RESUMEN
Glioblastoma (GBM) is a primary brain cancer that contains populations of stem-like cancer cells (GSCs) that home to specialized perivascular niches. GSC interactions with their niche influence self-renewal, differentiation and drug resistance, although the pathways underlying these events remain largely unknown. Here, we report that the integrin αvß8 and its latent transforming growth factor ß1 (TGFß1) protein ligand have central roles in promoting niche co-option and GBM initiation. αvß8 integrin is highly expressed in GSCs and is essential for self-renewal and lineage commitment in vitro. Fractionation of ß8high cells from freshly resected human GBM samples also reveals a requirement for this integrin in tumorigenesis in vivo. Whole-transcriptome sequencing reveals that αvß8 integrin regulates tumor development, in part, by driving TGFß1-induced DNA replication and mitotic checkpoint progression. Collectively, these data identify the αvß8 integrin-TGFß1 signaling axis as crucial for exploitation of the perivascular niche and identify potential therapeutic targets for inhibiting tumor growth and progression in patients with GBM.
Asunto(s)
Neoplasias Encefálicas/patología , Carcinogénesis/metabolismo , Glioblastoma/patología , Integrinas/metabolismo , Células Madre Neoplásicas/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Antígeno AC133/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Autorrenovación de las Células , Progresión de la Enfermedad , Humanos , Cadenas beta de Integrinas/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Ratones , Ratones SCID , Cultivo Primario de Células , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Sphingomyelinase C (SMase) inhibits CFTR chloride channel activity in multiple cell systems, an effect that could exacerbate disease in CF and COPD patients. The mechanism by which sphingomyelin catalysis inhibits CFTR is not known but evidence suggests that it occurs independently of CFTR's regulatory "R" domain. In this study we utilized the Xenopus oocyte expression system to shed light on how CFTR channel activity is reduced by SMase. We found that the pathway leading to inhibition is not membrane delimited and that inhibited CFTR channels remain at the cell membrane, indicative of a novel silencing mechanism. Consistent with an effect on CFTR gating behavior, we found that altering gating kinetics influenced the sensitivity to inhibition by SMase. Specifically, increasing channel activity by introducing the mutation K1250A or pretreating with the CFTR potentiator VX-770 (Ivacaftor) imparted resistance to inhibition. In primary bronchial epithelial cells, we found that basolateral, but not apical, application of SMase leads to a redistribution of sphingomyelin and a reduction in forskolin- and VX-770-stimulated currents. Taken together, these data suggest that SMase inhibits CFTR channel function by locking channels into a closed state and that endogenous CFTR in HBEs is affected by SMase activity.
Asunto(s)
Proteínas Bacterianas/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/farmacología , Animales , Proteínas Bacterianas/química , Catálisis , Membrana Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Activación Enzimática , Humanos , Potenciales de la Membrana , Modelos Moleculares , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Unión Proteica , Conformación Proteica , Esfingomielina Fosfodiesterasa/química , Staphylococcus aureus/enzimologíaRESUMEN
Reduced Paired box 5 (PAX5) levels have important roles in the pathogenesis of human B-cell acute lymphoblastic leukemia. However, the role of PAX5 in human lymphoma remains unclear. We generated PAX5-silenced cells using mantle cell lymphoma (MCL) as a model system. These PAX5(-) MCL cells exhibited unexpected phenotypes, including increased proliferation in vitro, enhanced tumor infiltration in vivo, robust adhesion to the bone marrow stromal cells and increased retention of quiescent stem-like cells. These phenotypes were attributed to alterations in the expression of genes including p53 and Rb, and to phosphoinositide 3-kinase/mammalian target of rapamycin and phosphorylated signal transducer and activator of transcription 3 pathway hyperactivation. On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6. Moreover, decreased PAX5 levels in CD19+ MCL cells correlated with their increased infiltration and progression; thus, PAX5 levels can be used as a prognostic marker independent of cyclin D1 in advanced MCL patients. Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells. Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.
Asunto(s)
Linfocitos B/metabolismo , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Linfoma de Células del Manto/genética , Factor de Transcripción PAX5/genética , Animales , Antineoplásicos/farmacología , Linfocitos B/patología , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Linfoma de Células del Manto/diagnóstico , Linfoma de Células del Manto/tratamiento farmacológico , Linfoma de Células del Manto/mortalidad , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Ratones , Factor de Transcripción PAX5/antagonistas & inhibidores , Factor de Transcripción PAX5/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Pronóstico , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Sweat Na(+) concentration ([Na(+)]) varies greatly among individuals and is particularly high in cystic fibrosis (CF). The purpose of this study was to determine whether excess sweat [Na(+)] differentially impacts thirst drive and other physiological responses during progressive dehydration via exercise in the heat. Healthy subjects with high-sweat [Na(+)] (SS) (91.0 ± 17.3 mmol/l), Controls with average sweat [Na(+)] (43.7 ± 9.9 mmol/l), and physically active CF patients with very high sweat [Na(+)] (132.6 ± 6.4 mmol/l) cycled in the heat without drinking until 3% dehydration. Serum osmolality increased less (P < 0.05) in CF (6.1 ± 4.3 mosmol/kgH(2)O) and SS (8.4 ± 3.0 mosmol/kgH(2)O) compared with Control (14.8 ± 3.5 mosmol/kgH(2)O). Relative change in plasma volume was greater (P < 0.05) in CF (-19.3 ± 4.5%) and SS (-18.8 ± 3.1%) compared with Control (-14.3 ± 2.3%). Thirst during exercise and changes in plasma levels of vasopressin, angiotensin II, and aldosterone relative to percent dehydration were not different among groups. However, ad libitum fluid replacement was 40% less, and serum NaCl concentration was lower for CF compared with SS and Control during recovery. Despite large variability in sweat electrolyte loss, thirst appears to be appropriately maintained during exercise in the heat as a linear function of dehydration, with relative contributions from hyperosmotic and hypovolemic stimuli dependent upon the magnitude of salt lost in sweat. CF exhibit lower ad libitum fluid restoration following dehydration, which may reflect physiological cues directed at preservation of salt balance over volume restoration.
Asunto(s)
Fibrosis Quística/metabolismo , Ejercicio Físico/fisiología , Calor , Sodio/metabolismo , Sudor/metabolismo , Sed/fisiología , Adulto , Aldosterona/sangre , Angiotensina II/sangre , Peso Corporal/fisiología , Estudios de Casos y Controles , Fibrosis Quística/fisiopatología , Deshidratación/fisiopatología , Femenino , Humanos , Masculino , Concentración Osmolar , Volumen Plasmático/fisiología , Vasopresinas/sangreRESUMEN
ClC chloride channels play essential roles in membrane excitability and maintenance of osmotic balance. Despite the recent crystallization of two bacterial ClC-like proteins, the gating mechanism for these channels remains unclear. In this study we tested scorpion venom for the presence of novel peptide inhibitors of ClC channels, which might be useful tools for dissecting the mechanisms underlying ClC channel gating. Recently, it has been shown that a peptide component of venom from the scorpion L. quinquestriatus hebraeus inhibits the CFTR chloride channel from the intracellular side. Using two-electrode voltage clamp we studied the effect of scorpion venom on ClC-0, -1, and -2, and found both dose- and voltage-dependent inhibition only of ClC-2. Comparison of voltage-dependence of inhibition by venom to that of known pore blockers revealed opposite voltage dependencies, suggesting different mechanisms of inhibition. Kinetic data show that venom induced slower activation kinetics compared to pre-venom records, suggesting that the active component(s) of venom may function as a gating modifier at ClC-2. Trypsinization abolished the inhibitory activity of venom, suggesting that the component(s) of scorpion venom that inhibits ClC-2 is a peptide.
Asunto(s)
Canales de Cloruro/antagonistas & inhibidores , Péptidos/fisiología , Venenos de Escorpión/farmacología , Animales , Canales de Cloruro CLC-2 , Células Cultivadas , Canales de Cloruro/biosíntesis , Canales de Cloruro/genética , Canales de Cloruro/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Oocitos/metabolismo , Técnicas de Placa-Clamp , Péptidos/química , Conejos , Venenos de Escorpión/química , Escorpiones/química , Escorpiones/fisiología , XenopusRESUMEN
The objective of the present study was to clarify the mechanism by which the sulfonylurea drug, glibenclamide, inhibits single CFTR channels in excised patches from Xenopus oocytes. Glibenclamide blocks the open pore of the channel via binding at multiple sites with varying kinetics. In the absence of glibenclamide, open-channel bursts exhibited a flickery intraburst closed state (C1); this is due to block of the pore by the pH buffer, TES. Application of 25 microM glibenclamide to the cytoplasmic solution resulted in the appearance of two drug-induced intraburst closed states (C2, C3) of widely different duration, which differed in pH-dependence. The kinetics of interaction with the C3 state, but not the C2 state, were strongly voltage-dependent. The durations of both the C2 and C3 states were concentration-dependent, indicating a non-linear reaction scheme. Application of drug also increased the burst duration, which is consistent with an open-channel blocking mechanism. A kinetic model is proposed. These results indicate that glibenclamide interacts with open CFTR channels in a complex manner, involving interactions with multiple binding sites in the channel pore.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Gliburida/farmacología , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Oocitos/fisiología , Xenopus/fisiología , Animales , Femenino , Concentración de Iones de Hidrógeno , Activación del Canal Iónico/efectos de los fármacos , Oocitos/efectos de los fármacos , Técnicas de Placa-ClampRESUMEN
Blockade of the CFTR chloride channel by glibenclamide was studied in Xenopus oocytes using two-electrode voltage-clamp recordings, macropatch recordings, and summations of single-channel currents, in order to test a kinetic model recently developed by us from single-channel experiments. Both the forward and reverse macroscopic reactions, at negative and positive membrane potential V(M), respectively, were slow in comparison to those reactions for other CFTR pore blockers such as DPC and NPPB, resulting in prominent relaxations on the order of tens of milliseconds. The rate of the reverse reaction was voltage-dependent, and dependent on the Cl(-) driving force, while that of the forward reaction was not. In inside-out macropatches, block and relief from block occurred in two distinct phases that differed in apparent affinity. The results are consistent with the presence of multiple glibenclamide binding sites in CFTR, with varying affinity and voltage dependence; they support the kinetic model and suggest experimental approaches for identification of those sites by mutagenesis.
Asunto(s)
Antiarrítmicos/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Gliburida/farmacología , Animales , Antiarrítmicos/metabolismo , Sitios de Unión , Gliburida/metabolismo , Cinética , Potenciales de la Membrana/efectos de los fármacos , Oocitos , Técnicas de Placa-Clamp , Unión Proteica , XenopusRESUMEN
The goal of the experiments described here was to explore the possible role of fixed charges in determining the conduction properties of CFTR. We focused on transmembrane segment 6 (TM6) which contains four basic residues (R334, K335, R347, and R352) that would be predicted, on the basis of their positions in the primary structure, to span TM6 from near the extracellular (R334, K335) to near the intracellular (R347, R352) end. Cysteines substituted at positions 334 and 335 were readily accessible to thiol reagents, whereas those at positions 347 and 352 were either not accessible or lacked significant functional consequences when modified. The charge at positions 334 and 335 was an important determinant of CFTR channel function. Charge changes at position 334--brought about by covalent modification of engineered cysteine residues, pH titration of cysteine and histidine residues, and amino acid substitution--produced similar effects on macroscopic conductance and the shape of the I-V plot. The effect of charge changes at position 334 on conduction properties could be described by electrodiffusion or rate-theory models in which the charge on this residue lies in an external vestibule of the pore where it functions to increase the concentration of Cl adjacent to the rate-limiting portion of the conduction path. Covalent modification of R334C CFTR increased single-channel conductance determined in detached patches, but did not alter open probability. The results are consistent with the hypothesis that in wild-type CFTR, R334 occupies a position where its charge can influence the distribution of anions near the mouth of the pore.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Metanosulfonato de Etilo/análogos & derivados , Animales , Aniones/metabolismo , Arginina/genética , Cisteína/genética , Cisteína/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Disulfuros/metabolismo , Conductividad Eléctrica , Metanosulfonato de Etilo/farmacología , Femenino , Humanos , Concentración de Iones de Hidrógeno , Lisina/genética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Mercaptoetanol/farmacología , Mesilatos/farmacología , Modelos Biológicos , Oocitos/fisiología , Técnicas de Placa-Clamp , Perfusión , XenopusRESUMEN
The variety of methods used to identify the structural determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator Cl(-) channel has made it difficult to assemble the data into a coherent framework that describes the three-dimensional structure of the pore. Here, we compare the relative importance of sites previously studied and identify new sites that contribute strongly to anion selectivity. We studied Cl(-) and substitute anions in oocytes expressing wild-type cystic fibrosis transmembrane conductance regulator or 12-pore-domain mutants to determine relative permeability and relative conductance for 9 monovalent anions and 1 divalent anion. The data indicate that the region of strong discrimination resides between T338 and S341 in transmembrane 6, where mutations affected selectivity between Cl(-) and both large and small anions. Mutations further toward the extracellular end of the pore only strongly affected selectivity between Cl(-) and larger anions. Only mutations at S341 affected selectivity between monovalent and divalent anions. The data are consistent with a narrowing of the pore between the extracellular end and a constriction near the middle of the pore.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Activación del Canal Iónico/fisiología , Alanina/genética , Sustitución de Aminoácidos , Animales , Aniones/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Femenino , Mutagénesis/fisiología , Oocitos/fisiología , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína , Agua/química , XenopusRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel with distinctive kinetics. At the whole-cell level, CFTR currents in response to voltage steps are time independent for wild type and for the many mutants reported so far. Single channels open for periods lasting up to tens of seconds; the openings are interrupted by brief closures at hyperpolarized, but not depolarized, potentials. Here we report a serine-to-phenylalanine mutation (S1118F) in the 11th transmembrane domain that confers voltage-dependent, single-exponential current relaxations and moderate inward rectification of the macroscopic currents upon expression in Xenopus oocytes. At steady state, the S1118F-CFTR single-channel conductance rectifies, corresponding to the whole-cell rectification. In addition, the open-channel burst duration is decreased 10-fold compared with wild-type channels. S1118F-CFTR currents are blocked in a voltage-dependent manner by diphenylamine-2-carboxylate (DPC); the affinity of S1118F-CFTR for DPC is similar to that of the wild-type channel, but blockade exhibits moderately reduced voltage dependence. Selectivity of the channel to a range of anions is also affected by this mutation. Furthermore, the permeation properties change during the relaxations, which suggests that there is an interaction between gating and permeation in this mutant. The existence of a mutation that confers voltage dependence upon CFTR currents and that changes kinetics and permeation properties of the channel suggests a functional role for the 11th transmembrane domain in the pore in the wild-type channel.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Activación del Canal Iónico/fisiología , Estructura Terciaria de Proteína/genética , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Activación del Canal Iónico/genética , Magnesio/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Permeabilidad/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Xenopus , ortoaminobenzoatos/farmacologíaRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) protein forms a Cl(-) channel found in the plasma membranes of many epithelial cells, including those of the kidney, gut and conducting airways. Mutation of the gene encoding CFTR is the primary defect in cystic fibrosis, a disease that affects approximately 30 000 individuals in the United States alone. Alteration of CFTR function also plays an important role in the pathophysiology of secretory diarrhea and polycystic kidney disease. The basic mechanisms of permeation in this channel are not well understood. It is not known which portions of the protein contribute to forming the pore or which amino acid residues in those domains are involved in the biophysical processes of ion permeation. In this review, I will discuss (i) the present understanding of ion transport processes in the wild-type CFTR channel, (ii) the experimental approaches currently being applied to investigate the pore, and (iii) a proposed structure that takes into account the present data on mechanisms of ion selectivity in the CFTR channel and on blockade of the pore by open-channel blockers.
Asunto(s)
Permeabilidad de la Membrana Celular , Canales de Cloruro , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Secuencia de Aminoácidos , Animales , Canales de Cloruro/química , Canales de Cloruro/fisiología , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Humanos , Modelos Moleculares , Datos de Secuencia MolecularRESUMEN
Blockers of CFTR with well-characterized kinetics and mechanism of action will be useful as probes of pore structure. We have studied the mechanism of block of CFTR by the arylaminobenzoates NPPB and DPC. Block of macroscopic currents by NPPB and DPC exhibited similar voltage-dependence, suggestive of an overlapping binding region. Kinetic analysis of single-channel currents in the presence of NPPB indicate drug-induced closed time constants averaging 2.2 msec at -100 mV. The affinity for NPPB calculated from single-channel block, K(D) = 35 microm, exceeds that for other arylaminobenzoates studied thus far. These drugs do not affect the rate of activation of wild-type (WT) channels expressed in oocytes, consistent with a simple mechanism of block by pore occlusion, and appear to have a single binding site in the pore. Block by NPPB and DPC were affected by pore-domain mutations in different ways. In contrast to its effects on block by DPC, mutation T1134F-CFTR decreased the affinity and reduced the voltage-dependence for block by NPPB. We also show that the alteration of macroscopic block by NPPB and DPC upon changes in bath pH is due to both direct effects (i.e., alteration of voltage-dependence) and indirect effects (alteration of cytoplasmic drug loading). These results indicate that both NPPB and DPC block CFTR by entering the pore from the cytoplasmic side and that the structural requirements for binding are not the same, although the binding regions within the pore are similar. The two drugs may be useful as probes for overlapping regions in the pore.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Cloruro/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Nitrobenzoatos/farmacología , ortoaminobenzoatos/farmacología , Animales , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Electrofisiología , Femenino , Humanos , Concentración de Iones de Hidrógeno , XenopusRESUMEN
Patients with cystic fibrosis (CF) display defects in airway ion transport, but the influence of airway transport phenotype on improved prognosis is not known. We studied airway bioelectric properties in five CF patients with the rare A455E mutation that is associated with mild pulmonary disease. We also evaluated five patients possessing premature truncation mutations (G542X and R553X) for which an association with mild pulmonary disease has not been as well established. We found no evidence in vivo that a mild lung disease mutation in the CF transmembrane regulator gene (CFTR) led to correction or partial correction of: (1) unstimulated Cl- secretion; (2) beta-agonist-activated Cl- secretion; (3) basal sodium reabsorption; or (4) amiloride-sensitive airway sodium transport. Early phase therapeutic trials in CF, including human gene transfer trials, rely heavily on improvements in airway potential difference to identify promising interventions and an improved prognosis. Based on our findings in a naturally occurring group of CF patients with an improved pulmonary prognosis (A455E), one can argue that marked clinical benefit might be possible without any improvement whatsoever in airway bioelectric phenotype. Moreover, if genetic modifiers exist that influence the severity of a particular CFTR mutation (e.g., A455E), these may be independent of human airway Cl-secretion in vivo, since we detected minimal Cl--secretory responses in patients with A455E.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Transporte Iónico , Mutación , Mucosa Nasal/metabolismo , Adolescente , Agonistas Adrenérgicos beta/farmacología , Adulto , Amilorida/farmacología , Animales , Células COS/metabolismo , Niño , Cloruros/metabolismo , AMP Cíclico/farmacología , Fibrosis Quística/fisiopatología , Femenino , Genotipo , Humanos , Transporte Iónico/efectos de los fármacos , Isoproterenol/farmacología , Masculino , Potenciales de la Membrana , Mucosa Nasal/fisiopatología , Pronóstico , Sodio/metabolismoRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) is both a member of the ATP-binding cassette superfamily and a Cl(-)-selective ion channel. We investigated the permeation pathway of human CFTR with measurements on conduction and open-channel blockade by diphenylamine-2-carboxylic acid (DPC). We used site-directed mutagenesis and oocyte expression to locate residues in transmembrane domain (TM) 6 and TM 12 that contact DPC and control rectification and single-channel conductances. Thus, TM 12 and the previously investigated TM 6 line the CFTR pore. In each TM, residues in contact with DPC are separated by two turns of an alpha helix. The contributions of TM 6 and TM 12 to DPC block and Cl- permeation, however, are not equivalent. The resulting structural model for the conduction pathway may guide future studies of permeation in other Cl- channels and ATP-binding cassette transporters.
Asunto(s)
Canales Iónicos/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Secuencia de Aminoácidos , Animales , Bloqueadores de los Canales de Calcio/farmacología , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Conductividad Eléctrica , Espacio Extracelular/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Permeabilidad , Ratas , ortoaminobenzoatos/metabolismo , ortoaminobenzoatos/farmacologíaRESUMEN
The gene defective in cystic fibrosis encodes a Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is blocked by diphenylamine-2-carboxylate (DPC) when applied extracellularly at millimolar concentrations. We studied the block of CFTR expressed in Xenopus oocytes by DPC or by a closely related molecule, flufenamic acid (FFA). Block of whole-cell CFTR currents by bath-applied DPC or by FFA, both at 200 microM, requires several minutes to reach full effect. Blockade is voltage dependent, suggesting open-channel block: currents at positive potentials are not affected but currents at negative potentials are reduced. The binding site for both drugs senses approximately 40% of the electric field across the membrane, measured from the inside. In single-channel recordings from excised patches without blockers, the conductance was 8.0 +/- 0.4 pS in symmetric 150 mM Cl-. A subconductance state, measuring approximately 60% of the main conductance, was often observed. Bursts to the full open state lasting up to tens of seconds were uninterrupted at depolarizing membrane voltages. At hyperpolarizing voltages, bursts were interrupted by brief closures. Either DPC or FFA (50 microM) applied to the cytoplasmic or extracellular face of the channel led to an increase in flicker at Vm = -100 mV and not at Vm = +100 mV, in agreement with whole-cell experiments. DPC induced a higher frequency of flickers from the cytoplasmic side than the extracellular side. FFA produced longer closures than DPC; the FFA closed time was roughly equal (approximately 1.2 ms) at -100 mV with application from either side. In cell-attached patch recordings with DPC or FFA applied to the bath, there was flickery block at Vm = -100 mV, confirming that the drugs permeate through the membrane to reach the binding site. The data are consistent with the presence of a single binding site for both drugs, reached from either end of the channel. Open-channel block by DPC or FFA may offer tools for use with site-directed mutagenesis to describe the permeation pathway.
Asunto(s)
Cloruros/metabolismo , Fibrosis Quística/metabolismo , Ácido Flufenámico/farmacología , Proteínas de la Membrana/metabolismo , ortoaminobenzoatos/farmacología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Canales de Cloruro , AMP Cíclico/metabolismo , Fibrosis Quística/genética , Electrofisiología , Espacio Extracelular/metabolismo , Femenino , Indicadores y Reactivos , Cinética , Oocitos/efectos de los fármacos , Oocitos/metabolismo , XenopusRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl- channel activated by phosphorylation, was expressed in Xenopus oocytes along with various combinations of several other components of the cAMP signalling pathway. Activation of the coexpressed beta 2 adrenergic receptor increased cAMP and led to CFTR activation. The activation of CFTR (1) requires only short (15 s) exposure to isoproterenol, (2) occurs for agonist concentrations 100-1000 fold lower than those that produce cAMP increases detectable by a radioimmunoassay, (3) requires injection of only 5 pg of receptor cRNA per oocyte, and (4) can be increased further by coexpression of cRNA for adenylyl cyclase type II or III or for Gs alpha. In addition, CFTR activation and cAMP increases by beta 2 activation were enhanced by activation of the coexpressed 5HT1A receptor, which is thought to couple to Gi. The additional activation by the 5HT1A receptor was enhanced by coexpression of adenylyl cyclase type II but not with type III and may proceed via the beta gamma subunits of a G protein. The sensitivity of the assay system is also demonstrated by responses to vasoactive intestinal peptide and to pituitary adenylate cyclase-activating polypeptide in oocytes injected with cerebral cortex mRNA.
Asunto(s)
AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de la Membrana/metabolismo , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Animales , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Femenino , Proteínas de Unión al GTP/genética , Expresión Génica , Humanos , Técnicas In Vitro , Proteínas de la Membrana/genética , Oocitos/metabolismo , ARN Complementario/genética , ARN Mensajero/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Serotonina/metabolismo , Receptores de Serotonina 5-HT1 , Xenopus laevisRESUMEN
Serotonin (5-HT) and other contractile agonists stimulate Na(+)-H+ exchange in vascular smooth muscle. Since intracellular alkalinization, per se, stimulates contraction, we tested whether 5-HT-induced contraction was associated with an increased pHi. In HCO3(-)-free buffer (pHo 7.4), 5-HT (10(-5) M) increased pHi, as measured by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence, from 7.10 +/- 0.03 to 7.34 +/- 0.03 (p < 0.01) in primary cultures of canine femoral artery vascular smooth muscle cells grown to confluence in the presence of 10% fetal calf serum. In HCO3- buffer (24 mM, pHo 7.4), resting pHi was 7.26 +/- 0.04 (p < 0.01 versus HCO3(-)-free buffer) but was not altered by 5-HT. In both types of buffer, 5-HT stimulated 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake (Na(+)-H+ exchange). In HCO3- buffer and in Na(+)- and HCO3(-)-free buffer, 5-HT increased 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 36Cl- uptake, suggesting that 5-HT stimulated Na(+)-independent Cl(-)-HCO3- and Cl(-)-Cl- exchange activities, respectively. Individual vascular smooth muscle cells were then cultured on rat tail tendon collagen gels in the presence of 0.5% fetal calf serum, and cell length and pHi were measured by video and epifluorescence microscopy. 5-HT contracted cells in a dose-dependent, reversible, and ketanserin-inhibitable manner. These cells, like cells grown in 10% fetal calf serum, exhibited Na(+)-H+ and Na(+)-independent Cl(-)-HCO3- exchange. In HCO3- buffer, 5-HT contracted cells without an associated change in pHi. We concluded the following: 1) 5-HT stimulated both Na(+)-H+ and Na(+)-independent Cl(-)-HCO3- exchange activities in cultured vascular smooth muscle cells in parallel. 2) As a result of enhanced H+ and HCO3- efflux, pHi was not altered. 3) In the presence of HCO3-, 5-HT-induced contraction was not associated with a change in pHi.
Asunto(s)
Concentración de Iones de Hidrógeno , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Serotonina/farmacología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Bicarbonatos/metabolismo , Células Cultivadas , Cloruros/metabolismo , Perros , Femenino , HEPES/farmacología , Masculino , Músculo Liso Vascular/metabolismo , Sodio/metabolismo , Estimulación QuímicaRESUMEN
It is evident from the present analysis that although a role for Ca2+ in controlling hypertonic cell volume regulation and RVI mechanisms has not been shown, Ca2+ plays a central role in activating and controlling hypotonic cell volume regulation and RVD mechanisms in most cells. However, this Ca2+ dependency is highly variable among cell types and tissues. Cells can be grouped into three general categories based on the relative dependency of RVD on Ca2+: 1) cells that are highly dependent on extracellular Ca2+ and the activation of Ca2+ influx, supposedly reflecting activation of Ca2+ channels, such as observed for the renal PST cells and osteosarcoma cells; 2) cells that are not dependent on extracellular Ca2+ and Ca2+ influx but that require at least a certain basal intracellular Ca2+ level or transient release of Ca2+ from internal stores, such as observed for the Ehrlich ascites tumor cells and medullary thick ascending limb cells; and 3) cells that display little if any Ca2+ dependency, such as the lymphocytes. There is initial evidence that this variable dependency of RVD on Ca2+ may reflect, in large part, a variable Ca2+ threshold of RVD processes, although this notion has not been fully investigated. The site and mechanism of Ca2+ dependency of RVD are poorly understood. Initial studies pointed to a possible direct control of K+ and/or Cl- channels by Ca2+ to modulate KCl efflux and, hence, RVD. This view appears to be too simplistic, however, as it is increasingly evident that the ion channels involved in RVD may not be directly Ca2+ dependent and that some other regulatory process controlling the channels, perhaps a phosphorylation step, may be the Ca(2+)-dependent event. Given the added complexity of the time-dependent variability of the action of Ca2+, i.e., the Ca2+ window, coupled with the variability of the RVD mechanisms among cell and tissue types, it is likely that the RVD mechanism is a highly complex process involving events and biochemical pathways throughout the cell rather than events simply localized to the inner face of the plasma membrane. It remains for future studies to determine the exact biochemical events that underly the RVD mechanism and its control, and the Ca2+ dependency of each step, before a full understanding will be attained of the role of Ca2+ in modulating RVD.
Asunto(s)
Calcio/fisiología , Tamaño de la Célula/fisiología , Animales , Transporte Biológico , Calcio/metabolismo , Canales de Calcio/fisiología , Membrana Celular/metabolismo , Membrana Celular/fisiología , Humanos , Transducción de Señal/fisiologíaRESUMEN
The mechanism of Ca(2+)-dependent control of hypotonic cell volume regulation was investigated in the isolated, nonperfused renal proximal straight tubule. When proximal tubules were exposed to hypotonic solution with 1 mM Ca2+, cells swelled rapidly and then underwent regulatory volume decrease (RVD). This treatment resulted in an increase in intracellular free calcium concentration ([Ca2+]i) by a mechanism that had two phases: the first was a transient increase from baseline (136 nM) to a peak (413 nM) that occurred in the first 15-20 sec, but was followed by a rapid decay toward the pre-swelling levels. The second phase was characterized by a sustained elevation of [Ca2+]i above the baseline (269 nM), which was maintained over several minutes. The dependence of these two phases on extracellular Ca2+ was determined. Reduction of bath [Ca2+] to 10 or 1 microM partially diminished the transient phase, but abolished the sustained phase completely, such that [Ca2+]i fell below the baseline levels during RVD. It was concluded that the transient increase resulted predominantly from swelling-activated release of intracellular Ca2+ stores and that the sustained phase was due to swelling-activated Ca2+ entry across the plasma membrane. Ca2+ entry probably also contributed to the transient increase in [Ca2+]i. The time dependence of swelling-activated Ca2+ entry was also investigated, since it was previously shown that RVD was characterized by a "calcium window" period (less than 60 sec), during which extracellular Ca2+ was required. Outside of this time period, RVD would inactivate and could not be reactivated by subsequent addition of Ca2+. It was found that the Ca2+ permeability did not inactivate over several minutes, indicating that the temporal dependence of RVD on extracellular Ca2+ is not due to the transient activation of a Ca2+ entry pathway.
Asunto(s)
Calcio/farmacología , Túbulos Renales Proximales/citología , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Calcio/farmacocinética , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , Femenino , Soluciones Hipotónicas , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Ionomicina/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Conejos , Sodio/farmacocinética , Factores de TiempoRESUMEN
The Ca2+ entry pathways in the basolateral plasma membrane of the isolated, nonperfused proximal straight tubule (PST) of rabbit kidney were investigated using fura-2 fluorescence microscopy. Under isotonic conditions, reduction of bath [Ca2+] from 1 mM to 1 microM caused intracellular free calcium concentration ([Ca2+]i) to fall close to zero. Treatment with 10 microM verapamil, a calcium channel blocker, had a similar effect. Treatment with verapamil or low Ca2+ also induced fluctuations in cell volume. However, isotonic treatment with 10 microM nifedipine, a dihydropyridine (DHP)-type calcium channel blocker, did not affect [Ca2+]i or cell volume, indicating that the endogenous Ca2+ entry pathway is verapamil-sensitive but DHP-insensitive. When cells were exposed to hypotonic solutions in the presence of 1 mM Ca2+, they swelled and underwent normal RVD while [Ca2+]i increased transiently to a peak before decreasing to a late phase plateau level above the baseline level (see McCarty, N.A., O'Neil, R.G. 1991. J. Membrane Biol. 123:149-160). When cells were swollen in the presence of verapamil or low bath [Ca2+], RVD was abolished and [Ca2+]i fell well below the baseline during the late phase response. In contrast, when cells were swollen in the presence of nifedipine, RVD and the late phase rise in [Ca2+]i were abolished, but [Ca2+]i did not fall below the baseline level in the late phase, indicating that nifedipine inhibited the swelling-induced Ca2+ entry but that Ca2+ entry by another pathway was undisturbed. It was concluded that PST cells are characterized by two Ca2+ permeability pathways in the basolateral membrane. Under both isotonic and hypotonic conditions, Ca2+ entry occurs at a slow rate via a verapamil-sensitive, DHP-insensitive "baseline" Ca2+ entry pathway. Cell swelling activates a separate DHP-sensitive, verapamil-sensitive Ca2+ entry pathway, which is responsible for the supply of Ca ions to the Ca(2+)-dependent mechanism by which cell volume regulation is achieved.