Molecular understanding of chronic pancreatitis: a perspective on the future.

Despite the recent development of medical imaging technology, chronic pancreatitis can only be diagnosed when the disease is fully established. This is due to the lack of specific and sensitive markers for this disease. The discovery of mutations in the cationic trypsinogen gene in patients with hereditary pancreatitis and a high incidence of mutations in the cystic fibrosis transmembrane conductance regulator gene in patients with chronic pancreatitis might be important clues to understanding the molecular mechanisms of this disease. The interaction between ethanol and ion channels might be the missing link between alcohol ingestion and chronic pancreatitis.     

Maximization of the rate of chloride conduction in the CFTR channel pore by ion-ion interactions

Multi-ion pore behaviour has been identified in many Cl(-) channel types but its biophysical significance is uncertain. Here, we show that mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel that disrupt anion-anion interactions within the pore are associated with drastically reduced single channel conductance. These results are consistent with models suggesting that rapid Cl(-) permeation in CFTR results from repulsive ion-ion interactions between Cl(-) ions bound concurrently inside the pore. Naturally occurring mutations that disrupt these interactions can result in cystic fibrosis.     

Cystic fibrosis transmembrane conductance regulator (CFTR): Making an ion channel out of an active transporter structure

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a member of the ATP-binding cassette (ABC) family of membrane transport proteins, most members of which function as ATP-dependent pumps. CFTR is unique among human ABC proteins in functioning not as a pump, but as an ion channel. Recent structural data has indicated that CFTR shares broadly similar overall architecture and ATP-dependent conformational changes as other ABC proteins. Functional investigations suggest that CFTR has a unique open portal connecting the cytoplasm to the transmembrane channel pore, that allows for a continuous pathway for Cl^? ions to cross the membrane in one conformation. This lateral portal may be what allows CFTR to function as an ion channel rather than as a pump, suggesting a plausible mechanism by which channel function may have evolved in CFTR.     

C-terminal truncations destabilize the cystic fibrosis transmembrane conductance regulator without impairing its biogenesis. A novel class of mutation.

Defective cAMP-stimulated chloride conductance of the plasma membrane of epithelial cell is the hallmark of cystic fibrosis (CF) and results from mutations in the cystic fibrosis transmembrane conductance regulator, CFTR. In the majority of CF patients, mutations in the CFTR lead to its misfolding and premature degradation at the endoplasmic reticulum (ER). Other mutations impair the cAMP-dependent activation or the ion conductance of CFTR chloride channel. In the present work we identify a novel mechanism leading to reduced expression of CFTR at the cell surface, caused by C-terminal truncations. The phenotype of C-terminally truncated CFTR, representing naturally occurring premature termination and frameshift mutations, were examined in transient and stable heterologous expression systems. Whereas the biosynthesis, processing, and macroscopic chloride channel function of truncated CFTRs are essentially normal, the degradation rate of the mature, complex-glycosylated form is 5- to 6-fold faster than the wild type CFTR. These experiments suggest that the C terminus has a central role in maintaining the metabolic stability of the complex-glycosylated CFTR following its exit from the ER and provide a plausible explanation for the severe phenotype of CF patients harboring C-terminal truncations.     

Applications of proteomic technologies for understanding the premature proteolysis of CFTR

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes an ATP-dependent anion channel. Disease-causing mutations can affect channel biogenesis, trafficking or function, and result in reduced ion transport at the apical surface of many tissues. The most common CFTR mutation is a deletion of phenylalanine at position 508 (ΔF508), which results in a misfolded protein that is prematurely targeted for degradation. This article focuses on how proteomic approaches have been utilized to explore the mechanisms of premature proteolysis in CF. Additionally, we emphasize the potential for proteomic-based technologies in expanding our understanding of CF pathophysiology and therapeutic approaches.     

Allele frequency for Cystic fibrosis in Indians vis-a/-vis global populations

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene. This gene encodes a protein involved in epithelial anion channel. Cystic fibrosis is the most common life-limiting genetic disorder in Caucasians; it also affects other ethnic groups like the Blacks and the Native Americans. Cystic fibrosis is considered to be rare among individuals from the Indian subcontinent. We analyzed a total of 29 world׳s populations for cystic fibrosis on the basis of gene frequency and heterozygosity. Among 29 countries Switzerland revealed the highest gene frequency and heterozygosity for CF (0.022, 0.043) whereas Japan recorded the lowest values (0.002, 0.004) followed by India (0.004, 0.008). Our analysis suggests that the prevalence of cystic fibrosis is very low in India.     

Cystic Fibrosis Transmembrane Conductance Regulator–associated ATP Release Is Controlled by a Chloride Sensor

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis, and has also been closely associated with ATP permeability in cells. Using a Xenopus oocyte cRNA expression system, we have evaluated the molecular mechanisms that control CFTR-modulated ATP release. CFTR-modulated ATP release was dependent on both cAMP activation and a gradient change in the extracellular chloride concentration. Activation of ATP release occurred within a narrow concentration range of external Cl⁻ that was similar to that reported in airway surface fluid. Mutagenesis of CFTR demonstrated that Cl⁻ conductance and ATP release regulatory properties could be dissociated to different regions of the CFTR protein. Despite the lack of a need for Cl⁻ conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl >> Br; R347P, Cl >> Br; R347E, Br >> Cl; R334W, Cl = Br). We hypothesize that residues R347 and R334 may contribute a Cl⁻ binding site within the CFTR channel pore that is necessary for activation of ATP efflux in response to increases of extracellular Cl⁻. In summary, these findings suggest a novel chloride sensor mechanism by which CFTR is capable of responding to changes in the extracellular chloride concentration by modulating the activity of an unidentified ATP efflux pathway. This pathway may play an important role in maintaining fluid and electrolyte balance in the airway through purinergic regulation of epithelial cells. Insight into these molecular mechanisms enhances our understanding of pathogenesis in the cystic fibrosis lung.     

Loss of the normal epicardial to endocardial gradient of cftr mRNA expression in the hypertrophied rabbit left ventricle

The electrical instability of hypertrophied and failing hearts is caused by delayed repolarisation, which is thought to be due in part to altered levels and/or patterns of expression of ion channel genes. The aim of this study was to investigate changes in the levels and pattern of cystic fibrosis transmembrane conductance regulator (cftr) mRNA expression in a combined pressure and volume overload model of heart failure in the rabbit, using in situ mRNA hybridisation. There was a decrease in cftr mRNA expression, primarily due to a decrease in epicardial expression and, hence, loss of the normal epicardial to endocardial gradient of cftr mRNA expression in the rabbit left ventricle. In contrast there was an increase in atrial natriuretic factor (anf) mRNA expression in the hypertrophied hearts with preferential reexpression in subendocardial regions. The patterns of both cftr and anf mRNA expression in the hypertrophied hearts were similar to those seen in embryonic hearts. This suggests that the reversion to an embryonic pattern of gene expression in cardiac hypertrophy applies to ion channel genes. The loss of the normal transmural gradient of repolarising ion channels is likely to contribute to instability of repolarisation in the hypertrophied heart and hence increased risk of cardiac arrhythmias in patients with heart failure.     

Cloning the mouse homolog of the human cystic fibrosis transmembrane conductance regulator gene.

The cystic fibrosis transmembrane conductance regulator is encoded by the gene known to be mutated in patients with cystic fibrosis. This paper reports the cloning and sequencing of cDNAs for the murine homolog of the human cystic fibrosis transmembrane conductance regulator gene. A clone that, by analogy to the human sequence, extends 3' from exon 9 to the poly(A) tail was isolated from a mouse lung cDNA library. cDNA clones containing exons 4 and 6b were also isolated and sequenced, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-9 were cloned by PCR from mouse RNA. The deduced mouse protein sequence is 78% identical to the human cystic fibrosis transmembrane regulator, with higher conservation in the transmembrane and nucleotide-binding domains. Amino acid sequences in which known cystic fibrosis missense mutations occur are conserved between man and mouse; in particular, the predicted mouse protein has a phenylalanine residue corresponding to that deleted in the most common human cystic fibrosis mutation (delta F508), which should allow the use of transgenic strategies to introduce this mutation in attempts to create a "cystic fibrosis mouse".     

Mukoviszidose – eine pleiotrope Ionenkanalerkrankung mit wesentlicher Lungenbeteiligung

The monogenic disease cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is inherited in an autosomal recessive fashion. Successful diagnosis of the disease is achieved by patient history, clinical assessment, genetic analysis of the CFTR gene and by in vivo measurement and ex vivo characterization of the basic defect in patient’s samples. Frequently, differential diagnosis of CFTR-related disorders such as congenital bilateral absence of the vas deferens (CBAVD), pancreatitis and bronchiectasis needs to be performed. Molecular therapeutics has been developed for some CFTR mutations and these will facilitate direct clinical use of the information provided by CFTR mutation analysis.     

An unstable transmembrane segment in the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel with 12 membrane-spanning sequences, undergoes inefficient maturation in the endoplasmic reticulum (ER). Potentially charged residues in transmembrane segments may contribute to this defect in biogenesis. We demonstrate that transmembrane segment 6 of CFTR, which contains three basic amino acids, is extremely unstable in the lipid bilayer upon membrane insertion in vitro and in vivo. However, two distinct mechanisms counteract this anchoring deficiency: (i) the ribosome and the ER translocon co-operate to prevent transmembrane segment 6 from passing through the membrane co- translationally; and (ii) cytosolic domains of the ion channel post-translationally maintain this segment of CFTR in a membrane-spanning topology. Although these mechanisms are essential for successful completion of CFTR biogenesis, inefficiencies in their function retard the maturation of the protein. It seems possible that some of the disease-causing mutations in CFTR may reduce the efficiency of proper membrane anchoring of the protein.     

Probing the basic defect in cystic fibrosis.

The concurrent developments in electrophysiology studies and the identification of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has provided a unique opportunity to probe the basic cellular defect underlying cystic fibrosis. Various properties of the CFTR protein have been deduced from its primary sequence, the variety of mutations in patients and genotype-phenotype correlations, as well as the results of more recent DNA transfection studies. The most exciting observation is the fact that CFTR acts like a cAMP-regulated Cl- channel.     

CFTR型氯离子通道研究进展

囊性纤维化跨膜传导调节因子（CFTR）是一种重要的氯离子通道,突变易引起囊性纤维化病变,故得名。一系列研究表明,CFTR由5个结构域组成：两个跨膜结构域形成氯离子通道;两个核苷酸结合结构域调节通道的开闭;一个调节结构域主要影响氯通道的活动。这些结构域通过协同作用共同控制了氯离子的跨膜流动,而一些突变可以影响细胞功能而导致囊性纤维化的发生。本文通过介绍CFTR基本结构、调节机制、与囊性纤维化病变的关系及针对CFTR的治疗而对CFTR型氯离子通道有一个的全面的理解。     

Cystic fibrosis-associated mutations at arginine 347 alter the pore architecture of CFTR. Evidence for disruption of a salt bridge

Arginine 347 in the sixth transmembrane domain of cystic fibrosis transmembrane conductance regulator (CFTR) is a site of four cystic fibrosis-associated mutations. To better understand the function of Arg-347 and to learn how mutations at this site disrupt channel activity, we mutated Arg-347 to Asp, Cys, Glu, His, Leu, or Lys and examined single-channel function. Every Arg-347 mutation examined, except R347K, had a destabilizing effect on the pore, causing the channel to flutter between two conductance states. Chloride flow through the larger conductance state was similar to that of wild-type CFTR, suggesting that the residue at position 347 does not interact directly with permeating anions. We hypothesized that Arg-347 stabilizes the channel through an electrostatic interaction with an anionic residue in another transmembrane domain. To test this, we mutated anionic residues (Asp-924, Asp-993, and Glu-1104) to Arg in the context of either R347E or R347D mutations. Interestingly, the D924R mutation complemented R347D, yielding a channel that behaved like wild-type CFTR. These data suggest that Arg-347 plays an important structural role in CFTR, at least in part by forming a salt bridge with Asp-924; cystic fibrosis-associated mutations disrupt this interaction.     

Heterogeneity in the severity of cystic fibrosis and the role of CFTR gene mutations

Cystic fibrosis is a common, fatal disorder caused by abnormalities in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encodes a chloride channel that regulates secretion in many exocrine tissues. The presentation of cystic fibrosis is highly variable as measured by the age of onset of disease, the presence of pancreatic insufficiency, or the progression of lung disease. Over 400 mutations in the CFTR gene have been described in cystic fibrosis patients and considerable effort has focused on the correlation between specific mutations and genotypes and clinical characteristics. Individual tissues display variation in their sensitivity to CFTR mutations. The vas deferens is functionally disrupted in nearly all males, whereas mild and severe pancreatic involvement is determined by the patient's genotype. The severity of pulmonary disease is poorly correlated with genotype, suggesting that there are other important genetic and/or environmental factors that contribute to lung infections and the subsequent disruption of lung function.     

The basic defect in cystic fibrosis.

Recent evidence strongly suggests that the cystic fibrosis gene product (CFTR) is a Cl- channel. Its properties, however, differ from those of a 30-50 pS outwardly rectifying channel previously implicated as defective in cystic fibrosis. It is still uncertain whether the pleiotropic effects of the CF defect, such as increased airway Na+ absorption and mucus sulfation, are secondary to reduced Cl- conductance, or reflect additional functions of CFTR.     

Mutations in the nucleotide binding domain 1 signature motif region rescue processing and functional defects of cystic fibrosis transmembrane conductance regulator delta f508

The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP binding cassette (ABC) transporter that functions as a phosphorylation- and nucleotide-regulated chloride channel, is mutated in cystic fibrosis (CF) patients. Deletion of a phenylalanine at amino acid position 508 (DeltaF508) in the first nucleotide binding domain (NBD1) is the most prevalent CF-causing mutation and results in defective protein processing and reduced CFTR function, leading to chloride impermeability in CF epithelia and heterologous systems. Using a STE6/CFTRDeltaF508 chimera system in yeast, we isolated two novel DeltaF508 revertant mutations, I539T and G550E, proximal to and within the conserved ABC signature motif of NBD1, respectively. Western blot and functional analysis in mammalian cells indicate that mutations I539T and G550E each partially rescue the CFTRDeltaF508 defect. Furthermore, a combination of both revertant mutations resulted in a 38-fold increase in CFTRDeltaF508-mediated chloride current, representing 29% of wild type channel activity. The G550E mutation increased the sensitivity of CFTRDeltaF508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTRDeltaF508 channel activity by 2 mm 3-isobutyl-1-methylxanthine. The data show that the DeltaF508 defect can be significantly rescued by second-site mutations in the nucleotide binding domain 1 region, that includes the LSGGQ consensus motif.     

Dual activity of aminoarylthiazoles on the trafficking and gating defects of the cystic fibrosis transmembrane conductance regulator chloride channel caused by cystic fibrosis mutations

A large fraction of mutations causing cystic fibrosis impair the function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel by causing reduced channel activity (gating defect) and/or impaired exit from the endoplasmic reticulum (trafficking defect). Such defects need to be treated with separate pharmacological compounds termed potentiators and correctors, respectively. Here, we report the characterization of aminoarylthiazoles (AATs) as compounds having dual activity. Cells expressing mutant CFTR were studied with functional assays (fluorescence-based halide transport and short circuit current measurements) to assess the effect of acute and chronic treatment with compounds. We found that AATs are effective on F508del, the most frequent cystic fibrosis mutation, which is associated with both a gating and a trafficking defect. AATs are also effective on mutations like G1349D and G551D, which cause only a gating defect. Evaluation of a panel of AAT analogs identified EN277I as the most effective compound. Incubation of cells expressing mutant CFTR with EN277I caused a strong stimulation of channel activity as demonstrated by single channel recordings. Compounds with dual activity such as AATs may be useful for the development of effective drugs for the treatment of cystic fibrosis.