During fetal development, the lung is filled with fluid that is secreted by an active Cltransport promoting lung growth. The basolateral Na+,K+,2Cl- cotransporter (NKCC1) participates in Cl- secretion. The apical Cl- channels responsible for secretion are unknown but studies suggest an involvement of the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is developmentally regulated with a high expression in early fetal development and a decline in late gestation. Perinatal lung transition is triggered by hormones that stimulate alveolar Na+ channels resulting in fluid absorption. Little is known on how hormones affect pulmonary Cl- channels. Since the rise of fetal cortisol levels correlates with the decrease in fetal CFTR expression, a causal relation may be assumed. The aim of this study was to analyze the influence of glucocorticoids on pulmonary Cl- channels. Alveolar cells from fetal and adult rats, A549 cells, bronchial Calu-3 and 16HBE14o- cells, and primary rat airway cells were studied with real-time quantitative PCR and Ussing chambers. In fetal and adult alveolar cells, glucocorticoids strongly reduced Cftr expression and channel activity, which was prevented by mifepristone. In bronchial and primary airway cells CFTR mRNA expression was also reduced, whereas channel activity was increased which was prevented by LY-294002 in Calu-3 cells. Therefore, glucocorticoids strongly reduce CFTR expression while their effect on CFTR activity depends on the physiological function of the cells. Another apical Cl- channel, anoctamin 1 showed a glucocorticoid-induced reduction of mRNA expression in alveolar cells and an increase in bronchial cells. Furthermore, voltage-gated chloride channel 5 and anoctamine 6 mRNA expression were increased in alveolar cells. NKCC1 expression was reduced by glucocorticoids in alveolar and bronchial cells alike. The results demonstrate that glucocorticoids differentially modulate pulmonary Clchannels and are likely causing the decline of CFTR during late gestation in preparation for perinatal lung transition.

Ausgangslage: Die genetischen Grundlagen der chronischen Pankreatitis sind zum heutigen Zeitpunkt nur unzureichend erforscht. Mutationen im Gen des Serinprotease-Inhibitors Kazal Type 1 (SPINK1) und heterozygote Mutationen im CFTR-Gen wurden in zahlreichen Untersuchungen gehäuft bei Patienten mit chronischer Pankreatitis nachgewiesen. Methodik: Es wurden retrospektiv anhand der Daten der Pankcourse Studie (2004-2007) Untersuchungen bei Patienten mit chronischer Pankreatitis zur Häufigkeit von SPINK1- und CFTR-Mutationen sowie zum Manifestationszeitpunkt der Erkrankung durchgeführt. In Fall-Kontroll-Analysen wurde untersucht, ob sich Unterschiede in den jeweiligen Krankheitsverläufen nachweisen lassen. Ergebnisse: Eine heterozygote SPINK1-Mutation (N34S) konnte bei 11,5% und eine CFTR-Mutationen bei 24% der untersuchten Patienten nachgewiesen werden. Bei Patienten mit SPINK1-Mutation fand sich im Gegensatz zu Patienten mit CFTR-Mutation eine signifikant frühere Krankheitsmanifestation als bei Patienten ohne Mutationsnachweis. Patienten mit SPINK1-Mutation mussten zudem seltener und später operiert werden als Patienten ohne Mutation. Bei Patienten mit CFTR-Mutation zeigte sich ein signifikant früheres Auftreten von Stenosierungen und Konkrementen des D. pancreaticus im Vergleich zur Kontrollgruppe. Schlussfolgerung: Die ätiologische Bedeutung von SPINK1- und CFTR-Mutationen konnte bestätigt werden. Es fanden sich einzelne Hinweise auf einen durch die jeweilige Mutation verursachten charakteristischen Krankheitsverlauf, was durch weitergehende Untersuchungen bestätigt werden muss.

Integral membrane proteins (IMPs) assume critical roles in cell biology and are key targets for drug discovery. Given their involvement in a wide range of diseases, the structural and functional characterization of IMPs are of significant importance. However, this remains notoriously challenging due to the difficulties of stably purifying membrane-bound, hydrophobic proteins. Compounding this, many diseases are caused by IMP mutations that further decrease their stability. One such example is cystic fibrosis (CF), which is caused by misfolding or dysfunction of the epithelial cell chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Roughly 70% of CF patients world-wide harbor the ΔF508-CFTR mutation, which interrupts CFTR’s folding, maturation, trafficking and function. No existing treatment sufficiently addresses the consequences of ΔF508, and the substantial instability that results from this mutation limits our ability to study ΔF508-CFTR in search of better treatments. To that end, my colleagues at Sanofi generated homology models of full-length wild-type and ΔF508-CFTR +/- second-site suppressor mutations (SSSMs) V510D and R1070W, and performed molecular dynamics (MD) simulations for each model. Using information obtained from this analysis, I tested several hypotheses on the mechanism by which ΔF508 destabilizes full-length CFTR and how SSSMs suppress this effect. Leveraging studies of the purified NBD1 subdomain and of full-length CFTR in a cellular context, I confirmed the prediction of a key salt-bridge interaction between V510D and K564 important to second-site suppression. Furthermore, I identified a novel class of SSSMs that support a key prediction from these analyses: that helical unraveling of TM10, within CFTR’s second transmembrane domain, is an important contributor to ΔF508-induced instability. In addition, I developed a detergent-free CFTR purification method using styrene-maleic acid (SMA) copolymer to extract the channel directly from its cell membrane along with the surrounding lipid content. The resulting particles were stable, monodisperse discs containing a single molecule of highly-purified CFTR. With this material, I optimized grid preparation techniques and carried out cryo-EM structural analysis of WT-hCFTR which resulted in 2D particle class averages which were consistent with an ABC transporter shape characteristic of CFTR, and a preliminary 3D reconstruction. This result establishes a foundation for future characterization of ΔF508-CFTR in its native state. I have also applied this SMA-based purification method to the facilitated glucose transporter GLUT1 (SLC2A1). SLC2A1 mutations contribute to a rare and developmentally debilitating disease called GLUT1-deficiency syndrome. Using SMA, I successfully extracted GLUT1 in its native state. With the application of this method, I was able to purify endogenous GLUT1 from erythrocytes, in complex with several associated proteins as well as the surrounding lipids, in its monomeric, dimeric and tetrameric forms without the use of cross-linking or chimeric mutations. These results point to the potential for studying isolated IMPs without the use of destabilizing detergents and thereby offer a pathway to analysis of wild-type and mutant membrane protein structure, function and pharmacodynamics.

No description available.

<p>Fluid secretion is essential to organ development and function, yet relatively little is known about the roles of fluid secretion <italic>in vivo</italic>. Early in development, fluid secretion plays important roles during the process of lumen formation and is necessary for organ homeostasis throughout life. A human disease, cystic fibrosis (CF) is caused by loss of cystic fibrosis transmembrane conductance regulator (CFTR) function, a chloride channel and key regulator of vertebrate fluid secretion. CFTR regulates fluid secretion by governing ion transport and osmotic gradients across epithelia. </p><p>To identify the developmental requirements for <italic>cftr</italic> function, we generated <italic>cftr</italic> mutant zebrafish using transcription activator like effector nucleases (TALENs). In <italic>cftr</italic> mutant zebrafish, we observed defects in the specification of left-right (LR) asymmetry. In the zebrafish, LR asymmetry is specified in part by directional fluid flow within a ciliated structure, Kupffer's vesicle (KV). Using live imaging of several transgenic markers in KV, we determined that lumen expansion is impaired in <italic>cftr</italic> mutants, which prevents directional fluid flow necessary for KV function. To examine <italic>cftr</italic> expression, we generated bacterial artificial chromosome (BAC) transgenic zebrafish expressing fluorescent Cftr fusion proteins under the control of the <italic>cftr</italic> promoter. These transgenes express Cftr within the KV epithelium and the protein localizes to the apical membrane. These transgenes rescue the KV function and the specification of LR asymmetry. These studies reveal a new role for <italic>cftr</italic> during KV morphogenesis and function in the zebrafish. </p><p>In the zebrafish pancreas, we found that loss of <italic>cftr</italic> function leads to defects reminiscent of CF including destruction of exocrine tissue and changes in islet morphology. Additionally, we observed exocrine pancreatic destruction by 3 weeks post fertilization (wpf). Analysis of <italic>cftr</italic> BAC expression in the adult and larval zebrafish pancreata revealed that <italic>cftr</italic> is expressed specifically within the ducts, localized to the apical membrane throughout life. Adult <italic>cftr</italic> mutant pancreata developed substantial degeneration of exocrine tissue and experienced reduced growth rates. In contrast, we found that <italic>cftr</italic> is not necessary for the specification or initial development of the larval pancreas. Exocrine and endocrine tissues developed similarly in WT and <italic>cftr</italic> mutant larvae. These results indicate that <italic>cftr</italic>-dependent fluid secretion is important for maintenance of the zebrafish pancreas. Altogether, these studies of <italic>cftr</italic> function in KV and the pancreas demonstrate that fluid secretion is an essential component of lumen morphogenesis and organ function.</p> / Dissertation

ABSTRACT INTRODUCTION: Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. The gene mutation profile is extremely heterogeneous and mutations show a variable distribution among population groups. In SA the 3120+1G->A splice site mutation has been found predominantly in Black and Coloured patients. It occurs in Black CF patients at an estimated frequency of 46%. The CF carrier frequency is estimated at 1/34 in Black and 1/55 in Coloured populations, and based on these rates, it is clear that a significant number of Black and Coloured patients remain undiagnosed. Point mutations account for the majority of the mutations that have been found in the CFTR gene. Copy number mutations are, however, increasingly being detected in CF patients through the use of gene dosage-dependant assays. These mutations have been found to occur in the CFTR gene in various African American families and exon rearrangements are thought to account for 1.3% of all CF chromosomes across all populations. AIMS: To use haplotypes to analyse the origin(s) of the 3120+1G->A mutation and the likely frequencies of the remaining unknown mutations. To increase mutation detection in the SA Black and Coloured groups by searching for CFTR gene exons for copy number mutations. METHODS: In patients with at least one copy of the 3120+1G>A mutation haplotype studies will be used to elucidate the origin(s) of this mutation in SA Black and Coloured CF patients, by analyzing pyrosequencing SNP genotype data. In patients with at least one unknown mutation, haplotype studies will reveal the likely relative frequencies of the unknown mutations in these populations. In Black and Coloured CF patients with at least one unknown mutation, a multiplex ligation dependant probe amplification (MLPA) CF kit will be used for the detection of exon copy number mutations. RESULTS: The results of the haplotype data show that there is a G-G-C-G-T-A haplotype, for markers MetD-KM19-J44-T854T-Tub18-J32, associated with the 3120+1G->A mutation in both Black and Coloured patients. Unknown mutation-associated haplotypes indicate that there are two relatively common unknown mutations in each of these populations. MLPA results show that one patient is a carrier of an exon 2 deletion. CONCLUSION: A single origin for the 3120+1G>A mutation in Black and Coloured CF patients is supported by the data. Exon copy number changes in the CFTR gene are not a major mutational mechanism leading to CF in SA Black and Coloured patients.

The most common cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation is δF508 and this causes cystic fibrosis (CF). Animal models that recapitulate the human disease phenotype are critical to understanding pathophysiologic mechanisms in CF and developing therapies. New CF models in the pig and ferret have been generated that develop lung, pancreatic, liver, and intestinal pathologies that reflect disease in CF patients. Species-specific biology in the processing of CFTR has demonstrated that pig and mouse δF508-CFTR proteins are more effectively processed to the apical membrane of airway epithelia than human δF508-CFTR. The processing behavior of ferret wild-type (WT) and δF508-CFTR proteins remain unknown and such information is important to predicting the utility of a δF508-CFTR ferret. To this end, we sought to compare processing, membrane stability, and function of human and ferret WT- and δF508-CFTR proteins in a heterologous expression system using HT1080, HEK293T, BHK21, and Cos7 cells, as well as human and ferret CF polarized airway epithelia. Analysis of the protein processing and stability by metabolic pulse-chase and surface On-Cell Western blots revealed that WT-fCFTR half-life and membrane stability were increased relative to WT-hCFTR. Furthermore, in BHK21, Cos7, and CuFi cells, human and ferret δF508-CFTR processing was negligible, while low levels of processing of δF508-fCFTR could be seen in HT1080 and HEK293T cells. Only the WT-fCFTR, but not δF508-fCFTR, produced functional cAMP-inducible chloride currents in both CF human and ferret airway epithelia. Further elucidation of the mechanism responsible for elevated fCFTR protein stability may lead to new therapeutic approaches to augment CFTR function. These findings also suggest that generation of a ferret CFTRδF508/δF508 animal model may be useful. Furthermore, in the CFTR and CFTR+/+ ferret model we have characterized abnormalities in the bioelectric properties of the trachea, stomach, intestine and gallbladder of newborn CF ferrets. Short circuit current (ISC) analysis of CF and WT tracheas revealed the following similarities and differences: 1) amiloride sensitive sodium currents were similar between genotypes, 2) responses to 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid (DIDS) were ~4-fold greater in CF animals, suggesting elevated baseline chloride transport through non-CFTR channels, and 3) as expected, there was a lack of IBMX/forskolin-stimulated and GlyH-101-inhibited currents in CF animals due to the lack of CFTR. CFTR mRNA and protein was present throughout all levels of the WT ferret and IBMX/forskolin-inducible ISC was only observed in WT animals. Interestingly, IBMX/forskolin-inducible intestinal ISC in WT animals was not inhibited by the CFTR inhibitor GlyH-101 or bumetanide. The luminal pH of the CF ferret stomach was significantly decreased relative to the controls, while both genotypes maintained near neutral pH along the length of the intestine. The WT stomach and gallbladder exhibited significantly enhanced IBMX/forskolin ISC responses and inhibition by GlyH-101 relative to CF samples. These findings demonstrate that multiple organs affected by disease in the CF ferret have bioelectric abnormalities consistent with the lack of cAMP-mediated chloride transport.

Misfolding of membrane proteins as a result of mutations that disrupt their functions in substrate transport across the membrane or signal transduction is the cause of many significant human diseases. Yet, we still have a limited understanding of the direct consequences of these mutations on folding and function - a necessary step toward the rational design of corrective therapeutics. This thesis addresses the gap in understanding the residue-specific implications for folding through a series of experiments that utilize the cystic fibrosis transmembrane conductance regulator (CFTR) as a model in various contexts. We first examined the thermodynamic implications of mutations in the soluble nucleotide binding domain 1 (NBD1) of CFTR. We found that mutations can have a significant effect on thermodynamic stability that is masked in non-physiological conditions. Our studies were then focussed on a membrane-embedded hairpin CFTR fragment comprised of transmembrane segments 3 (TM3) and 4 (TM4) to evaluate the direct effects of mutations on folding in a systematic manner. It was found that the translocon-mediated membrane insertion of helices closely parallels a basic hydrophobic-aqueous partitioning event. This study was then extended to determine residue-specific effects on helix-helix association. We found that this process is not solely dependent on hydropathy, but there is a context dependence of these results with regard to residue position within the helix. Overall, these findings constitute a key step in relating mutation-derived effects on membrane protein folding to the underlying basis of human disease such as cystic fibrosis.

La Fibrosi Quística (FQ; MIM# 219700) és una de les malalties letals més comuns en les poblacions d'origen caucàsic, amb herència autosòmica recessiva. El seu fenotip clínic es caracteritza per l'acumulació de moc viscós en òrgans exocrins, amb conseqüències patològiques en el tracte respiratori, gastrointestinal i urogenital. Mutacions al gen CFTR (cystic fibrosis transmembrane conductance regulator ABCC7; MIM# 602421) són responsables de la malaltia. Fins al moment, ja s'han descrit més de 1600 mutacions en el gen CFTR. La gran majoria són mutacions puntuals de les quals: 42% són mutacions de sentit erroni (missense).El gen CFTR codifica per una proteïna de membrana de 1480 aminoàcids, que pertany a la gran família dels transportadors ABC. La proteïna CFTR presenta una estructura simètrica, formada per dos motius repetits, cadascun d'ells format per una regió transmembrana (TMD), composada per sis &#945;-hèlix, i una regió citosòlica hidrofílica d'unió a ATP (NBD). Aquests dos motius queden units mitjançant un domini regulador (R), citosòlic i hidrofílic, que és fosforila per PKA i PKC. El paper principal que desenvolupa el canal CFTR en la part apical de les cèl·lules epitelials és el transport de fluid transepitelial i electròlits a través de la membrana, activitat que ve regulada directament per la fosforilació de PKA. Aquest canal permet el trànsit d'ions de clorur a través d'ell en qualsevol direcció, i es caracteritza per tenir una baixa conductància unitària (la seva conductància és de 7-10 pS).Existeixen moltes evidències en la literatura on les mutacions de sentit erroni, independentment de la seva localització en el gen CFTR, poden afectar a la funció del canal CFTR a diferents nivells: biogènesi de la proteïna, transport i localització, propietats electrofisiològiques del canal.Així que l'objectiu general de la tesi va ser l'anàlisi molecular i funcional de vuit mutacions amb sentit erroni (p.P5L, p.S50P, p.E60K, p.R75Q, p.G85E, p.G85V, p.Y89C, p.E92K) localitzades en la regió N-terminal de CFTR, i identificades majoritàriament a la població espanyola.Per portar a terme els objectius de la tesi, es van generar les vuit mutacions per mutagènesi dirigida en el vector pCMVCFTRNot6.2wt, i es van expressar en cèl·lules epitelials HEK293. Es va analitzar el processament i patró de maduració de les proteïnes mutants per Western blot i la seva localització cel·lular per immunocitoquímica. I posteriorment, es van caracteritzar funcionalment els canals mutants mitjançant la tècnica de Patch clamp en configuració whole cell i excised inside-out.Vam observar que cinc de les mutacions analitzades, p.S50P, p.E60K, p.G85E, p.G85V i p.E92K, provoquen un processament i una maduració incorrecte de la proteïna. Totes cinc presenten un comportament molt similar a la mutació p.F508del, podent-se classificar aquestes mutacions dins la classe II.La mutació p.P5L afecta a la funció de CFTR a dos nivells: a nivell de la biogènesi i a nivell de l'activitat del canal. Aquesta mutació provoca una disminució de la densitat de canals a la membrana citoplasmàtica. La conductància i la cinètica del canal també s'alteren. La proteïna mutant p.Y89C afecta a l'activitat del canal. Aquesta mutació altera específicament la cinètica del canal. El canal CFTR corresponent a la mutació p.R75Q presenta un patró de maduració i un comportament funcional del canal anàleg a la proteïna CFTR no mutada. Els nostres estudis corroboren la classificació de p.R75Q com a mutació sense expressió clínica.El comportament sever de la majoria de les mutacions avaluades, afectant la biogènesi i/o l'activitat del canal, ressalta la importància funcional de la cua N-terminal i del segment TM1 en la fisiologia de CFTR. / Over 1,600 cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence variations have been identified in patients with cystic fibrosis (CF) and related disorders involving an impaired function of the CFTR chloride channel. However, detailed structure-function analyses have only been established for a few of them.This study aimed evaluating the impact of eight N-terminus CFTR natural missense changes on channel behavior. By site-directed mutagenesis, we generated four CFTR variants in the N-terminal cytoplasmic tail (p.P5L, p.S50P, p.E60K, and p.R75Q) and four in the first transmembrane segment of membrane-spanning domain 1 (p.G85E/V, p.Y89C, and p.E92K). Immunoblot analysis revealed that p.S50P, p.E60K, p.G85E/V, and p.E92K produced only core-glycosylated proteins. Immunofluorescence and whole cell patch-clamp confirmed intracellular retention, thus reflecting a defect of CFTR folding and/or trafficking. In contrast, both p.R75Q and p.Y89C had a glycosylation pattern and a subcellular distribution comparable to the wild-type CFTR, while the percentage of mature p.P5L was considerably reduced, suggesting a major biogenesis flaw on this channel. Nevertheless, whole-cell chloride currents were recorded for all three variants. Single-channel patch-clamp analyses revealed that the channel activity of p.R75Q appeared similar to that of the wild-type CFTR, while both p.P5L and p.Y89C channels displayed abnormal gating. Overall, our results predict a major impact of theCFTR missense variants analyzed, except p.R75Q, on the CF phenotype and highlight the importance of the CFTR N-terminus on channel physiology.

No description available.