The cystic fibrosis transmembrane conductance regulator (CFTR): three-dimensional structure and localization of a channel gate

Cystic fibrosis affects about 1 in 2500 live births and involves loss of transmembrane chloride flux due to a lack of a membrane protein channel termed the cystic fibrosis transmembrane conductance regulator (CFTR). We have studied CFTR structure by electron crystallography. The data were compared with existing structures of other ATP-binding cassette transporters. The protein was crystallized in the outward facing state and resembled the well characterized Sav1866 transporter. We identified regions in the CFTR map, not accounted for by Sav1866, which were potential locations for the regulatory region as well as the channel gate. In this analysis, we were aided by the fact that the unit cell was composed of two molecules not related by crystallographic symmetry. We also identified regions in the fitted Sav1866 model that were missing from the map, hence regions that were either disordered in CFTR or differently organized compared with Sav1866. Apart from the N and C termini, this indicated that in CFTR, the cytoplasmic end of transmembrane helix 5/11 and its associated loop could be partly disordered (or alternatively located).     

Multiple membrane-cytoplasmic domain contacts in the cystic fibrosis transmembrane conductance regulator (CFTR) mediate regulation of channel gating

The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ATP-binding cassette (ABC) ion channel mutated in patients with cystic fibrosis. The most common mutation, deletion of phenylalanine 508 (DeltaF508) and many other disease-associated mutations occur in the nucleotide binding domains (NBD) and the cytoplasmic loops (CL) of the membrane-spanning domains (MSD). A recently constructed computational model of the CFTR three-dimensional structure, supported by experimental data (Serohijos, A. W., Hegedus, T., Aleksandrov, A. A., He, L., Cui, L., Dokholyan, N. V., and Riordan, J. R. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 3256-3261) revealed that several of these mutations including DeltaF508 disrupted interfaces between these domains. Here we have used cysteine cross-linking experiments to verify all NBD/CL interfaces predicted by the structural model and observed that their cross-linking has a variety of different effects on channel gating. The interdomain contacts comprise aromatic clusters important for stabilization of the interfaces and also involve the Q-loops and X-loops that are in close proximity to the ATP binding sites. Cross-linking of all domain-swapping contacts between NBDs and MSD cytoplasmic loops in opposite halves of the protein rapidly and reversibly arrest single channel gating while those in the same halves have lesser impact. These results reinforce the idea that mediation of regulatory signals between cytoplasmic- and membrane-integrated domains of the CFTR channel apparently relies on an array of precise but highly dynamic interdomain structural joints.     

Plasma membrane lipids of human diploid fibroblasts from normal individuals and patients with cystic fibrosis.

The lipid composition of isolated plasma membranes of human skin fibroblasts is described for the first time. Plasma membranes from a number of strains of fibroblasts from patients with cystic fibrosis and matched normals were isolated by a recently described procedure and analysed for major phospholipid classes, cholesterol and fatty acids. No differences in the quantities of these compounds were detected between cells of the two different origins. The fetal calf serum used to supplement the growth medium contained relatively more palmitoleate and oleate but less stearate than the membranes. There were also no consistent differences between cystic fibrosis and normal membranes in terms of the fatty acid compositions of their individual phospholipid classes. Consistent with this lack of chemical change in the lipids of membranes of cystic fibrosis cells, the degree of fluorescence polarization of diphenylhexatriene, an index of fluidity, was also unchanged.     

Equilibrium, kinetic and photoaffinity labeling studies of daunomycin binding to P-glycoprotein-containing membranes of multidrug-resistant Chinese hamster ovary cells

The binding of daunomycin and its Bolton-Hunter derivative iodomycin to plasma membranes isolated from multidrug-resistant Chinese hamster ovary cells (CHO B30) and their drug-sensitive parents (B1) was investigated. The thermodynamics and kinetics of equilibrium binding monitored by fluorescence titrations and temperature-jump relaxation spectrometry were compared with the specificity of covalent photolabeling with [3H]daunomycin and [125I]iodomycin. The facts that the uptake of anthracycline from aqueous solution into the CHO membranes was not accompanied by any substantial increase of fluorescence anisotropy nor by any spectral shift of the fluorescence emission spectrum and that the partition ratio into the membrane was 20-30-fold higher when compared to a lecithin bilayer, provided evidence that the non-covalent drug binding sites are constituted by polar protein domains without any substantial contribution from the surrounding lipids. Photoaffinity labeling with nanomolar concentrations of anthracycline and equilibrium binding curves independently showed that a 150-170-kDa plasma membrane glycoprotein (P-glycoprotein), whose overexpression is the major difference between B1 and B30 membranes, provides the binding sites of highest affinity for daunomycin and iodomycin (K approximately equal to 4 x 10(7) M-1). Comparison of photolabeling and equilibrium data suggested that the same binding sites on P-glycoprotein were most probably being monitored. The photolabeling of P-glycoprotein by iodomycin was inhibited in a dose-dependent manner by other compounds to which multi-drug-resistant cells are either resistant or collaterally sensitive with the following orders of effectiveness: vinblastine greater than verapamil greater than nitrendipine greater than daunomycin much greater than colchicine. Temperature-jump experiments covering the time range of 1 microseconds to 1 s revealed a single concentration-dependent relaxation time of 10-30 microseconds. The association of daunomycin with its binding sites in the membranes was found to be a diffusion-controlled process with kon rates of 2-4 X 10(9) M-1 s-1. Therefore, the selectivity of drug binding was entirely reflected in the dissociation rates.     

Affinity chromatographic sorting of carboxypeptidase a and its chemically modified derivatives

Carboxypeptidase A and derivatives obtained by chemical modification of various active center components were subjected to affinity chromatography on a p-aminobenzylsuccinic acid-Sepharose 4B conjugate. Tetardation of the enzyme on the column was dependent on the residue modified when elution was carried out with 0.3 m NaCl at pH 7.0. Both the functional zinc atom and the active site residue Glu-270 are essential for effective adsorption while alteration of residues involved in hydrophobic interaction with substrate or in recognition of its terminal carboxyl group decreased retention on the affinity matrix. Elution of native carboxypeptidase with competing soluble benzylsuccinic acid indicated that only active center binding of the immobilized inhibitor accounts for retardation of the enzyme on the column. Hence, affinity chromatography on this biospecific adsorbent using mild elution conditions (which do not distort protein structure) provides an excellent tool for the rapid isolation and purification of active center modified enzyme even from a complex mixture of reaction products.     

Modeling progressive non-alcoholic fatty liver disease in the laboratory mouse

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world and its prevalence is rising. In the absence of disease progression, fatty liver poses minimal risk of detrimental health outcomes. However, advancement to non-alcoholic steatohepatitis (NASH) confers a markedly increased likelihood of developing severe liver pathologies, including fibrosis, cirrhosis, organ failure, and cancer. Although a substantial percentage of NAFLD patients develop NASH, the genetic and molecular mechanisms driving this progression are poorly understood, making it difficult to predict which patients will ultimately develop advanced liver disease. Deficiencies in mechanistic understanding preclude the identification of beneficial prognostic indicators and the development of effective therapies. Mouse models of progressive NAFLD serve as a complementary approach to the direct analysis of human patients. By providing an easily manipulated experimental system that can be rigorously controlled, they facilitate an improved understanding of disease development and progression. In this review, we discuss genetically- and chemically-induced models of NAFLD that progress to NASH, fibrosis, and liver cancer in the context of the major signaling pathways whose disruption has been implicated as a driving force for their development. Additionally, an overview of nutritional models of progressive NAFLD is provided.     

Fluorescent modified phosphatidylcholine floppase activity of reconstituted multidrug resistance-associated protein MRP1

Multidrug resistance-associated protein (MRP1) may function as a floppase in human red blood cells to translocate phosphatidylserine and/or phosphatidylcholine from inner membrane leaflet to outer leaflet. Here we report that the purified and reconstituted MRP1 protein into asolectin proteoliposomes is mainly in an inside-out configuration and possesses the ability to flop a fluorescent labeled phosphatidylcholine (NBD-PC) from outer leaflet (protoplasmic) to inner leaflet (extracytoplasmic). The reconstituted MRP1 protein retains endogenous ATPase activity. ATP hydrolysis is required for the flopping since removal of ATP and/or Mg2+ inhibits the translocation of NBD-PC. Further evidence to support this conclusion is that the translocation of NBD-PC is inhibited by vanadate, which traps ATP hydrolysis product ADP in the nucleotide binding domains. In addition, the translocation of NBD-PC by proteoliposomes containing MRP1 protein is in a glutathione-dependent manner, similar to the process of translocating anticancer drugs such as daunorubicin. verapamil, vincristine, vinblastine, doxorubicin and oxidized glutathione partially inhibited the translocation of NBD-PC, whereas MK 571, an inhibitor of MRP1 protein, inhibited the translocation almost completely. Taken together, the purified and reconstituted MRP1 protein possesses the ability to flop NBD-PC from outer to inner leaflet of the proteoliposomes.