Here we expressed wild-type human CFTR as a FLAG-fused protein in HEK293 cells heterologously and purified it in three actions: anti-FLAG and wheat germ agglutinin affinity chromatographies and size exclusion chromatography. to be an ellipsoidal particle with dimensions of 120 106 162?. It comprises a small dome-shaped extracellular and membrane-spanning domain name and WAY 181187 a large cytoplasmic domain name with WAY 181187 orifices beneath the putative transmembrane domain name. EM observation of CFTRanti-regulatory domain name antibody complex confirmed that two regulatory domains are located around the bottom end of the larger oval cytoplasmic domain name. The cystic fibrosis transmembrane conductance regulator (CFTR)3 (also termed ABCC7) is usually a unique member of the ATP-binding cassette (ABC) superfamily in that CFTR functions as an anion channel, whereas most other members function as active transporters. CFTR is usually expressed in the luminal membranes of secreting and absorbing epithelia and plays a critical role in transepithelial salt and water transport. Dysfunction of CFTR leads to cystic fibrosis, the most common lethal autosomal recessive disorder in Caucasians (1-3). On the other hand, extremely high activity of WAY 181187 CFTR, usually caused by bacterial toxins, results in secretory diarrhea (4, 5), killing millions of infants in developing countries every year (6). Understanding the structure/function relationship and the underlying mechanisms of CFTR are essential for developing novel therapeutics for CFTR-mediated diseases. Like other ABC transporters, CFTR is usually formed by two repeated motifs, each of which has a membrane-spanning domain name (MSD) and a cytoplasmic nucleotide-binding domain name (NBD). However, a regulatory domain name (R domain name) located between the first NBD (NBD1) and the second MSD (MSD2) is unique in CFTR among ABC transporters. This domain name contains several phosphorylation sites for protein kinase A and protein kinase C, and the level of their phosphorylation controls CFTR channel activity. Once they are phosphorylated, opening and closing (gating) of the CFTR channel is controlled by ATP binding and hydrolysis at its two NBDs (7). Each NBD contains the Walker A and Walker B nucleotide-binding motifs and the signature sequence LSGGQ, which defines the ABC superfamily. It is generally accepted that NBD1 and -2 are dimerized in a head to tail configuration with two ATP molecules sandwiched between the Walker A/B motifs of one NBD and the signature sequences of the counterpart NBD (8). There are lines of convincing evidence that the opening of CFTR chloride channel is associated with this NBD dimerization (9). The detailed dynamics of ATP binding and NBD dimerization in CFTR gating has been intensively investigated (10-12). Despite a plethora of biochemical and electrophysiological results, structural information of CFTR is limited because of the lack of abundant CFTR protein sources and the difficulties in protein purification and crystallization. So far, only the structure of the NBD1 domain name of CFTR has been determined by x-ray crystallography using mouse (13) and human (14) proteins. Ford and co-workers (15, 16) described low resolution structures of wild-type human CFTR by two-dimensional electron crystallography (15) and by the single particle reconstruction technique (16). The two-dimensional crystallography showed two different monomeric structures for CFTR (15). On the other hand, the single particle analysis suggested WAY 181187 a structure whose particle size is compatible with a dimeric association by two CFTR molecules (16). In this study, we purified glycosylated mature CFTR and reconstructed its three-dimensional structure from negatively stained EM images. Our data suggest that two CFTR proteins form an ellipsoidal tail to tail dimeric configuration with side orifices in the cytoplasm and that at least part of the R domain name is located around the bottom end of the larger cytoplasmic domain name. EXPERIMENTAL PROCEDURES for 10 min to remove debris. The supernatant was centrifuged at 100,000 for 60 min to obtain the membrane fraction. intracellular answer) made up of 150 mm NMDG-Cl, 2 mm MgCl2, 10 mm EGTA, and 8 mm Tris, pH 7.4 with NMDG. Patches were held at -50 mV in all experiments. CFTR channel currents were recorded at room heat. The currents were Rabbit Polyclonal to ABHD8 filtered at 100 Hz with an eight-pole Bessel filter (model LPF-8; Warner Devices) and digitized on line at 500 Hz. CFTR channels were activated by the combined application of 1 1 mm MgATP and 25 models/ml cAMP-dependent protein kinase. MgATP and cAMP-dependent protein kinase were purchased from Sigma. for 30 min, the supernatant made up of solubilized FLAG-tagged CFTR was loaded onto an anti-FLAG affinity column (Sigma) equilibrated in advance. The column was then.
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