric HCl secretion.

Asunto(s)

Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Clonación Molecular , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Mucosa Gástrica/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Catálisis , Canales de Cloruro/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Oocitos/metabolismo , ARN Mensajero/metabolismo , Conejos , Ratas , Proteínas Recombinantes , Xenopus laevis

RESUMEN

Murine lung epithelial (MLE) cell lines were produced from lung tumors derived from transgenic mice bearing the viral oncogene, SV40 large T antigen, under transcriptional control of the promoter-enhancer region of the human surfactant protein C (SP-C) gene. Cells were selected on the basis of increased murine cystic fibrosis transmembrane conductance regulator (mCFTR) mRNA content and were dilution cloned to produce distinct immortalized epithelial cell lines. MLE-13a3 cell lines expressing high levels of mCFTR mRNA also expressed apolipoprotein J (apoJ) mRNA, a developmentally regulated glycoprotein expressed preferentially in fetal lung. SP-A, -B, and -C were not detected or were present at low levels in the MLE cells that contained abundant CFTR and apoJ mRNA. In contrast, MLE cells, cloned on the basis of abundant surfactant protein mRNAs, expressed apoJ and mCFTR mRNAs at low levels. Forskolin-stimulated short-circuit current, typical of CFTR-mediated chloride transport activity, was generated by monolayers of subclones of the MLE-13a3 cell lines. Tumor necrosis factor-alpha stimulated mCFTR mRNA, whereas dexamethasone, retinoic acid, and phorbol ester had no effect on the levels of mCFTR mRNA in MLE-13a3 cells.

Asunto(s)

Antígenos Transformadores de Poliomavirus/análisis , Transformación Celular Viral , Pulmón/patología , Ratones Transgénicos/inmunología , Chaperonas Moleculares , Animales , Separación Celular , Clusterina , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Epitelio/metabolismo , Epitelio/patología , Glicoproteínas/genética , Pulmón/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos/genética , ARN Mensajero/metabolismo , Células Tumorales Cultivadas

RESUMEN

Cystic fibrosis (CF) is a common, fatal recessive disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene manifested by abnormalities in the regulation of chloride ion (Cl-) secretion across the apical membrane of epithelial cells throughout the body. Adenovirus-mediated delivery of the normal CFTR cDNA and correction of the CF epithelial cell Cl- secretory phenotype suggests the feasibility of gene therapy for CF lung disease. Few studies, however, have focused on the evaluation of the safety of the adenovirus-mediated gene transfer approach. This study presents in vitro data on the efficacy and safety of adenovirus-mediated transfer of the human CFTR cDNA using Av1Cf2. Av1Cf2-mediated transfer of the human CFTR cDNA complemented the abnormal cAMP-regulated Cl- permeability of cells with the CF epithelial phenotype. Av1 vectors did not replicate infectious virus in HeLa cells infected in vitro, although trace vector DNA synthesis was observed at very high multiplicity of infection. Expression of the adenoviral late gene for the hexon capsid protein was observed at trace levels in Av1 vector-infected HeLa cells, but not in freshly isolated human bronchial epithelial cells, consistent with the pattern of DNA synthesis observed in these different target cells. Although, these observations support the efficacy and safety of use of Av1Cf2 for treatment of the fatal pulmonary component of CF.

Asunto(s)

Adenovirus Humanos/genética , Fibrosis Quística/terapia , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos , Línea Celular , ADN Recombinante/genética , Células Epiteliales , Eliminación de Gen , Terapia Genética/efectos adversos , Humanos , Proteínas de la Membrana/genética , Fenotipo

RESUMEN

Cystic fibrosis transmembrane conductance regulator (CFTR) is present in acidic intracellular vesicles. Human normal and delta F508 CFTR Cl- channel characteristics at pH 7.4 and pH 4.5 were determined by fusing Xenopus laevis oocyte plasma membranes containing the expressed channels to planar lipid bilayers. At pH 7.4, both channels exhibited linear current-voltage curves, a 10 +/- 0.3-pS conductance using 800 mM CsCl, and a 9:1 Cl-/Cs+ discrimination ratio obtained from a 32 +/- 2 mV reversal potential with a fivefold gradient. At -80 mV, the open probability (Po) of mutant CFTR was 53% that of normal CFTR. Reduction of the trans-pH from 7.4 to 4.5 had no effect on the above characteristics except for Po, where it caused a 47% reduction in normal CFTR Po (due to a 75% decrease in mean open time) and a 75% reduction in delta F508 CFTR Po (due to a 6-fold increase in mean closed time). Normal CFTR can thus function in the environment of acidic intracellular organelles, whereas activity of mutant CFTR would be greatly reduced. These results may be of significance to understanding the cystic fibrosis defect.

Asunto(s)

Ácidos/farmacología , Canales de Cloruro/efectos de los fármacos , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/genética , Animales , Canales de Cloruro/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Electrofisiología , Eliminación de Gen , Humanos , Concentración de Iones de Hidrógeno , Membrana Dobles de Lípidos , Proteínas de la Membrana/fisiología , Mutación , Oocitos , Xenopus laevis

RESUMEN

HCl secretion across mammalian gastric parietal cell apical membrane may involve Cl- channels. H(+)-K(+)-ATPase-containing membranes isolated from gastric mucosa of histamine-stimulated rabbits were fused to planar lipid bilayers. Channels were recorded with symmetric 800 mM CsCl solutions, pH 7.4. A linear current-voltage (I-V) relationship was obtained, and conductance was 28 +/- 1 pS at 800 mM CsCl. Conductance was 6.9 +/- 2 pS at 150 mM CsCl. Reversal potential was +22 mV with a fivefold cis-trans CsCl concentration gradient, indicating that the channel was anion selective with a discrimination ratio of 6:1 for Cl- over Cs+. Anion selectivity of the channel was I- > Cl- > or = Br- > NO3-, and gluconate was impermeant. Channels obtained at pH 7.4 persisted when pH of medium bathing the trans side of the bilayer (pHtrans) was reduced to pH 3, without a change in conductance, linearity of I-V relationship, or ion selectivity. In contrast, asymmetric reduction of pH of medium bathing the cis side of the bilayer from 7.4 to 3 always resulted in loss of channel activity. At pH 7.4, open probability (Po) of the channel was voltage dependent, i.e., predominantly open at +80 mV but mainly closed at -80 mV. In contrast, with low pHtrans, channel Po at -80 mV was increased 3.5-fold. The Cl- channel was Ca2+ indifferent. In absence of ionophores, ion selectivity for support of H(+)-K(+)-ATPase activity and H+ transport was consistent with that exhibited by the channel and could be limited by substitution with NO3-, whereas maximal H(+)-K(+)-ATPase activity was indifferent to anion present, demonstrating that anion transport can be rate limiting. Cl- channels with similar characteristics (conductance, linear I-V relationship, and ion selectivity) were also present in H(+)-K(+)-ATPase-containing vesicles isolated from resting (cimetidine-treated) gastric mucosa, exhibiting at -80 mV a pH-independent approximately 3.5-fold lower Po than stimulated vesicle channels. At -80 mV, reduction of pHtrans increased Po of both resting and stimulated Cl- channels by five- to sixfold. Changing membrane potential from 0 to -80 mV across stimulated vesicles increased Cl- channel activity an additional 10-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

Asunto(s)

Proteínas de la Membrana/metabolismo , Células Parietales Gástricas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Aniones/metabolismo , Aniones/farmacología , Transporte Biológico/efectos de los fármacos , Calcio/farmacología , Canales de Cloruro , Electrofisiología , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Proteínas de la Membrana/fisiología , Descanso

RESUMEN

The bee venom polypeptide melittin binds to and inhibits the gastric hydrogen-potassium-adenosinetriphosphatase (H(+)-K(+)-ATPase). A search for parietal cell proteins with a melittin-like structure was carried out. A 67-kDa (doublet) protein, which reacted with a polyclonal antimelittin antibody, was found in purified rabbit parietal cells. The protein exhibited reversible stimulus-dependent redistribution from cytosol to (total) membranes. It was also found to be associated with H(+)-K(+)-ATPase-containing membranes when isolated from the gastric mucosae of rabbits treated with histamine, but not with cimetidine. The presence of the protein correlated with the ability of the membrane preparations to exhibit ionophore-independent HCl accumulation, a characteristic of gastric membranes from histamine-stimulated animals. The 67-kDa melittin-like protein may play a role in the functional changes in the gastric parietal cell that are involved in stimulation of HCl secretion.

Asunto(s)

Anticuerpos , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Meliteno/inmunología , Proteínas de la Membrana/metabolismo , Células Parietales Gástricas/metabolismo , Proteínas/metabolismo , Aminopirina/metabolismo , Animales , Membrana Celular/metabolismo , Cimetidina/farmacología , Citosol/metabolismo , Electroforesis en Gel de Poliacrilamida , ATPasa Intercambiadora de Hidrógeno-Potásio/aislamiento & purificación , Histidina/farmacología , Immunoblotting , Técnicas In Vitro , Cinética , Proteínas de la Membrana/aislamiento & purificación , Peso Molecular , Células Parietales Gástricas/efectos de los fármacos , Proteínas/aislamiento & purificación , Conejos , Factores de Tiempo

Asunto(s)

Mucosa Gástrica/enzimología , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Meliteno/química , Meliteno/farmacología , Estructura Secundaria de Proteína , Venenos de Avispas/química , Venenos de Avispas/farmacología , Animales , Péptidos y Proteínas de Señalización Intercelular , Sustancias Macromoleculares , Péptidos , Inhibidores de la Bomba de Protones , Porcinos

RESUMEN

The 26 amino acid bee venom toxin, melittin, is an amphipathic helical polypeptide which inhibits the gastric (H+ + K+)ATPase. The site of interaction with the (H+ + K+)ATPase was shown to be the alpha subunit of the (H+ + K+)ATPase in studies using [125I]azidosalicylyl melittin, a radioactive photoaffinity analog of melittin. A synthetic amphipathic polypeptide (Trp3) containing tryptophan, which exhibits a structure similar to that of melittin, also inhibited the gastric (H+ + K+)ATPase, and prevented labeling by [125I]azidosalicylyl melittin. These findings suggested that melittin and the synthetic amphipathic helical polypeptide were bound to the same or overlapping site(s). In the present studies, novel tritiated photoaffinity analogs of Trp3 containing benzoylphenylalanine (in place of tryptophan) were used to photoaffinity label the (H+ + K+)ATPase. These studies help to establish that the (H+ + K+)ATPase contains a binding site for polypeptides which exhibit an amphipathic helical motif. The precise amino acid sequence of the polypeptide appears to be of secondary importance for interaction with the (H+ + K+)ATPase as long as the alpha helical motif is present. The benzoylphenylalanine containing polypeptides are ideal for mapping the binding site on the (H+ + K+)ATPase. Using an antibody which recognizes this amphipathic helical ('melittin-like') motif, we have demonstrated that the gastric parietal cell contains a 67 kDa 'melittin-like' protein. This protein was associated with the gastric parietal cell apical membrane in the stimulated (secreting) state, but not in the resting (non-secreting) state. The binding site for the gastric 'melittin-like' protein appears to overlap with the melittin binding site on the alpha subunit of the (H+ + K+)ATPase. The potential physiological significance of the melittin binding site and the overlapping binding site for this newly identified endogenous 'melittin-like' protein on the (H+ + K+)ATPase to regulated HCl secretion by the parietal cell is presently under investigation.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

ATPasa Intercambiadora de Hidrógeno-Potásio/química , Meliteno/química , Estómago/enzimología , Marcadores de Afinidad , Secuencia de Aminoácidos , Animales , Sitios de Unión , Transporte Biológico Activo , Datos de Secuencia Molecular , Quinasa de Cadena Ligera de Miosina/química , Péptidos/química , Solubilidad , Relación Estructura-Actividad , Porcinos