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1.
J Cyst Fibros ; 10 Suppl 2: S53-66, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21658643

ABSTRACT

In the majority of cases, there is no difficulty in diagnosing Cystic Fibrosis (CF). However, there may be wide variation in signs and symptoms between individuals which encourage the scientific community to constantly improve the diagnostic tests available and develop better methods to come to a final diagnosis in patients with milder phenotypes. This paper is the result of discussions held at meetings of the European Cystic Fibrosis Society Diagnostic Network supported by EuroCareCF. CFTR bioassays in the nasal epithelium (nasal potential difference measurements) and the rectal mucosa (intestinal current measurements) are discussed in detail including efforts to standardize the techniques across Europe. New approaches to evaluate the sweat gland, future of genetic testing and methods on the horizon like CFTR expression in human leucocytes and erythrocytes are discussed briefly.


Subject(s)
Cystic Fibrosis/diagnosis , Diagnostic Techniques, Respiratory System/trends , Medicine/trends , Europe , Humans
2.
J Cyst Fibros ; 10 Suppl 2: S86-102, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21658649

ABSTRACT

Several diseases have been clinically or genetically related to cystic fibrosis (CF), but a consensus definition is lacking. Here, we present a proposal for consensus guidelines on cystic fibrosis transmembrane conductance regulator (CFTR)-related disorders (CFTR-RDs), reached after expert discussion and two dedicated workshops. A CFTR-RD may be defined as "a clinical entity associated with CFTR dysfunction that does not fulfil diagnostic criteria for CF". The utility of sweat testing, mutation analysis, nasal potential difference, and/or intestinal current measurement for the differential diagnosis of CF and CFTR-RD is discussed. Algorithms which use genetic and functional diagnostic tests to distinguish CF and CFTR-RDs are presented. According to present knowledge, congenital bilateral absence of vas deferens (CBAVD), acute recurrent or chronic pancreatitis and disseminated bronchiectasis, all with CFTR dysfunction, are CFTR-RDs.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/classification , Cystic Fibrosis/genetics , Medicine/standards , Practice Guidelines as Topic , Cystic Fibrosis/physiopathology , Europe , Humans
3.
Br J Pharmacol ; 153(6): 1311-23, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18223673

ABSTRACT

BACKGROUND AND PURPOSE: Cystic fibrosis (CF) is caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. In the search for new CF therapies, small molecules have been identified that rescue the defective channel gating of CF mutants (termed CFTR potentiators). Here, we investigate the long-term effects of genistein, the best-studied CFTR potentiator, on the expression and function of CFTR. EXPERIMENTAL APPROACH: We pre-treated baby hamster kidney (BHK) cells expressing wild-type or F508del-CFTR (the most common CF mutant) with concentrations of genistein that potentiate (30 microM) or inhibit (100 microM) CFTR function for 2 or 24 h at 37 degrees C before examining CFTR maturation, expression and single-channel activity. KEY RESULTS: Using the iodide efflux technique, we found that genistein pre-treatment failed to restore function to F508del-CFTR, but altered that of wild-type CFTR. Pre-treatment of cells with genistein for 2 h had little effect on CFTR processing, whereas pre-treatment for 24 h either augmented (30 microM genistein) or impaired (100 microM genistein) CFTR maturation. Using immunocytochemistry, we found that all genistein pre-treatments increased the localization of CFTR protein to the cell surface. However, following the incubation of cells with genistein (100 microM) for 2 h, individual CFTR Cl(-) channels exhibited characteristics of channel block upon channel activation. CONCLUSIONS AND IMPLICATIONS: Genistein pre-treatment alters the maturation, cell surface expression and single-channel function of CFTR in ways distinct from its acute effects. Thus, CFTR potentiators have the potential to influence CFTR by mechanisms distinct from their effects on channel gating.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/drug effects , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Genistein/pharmacology , Animals , Cell Line , Cricetinae , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Dose-Response Relationship, Drug , Genistein/administration & dosage , Humans , Immunohistochemistry , Iodides/metabolism , Ion Channel Gating/drug effects , Kidney , Protein Transport/drug effects , Time Factors
4.
Mol Membr Biol ; 21(1): 27-38, 2004.
Article in English | MEDLINE | ID: mdl-14668136

ABSTRACT

Niflumic acid is widely used to inhibit Ca(2+) -activated Cl(-) channels. However, the chemical structure of niflumic acid resembles that of diphenylamine-2-carboxylate, a drug that inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. To investigate how niflumic acid inhibits CFTR Cl(-) channel, we studied recombinant wild-type human CFTR in excised inside-out membrane patches. When added to the intracellular solution, niflumic acid caused a concentration- and voltage-dependent decrease of CFTR Cl(-) current with half-maximal inhibitory concentration (K(i)) of 253 microM and Hill co-efficient of approximately 1, at -50 mV. Niflumic acid inhibition of single CFTR Cl(-) channels was characterized by a very fast, flickery block that decreased dramatically current amplitude without altering open-probability. Consistent with these data, spectral analysis of CFTR Cl(-) currents suggested that channel block by niflumic acid was described by the closed <--> open <--> blocked kinetic scheme with blocker on rate (k(on)) = 13.9 x 10(6) M(-1)s(-1), off rate (k(off))=3348 s(-1) and dissociation constant (K(d)) = 241 microM, at -50 mV. Based on these data, we tested the effects of niflumic acid on transepithelial Cl(-) secretion and cyst growth using type I MDCK epithelial cells. Niflumic acid (200 microM) inhibited cAMP-stimulated, bumetanide-sensitive short-circuit current by 55%. Moreover, the drug potently retarded cyst growth. We conclude that niflumic acid is an open-channel blocker of CFTR that inhibits Cl(-) permeation by plugging the channel pore. It or related agents might be of value in the development of new therapies for autosomal dominant polycystic kidney disease.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors , Ion Channel Gating/drug effects , Niflumic Acid/pharmacology , Recombinant Proteins/antagonists & inhibitors , Animals , Cell Line , Chlorides/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Dogs , Electrophysiology , Humans , Ion Channel Gating/genetics , Membrane Potentials/drug effects , Molecular Structure , Patch-Clamp Techniques , Recombinant Proteins/chemistry , Recombinant Proteins/genetics
5.
Cell Physiol Biochem ; 10(5-6): 321-8, 2000.
Article in English | MEDLINE | ID: mdl-11125212

ABSTRACT

Cystic fibrosis (CF) is caused by mutations in the secretory Cl(-) channel CFTR (cystic fibrosis transmembrane conductance regulator). Variation in the severity of disease has been attributed to mutations in the CFTR gene that cause different degrees of dysfunction of the CFTR Cl(-) channel. However, studies of mouse models of CF indicate that the severity of intestinal pathology is not correlated with activity of the CFTR chloride channel. This observation suggests that other 'environmental' factors might be important in determining the severity of disease. In this respect, we have identified and characterised an additional cellular defect in intestinal epithelial cells of CF mice, the inability of these cells to regulate their volume after hypotonic challenge. Here, we review the function of murine CFTR as both a Cl(-) channel and as a regulator of volume-dependent homeostatic cell mechanisms.


Subject(s)
Cell Size , Chloride Channels/physiology , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Intestine, Small/cytology , Animals , Ion Channel Gating , Mice
6.
Am J Physiol Lung Cell Mol Physiol ; 279(4): L766-78, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11000138

ABSTRACT

The goal of this study was to develop a primary culture model of differentiated murine tracheal epithelium. When grown on semipermeable membranes at an air interface, dissociated murine tracheal epithelial cells formed confluent polarized epithelia with high transepithelial resistances ( approximately 12 kOmega. cm(2)) that remained viable for up to 80 days. Immunohistochemistry and light and electron microscopy demonstrated that the cells were epithelial in nature (cytokeratin positive, vimentin and alpha-smooth muscle actin negative) and differentiated to form ciliated and secretory cells from day 8 after seeding onward. With RT-PCR, expression of the cystic fibrosis transmembrane conductance regulator (Cftr) and murine beta-defensin (Defb) genes was detected (Defb-1 was constitutively expressed, whereas Defb-2 expression was induced by exposure to lipopolysaccharide). Finally, Ussing chamber experiments demonstrated an electrophysiological profile compatible with functional amiloride-sensitive sodium channels and cAMP-stimulated CFTR chloride channels. These data indicate that primary cultures of murine tracheal epithelium have many characteristics similar to those of murine tracheal epithelium in vivo. This method will facilitate the establishment of primary cultures of airway epithelium from transgenic mouse models of human diseases.


Subject(s)
Cell Differentiation , Respiratory Mucosa/cytology , Respiratory Mucosa/physiology , Amiloride/pharmacology , Animals , Bumetanide/pharmacology , Cell Culture Techniques/methods , Cell Polarity , Cell Separation/methods , Cells, Cultured , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Defensins/genetics , Female , Humans , Keratins/analysis , Male , Membrane Potentials/drug effects , Mice , Mice, Inbred C57BL , Respiratory Mucosa/ultrastructure , Reverse Transcriptase Polymerase Chain Reaction , Trachea , Vimentin/analysis
7.
J Physiol ; 524 Pt 2: 317-30, 2000 Apr 15.
Article in English | MEDLINE | ID: mdl-10766914

ABSTRACT

1. The isoflavone genistein may either stimulate or inhibit cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. To investigate how genistein inhibits CFTR, we studied CFTR Cl- channels in excised inside-out membrane patches from cells expressing wild-type human CFTR. 2. Addition of genistein (100 microM) to the intracellular solution caused a small decrease in single-channel current amplitude (i), but a large reduction in open probability (Po). 3. Single-channel analysis of channel block suggested that genistein (100 microM) may inhibit CFTR by two mechanisms: first, it may slow the rate of channel opening and second, it may block open channels. 4. Acidification of the intracellular solution relieved channel block, suggesting that the anionic form of genistein may inhibit CFTR. 5. Genistein inhibition of CFTR Cl- currents was weakly voltage dependent and unaffected by changes in the extracellular Cl- concentration. 6. Channel block was relieved by pyrophosphate (5 mM) and ATP (5 mM), two agents that interact with the nucleotide-binding domains (NBDs) of CFTR to greatly stimulate channel activity. 7. ATP (5 mM) prevented the genistein-induced decrease in Po, but was without effect on the genistein-induced decrease in i. 8. The genistein-induced decrease in i was voltage dependent, whereas the genistein-induced decrease in Po was voltage independent. 9. The data suggest that genistein may inhibit CFTR by two mechanisms. First, it may interact with NBD1 to potently inhibit channel opening. Second, it may bind within the CFTR pore to weakly block Cl- permeation.


Subject(s)
ATP-Binding Cassette Transporters/antagonists & inhibitors , Chloride Channels/antagonists & inhibitors , Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Genistein/pharmacology , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Algorithms , Animals , Cell Line , Chlorides/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Diphosphates/pharmacology , Electric Stimulation , Electrophysiology , Humans , Ion Channel Gating/drug effects , Membrane Potentials/physiology , Mice , Patch-Clamp Techniques , Phosphorylation
8.
Trends Pharmacol Sci ; 20(11): 448-53, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10542444

ABSTRACT

Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is associated with a wide spectrum of disease. In the search for modulators of CFTR, pharmacological agents that interact directly with the CFTR Cl- channel have been identified. Some agents stimulate CFTR by interacting with the nucleotide-binding domains that control channel gating, whereas others inhibit CFTR by binding within the channel pore and preventing Cl- permeation. Knowledge of the molecular pharmacology of CFTR might lead to new treatments for diseases caused by the dysfunction of CFTR.


Subject(s)
ATP-Binding Cassette Transporters/drug effects , Chloride Channels/drug effects , Cystic Fibrosis Transmembrane Conductance Regulator/drug effects , ATP-Binding Cassette Transporters/chemistry , Animals , Chloride Channels/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Humans
9.
Br J Pharmacol ; 128(1): 108-18, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10498841

ABSTRACT

1. Hypoglycaemia-inducing sulphonylureas, such as glibenclamide, inhibit cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In search of modulators of CFTR, we investigated the effects of the non-sulphonylurea hypoglycaemic agents meglitinide, repaglinide, and mitiglinide (KAD-1229) on CFTR Cl- channels in excised inside-out membrane patches from C127 cells expressing wild-type human CFTR. 2. When added to the intracellular solution, meglitinide and mitiglinide inhibited CFTR Cl- currents with half-maximal concentrations of 164+/-19 microM and 148+/-36 microM, respectively. However, repaglinide only weakly inhibited CFTR Cl- currents. 3. To understand better how non-sulphonylurea hypoglycaemic agents inhibit CFTR, we studied single channels. Channel blockade by both meglitinide and mitiglinide was characterized by flickery closures and a significant decrease in open probability (Po). In contrast, repaglinide was without effect on either channel gating or Po, but caused a small decrease in single-channel current amplitude. 4. Analysis of the dwell time distributions of single channels indicated that both meglitinide and mitiglinide greatly decreased the open time of CFTR. Mitiglinide-induced channel closures were about 3-fold longer than those of meglitinide. 5. Inhibition of CFTR by meglitinide and mitiglinide was voltage-dependent: at positive voltages channel blockade was relieved. 6. The data demonstrate that non-sulphonylurea hypoglycaemic agents inhibit CFTR. This indicates that these agents have a wider specificity of action than previously recognized. Like glibenclamide, non-sulphonylurea hypoglycaemic agents may inhibit CFTR by occluding the channel pore and preventing Cl- permeation.


Subject(s)
Chloride Channels/antagonists & inhibitors , Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors , Hypoglycemic Agents/pharmacology , Adenosine Triphosphate/metabolism , Animals , Benzamides/chemistry , Benzamides/pharmacology , Carbamates/chemistry , Carbamates/pharmacology , Cell Line , Chloride Channels/genetics , Chloride Channels/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Electric Conductivity , Epithelial Cells/drug effects , Epithelial Cells/enzymology , Epithelial Cells/metabolism , Glyburide/pharmacology , Humans , Indoles/chemistry , Indoles/pharmacology , Ion Channel Gating/drug effects , Isoindoles , Kinetics , Mice , Patch-Clamp Techniques , Piperidines/chemistry , Piperidines/pharmacology , Sulfonylurea Compounds/pharmacology , Transfection
10.
Physiol Rev ; 79(1 Suppl): S23-45, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9922375

ABSTRACT

Structure and Function of the CFTR Chloride Channel. Physiol. Rev. 79, Suppl.: S23-S45, 1999. - The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ABC transporter family that forms a novel Cl- channel. It is located predominantly in the apical membrane of epithelia where it mediates transepithelial salt and liquid movement. Dysfunction of CFTR causes the genetic disease cystic fibrosis. The CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. Here we review the structure and function of this unique channel, with a focus on how the various domains contribute to channel function. The MSDs form the channel pore, phosphorylation of the R domain determines channel activity, and ATP hydrolysis by the NBDs controls channel gating. Current knowledge of CFTR structure and function may help us understand better its mechanism of action, its role in electrolyte transport, its dysfunction in cystic fibrosis, and its relationship to other ABC transporters.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Humans , Hydrolysis , Ion Channel Gating , Phosphorylation , Structure-Activity Relationship
11.
J Physiol ; 512 ( Pt 3): 751-64, 1998 Nov 01.
Article in English | MEDLINE | ID: mdl-9769419

ABSTRACT

1. We investigated the effect of protein kinases and phosphatases on murine cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, expressed in Chinese hamster ovary (CHO) cells, using iodide efflux and the excised inside-out configuration of the patch-clamp technique. 2. The protein kinase C (PKC) activator, phorbol dibutyrate, enhanced cAMP-stimulated iodide efflux. However, PKC did not augment the single-channel activity of either human or murine CFTR Cl- channels that had previously been activated by protein kinase A. 3. Fluoride, a non-specific inhibitor of protein phosphatases, stimulated both human and murine CFTR Cl- channels. However, calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, did not enhance cAMP-stimulated iodide efflux. 4. The alkaline phosphatase inhibitor, (-)-bromotetramisole augmented cAMP-stimulated iodide efflux and, by itself, stimulated a larger efflux than that evoked by cAMP agonists. However, (+)-bromotetramisole, the inactive enantiomer, had the same effect. For murine CFTR, neither enantiomer enhanced single-channel activity. In contrast, both enantiomers increased the open probability (Po) of human CFTR, suggesting that bromotetramisole may promote the opening of human CFTR. 5. As murine CFTR had a low Po and was refractory to stimulation by activators of human CFTR, we investigated whether murine CFTR may open to a subconductance state. When single-channel records were filtered at 50 Hz, a very small subconductance state of murine CFTR was observed that had a Po greater than that of human CFTR. The occupancy of this subconductance state may explain the differences in channel regulation observed between human and murine CFTR.


Subject(s)
Chloride Channels/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Algorithms , Animals , CHO Cells , Cricetinae , Electric Stimulation , Electrophysiology , Enzyme Activation/drug effects , Enzyme Activation/physiology , Enzyme Inhibitors/pharmacology , Fluorides/pharmacology , Humans , Iodides/metabolism , Membrane Potentials/physiology , Mice , Patch-Clamp Techniques , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoprotein Phosphatases/pharmacology , Protein Kinase C/metabolism , Protein Kinase Inhibitors , Protein Kinases/pharmacology
12.
J Physiol ; 508 ( Pt 2): 379-92, 1998 Apr 15.
Article in English | MEDLINE | ID: mdl-9508803

ABSTRACT

1. To investigate the function of the murine cystic fibrosis transmembrane conductance regulator (CFTR), a full-length cDNA encoding wild-type murine CFTR was assembled and stably expressed in Chinese hamster ovary (CHO) cells. 2. Like human CFTR, murine CFTR formed Cl- channels that were regulated by cAMP-dependent phosphorylation and intracellular ATP. However, murine CFTR Cl- channels had a reduced single-channel conductance and decreased open probability (Po) compared with those of human CFTR. 3. Analysis of the dwell time distributions of single channels suggested that the reduced Po of murine CFTR was caused by both decreased residence in the open state and transitions to a new closed state, described by an intermediate closed time constant. 4. For both human and murine CFTR, ATP and ADP regulated the rate of exit from the long-lived closed state. 5. 5'-Adenylylimidodiphosphate (AMP-PNP) and pyrophosphate, two compounds that disrupt cycles of ATP hydrolysis, stabilized the open state of human CFTR. However, neither agent locked murine CFTR Cl- channels open, although AMP-PNP increased the Po of murine CFTR. 6. The data indicate that although human and murine CFTR have many properties in common, some important differences in function are observed. These differences could be exploited in future studies to provide new understanding about CFTR.


Subject(s)
Chloride Channels/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Ion Channel Gating/physiology , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Adenylyl Imidodiphosphate/pharmacology , Animals , Cells, Cultured , Chloride Channels/drug effects , Cricetinae , Cyclic AMP/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , DNA/genetics , Diphosphates/pharmacology , Electrophysiology , Humans , Ion Channel Gating/drug effects , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Patch-Clamp Techniques
13.
J Physiol ; 503 ( Pt 2): 333-46, 1997 Sep 01.
Article in English | MEDLINE | ID: mdl-9306276

ABSTRACT

1. The sulphonylurea drug glibenclamide is a widely used inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR). To investigate how glibenclamide inhibits CFTR, we studied CFTR Cl- channels using excised inside-out membrane patches from cells expressing wild-type human CFTR. 2. Addition of glibenclamide (10-100 microM) to the intracellular solution caused a concentration-dependent decrease in the open time of CFTR Cl- channels, but closed times did not change. This suggests that glibenclamide is an open-channel blocker of CFTR. 3. Glibenclamide is a weak organic acid. Acidification of the intracellular solution relieved glibenclamide inhibition of CFTR, suggesting that the anionic form of glibenclamide inhibits CFTR. 4. To begin to identify the glibenclamide binding site in CFTR, we investigated whether glibenclamide competes with either MgATP or Cl- ions for a common binding site. Glibenclamide inhibition of CFTR was unaffected by nucleotide-dependent stimulation of CFTR, suggesting that glibenclamide and intracellular MgATP interact with CFTR at distinct sites. 5. Glibenclamide inhibition of CFTR was voltage dependent and enhanced when the external Cl- concentration was decreased. The data suggest that glibenclamide and Cl- ions may compete for a common binding site located within a large intracellular vestibule that is part of the CFTR pore.


Subject(s)
Chloride Channels/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Glyburide/pharmacology , Hypoglycemic Agents/pharmacology , Animals , Cell Line , Chloride Channels/drug effects , Diphosphates/antagonists & inhibitors , Diphosphates/pharmacology , Electric Stimulation , Electrophysiology , Glyburide/chemistry , Glyburide/metabolism , Humans , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/metabolism , Ion Channel Gating/drug effects , Kinetics , Membrane Potentials/physiology , Mice , Patch-Clamp Techniques
14.
J Biol Chem ; 271(41): 25184-91, 1996 Oct 11.
Article in English | MEDLINE | ID: mdl-8810276

ABSTRACT

To explore the relationship between structure and function in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, we studied Xenopus CFTR. We found that the anion permeability sequence of cAMP-activated Cl- currents in the apical membrane of Xenopus A6 epithelia differed from that of cAMP-activated Cl- currents in human epithelia expressing CFTR. To understand the molecular basis for this difference and to learn whether CFTR from another species would have properties similar to human CFTR, we assembled a full-length Xenopus CFTR cDNA from A6 cells. Expression of Xenopus CFTR in HeLa cells generated cAMP-activated whole-cell currents and cAMP-dependent protein kinase-activated single channels that resembled those of human CFTR with the exception that the anion permeability sequence was different (Br- = I- > Cl- in Xenopus CFTR and Br- = Cl- > I- in human). In addition, the single-channel conductance of Xenopus CFTR was increased. To investigate protein regions that account for these differences, we constructed chimeric proteins by replacing either the first or second membrane-spanning domain of human CFTR with the equivalent region of Xenopus CFTR (hX1-6 and hX7-12, respectively) and examined their function in HeLa cells. We found that the anion permeability sequence (Br- = I- > Cl-) and single-channel conductance of hX1-6 resembled that of Xenopus CFTR expressed in HeLa cells, whereas hX7-12 had properties like those of human CFTR. However, the gating of hX1-6 showed a flickery behavior. The altered gating of hX1-6 was attributed to residues in the first extracellular loop of Xenopus CFTR because mutation of residues in that region to the corresponding residues of human CFTR produced gating behavior similar to that of human CFTR. These data suggest that sequence differences in the first membrane-spanning domains are responsible for the differences in the permeation properties of human and Xenopus CFTR and that the first extracellular loop influences channel gating.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Amino Acid Sequence , Animals , Cell Membrane/physiology , Cell Membrane Permeability , Cyclic AMP-Dependent Protein Kinases/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/biosynthesis , Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , HeLa Cells , Humans , Membrane Potentials , Molecular Sequence Data , Patch-Clamp Techniques , Phosphorylation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Regression Analysis , Sequence Homology, Amino Acid , Xenopus
15.
Mol Med Today ; 2(7): 290-7, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8796909

ABSTRACT

Defective epithelial Cl- secretion is the hallmark of the lethal genetic disease cystic fibrosis (CF). This abnormality is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a regulated Cl- channel. Since the identification of the single gene encoding CFTR, several hundred disease-causing mutations, associated with a wide variety of clinical phenotypes, have been reported. To understand the relationship between genotype and clinical phenotype, researchers have investigated how mutations in CFTR disrupt its function. Here, we review the recent progress in understanding how CF-associated mutations in CFTR produce defective Cl- channels, and discuss the implications of this knowledge for the development of therapy for CF.


Subject(s)
Chlorides/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/genetics , Cystic Fibrosis/metabolism , Cystic Fibrosis/therapy , Genotype , Humans , Models, Molecular , Mutation , Patch-Clamp Techniques
16.
J Biol Chem ; 271(25): 14995-5001, 1996 Jun 21.
Article in English | MEDLINE | ID: mdl-8663008

ABSTRACT

Proline residues located in membrane-spanning domains of transport proteins are thought to play an important structural role. In the cystic fibrosis transmembrane conductance regulator (CFTR), the predicted transmembrane segments contain four prolines: Pro99, Pro205, Pro324, and Pro1021. These residues are conserved across species, and mutations of two (P99L and P205S) are associated with cystic fibrosis. To evaluate the contribution of these prolines to CFTR Cl- channel function, we mutated each residue individually to either alanine or glycine or mutated all four simultaneously to alanine (P-Quad-A). We also constructed the two cystic fibrosis-associated mutations. cAMP agonists stimulated whole cell Cl- currents in HeLa cells expressing the individual constructs that resembled those produced by wild-type CFTR. However, the amount of current was decreased in the rank order: wild-type CFTR = Pro324 > Pro1021 > Pro99 >/= Pro205 mutants. The anion selectivity sequence of the mutants (Br- >/= Cl- > I-) resembled wild-type except for P99L (Br- >/= Cl- = I-). Although the Pro99, Pro324, and Pro1021 mutants produced mature protein, the amount of mature protein was much reduced with the Pro205 mutants, and the P-Quad-A made none. Because the Pro99 constructs produced mature protein but had altered whole cell currents, we investigated their single-channel properties. Mutant channels were regulated like wild-type CFTR; however, single-channel conductance was decreased in the rank order: wild-type CFTR >/= P99G > P99L >/= P99A. These results suggest that proline residues in the transmembrane segments are important for CFTR function, Pro205 is critical for correct protein processing, and Pro99 may contribute either directly or indirectly to the Cl- channel pore.


Subject(s)
Chloride Channels/physiology , Cyclic AMP/physiology , Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Proline , Protein Structure, Secondary , Adenosine Triphosphate/pharmacology , Amino Acid Sequence , Cyclic AMP/analogs & derivatives , Cyclic AMP/pharmacology , Cystic Fibrosis/genetics , Cystic Fibrosis/physiopathology , Electrophysiology , HeLa Cells , Humans , Membrane Potentials/drug effects , Models, Structural , Point Mutation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
17.
EMBO J ; 14(5): 876-83, 1995 Mar 01.
Article in English | MEDLINE | ID: mdl-7534226

ABSTRACT

Variability in the severity of cystic fibrosis (CF) is in part due to specific mutations in the CF transmembrane conductance regulator (CFTR) gene. To understand better how mutations in CFTR disrupt Cl- channel function and to learn about the relationship between genotype and phenotype, we studied two CF mutants, A455E and P574H, that are associated with pancreatic sufficiency. A455E and P574H are located close to conserved ATP binding motifs in CFTR. Both mutants generated cAMP-stimulated apical membrane Cl- currents in heterologous epithelial cells, but current magnitudes were reduced compared with wild-type. Patch-clamp analysis revealed that both mutants had normal conductive properties and regulation by phosphorylation and nucleotides. These mutants had normal or increased Cl- channel activity: A455E had an open-state probability (Po) similar to wild-type, and P574H had an increased Po because bursts of activity were prolonged. However, both mutants produced less mature glycosylated protein, although levels were greater than observed with the delta F508 mutant. These changes in channel activity and processing provide a quantitative explanation for the reduced apical Cl- current. These data also dissociate structural requirements for channel function from features that determine processing. Finally, the results suggest that the residual function associated with these two mutants is sufficient to confer a milder clinical phenotype and infer approaches to developing treatments.


Subject(s)
Adenosine Triphosphate/metabolism , Chloride Channels/metabolism , Membrane Proteins/genetics , Mutation/physiology , Animals , Cyclic AMP/agonists , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator , Epithelial Cells , Epithelium/metabolism , HeLa Cells , Humans , Ion Channel Gating , Membrane Proteins/metabolism , Pancreas/metabolism , Patch-Clamp Techniques , Phosphorylation , Rats , Rats, Inbred F344 , Thyroid Gland/physiology
18.
Biophys J ; 67(5): 1867-75, 1994 Nov.
Article in English | MEDLINE | ID: mdl-7532021

ABSTRACT

Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels appear to be regulated by hydrolysis of ATP and are inhibited by a product of hydrolysis, ADP. We assessed the effect of the other product of hydrolysis, inorganic phosphate (P(i)), on CFTR Cl- channel activity using the excised inside-out configuration of the patch-clamp technique. Millimolar concentrations of P(i) caused a dose-dependent stimulation of CFTR Cl- channel activity. Single-channel analysis demonstrated that the increase in macroscopic current was due to an increase in single-channel open-state probability (po) and not single-channel conductance. Kinetic modeling of the effect of P(i) using a linear three-state model indicated that the effect on po was predominantly the result of an increase in the rate at which the channel passed from the long closed state to the bursting state. P(i) also potentiated activity of channels studied in the presence of 10 mM ATP and stimulated Cl- currents in CFTR mutants lacking much of the R domain. Binding studies with a photoactivatable ATP analog indicated that Pi decreased the amount of bound nucleotide. These results suggest that P(i) increased CFTR Cl- channel activity by stimulating a rate-limiting step in channel opening that may occur by an interaction of P(i) at one or both nucleotide-binding domains.


Subject(s)
Chloride Channels/drug effects , Chloride Channels/metabolism , Membrane Proteins/metabolism , Phosphates/pharmacology , 3T3 Cells , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Animals , Azides/metabolism , Biophysical Phenomena , Biophysics , Cell Line , Chloride Channels/genetics , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator , HeLa Cells , Humans , In Vitro Techniques , Membrane Proteins/genetics , Mice , Models, Biological , Mutation , Sulfates/pharmacology , Transfection
19.
Biophys J ; 66(5): 1398-403, 1994 May.
Article in English | MEDLINE | ID: mdl-7520292

ABSTRACT

Phosphorylated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels require nucleoside triphosphates, such as ATP, to open. As the concentration of intracellular ATP increases, the probability of the channel being open (Po) increases. To better understand how ATP regulates the channel, we studied excised inside-out membrane patches that contained single, phosphorylated CFTR Cl- channels and examined the kinetics of gating at different concentrations of ATP. As the ATP concentration increased from 0.1 to 3 mM the mean closed time decreased, but mean open time did not change. Analysis of the data using histograms of open- and closed-state durations, the maximum likelihood method, and the log-likelihood ratio test suggested that channel behavior could be described by a model containing one open and two closed states (C1<==>C2<==>O). ATP regulated phosphorylated channels at the transition between the closed states C1 and C2: as the concentration of ATP increased, the rate of transition from C1 to C2 (C1-->C2) increased. In contrast, transitions from C2 to C1 and between C2 and the open state (O) were not significantly altered by ATP. Addition of ADP in the presence of ATP decreased the transition rate from C1 to C2 without affecting other transition rates. These data suggest that ATP regulates CFTR Cl- channels through an interaction that increases the rate of transition from the closed state to a bursting state in which the channel flickers back and forth between an open and a closed state (C2). This transition may reflect ATP binding or perhaps a step subsequent to binding.


Subject(s)
Adenosine Triphosphate/metabolism , Chloride Channels/metabolism , Membrane Proteins/metabolism , 3T3 Cells/drug effects , 3T3 Cells/metabolism , Adenosine Diphosphate/pharmacology , Adenosine Triphosphate/pharmacology , Animals , Biophysical Phenomena , Biophysics , Chloride Channels/drug effects , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator , HeLa Cells , Humans , In Vitro Techniques , Ion Channel Gating , Kinetics , Likelihood Functions , Mice , Models, Biological
20.
Am J Physiol ; 266(4 Pt 1): L405-13, 1994 Apr.
Article in English | MEDLINE | ID: mdl-7513963

ABSTRACT

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel regulated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphorylation and by intracellular nucleotides. The function of CFTR, like other recombinant ion channels, has generally been studied in single cells using voltage-clamp techniques. However, because CFTR is normally located in the apical membrane of epithelia we wanted to develop a system to study the function of recombinant CFTR expressed in an epithelium. We chose Fischer rat thyroid (FRT) epithelia for two reasons. First, when grown on permeable filter supports, FRT cells form polarized epithelia with a high transepithelial resistance. Second, they have no endogenous cAMP-regulated Cl- channels in their apical membrane. We expressed CFTR in FRT epithelia either transiently, using recombinant vaccinia virus, or stably, using a retrovirus. To measure apical membrane Cl- currents, we permeabilized the basolateral membrane to monovalent ions with nystatin and imposed a large transepithelial Cl- concentration gradient. cAMP agonists stimulated apical membrane Cl- currents in FRT epithelia infected with wild-type CFTR (vTF-CFTR) but not in FRT epithelia infected with either control virus (vTF7-3) or CFTR containing the delta F508 mutation (vTF-delta F508). These Cl- currents had properties similar to those of cAMP-activated Cl- currents in cells expressing endogenous or recombinant CFTR.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Genetic Techniques , Membrane Proteins/metabolism , Thyroid Gland/metabolism , Animals , Cell Membrane/metabolism , Cells, Cultured , Chloride Channels/physiology , Cystic Fibrosis Transmembrane Conductance Regulator , Electric Conductivity , Epithelial Cells , Epithelium/metabolism , Epithelium/physiology , Rats , Rats, Inbred F344 , Thyroid Gland/cytology , Thyroid Gland/physiology
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