The DF508 Mutation Causes CFTR Misprocessing and Cystic
The most common CF-associated mutation is DF508, which deletes a phenylalanine in position 508 In vitro s tudies indicate that the resultant protein, CFTR- DF508, is misprocessed, although the in vivo consequences of this mutation remain uncertain To better understand the effects of the DF508 mutation in vivo, we produced CFTRDF508/DF508 pigs
REVIEW ARTICLE THE CRADLE OF THE ΔF508 MUTATION
mutation from the Baluch ethnicity residing in Pakistan Keywords: CFTR, Delta F508, Mutation Cystic fibrosis (CF) is the most common life-limiting autosomal recessive disorder; it affects nearly 1/2500 live births in Caucasians 1 CF is a multi-system disease and can involve secretory cells, sinuses, lungs,
CFTR MUTATION CLASSES Normal Class I Class II Class III Class
MUTATION EXAMPLES F508del N1303K I507del D1152H R347P R117H 3849+10kbC T 2789+5G A A455E G542X W1282X R553X aka “nonsense mutations, splice mutations or deletions” G551D S549N aka “gating mutations” Correctors such as lumacaftor or tezacaftor help defective CFTR fold correctly Potentiators such as ivacaftor help open the CFTR channel,
Cystic fibrosis: New compounds display strong therapeutic
despite its mutation, the delta-F508-CFTR protein may satisfactorily fulfill its function The problem is that once it is synthesized, it is recognized as being
In vitro pharmacologic restoration of CFTR-mediated chloride
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
Cystic Fibrosis
Nov 30, 2004 · panel that includes a mutation not included in the father's carrier test However, in this case, the baby carries the delta F508, inherited from his mother and the G542X mutation, inherited from his father; the G542X mutation is included in the 23-mutation panel used to test Mr R Mr R's carrier test should have detected this mutation
Current Clinical Trials at UNC CF Drug Development Highlights
most common CFTR mutation – delta F508 In a recently reported study, VX-809 was found to be generally safe, but was not able to restore CFTR function in patients with two copies of the delta F508 mutation There is a strong scientific rationale to expect that combining VX-809 with VX770 could be more effective, however An in-
R553X and W1316X in respiratory epithelial messenger RNA
patient (no 528) homozygous for the common CF mutation (delta F508); and a CF patient (no 272) who carries the R553X mutation and a missense mutation, S549N When mRNA from bronchial cells of the normal individual, the delta F508 homozygote, and the S549N/R553X compound heterozygote was reverse transcribed and amplified by
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Pharmacologic Correction of
F508-CFTR
2457J. Clin. Invest.
© The American Society for Clinical Investigation, Inc.0021-9738/97/11/2457/09 $2.00
Volume 100, Number 10, November 1997, 2457-2465
http://www.jci.org In Vitro Pharmacologic Restoration of CFTR-mediated Chloride Transport with Sodium 4-Phenylbutyrate in Cystic Fibrosis Epithelial CellsContaining
F508-CFTR
Ronald C. Rubenstein, Marie E. Egan,* and Pamela L. ZeitlinEudowood Division of Pediatric Respiratory Sciences, The Johns Hopkins Hospital, Baltimore, Maryland 21287; and
Section of
Pediatric Respiratory Medicine, Yale University, New Haven, Connecticut 06520Abstract
The most common cystic fibrosis transmembrane conduc- tance regulator mutation,F508-CFTR, is a partially func-
tional 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 ofF508-CFTR to escape deg-
radation and appear at the cell surface. Primary cultures of nasal polyp epithelia from CF patients (F508 homozygous
or heterozygous), or the CF bronchial epithelial cell lineIB3-1 (
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 se- cretion. 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 with5 mM 4PBA also resulted in the appearance of higher mo-
lecular 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 F508-CFTR. These data indicate that 4PBA is a prom- ising pharmacologic agent for inducing correction of the CF phenotype in CF patients carrying theF508 mutation. (
J.Clin. Invest.
1997. 100:2457-2465.) Key words: cystic fibro-
sis CFTR phenylbutyrate pharmacotherapyIntroduction
Cystic fibrosis (CF)
1 is a systemic disorder that results when mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an apical membrane glycoprotein, lead to a reduction in apical membrane chloride transport. CFTR is acAMP-dependent chloride channel that regulates fluid compo-sition in the respiratory and gastrointestinal tracts. In CF, un-
usually thick mucus leads to recurrent airway and intestinal obstruction, as well as chronic respiratory infection.70% of the mutant CFTR alleles in the Caucasian popula-
tion result from deletion of phenylalanine at position 508 F508-CFTR). This mutation results in a protein capable of conducting chloride (1-4), but absent from the plasma mem- brane because of aberrant intracellular processing (5). Under usual conditions at 37 C,F508-CFTR is retained in the endo-
plasmic reticulum (ER), perhaps by prolonged association with the ER chaperones calnexin (6) and hsp70 (7), and is then targeted for degradation by the ubiquitin/proteasome pathway (8, 9). There is one published report that by overexpression ofF508-CFTR, functional
F508-CFTR protein can be mea-
sured at the cell surface. This work usedF508-homozygous
CF airway and immortalized pancreatic cells treated with the transcriptional regulator butyrate (10), a prototype drug with considerable toxicity and a half-life necessitating intravenous administration (11). The F508 "trafficking" block is also reversible by incuba- tion of cultured CF epithelial cells at reduced temperatures (25-27 C). In a time-dependent manner, lowered temperature results in the appearance of CFTR protein and channel activ- ity at the cell surface (12). This observation suggests an intrin- sic thermodynamic instability inF508-CFTR at 37
C that
leads to recognition of the mutant protein by the ER quality control mechanism, prevents further trafficking, and results in protein degradation. Thermal stabilization may allow the pro- tein to assume the appropriate conformation for normal traf- ficking. High concentrations of glycerol ( or 10%), a pro- tein stabilizing agent or "chemical chaperone," also appears to facilitate movement ofF508-CFTR from the ER to the
plasma membrane (13, 14). Our goal is to identify nontoxic, clinically useful com- pounds that will increase the amount ofF508-CFTR on the
surface epithelium of patients with CF. Sodium 4-phenylbu- tyrate (4PBA) is a butyrate analogue that is approved for clini- cal use as an ammonia scavenger in subjects with urea cycle disorders. It is also a known transcriptional regulator of - and -globin (15) that may act by inhibiting histone deactylase (16). The hypothesis of this study is that 4PBA will promoteF508-CFTR trafficking to the cell surface.
Address correspondence to Ronald C. Rubenstein, M.D., Ph.D., Pe- diatric Pulmonary, The Johns Hopkins Hospital - Park 316, 600 N. Wolfe St., Baltimore, MD 21287. Phone: 410-955-2035; FAX: 410-955-1030; E-mail: rrubenst@welchlink.welch.jhu.edu
Received for publication 28 May 1997 and accepted in revised form16 September 1997.
1.Abbreviations used in this paper:
CF, cystic fibrosis; CFTR, cystic fi-
brosis transmembrane conductance regulator;F508-CFTR, cystic fi-
brosis transmembrane conductance regulator containing a deletion of phenylalanine at position 508; ER, endoplasmic reticulum; 4PBA, sodium 4-phenylbutyrate; PKA, cAMP-dependent protein kinase; W1282X, mutation of tryptophan at position 1282 to a termination codon. 2458Rubenstein et al.
Methods
Cell culture.
Primary CF nasal epithelial cells were isolated from sur- gical specimens and grown in tissue culture flasks coated with vitro- gen/fibronectin/bovine serum albumin using modifications of previ- ously described procedures (17). IB3-1 cells (17) were grown on uncoated plastic supports or, for immunocytochemistry, on coated glass cover slips. Cells were routinely cultured in 5% CO 2 incubators in LHC-8 (Biofluids, Inc., Rockville, MD) supplemented with 5% fe- tal bovine serum (Sigma Chemical Co., St. Louis, MO), 100 U/ml pen- icillin/streptomycin (GIBCO BRL, Gaithersburg, MD), 0.2 mg/ml Pri- maxin (imipenim; Merck and Co., West Point, PA), 80 g/ml tobramycin (Eli Lilly & Co., Indianapolis, IN), and 2.5 g/ml fungi- zone (Biofluids, Inc.). Cells for control experiments were cultured un- der routine conditions described above. Growth medium for the treated cells was comprised of the indicated agent at the indicated concentration added to the routine growth medium and incubated in a 5% CO 2 atmosphere.Chloride efflux.
Chloride efflux experiments were performed es-
sentially as described by Trapnell et al. (18). Briefly, cells were seeded in coated 35-mm dishes under the indicated conditions and grown to 80-90% confluence. Cells were loaded with 3Ci/dish of
Na 36Cl under equilibrium conditions in bicarbonate-free Ringer's so- lution buffered with 10 mM Hepes, pH 7.4, for 2-4 h at 37
C for con-
trol and 4PBA-treated cells and at 27C for cells incubated at the
27C condition. Cells were washed three times with 1 ml of ice cold Ringer's solution before addition of 1 ml Ringer's solution with (stim- ulated) or without (basal) 13.5
M forskolin (Sigma Chemical Co.) at
room temperature. The Ringer's solution was changed every 15 s and counted by liquid scintillation in CytoScint (ICN Research Products, Costa Mesa, CA). Cells were lysed after 3 min with 0.2 N NaOH and residual 36Cl was determined by liquid scintillation. Apparent first or- der rate constants for chloride secretion were determined from three to four independent time courses for each condition using a nonlinear least squares method. Statistical comparison of these rate constants was performed using a two-tailed t test.
Single channel/patch clamp recording.
Single channel patch clamp
studies were performed in the excised inside-out patch configuration using conventional procedures (19). Recording pipettes were con- structed from borosilicate glass capillaries (Garner Glass, Claremont, CA) using a microelectrode puller (PP83; Narishige Scientific Instru- ment Laboratory, Tokyo, Japan). The pipettes were partially filled with a standard pipette solution and had tip resistance of 2-5 M Recording was performed at room temperature (20-22C). Single
channel currents were recorded with a patch clamp amplifier (Axo- patch 200A; Axon Instruments Inc., Foster City, CA), low pass fil- tered at 1 kHz using an 8-pole Bessel filter, and stored on videotape after pulse code modulation (model PCM-501ES; Sony, Tokyo, Ja- pan). Data were redigitized (4 kHz) for analysis, transferred to a desktop computer, and analyzed using pCLAMP6 (Axon Instru- ments Inc.).The bath solution contained (mM): NaCl, 140; MgCl
2 , 2; EGTA,1; Hepes, 5; and CaCl
2 , 0.5 (free Ca 2110 nM as measured by Fura-2),
pH 7.3. The pipette solution contained (mM): NaCl, 140; CaCl 2 , 2; and Hepes, 5, pH 7.3. All solutions were filtered through 0.2- m fil- ters. Chloride channels were activated by the addition of the catalytic subunit of protein kinase A (PKA; Promega, Madison, WI) and Mg- ATP at final concentrations of 50 nM and 1 mM, respectively. To pre- vent CFTR channel rundown in excised patches, 1 mM Mg-ATP was added to the bath solution.Antibodies.
Antisera 169 and 181 directed against human CFTR
peptides in the R domain and before NBF-1, respectively, were gen- erated in rabbits and described previously in publications from our laboratory (20). Antiserum 169 was used at a 1:500 dilution for immu- nocytochemistry and antiserum 181 was used at 1:2,000 dilution for immunoblot detection. The secondary antibody for both immunocy-tochemistry and immunoblot analyses was a donkey anti-rabbit IgG-horseradish peroxidase conjugate (Amersham, Arlington Heights, IL)
used at 1:1,000 dilution for immunocytochemistry and 1:10,000 dilu- tion for immunoblots. Incubation times for both primary antisera and secondary antibodies were 1 h at room temperature.