Volume-regulated chloride channels associated with the human multidrug-resistance P-glycoprotein.

Expression of P-glycoprotein, the product of the MDR1 gene, confers multidrug resistance on cell lines and human tumours (reviewed in refs 1,2). P-glycoprotein (relative molecular mass 170,000) is an ATP-dependent, active transporter which pumps hydrophobic drugs out of cells, but its normal physiological role is unknown. It is a member of the ABC (ATP-binding cassette) superfamily of transporters, which includes many bacterial transport systems, the putative peptide transporter from the major histocompatibility locus, and the product of the cystic fibrosis gene (the cystic fibrosis transmembrane regulator, CFTR). CFTR is located in the apical membranes of many secretory epithelia and is associated with a cyclic AMP-regulated chloride channel. At least two other chloride channels are present in epithelial cells, regulated by cell volume and by intracellular Ca2+, respectively. Because of the structural and sequence similarities between P-glycoprotein and CFTR, and because P-glycoprotein is abundant in many secretory epithelia, we examined whether P-glycoprotein might be associated with one or other of these channels. We report here that expression of P-glycoprotein generates volume-regulated, ATP-dependent, chloride-selective channels, with properties similar to channels characterized previously in epithelial cells.     

A cluster of cystic fibrosis mutations in the first nucleotide-binding fold of the cystic fibrosis conductance regulator protein.

The gene responsible for cystic fibrosis (CF) has recently been identified and is predicted to encode a protein of 1,480 amino acids called the CF transmembrane conductance regulator (CFTR). Several functional regions are thought to exist in the CFTR protein, including two areas for ATP-binding, termed nucleotide-binding folds (NBFs), a regulatory (R) region that has many possible sites for phosphorylation by protein kinases A and C, and two hydrophobic regions that probably interact with cell membranes. The most common CF gene mutation leads to omission of phenylalanine residue 508 in the putative first NBF, indicating that this region is functionally important. To determine whether other mutations occur in the NBFs of CFTR, we determined the nucleotide sequences of exons 9, 10, 11 and 12 (encoding the first NBF) and exons 20, 21 and 22 (encoding most of the second NBF) from 20 Caucasian and 18 American-black CF patients. One cluster of four mutations was discovered in a 30-base-pair region of exon 11. Three of these mutations cause amino-acid substitutions at residues that are highly conserved among the CFTR protein, the multiple-drug-resistance proteins and ATP-binding membrane-associated transport proteins. The fourth mutation creates a premature termination signal. These mutations reveal a functionally important region in the CFTR protein and provide further evidence that CFTR is a member of the family of ATP-dependent transport proteins.     

CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains

ABC (ATP-binding cassette) proteins constitute a large family of membrane proteins that actively transport a broad range of substrates. Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ABC proteins in that its transmembrane domains comprise an ion channel. Opening and closing of the pore have been linked to ATP binding and hydrolysis at CFTR's two nucleotide-binding domains, NBD1 and NBD2 (see, for example, refs 1, 2). Isolated NBDs of prokaryotic ABC proteins dimerize upon binding ATP, and hydrolysis of the ATP causes dimer dissociation. Here, using single-channel recording methods on intact CFTR molecules, we directly follow opening and closing of the channel gates, and relate these occurrences to ATP-mediated events in the NBDs. We find that energetic coupling between two CFTR residues, expected to lie on opposite sides of its predicted NBD1-NBD2 dimer interface, changes in concert with channel gating status. The two monitored side chains are independent of each other in closed channels but become coupled as the channels open. The results directly link ATP-driven tight dimerization of CFTR's cytoplasmic nucleotide-binding domains to opening of the ion channel in the transmembrane domains. This establishes a molecular mechanism, involving dynamic restructuring of the NBD dimer interface, that is probably common to all members of the ABC protein superfamily.     

The ABC protein turned chloride channel whose failure causes cystic fibrosis

CFTR chloride channels are encoded by the gene mutated in patients with cystic fibrosis. These channels belong to the superfamily of ABC transporter ATPases. ATP-driven conformational changes, which in other ABC proteins fuel uphill substrate transport across cellular membranes, in CFTR open and close a gate to allow transmembrane flow of anions down their electrochemical gradient. New structural and biochemical information from prokaryotic ABC proteins and functional information from CFTR channels has led to a unifying mechanism explaining those ATP-driven conformational changes.     

Sequences encoded in the class II region of the MHC related to the 'ABC' superfamily of transporters

Class I molecules of the major histocompatibility complex (MHC) bind and present peptides derived from the degradation of intracellular, often cytoplasmic, proteins, whereas class II molecules usually present proteins from the extracellular environment. It is not known how peptides derived from cytoplasmic proteins cross a membrane before presentation at the cell surface. But certain mutations in the MHC can prevent presentation of antigens with class I molecules. In addition, mutations possibly in the MHC can affect presentation by class II molecules. Here we report the finding of a new gene in the MHC that might have a role in antigen presentation and which is related to the ABC (ATP-binding cassette) superfamily of transporters. This superfamily includes the human multidrug-resistance protein, and a series of transporters from bacteria and eukaryotic cells capable of transporting a range of substrates, including peptides.     

Cystic fibrosis in the mouse by targeted insertional mutagenesis.

Cystic fibrosis is a fatal genetic disorder which afflicts 50,000 people worldwide. A viable animal model would be invaluable for investigating and combating this disease. The mouse cystic fibrosis transmembrane conductance regulator gene was disrupted in embryonal stem cells using an insertional gene targeting vector. Germ-line chimaeras were derived and the offspring of heterozygous crosses studied. These homozygous mutant mice survive beyond weaning. In vivo electrophysiology demonstrates the predicted defect in chloride ion transport in these mice and can distinguish between each genotype. Histological analysis detects important hallmarks of human disease pathology, including abnormalities of the colon, lung and vas deferens. This insertional mouse mutation provides a valid model system for the development and testing of therapies for cystic fibrosis patients.     

Control of CFTR chloride conductance by ATP levels through non-hydrolytic binding.

Site-specific mutation and membrane reconstitution experiments provide compelling evidence that the product of the gene which is at fault in the disease cystic fibrosis, termed the cystic fibrosis transmembrane conductance regulator (CFTR), is a small-conductance chloride channel activated by phosphorylation. As transport of chloride ions is passive, the predicted presence of two nucleotide-binding domains in CFTR seems as puzzling as a report that ATP hydrolysis is essential to activate the channel. We now find that in the sweat duct, which expresses high levels of CFTR and has a very high Cl- conductance, intracellular concentrations of ATP must be about normal (5 mM) for activation of this conductance, apparently by a non-hydrolytic, perhaps allosteric, mechanism. This passive dependence on ATP should mean that even a modest depletion of cell energy levels will significantly lower the energy demands of electrolyte transport by decreasing chloride conductance. We believe this direct coupling between cellular ATP levels and chloride channel activity is an adaptive mechanism to protect the tissue from damage resulting from excessive energy depletion.     

Crystal structure of a catalytic intermediate of the maltose transporter

The maltose uptake system of Escherichia coli is a well-characterized member of the ATP-binding cassette transporter superfamily. Here we present the 2.8-A crystal structure of the intact maltose transporter in complex with the maltose-binding protein, maltose and ATP. This structure, stabilized by a mutation that prevents ATP hydrolysis, captures the ATP-binding cassette dimer in a closed, ATP-bound conformation. Maltose is occluded within a solvent-filled cavity at the interface of the two transmembrane subunits, about halfway into the lipid bilayer. The binding protein docks onto the entrance of the cavity in an open conformation and serves as a cap to ensure unidirectional translocation of the sugar molecule. These results provide direct evidence for a concerted mechanism of transport in which solute is transferred from the binding protein to the transmembrane subunits when the cassette dimer closes to hydrolyse ATP.     

Defective acidification of intracellular organelles in cystic fibrosis.

The phenotype of cystic fibrosis (CF) includes abnormalities in transepithelial transport of Cl- (refs 1-5), decreased sialylation and increased sulphation and fucosylation of glycoproteins, and lung colonization with Pseudomonas. It is not apparent how these abnormalities are interrelated, nor how they result from loss of function of the CF gene-encoded transmembrane regulator (CFTR). We have previously shown that that the pH of a secretory granule is regulated by the vesicular conductance for Cl- (ref. 11). Here we find defective acidification in CF cells of the trans-Golgi/trans-Golgi network, of prelysosomes and of endosomes as a result of diminished Cl- conductance. Sialytation of proteins and lipids is reduced and ligand traffic altered. These abnormalities can result from defective acidification because vacuolar pH regulates glycoprotein processing and ligand transport. The CF phenotype is similar to that of alkalinized cells and acidification-defective mutatants.     

Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms

The bacterium Pseudomonas aeruginosa permanently colonizes cystic fibrosis lungs despite aggressive antibiotic treatment. This suggests that P. aeruginosa might exist as biofilms--structured communities of bacteria encased in a self-produced polymeric matrix--in the cystic fibrosis lung. Consistent with this hypothesis, microscopy of cystic fibrosis sputum shows that P. aeruginosa are in biofilm-like structures. P. aeruginosa uses extracellular quorum-sensing signals (extracellular chemical signals that cue cell-density-dependent gene expression) to coordinate biofilm formation. Here we found that cystic fibrosis sputum produces the two principal P. aeruginosa quorum-sensing signals; however, the relative abundance of these signals was opposite to that of the standard P. aeruginosa strain PAO1 in laboratory broth culture. When P. aeruginosa sputum isolates were grown in broth, some showed quorum-sensing signal ratios like those of the laboratory strain. When we grew these isolates and PAO1 in a laboratory biofilm model, the signal ratios were like those in cystic fibrosis sputum. Our data support the hypothesis that P. aeruginosa are in a biofilm in cystic fibrosis sputum. Moreover, quorum-sensing signal profiling of specific P. aeruginosa strains may serve as a biomarker in screens to identify agents that interfere with biofilm development.     

In vivo cell-specific expression of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The principal manifestations of CF include increased concentration of Cl- in exocrine gland secretions, pancreatic insufficiency, chronic lung disease, intestinal blockage and malabsorption of fat, and male and female infertility. Insight into the function of CFTR can be gained by correlating its cell-specific expression with the physiology of those cells and with CF pathology. Determination of CFTR messenger RNA in rat tissues by in situ hybridization shows that it is specifically expressed in the ductal cells of the pancreas and the salivary glands. In the intestine, decreasing gradients of expression of the CFTR gene are observed on both the crypt-villus and the proximal-distal axes. This expression is consistent with CFTR being responsible for bidirectional Cl- transport, secretion in the intestinal crypts and reabsorption in the silivary gland ducts, and suggests that in these tissues CFTR functions as a regulated Cl- channel. In the lung, a broad band of hybridization includes the mucosa and submucosa of the bronchi and bronchioles. In the testis, CFTR expression is regulated during the cycle of the seminiferous epithelium. Postmeiotic expression is maximal in the round spermatids of stages VII and VIII, suggesting that CFTR plays a critical role in spermatogenesis and that deficiency of this function contributes to CF male infertility.     

Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in cultured cystic fibrosis airway epithelial cells and Cl- channel activation assessed in single cells using a fluorescence microscopic assay and the patch-clamp technique. Expression of CFTR, but not of a mutant form of CFTR (delta F508), corrected the Cl- channel defect. Correction of the phenotypic defect demonstrates a causal relationship between mutations in the CFTR gene and defective Cl- transport which is the hallmark of the disease.     

Immuno-isolation of Sec7p-coated transport vesicles from the yeast secretory pathway.

The transport of proteins destined for post-endoplasmic reticulum locations in the secretory pathway is mediated by small vesicular carriers. Transport vesicles have been generated in cell-free assays from the yeast Saccharomyces cerevisiae, and mammalian systems. Yeast genes encoding cytosolic components that participate in vesicular traffic were first identified from the collection of conditional-lethal sec-(secretory) mutants. Mutations in the yeast SEC7 gene disrupt protein transport in the secretory pathway at the nonpermissive temperature. The SEC7 gene product is a phosphoprotein of relative molecular mass 230,000 that functions from the cytoplasmic aspect of intracellular membranes. We report that in a yeast cell-free transport assay, the introduction of antibodies to Sec7 protein (Sec7p) results in the accumulation of transport vesicles. These vesicles are retrieved with Sec7p-specific antibodies by immuno-isolation for biochemical and electron microscopic characterization. Sec7p on the surface of the accumulated transport vesicles, in combination with previous genetic and biochemical studies, implicate Sec7p as part of a (non-clathrin) vesicle coat. This Sec7p-containing coat structure is proposed to be essential for vesicle budding at multiple stages in the yeast secretory pathway.     

A candidate for the cystic fibrosis locus isolated by selection for methylation-free islands

A genomic sequence close to the cystic fibrosis locus with the characteristics of an HTF island has been selectively cloned and characterized. Two markers flanking this sequence, which is conserved throughout mammalian evolution, show a very much greater disequilibrium than that found with any existing marker. A single mutational event accounts for most cases of cystic fibrosis. The sequence is expressed, and is a candidate for the cystic fibrosis gene.     

Chloride impermeability in cystic fibrosis

Cystic fibrosis is the most common fatal genetic disease affecting caucasians and is perhaps best characterized as an exocrinopathy involving a disturbance in fluid and electrolyte transport. A high NaCl concentration in the sweat is characteristic of patients with this disease; the basic physiological reason for this abnormality is unknown. We have microperfused isolated sweat ducts from control subjects and cystic fibrosis patients, and report here results which suggest that abnormally low Cl- permeability in cystic fibrosis leads to poor reabsorption of NaCl in the sweat duct, and hence to a high concentration of NaCl in the sweat.     

A new member of the immunoglobulin superfamily--CTLA-4

The immunoglobulin superfamily is a group of proteins, each made of one or several domains sharing key structural features with either the variable (V) or the constant (C) immunoglobulin domains. It includes such functionally important members as the immunoglobulins themselves, major histocompatibility complex (MHC) class I and class II and T-cell receptor (TCR) molecules. Several members of this superfamily are expressed on lymphocytes where they are membrane-bound and capable of interactions with other members of the family, thus taking part in cell-cell recognition. In screening mouse cytolytic-T-cell-derived cDNA libraries, we came across cDNA clones defining a sequence, CTLA-4, which could encode a 223-amino-acid protein clearly belonging to the immunoglobulin superfamily. It consists of one V-like domain flanked by two hydrophobic regions, one of which has a structure suggestive of membrane anchoring. CTLA-4 is mainly expressed in activated lymphocytes and is coinduced with T-cell-mediated cytotoxicity in inducible models of this process. The mouse ctla-4 gene maps to band C of chromosome 1.     

Mediation of meiotic and early mitotic chromosome segregation in Drosophila by a protein related to kinesin.

Contrary to the traditional view that microtubules pull chromosomes polewards during the anaphase stage of meiotic and mitotic cell divisions, new evidence suggests that the chromosome movements are driven by a motor located at the kinetochore. The process of chromosome segregation involves proper arrangement of kinetochores for spindle attachment, followed by spindle attachment and chromosome movement. Mechanisms in Drosophila for chromosome segregation in meiosis differ in males and females, implying the action of different gene products in the two sexes. A product encoded at the claret locus in Drosophila is required for normal chromosome segregation in meiosis in females and in early mitotic divisions of the embryo. Here we show that the predicted amino-acid sequence of this product is related to the heavy chain of kinesin. The conserved region corresponds to the kinesin motor domain and includes the ATP-binding site and a region that can bind microtubules. A second region contains a leucine repeat motif which may mediate protein-subunit interactions necessary for attachment of chromosomes to the spindle. The mutant phenotype of chromosome nondisjunction and loss, and its similarity to the kinesin ATP-binding domain, suggest that the product encoded at claret not only stabilizes chromosome attachments to the spindle, but may also be a motor that drives chromosome segregation in female meiosis.     

Tissue localization and chromosomal assignment of a serum protein that tracks the cystic fibrosis gene

The basic gene defect in the autosomal recessive disorder cystic fibrosis has not been identified, and no firm linkage of the disorder to any other marker has been reported. However, a serum protein abnormality present in unaffected heterozygotes as well as in affected homozygotes has been described, and immunological quantitation of this protein, termed cystic fibrosis antigen, allows the three genotypes to be distinguished. We show here that an immunologically indistinguishable protein is present at high concentrations in granulocytes from normal and cystic fibrosis individuals as well as in myeloid leukaemia cells. Somatic cell hybrids between the mouse myeloid stem-cell line WEHI-TG and myeloid leukaemia cells express cystic fibrosis antigen only when human chromosome I is present.