RESUMEN
CFTR is a cyclic AMP and nucleotide-related chloride-selective channel with a low unitary conductance. Many of the physiological roles of CFTR are effectively studied in intact cells and tissues. However, there are also several clear advantages to the application of cell-free technologies to the study of the biochemical and biophysical properties of CFTR. When expressed in heterologous cells, CFTR is processed relatively poorly, depending, however, on the cell-type analysed. In some cells, only 20-25% of the protein which is initially synthesized exits the endoplasmic reticulum to insert into the cell membrane [Cell 83 (1995) 121; EMBO J. 13 (1994) 6076]. Further, many of the disease-causing mutants of CFTR result in even lower processing efficiencies. Therefore, several procedures have been developed to study regulated CFTR channel function expressed in microsomal membranes and following its purification and reconstitution. These experimental approaches and their application are discussed here.
Asunto(s)
Membrana Celular/fisiología , Técnicas de Laboratorio Clínico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/biosíntesis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Humanos , Técnicas In Vitro , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiologíaRESUMEN
Using the patch-clamp (PC) and planar lipid bilayer (PLB) techniques the molecular behaviour of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel can be visualised in real-time. The PC technique is a highly powerful and versatile method to investigate CFTR's mechanism of action, interaction with other proteins and physiological role. Using the PLB technique, the structure and function of CFTR can be investigated free from the influence of other proteins. Here we discuss how these techniques are employed to investigate the CFTR Cl- channel with special emphasis on its permeation, conduction and gating properties.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Membrana Dobles de Lípidos , Técnicas de Placa-Clamp/métodos , Humanos , Activación del Canal Iónico/fisiologíaRESUMEN
Flux studies using either radioisotopes or ion-selective electrodes are a convenient method to assay the function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Here, we described three different protocols to study the properties, regulation and pharmacology of the CFTR Cl- channel in populations of cells and artificial vesicles. These techniques are widely used to evaluate the function of wild-type and mutant CFTR prior to detailed analyses using the patch-clamp technique. Moreover, they have proved especially valuable in the search for new drugs to treat cystic fibrosis.
Asunto(s)
Membrana Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Técnicas Citológicas , Transporte Iónico/fisiología , Técnicas de Cultivo de Célula , Membrana Celular/efectos de los fármacos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/efectos de los fármacos , Humanos , Electrodos de Iones Selectos , Liposomas/farmacología , Radioisótopos/farmacologíaRESUMEN
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a phosphorylation and nucleotide regulated chloride channel. CFTR also directly mediates the hydrolysis of ATP and this catalytic activity is loosely coupled to CFTR channel gating. However, mechanistic detail regarding the role of ATP hydrolysis in channel function is lacking. Our further understanding of the molecular basis for normal channel activity requires kinetic analysis of the ATPase activity by the full-length protein. This article describes an effective assay of ATPase activity by purified, reconstituted CFTR protein.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Técnicas de Laboratorio Clínico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/aislamiento & purificación , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Activación del Canal Iónico/fisiología , Adenosina Trifosfatasas/fisiología , Humanos , Hidrólisis , CinéticaAsunto(s)
Canales de Cloruro/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Secuencia de Aminoácidos , Canales de Cloruro/fisiología , Cloro/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Sistema Digestivo/metabolismo , Electrofisiología , Hidrólisis , Canales Iónicos/fisiología , Iones , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conductos Pancreáticos/fisiología , Fosforilación , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Sistema Respiratorio/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Studies have shown that expression of cystic fibrosis transmembrane conductance regulator (CFTR) is associated with enhanced glutathione (GSH) efflux from airway epithelial cells, implicating a role for CFTR in the control of oxidative stress in the airways. To define the mechanism underlying CFTR-associated GSH flux, we studied wild-type and mutant CFTR proteins expressed in Sf9 membranes, as well as purified and reconstituted CFTR. We show that CFTR-expressing membrane vesicles mediate nucleotide-activated GSH flux, which is disrupted in the R347D pore mutant, and in the Walker A K464A and K1250A mutants. Further, we reveal that purified CFTR protein alone directly mediates nucleotide-dependent GSH flux. Interestingly, although ATP supports GSH flux through CFTR, this activity is enhanced in the presence of the non-hydrolyzable ATP analog AMP-PNP. These findings corroborate previous suggestions that CFTR pore properties can vary with the nature of the nucleotide interaction. In conclusion, our data demonstrate that GSH flux is an intrinsic function of CFTR and prompt future examination of the role of this function in airway biology in health and disease.
