Delta F508 Mutation Research Articles

Neonatal screening for the cystic fibrosis main mutation delta F508 in Estonia.

In this pilot study the frequency of delta F508 mutation carriers, their geographic distribution, and the prevalence of cystic fibrosis (CF) in Estonia were investigated. During the screening programme 7396 newborns were tested for delta F508 mutation and 88 were found to carry this deletion. The mean frequency of delta F508 mutation carriers in Estonia was thus estimated as 1 out of 84 live births. In eight separate districts of Estonia the heterozygote frequencies differed significantly (p = 0.0369), with the highest incidence (1:36) on the Baltic Sea islands and western coastal region and with the lowest in south eastern parts of Estonia.

Limited proteolysis as a probe for arrested conformational maturation of delta F508 CFTR.

Deletion of phenylalanine 508 (delta F508) in the cystic fibrosis transmembrane-conductance regulator (CFTR) prevents the otherwise functional protein from reaching the plasma membrane and is the leading cause of cystic fibrosis. Indirect evidence suggests that the mutant protein, delta F508 CFTR, is misfolded. We address this issue directly, using comparative limited proteolysis of CFTR at steady steady state and during biosynthesis in the native microsomal environment. Distinct protease susceptibilities suggest that cytosolic domain conformations of wild type and delta F508 CFTR differ, not only near F508, but globally. Moreover, delta F508 CFTR proteolytic cleavage patterns were indistinguishable from those of the early folding intermediate of wild type CFTR. The results suggest that the delta F508 mutation causes the accumulation of a form of the protein that resembles an intermediate in the biogenesis of the wild type CFTR, rather than induces the production of non-native variant.

Antibiotic accumulation and membrane trafficking in cystic fibrosis cells.

Cystic fibrosis (CF) results from mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) which is a regulated chloride channel. The deltaF508 mutation prevents the post-translational glycosylation and membrane insertion of the protein. Severe disease follows, with the formation of a viscous mucus and subsequent chronic bacterial infection of the lungs, necessitating frequent, and often long, periods of antibiotic treatment. The pharmacokinetics of antibiotics in CF patients are abnormal, with lower blood serum levels and higher clearance rates which have never been satisfactorily explained. We found that accumulation of gentamicin in nasal polyp tissue non-CF cells was subject to regulation by the effectors and inhibitors of CFTR function; regulation was lost in deltaF508 CF cells and accumulation was more than doubled because of the inhibition of exocytosis.

Allele dropout in polar bodies and blastomeres.

Because allele dropout (ADO) is frequently observed in single-cell polymerase chain reaction analysis, it is important to develop a method for efficient detection of ADO, in order to avoid possible misdiagnosis in preimplantation diagnosis. We introduced a simultaneous amplification of mutant genes and linked polymorphic markers, such as a 4-bp repeat (GATT) at the 3' end of intron 6 in the cystic fibrosis (CF) gene and a short tandem repeat at the 5' end of the beta-globin gene. Three types of single heterozygous cells were studied for the amplification of both alleles, including 150 blastomeres, 1615 fibroblasts, and 170 first polar bodies, obtained from patients at risk for having children with cystic fibrosis (delta F-508 mutation) or sickle cell disease. ADO rates of as high as 33.3% for delta F-508 mutation and 22.8% for beta-globin gene were observed in single blastomeres, compared to 7.1 and 7.7% in single fibroblasts and 5.9 and 9.6% in first polar bodies, respectively. The application of simultaneous amplification of the above linked polymorphic markers allowed detection of more than half of the cases of ADO in blastomeres (19.4% for cystic fibrosis and 12.3% for beta-globin gene) and almost all ADOs in polar bodies, particularly when the two-step sequential analysis of the first and second polar body was applied in preimplantation diagnosis of single gene disorders. Simultaneous amplification of linked polymorphic markers in single-cell DNA analysis of single-gene defects is an efficient method for avoiding the risk of misdiagnosis in preimplantation diagnosis.

Dysregulation of proteoglycan production by intrahepatic biliary epithelial cells bearing defective (delta-f508) cystic fibrosis transmembrane conductance regulator.

Hepatic dysfunction in cystic fibrosis (CF) has been attributed to accumulation of viscous mucoid secretions in intrahepatic bile ducts. The purpose of our study was to compare glycoconjugate secretion by intrahepatic biliary epithelial (IBE) cells derived from normal livers and livers of CF patients with the delta F508 mutation of the cystic fibrosis transmembrane conductance regulator (CFTR). Confluent cells were incubated with 3H-glucosamine (GlcN) for 16 hours, and radiolabeled macromolecules were analyzed for the amount and type of glycoconjugates. Incorporation of 3H-GlcN into macromolecular glycoconjugates was two- to threefold higher in CF cells versus normals, as was uptake of 3H-Glcn into the cytoplasm of CF cells. Gel exclusion chromatography on Sepharose Cl 4B revealed that the secreted glycoconjugates from CF cells eluted entirely in the excluded fraction (molecular weight > 2 x 10(6)), while, in the normal cells, 60% of the glycoconjugates eluted as lower-molecular-weight species. The high-molecular-weight glycoconjugates in both CF and normal cells were identified as chondroitin sulfates, as evidenced by susceptibility to beta elimination, chondroitinase digestion, and amino acid composition. Western blotting of IBE cell secretions with a polyclonal antibody to chondroitin sulfate revealed proteoglycan bands at 100 and 210 kd. Our results indicate that secretion of chondroitin sulfate is markedly increased in CF biliary epithelium in vitro compared with non-CF cells. Increased uptake of precursor 3H-GlcN may contribute to enhanced glycosylation of chondroitin sulfate in CF cells.

Preimplantation Diagnosis of Single Gene Disorders by Two-Step Oocyte Genetic Analysis Using First and Second Polar Body

Previous work on preimplantation genetic diagnosis (PGD) of single gene disorders by the first polar body (IPB) analysis has demonstrated that the genotype of a considerable number of embryos resulting from heterozygous oocytes cannot be predicted without testing their second PB (IIPB). To overcome this limitation we introduce a two-step DNA analysis of oocytes using both IPB and IIPB to identify hemizygous mutation-free oocytes following the second meiotic division. In the application of the approach to PGD of cystic fibrosis (CF) Delta F-508 mutation, sickle cell disease, and hemophilia B, 80 oocytes were studied by both PBs, resulting in the identification and transfer of 32 homozygous normal embryos. A follow-up genotyping of 52 embryos, resulting from oocytes tested by both IPB and IIPB demonstrated the accuracy of the predicted genotypes. In addition to a nested PCR analysis of the mutant genes in PBs and resulting embryos, simultaneous amplification of different polymorphic markers was performed, demonstrating the reliability of the two-step polar body analysis of oocytes.

7a The use of epididymal sperm for the treatment of male infertility

Congenital absence of the vas deferens (CAV), failed vasoepididymostomy, and all irreparable obstructions were once very frustrating conditions, because these patients have normal spermatogenesis, but were previously untreatable (Silber SJ, Ord T, Balmaceda J et al (1990) New England Journal of Medicine 323: 1788-1792). Microsurgical epididymal sperm aspiration (MESA) together with in vitro fertilization was introduced in 1985 and in 1988 to treat these cases, but only modest success was achieved (Temple-Smith PD, Southwick GJ, Yates CA et al (1985) Journal of In Vitro Fertilization and Embryo Transfer 2: 119-122; Silber SJ, Asch R, Balmaceda J et al (1988) Fertility and Sterility 500: 525-528; Silber SJ, Ord T, Balmaceda J et al (1990) New England Journal of Medicine 323: 1788-1792). It is very difficult to predict in which cases epididymal sperm will fertilize and in which cases it will not. The reason for this problem may be either sperm maturation defects that are poorly defined or senescent and pathological changes caused by the obstruction. Thus, intracytoplasmic sperm injection (ICSI) became necessary to achieve consistently good results with MESA. The only factor in these couples which affected success had nothing to do with the sperm origin or quality but rather was simply the age and ovarian reserve of the wife. Clearly, whether sperm was from the epididymis or the testis, frozen or fresh, or whether the male had CAV, or irreparable obstruction from a variety of other causes, made no meaningful difference. The cystic fibrosis genotype, the sperm morphology and the quality of motility had no impact. Furthermore, the only factor in the wife that mattered was her age. It is crucial to screen for CF in both the husband with CAV and also his wife. If the wife is negative for any of the 36 common CF mutations, we feel that it is quite safe to perform MESA-ICSI on the couple. The chances of a male offspring's having CAV are very remote, and the chances of the child's having cystic fibrosis are probably less than in a normal, unscreened population. However, if the wife turns out to be a CF carrier herself (4% incidence in the general population), the couple can still undergo MESA-ICSI, but pre-implantation embryo diagnosis would then be mandatory. We have published the first case of successful pre-implantation embryo diagnosis in a CAV-MESA case in which both partners were carriers of the delta F508 mutation (Liu J, Lissens W, Silber SJ et al (1994) Journal of the American Medical Association 23: 1858-1860. We require this as a routine approach whenever the female is discovered, on screening, to be a CF carrier.

In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing delta F508-CFTR.

The most common cystic fibrosis transmembrane conductance regulator mutation, delta F508-CFTR, is a partially functional chloride channel that is retained in the endoplasmic reticulum and degraded. We hypothesize that a known transcriptional regulator, sodium 4-phenylbutyrate (4PBA), will enable a greater fraction of delta F508-CFTR to escape degradation and appear at the cell surface. Primary cultures of nasal polyp epithelia from CF patients (delta F508 homozygous or heterozygous), or the CF bronchial epithelial cell line IB3-1 (delta F508/W1282X) were exposed to 4PBA for up to 7 d in culture. 4PBA treatment at concentrations of 0.1 and 2 mM resulted in the restoration of forskolin-activated chloride secretion. Protein kinase A-activated, linear, 10 pS chloride channels appeared at the plasma membrane of IB3-1 cells at the tested concentration of 2.5 mM. Treatment of IB3-1 cells with 0.1-1 mM 4PBA and primary nasal epithelia with 5 mM 4PBA also resulted in the appearance of higher molecular mass forms of CFTR consistent with addition and modification of oligosaccharides in the Golgi apparatus, as detected by immunoblotting of whole cell lysates with anti-CFTR antisera. Immunocytochemistry in CF epithelial cells treated with 4PBA was consistent with increasing amounts of delta F508-CFTR. These data indicate that 4PBA is a promising pharmacologic agent for inducing correction of the CF phenotype in CF patients carrying the delta F508 mutation.

In vivo activation of CFTR-dependent chloride transport in murine airway epithelium by CNP.

Inhibitors of guanosine 3',5'-cyclic monophosphate (cGMP)-inhibited phosphodiesterases stimulate Cl- transport across the nasal epithelia of cystic fibrosis mice carrying the delta F508 mutation [cystic fibrosis transmembrane conductance regulator (CFTR) (delta F/delta F)], suggesting a role for cGMP in regulation of epithelial ion transport. Here we show that activation of membrane-bound guanylate cyclases by C-type natriuretic peptide (CNP) stimulates hyperpolarization of nasal epithelium in both wild-type and delta F508 CFTR mice in vivo but not in nasal epithelium of mice lacking CFTR [CFTR(-/-)]. With the use of a nasal transepithelial potential difference (TEPD) assay, CNP was found to hyperpolarize lumen negative TEPD by 6.1 +/- 0.6 mV in mice carrying wild-type CFTR. This value is consistent with that obtained with 8-bromoguanosine 3',5'-cyclic monophosphate (6.2 +/- 0.9 mV). A combination of the adenylate cyclase agonist forskolin and CNP demonstrated a synergistic ability to induce Cl- secretion across the nasal epithelium of CFTR(delta F/delta F) mice. No effect on TEPD was seen with this combination when used on CFTR(-/-) mice, implying that the CNP-induced change in TEPD in CFTR(delta F/delta F) mice is CFTR-dependent.

Differential stimulation of cytosolic phospholipase A2 by bradykinin in human cystic fibrosis cell lines.

Tracheal epithelial cells and skin fibroblasts from different cystic fibrosis (CF) patients bearing the deltaF508 mutation of cystic fibrosis transmembrane conductance regulator (CFTR) released more arachidonic acid in response to bradykinin than do other CF and normal cells. Immortalized tracheal epithelial cell lines were used as models to study the mechanisms of this dysregulation. An 85 kD cytosolic phospholipase A2 (cPLA2) was found in these cells and bradykinin increased its binding to membranes of deltaF508 cells (CFT-2) but not to those of a double heterozygous CF cells (CFT-1), or of control cells (NT-1). The expression of G alpha(q)/11 protein was also increased in deltaF508 cells, with increased stimulation of phosphatidylinositol diphosphate-specific phospholipase C (PLC) by bradykinin, and an early, transient activation of mitogen-activated protein (MAP) kinase. As the binding of cPLA2 to membranes is Ca2+-dependent, the increased coupling to PLC could cause the hypersensitivity to bradykinin. Comparison of the effects of bradykinin to those observed with thapsigargin, an inhibitor of calcium reuptake, suggests that the increase of intracellular calcium is not the only mechanism involved in arachidonic acid release by bradykinin in deltaF508 cells. The lack of effect of calcium ionophore A23187 or TPA on arachidonic acid release from any of the cell lines suggested that activation needs a PKC-independent cPLA2 phosphorylation step, perhaps via MAP kinase activation. The binding of cPLA2 to membranes after bradykinin stimulation still occurred in CFT2 cells (deltaF508) homogenized in EDTA, suggesting that a membrane component plus increased intracellular calcium influenced cPLA2 anchoring to membranes. The defective processing of deltaF508 CFTR seems to increase cPLA2 stimulation by bradykinin, since the bradykinin-stimulated release of arachidonic acid is reversed by growing cells at 28 degrees C for 48 h. The deltaF508 mutation of CFTR appears to increase the stimulation of cPLA2 by Gq-mediated receptors in a PKC-independent and MAP kinase-dependent manner. Hence normal CFTR, or normally processed deltaF508 CFTR, inhibit cPLA2 stimulation. The greater reactivity of deltaF508 CFTR cells to inflammatory mediators might be part of the increased sensitivity of CF patients to lung inflammation.

Sensitive bioaffinity assays with individual microparticles and time-resolved fluorometry

Future immunoassays and nucleic acid hybridization assays will be performed in miniaturized formats that utilize microchips or microparticles. This will require a sensitive detection technology that allows spatial resolution. By using fluorescent europium chelates and time-resolved microfluorometry, one can detect 11,000 europium molecules on individual microparticles. In a miniaturized noncompetitive immunoassay of prostate-specific antigen (PSA), we quantitatively detected 5 ng/L (0.05 amol per particle) of the analyte on an individual microparticle with excellent precision over the whole measurement range (CV <10%). Using a hybridization assay, we also could detect the deltaF508 mutation for cystic fibrosis on individual microparticles. Consequently, fluorescent lanthanide chelate labels and time-resolved microfluorometry qualify as the next generation of technology in this field.

Detection of 100% of the CFTR mutations in 63 CF families from Tyrol.

We identified 100% of the CFTR gene mutations, including three novel mutations, in 126 unrelated cystic fibrosis chromosomes from Tyrol, Austria. The frequency of the major mutation deltaF508 (74.6%) was not significantly different in Tyrolian CF-patients than in patients from Bavaria (71.0%) and Middle- and Northern Germany (71.9%), but was significantly higher than in patients from Styria (58.1%) or Northern Italy (47.6%). Interestingly, the distribution of the next most frequent mutations, R1162X (8.7%) 2183AA-->G, 2789+5G-->A and G542X (2.4% each), was more similar to the distribution of these mutations among CF-patients from Northern Italy than to those from Styria, Bavaria or Middle- and Northern Germany. Nine further mutations occurred once or twice. One of these, the missense mutation M1101K, is rare worldwide but very frequent in the Hutterite brethren, a small founder population which came from Southern Austria to Northern America. Three other different mutations (deltaL453, 1874insT and 4108delT) were present in single Tyrolian families and have not been described before. The identification of 100% of CFTR gene mutations in a particular CF population demonstrates the power of genetic analysis for the diagnosis and counselling of CF families in this restricted geographical area of Austria. Our study provides evidence for a closer genetic relation between CF patients from Tyrol and those from Bavaria or Middle- and Northern Germany as well as Northern Italy, than between CF patients from the two Austrian states Tyrol and Styria.

Cystic fibrosis gene mutation (deltaF508) is associated with an intrinsic abnormality in Ca2+-induced arachidonic acid release by epithelial cells.

The mechanism(s) of chronic airway inflammation in cystic fibrosis (CF) remains poorly understood. We studied Ca2+-induced release of arachidonic acid (AA), a precursor of proinflammatory lipid mediators, in epithelial cell lines with the deltaF508 mutation in CF transmembrane conductance regulator (CFTR) gene and in those lacking this mutation or cells in which this mutation was corrected by a functional CFTR gene transfer. We found that: (i) the mutant cells manifested an abnormally high Ca2+-induced AA release as compared to controls, (ii) AA release appeared to be catalyzed by a phospholipase A2 (PLA2) but not by phospholipase C followed by diacylglycerol lipase, and (iii) either correction of the CFTR-mutation or inhibition of PLA2 activity rectified this AA release abnormality. Taken together, our results suggest that CFTR mutation is associated with an intrinsic abnormality in AA release by epithelial cells carrying the deltaF508 mutation and suggest that the mechanism of chronic airway inflammation in CF, at least in part, involves this abnormality. These results also partly explain the effectiveness of high-dose ibuprofen therapy in arresting the progression of destructive lung disease in CF. Furthermore, they raise the possibility that correction of abnormal AA release by inhibiting PLA2 activity may improve the therapeutic benefits of ibuprofen.

Localization and Suppression of a Kinetic Defect in Cystic Fibrosis Transmembrane Conductance Regulator Folding

A growing body of evidence indicates that the most common cystic fibrosis-causing mutation, DeltaF508, alters the ability of the cystic fibrosis transmembrane conductance regulator (CFTR) protein to fold and transit to the plasma membrane. Here we present evidence that the DeltaF508 mutation affects a step on the folding pathway prior to formation of the ATP binding site in the nucleotide binding domain (NBD). Notably, stabilization of the native state with 4 mM ATP does not alter the temperature-dependent folding yield of the mutant DeltaF508 NBD1 in vitro. In contrast, glycerol, which promotes DeltaF508-CFTR maturation in vivo, increases the folding yield of NBD1DeltaF and reduces the off pathway rate in vitro, although it does not significantly alter the free energy of stability. Likewise a second site mutation, R553M, which corrects the maturation defect in vivo, is a superfolder which counters the effects of DeltaF508 on the temperature-dependent folding yield in vitro, but does not significantly alter the free energy of stability. A disease-causing mutation, G551D, which does not alter the maturation of CFTR in vivo but rather its function as a chloride channel, and the S549R maturation mutation have no discernible effect on the folding of the domain. These results demonstrate that DeltaF508 is a kinetic folding mutation that affects a step early in the process, and that there is a significant energy barrier between the native state and the step affected by the mutation precluding the use of native state ligands to promote folding. The implications for protein folding in general are that the primary sequence may not necessarily simply define the most stable native structure, but rather a stable structure that is kinetically accessible.

The nucleotide binding folds of the cystic fibrosis transmembrane conductance regulator are extracellularly accessible.

Analysis of the primary sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) has suggested the presence of two predicted cytoplasmic regions of the protein which are thought to be nucleotide binding folds (NBF1 and NBF2). Previous studies have shown that purified recombinant NBF1 can form anion conducting channels in planar phospholipid bilayers [Arispe et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1539-1543] and that the bacterial His P protein (analogous to a NBF) can be extracellularly labeled with a membrane-impermeant reagent [Baichwal et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 620-624]. Based on these observations, it is reasonable to hypothesize that the NBFs from the CFTR are associated with the plasma membrane and have extracellularly-accessible regions. Direct biochemical evidence for this was obtained by determining the ability of the individual NBFs, expressed in intact Hi5 cells, to be chemically modified with the membrane-impermeant reagent NHS-biotin. The results indicate that both NBF1 and NBF2, in intact cells, can be chemically modified by extracellular NHS-biotin. The negative control, the cytosolic enzyme beta-galactosidase, was not significantly labeled under these conditions, verifying the extracellular nature of the labeling reaction. When the surface-accessibility of a NBF1 construct containing the CF-causing mutation deltaF508 was analyzed, similar labeling was observed, suggesting that the mutation does not affect this aspect of the CFTR's structure. These data support the conclusion that, under certain conditions, the NBFs are capable of spanning the plasma membrane, perhaps constituting a portion of the CFTR's ion conducting channel.

8-Cyclopentyl-1,3-dipropylxanthine and Other Xanthines Differentially Bind to the Wild-Type and ΔF508 Mutant First Nucleotide Binding Fold (NBF-1) Domains of the Cystic Fibrosis Transmembrane Conductance Regulator

Cystic fibrosis is an autosomal recessive disorder affecting chloride transport in pancreas, lung, and other tissues, which is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Certain alkyl xanthines such as CPX (8-cyclopentyl-1,3-dipropylxanthine) stimulate Cl- efflux from cells bearing the delta F508 genotype common to most cases of cystic fibrosis. We have hypothesized that the CFTR molecule itself might be the site for CPX action, perhaps in the region of the first nucleotide binding fold (NBF-1) domain. Therefore, to test this hypothesis directly we have used a rapid membrane filtration assay to measure the kinetics of association and dissociation of [3H]CPX to both recombinant NBF-1 and recombinant NBF-1 bearing the delta F508 mutation. We report that [3H]CPX binds with higher affinity to the delta F508-NBF-1 of CFTR (Kd = 1.0 nM) than to the wild-type NBF-1 of CFTR (Kd = 17.0 nM). These Kd values were calculated from direct measurements of the association and dissociation rate constants. The rate constants for the dissociation reaction of the wild-type NBF-1 and delta F508-NBF-1 of CFTR were not different from each other. However, the corresponding rate constants for the association reaction were k(+1) (NBF-1) = 4.7 +/- 0.9 x 10(4) M(-1) s(-1) and k(+1) (delta F508-NBF-1) = 1.6 +/- 0.3 x 10(5) M(-1) s(-1), respectively. These Kd values were corroborated by equilibrium-binding experiments, which gave very similar values. We have also measured the relative displacement of various xanthines from both wild-type NBF-1 and delta F508-NBF-1, in anticipation that the order of potencies for binding might parallel the action of the different xanthines on CF cells. For wild-type NBF-1, the rank order was DA-CPX > DAX > CPX > caffeine > adenosine >> IBMX > 2-thioCPX. For delta F508-NBF-1, the rank order was DAX > CPX > caffeine > DA-CPX > adenosine >> IBMX > 2-thioCPX. These relative potencies show close parallels with previously observed relative potencies of these drugs on CF cells, and thus lend strong support to the hypothesis that the mechanism of action on CF cells may involve direct interaction with the CFTR molecule itself.

Analysis of 16 cystic fibrosis mutations in Mexican patients

We carried out molecular analysis of 80 chromosomes from 40 unrelated Mexican patients with a diagnosis of cystic fibrosis. The study was performed in two PCR steps: a preliminary one to identify mutation delta F508, the most frequent cause of cystic fibrosis worldwide, and the second a reverse dot-blot with allele-specific oligonucleotide probes to detect 15 additional common mutations in the Caucasian population. A frequency of 45% for delta F508 was found, making it the most common in our sample of Mexican patients. Another five mutations (G542X, 3,849 + 10 kb C-->T, N1303K, SN549N, and 621 + 1 G-->T) were detected, and those accounted for 11.25%. The remaining mutations (43.75%) were undetectable with the methodology used.

Cystic fibrosis transmembrane conductance regulator: the first nucleotide binding fold targets the membrane with retention of its ATP binding function.

Most cases of cystic fibrosis are caused by a single deletion mutation (deltaF508) within the first nucleotide binding fold (NBF1) of the CFTR protein (cystic fibrosis transmembrane conductance regulator). NBF1 is classically defined as amino acid residues phenylalanine 433 through serine 589, encoded by exons 10-12, and only part of exon 9, of the CFTR gene. This assignment is based on sequence homology of this region of the CFTR protein with that of other nucleotide binding proteins. Here, we report that when the complete modular unit encoded precisely by exons 9-12 is expressed in Escherichia coli as glycine 404 through serine 589, i.e., as [G404-N432]NBF1 or as deltaF508[G404-N432]NBF1, the resultant proteins target the cytoplasmic membrane. Significantly, [G404-N432]NBF1 is readily labeled from the outside of intact E. coli spheroplasts with the water soluble, membrane impermeable probe Biotin-X-NHS, sulfosuccinimidyl-6-(biotinamido)-hexanoate. Similar findings were observed with the disease causing mutant deltaF508[G404-N432]NBF1. Three different control experiments which involved (1) assays for known cytosolic E. coli enzymes, (2) immuno-gold electron microscopy with antibody having an epitope for the biotin moiety, and (3) tests for biotinylation of the cytosolic component, Enzyme 1 of the glucose phosphotransferase system, demonstrated that the spheroplasts used in this study are neither leaky nor permeable to Biotin-X-NHS. In addition, membrane-associated [G404-N432]NBF1, upon solubilization with Triton X-100, was found to bind to an ATP-agarose column and be released therefrom by elution with ATP, emphasizing retention of a native-like structure. In sharp contrast, NBF1 localizes to the cytosol when the [G404-N432]-N-terminal region is replaced with the maltose binding protein. The novel findings reported here implicate a role of the N-terminal region of NBF1 in its subcellular localization and are directly relevant to our understanding of the membrane structure, function, and trafficking of CFTR.

CAMP and genistein stimulate HCO3- conductance through CFTR in human airway epithelia.

We studied the role of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel as an HCO3- conductor during adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation in human airway epithelial cell lines. HCO3- or Cl- currents across the apical membrane were measured in the presence of an HCO3- or Cl- gradient under short-circuit conditions in intact and alpha-toxin-permeabilized monolayers, which allowed manipulation of the intracellular regulators cAMP and ATP. CFTR as the current carrier for HCO3- was identified by 1) stimulation by cAMP, 2) ATP dependence, 3) blocker sensitivity, 4) stimulation by genistein, and 5) lack of stimulation in CF epithelia bearing mutated delta F508 CFTR. In pulmonary alpha-toxin-permeabilized Calu-3 monolayers, cytosolic addition of 100 microM cAMP stimulated apical HCO3- currents from -9.4 +/- 1.6 to -31.1 +/- 3.9 microA/cm2 (n = 18), and apical Cl- currents increased from -54.1 +/- 7.1 to -203.2 +/- 15.4 microA/cm2 (n = 27). Average relative permselectivity for HCO3- vs. Cl- was approximately 15%. Absence of cytosolic ATP resulted in loss of cAMP stimulation of HCO3- and Cl- currents. Genistein (50 microM), which has been proposed to inhibit phosphatases controlling apical CFTR, as well as the alkaline phosphatase inhibitor (-)-p-bromotetramisole (1 mM) further activated cAMP-stimulated HCO3- and Cl- currents. Activated currents remained stimulated on removal of cAMP, suggesting inhibition of a protein phosphatase by genistein and bromotetramisole. The Cl- channel blockers glibenclamide (300 microM) and N-phenylanthranilic acid (5 mM), but not 4,4'-dinitro-2,2'-stilbenedisulfonic acid (100 microM), inhibited cAMP- and genistein-stimulated HCO3- and Cl- currents. Blocker effects were absent in human CF tracheal cells homozygous for the delta F508 mutation of CFTR (CFT1); Cl- and HCO3- currents were rescued in CFT1 cells recombinantly expressing wild-type CFTR. Thus CFTR functions as a HCO3- and Cl- conductor, and genistein and bromotetramisole maximize CFTR activity in airway epithelial cells.

Molecular genetic studies in monogenic and polygenic human diseases

The main goal of this study was to determine and characterise the types of mutations in two monogenic human disorders: cystic fibrosis (CF) and Duchenne/Becker muscular dystrophy (DMD, BMD) and the susceptibility allele frequency in a polygenic disease: type I insulin-dependent diabetes mellitus (IDDM). After analysing 220 chromosomes for mutations in the CF (Cystic Fibrosis Transmembrane Conductance Regulator = CFTR) gene, delta F508 mutation was most abundant (41%) and out of the non-delta F508 CF mutations 5% was identified as G542X, G551D, R553X, N1303K and W1282X. The CF haplotype analysis by using linked markers to the CFTR gene revealed that the CF "B" haplotype occurred in 66.7% of patients, and this haplotype was 57.2% in patients carrying the delta F508 mutation. Prenatal genetic diagnosis for CF was performed in 10 fetuses: 3 were affected, 6 were carriers, and 1 without any CF mutation. Fifty % of 66 patients with DMB/BMD muscular dystrophy had one or more exon deletions in the dystrophin gene. Eighty-five % of the deletions occurred at the 3' and 15% at the 5' end of the gene. Out of the three prenatal diagnosis in one case DMD was substantiated. Thirty-six % of 50 patients with IDDM possessed four, 44% three and 20% two susceptibility markers in the HLA-DQA1, -DQB1 region. The onset of the disease correlated with the number of susceptibility alleles.