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Originally published In Press as doi:10.1074/jbc.M205644200 on July 10, 2002

J. Biol. Chem., Vol. 277, Issue 39, 35896-35905, September 27, 2002
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Mutations in the Nucleotide Binding Domain 1 Signature Motif Region Rescue Processing and Functional Defects of Cystic Fibrosis Transmembrane Conductance Regulator Delta F508*

Ana C. V. deCarvalho, Lisa J. Gansheroff, and John L. TeemDagger

From the Department of Biological Science, Florida State University, Tallahassee, Florida 32306

Received for publication, June 7, 2002, and in revised form, July 9, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (Delta F508) 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/CFTRDelta F508 chimera system in yeast, we isolated two novel Delta F508 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 CFTRDelta F508 defect. Furthermore, a combination of both revertant mutations resulted in a 38-fold increase in CFTRDelta F508-mediated chloride current, representing 29% of wild type channel activity. The G550E mutation increased the sensitivity of CFTRDelta F508 and wild type CFTR to activation by cAMP agonists and blocked the enhancement of CFTRDelta F508 channel activity by 2 mM 3-isobutyl-1-methylxanthine. The data show that the Delta F508 defect can be significantly rescued by second-site mutations in the nucleotide binding domain 1 region, that includes the LSGGQ consensus motif.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cystic fibrosis (CF)1 is the most frequent lethal genetic disease associated with a single gene in Caucasians (1). CF results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an ATP binding cassette (ABC) transporter that functions as a phosphorylation and nucleotide-regulated chloride channel located in the apical membrane of epithelial cells (2, 3). The ABC transporters constitute a large family of ubiquitously expressed proteins, mostly involved in ATP-driven translocation of diverse substrates across biological membranes (4, 5). It has been proposed that a functional ABC transporter has a minimal structural requirement of two membrane-spanning domains and two nucleotide binding domains (NBDs) (5). The NBDs, or ABC cassettes, share 30-50% sequence identity (6) and are characterized by the presence of three conserved motifs; Walker A and Walker B motifs are present in several nucleotide binding and hydrolyzing proteins (7), and the ABC-signature motif, located just upstream of the Walker B, is diagnostic of ABC cassettes (5, 6).

The deletion of the Phe-508 (Delta F508) in the first nucleotide binding domain (NBD1) of CFTR is the most frequent CF-causing mutation, present in 90% of CF chromosomes. Delta F508 impairs normal protein maturation and trafficking to the plasma membrane (8, 9), presumably through a localized effect on the folding of the NBD1 domain (10-12). This misfolding results in retention of CFTRDelta F508 by the endoplasmic reticulum-associated quality control and in subsequent degradation with the participation of the cytoplasmic proteasome (13). The CFTRDelta F508 biosynthetic processing defect can be partially rescued by low temperature (14), high concentrations of glycerol (15), and other low molecular weight compounds that affect the cellular folding environment (16).

CFTR channel is regulated by phosphorylation by cAMP-dependent protein kinase (PKA) at multiple sites in the regulatory (R) domain and by ATP binding and hydrolysis at the NBDs (3, 17). Maximal phosphorylation of PKA sites in the R domain controls the channel bursting rate and open probability (Po) of CFTR wt channels by increasing the apparent affinity of NBDs for ATP (18, 19). CFTR is modified in vivo by different levels of phosphorylation, resulting in channels with corresponding different biophysical characteristics (3, 17, 20). The Delta F508 mutation alters CFTR function by decreasing the channel open probability (Po) (21, 22). The defective activity of CFTRDelta F508 channel can be ameliorated pharmacologically (23-25).

To identify regions in the NBD1 that are affected by Delta F508, we isolated point mutations that rescued the functional and processing defects of CFTRDelta F508. Making use of the sequence homology of NBDs of CFTR and the Saccharomyces cerevisiae-mating peptide pheromone transporter, Ste6p (26, 27), a STE6/CFTR chimera was previously developed to study the Delta F508 mutation in yeast (28, 29). Mutations analogous to Delta F508 disrupt the function of other ABC transporters (30-34). The mutation equivalent to Delta F508 in the STE6 gene, Delta L455, did not result in a defective phenotype (35), but in the context of the CFTR sequences, Delta F508 disrupts the a-factor transport function of STE6/CFTR chimera (28, 36), providing a yeast system for identification of Delta F508 revertant mutations within CFTR sequences. Here we used the STE/CFTRDelta F508 chimera system to identify novel amino acid substitutions just upstream (I539T) and within (G550E) the ABC signature motif of CFTR NBD1 that partially suppressed the CFTRDelta F508 defect in HeLa cells and in Fischer rat thyroid (FRT) cells. The G550E mutation introduces a negatively charged amino acid in the highly conserved LSGGQ core signature motif of CFTR NBD1. Interestingly, the site of this mutation is flanked by two residues where CF-causing mutations have been identified that impair folding/trafficking of CFTR (S549R) or ATP-dependent channel gating (G551D) (37-41). We assessed the effect of the G550E mutation on the PKA-dependent activation of wild type and mutant CFTR chloride channel. We also investigated the effect of two compounds known to optimize PKA-dependent CFTRDelta F508 activity, genistein and 3-isobutyl-1-methylxanthine (IBMX) (23-25), on the Delta F508 revertant channels.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mutagenesis and Screen for Delta F508 Revertants in Yeast

The construction of the plasmids for expression of the STE6/CFTR and STE6/CFTRDelta F508 chimeras in yeast has been previously described (28). Derived from JTS6 plasmid carrying the STE6 gene, JTS6-H5 is a single copy CEN plasmid that carries the selectable marker URA3 and the STE6/CFTR hybrid gene H5, where sequences corresponding to Ste6p amino acid residues Arg-441-Ile-516 were replaced by the corresponding region from CFTR (F494-L558). Similarly, the CFTRDelta F508 sequence was used to make plasmid H5-Delta F508. A fragment containing 193 bp of CFTRDelta F508 DNA flanked by STE6 sequences, 101 bp on the amino terminus and 100 bp on the carboxyl terminus, was generated by PCR amplification of plasmid H5-Delta F508 using the following primers: forward (5'-GTTCTACGATAGCTATAATGGAT-3'), and reverse (5'-GCCTAATTGCCTTCATCAACAG-3'). To generate random point mutations within this fragment, PCR reactions were performed under mutagenic conditions (42, 43) using Taq DNA polymerase (Promega). For site-directed mutagenesis at the position corresponding to CFTR G550, degenerate DNA oligos were used in the PCR reactions to generate multiple codons. Yeast strain JPY201 (MATa, STE6Delta ::HIS3, gal2, ura5-52, lys2-801, trp1, leu2-3,112, his3Delta 200) (26) was co-transformed with the linearized H5-Delta F508 plasmid and the mutagenized 394-bp DNA fragments using the lithium acetate method. Mutations were inserted into H5-Delta F508 as a result of homologous recombination (44). The transformed JPY201 cells were plated in Sc-Ura medium, selective for transformants expressing the URA3 gene, and then mated with the yeast strain 22-2D4 (MATalpha , ura3-52, leu2-3,112, trp1). The yeast mating assays were performed as previously described (28, 29). Briefly, a lawn of 22-2D4 cells and transformed JPY201 colonies were replica-printed to a non-selective medium (yeast extract/peptone/dextrose) and incubated at 30 °C to allow mating. After 8 h, cells were replica-printed to a medium selective for diploids (yeast nitrogen base supplemented with leucine). After a retest, plasmid DNA was isolated from single haploid JPY201 colonies that gave rise to diploid colonies and sequenced. For a quantitative mating assay, JPY201 cells transformed with each H5 variant were grown to log phase in 0.1% glucose Sc-URA medium. From each culture, 3 × 106 cells were mixed with an equal number of 22-2D4 cells and collected by filtration onto a filter that was then placed on a yeast extract/peptone/dextrose plate for 4 h at 30 °C. Cells were resuspended, sonicated briefly, and plated from serial dilutions onto selective plates. Plates were incubated for 3 days at 30 °C, and diploid colonies were counted (45).

Vector Construction and CFTR Expression in Mammalian Cells

For the expression of CFTR variants in HeLa cells, pTM CFTR (28) digested at unique SmaI and XhoI sites flanking the Phe-508 region was used to clone the sequences from the H5-Delta F508 variants amplified by PCR. For expression in FRT cells, the pSwick (pMT3-Swick) vector (46) was similarly constructed. The CFTR region of the H5-Delta F508 hybrid gene containing second site mutations was amplified by PCR using the oligos Sma-L (forward, 5'-GGATTATGCCCGGGACCATTAAAG-3') and Xho-R (reverse, 5'-GATGAATGCTCGAGCTAAAGAAA-3'). The PCR product was digested with SmaI and XhoI, resulting in a 178-bp fragment corresponding to CFTR cDNA nucleotide residues 1630-1808 and introduced in-frame into pTM-CFTR and pSwick CFTR. Constructs were verified by DNA sequencing.

Transient Expression of CFTR in HeLa Cells-- HeLa cells were maintained at 37 °C in a humidified, 5% carbon dioxide atmosphere. Growth medium was minimum essential medium with Earle's salts (Sigma) supplemented with 10% fetal bovine serum (Summit Biotechnology) and 100 units/ml penicillin G sodium, 100 units/ml streptomycin sulfate, and 0.25 µg/ml amphotericin B (Invitrogen). The vaccinia virus/bacteriophage T7 hybrid expression system (47) was used for transient expression of the CFTR variants in HeLa cells, as described previously (28). Briefly, sub-confluent 100-mm plates of HeLa cells were infected with recombinant vaccinia virus expressing T7 RNA polymerase (10 MOI) and transfected with pTM plasmids carrying each CFTR variant cDNA under the control of the T7 promoter. Cells were incubated for 18 h and lysed.

Transient Expression of CFTR in FRT Cells-- FRT epitheloid cells and FRT cell lines stably expressing CFTR wt and CFTR Delta F508 (48) were gifts from Michael Welsh, University of Iowa. FRT cells were maintained at 37 °C in a humidified, 5% carbon dioxide atmosphere. Growth medium was Coon's modification of Ham's F-12 (Sigma) supplemented with 5% fetal bovine serum (Summit Biotechnology) and 100 units/ml penicillin G sodium, 100 units/ml streptomycin sulfate, and 0.25 µg/ml amphotericin B (Invitrogen). Dividing FRT cells were trypsinized in a solution containing 0.25% trypsin and 0.1% EDTA, pelleted, and resuspended in serum-free F-12 Coon's medium. Aliquots of cell suspension equivalent to 5 × 105 cells were transferred to microcentrifuge tubes, pelleted, and resuspended in F-12 Coon's medium containing DMRIE-cholesterol reagent (Invitrogen) complexed with pSwick plasmid, carrying each CFTR variant cDNA. Cells were transfected at 37 °C for 2 h with slow rotation and plated on Millicell hemagglutinin-permeable cell culture inserts (pore size, 0.45 µm, Millipore Co.). Transepithelial chloride currents were recorded 4 days after transfection.

Generation of FRT Stable Cell Lines-- FRT cells were co-transfected with pSwick CFTR and pcDNA plasmid (Invitrogen) using the DMRIE-cholesterol reagent. Clonal cell lines resistant to zeocin (Invitrogen) were expanded and screened for CFTR expression using short circuit chloride current measurements in Ussing chambers.

Western Blot Analysis of CFTR Protein

Cell Lysates-- For analysis of CFTR processing, transiently transfected HeLa cells were washed 3 times with phosphate-buffered saline and lysed with radioimmune precipitation assay buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0) containing a protease inhibitor mixture (1 mM benzamidine, 5 µg/ml pepstatin, 5 µg/ml leupeptin, 1 µg/ml aprotinin, and 17.4 µg/ml phenylmethylsulfonyl fluoride). After unbroken cells and nuclei were discarded by low speed centrifugation, proteins in the lysates were denatured in SDS-gel loading buffer and stored at -20 °C.

Protein-enriched Fraction-- Cells growing on 100-mm plates were washed 3 times with phosphate-buffered saline, collected in 2 ml of phosphate-buffered saline, pelleted, and resuspended in ice-cold 1 ml TEAA buffer (20 mM Tris/HCl, pH 8.0, 1 mM EDTA, 3 mM EGTA) supplemented with protease inhibitor mixture (1 mM benzamidine, 5 µg/ml pepstatin, 5 µg/ml leupeptin, 1 µg/ml aprotinin, and 17.4 µg/ml phenylmethylsulfonyl fluoride). After a 5-min incubation in TEAA, the cell suspension was passed 10 times through a 27-gauge needle. Nuclei and cell debris were pelleted (5 min/4,000 × g) and discarded, and the cleared supernatant was submitted to high speed centrifugation (100,000 × g/30 min). The pelleted membranes were resuspended in SDS-gel loading buffer and stored at -20 °C.

Immunoblot Analysis-- Fifty micrograms of total protein were separated by SDS-PAGE on 6% gels. Proteins were electrophoretically transferred to nitrocellulose membranes (Micron Separations Inc.). CFTR variants were probed with the monoclonal antibody M3A7, which recognizes an epitope within the CFTR NBD2 (9). A secondary anti-mouse IgG antibody peroxidase conjugate (Jackson ImmunoResearch Laboratories Inc.) was added, and protein bands were visualized using ECL (Amersham Biosciences).

Electrophysiology

FRT cell lines stably expressing CFTR variants or transiently transfected were plated at 2.5 × 105 cells/cm2 on Millicell-hemagglutinin cell culture inserts. Transepithelial resistance was monitored daily (Millicell Electrical Resistance System, Millipore Co.) and was typically greater than 3000 ohms/cm2 after 4 days. FRT monolayers were mounted in modified Ussing chambers (Jim's Instruments, Iowa City, IA) and continually gassed with O2. Temperature was maintained at 37 °C. Transepithelial chloride gradient was imposed by bathing the basolateral surface with a recording solution containing 135 mM NaCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 2.4 mM K2HPO4, 0.6 mM KH2PO4, 10 mM HEPES, and 10 mM dextrose, pH 7.4, and the apical surface with a similar recording solution, except that 135 mM sodium gluconate replaced the 135 mM NaCl, bringing the chloride concentration to 4.8 mM. The potential difference between the potential sensing electrodes was compensated. The transepithelial voltage was clamped to zero (voltage clamp channel module, model 558C-5, Dept. of Bioengineering, University of Iowa), and transepithelial resistance was monitored by recording current deflections in response to 2-s pulses of 1 or 5 mV every 50 s. The short circuit currents were recorded continuously on a charter recorder (model SR6335, Western Graphtec, Inc.). After a stable base-line current was observed (usually within less than 10 min), IBMX, forskolin, and genistein (Sigma) were added to the apical chamber, as described for each experiment, and the current, reflecting the flow of chloride promoted by its concentration gradient (Isc), was recorded as a downward deflection. The Isc was calculated as the difference between the base-line and the sustained phase of the response (plateau), or the peak current. Values were normalized by the area of the insert (0.6 cm2), and results were then expressed in µA/cm2.

Statistical Analysis

Data are expressed as the mean ± S.E. Current values were compared before normalization by the Student's t test or by one-way ANOVA with a Student-Newman-Keuls follow-up test at a 95% (p < 0.05) or greater confidence level.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of Delta F508 Suppressor Mutations Using the STE/CFTR Chimera System in Yeast-- A STE6/CFTR hybrid gene (H5-wt), in which a region coding for 74 amino acid residues within NBD1 of STE6 (R441 to I516) was replaced by the corresponding region of the CFTR NBD1 (F494 to L558), has been previously described (28). H5-wt complements the yeast ste6Delta mutation (JPY201 strain), restoring a-factor transport and consequent mating. However, when the Delta F508 mutation was introduced in the CFTR portion of H5 (H5-Delta F508), the mating efficiency, assessed by a quantitative mating assay, was reduced to 0.28% of H5-wt (Table I) (28). Because the Delta F508 mutation can be modeled in yeast, this system can be used for the identification of second site mutations within the CFTR NBD1 region that restore a-factor transport function to the H5-Delta F508 chimera. To generate random point mutations, the entire CFTR portion of H5-Delta F508 was subjected to in vitro PCR mutagenesis and introduced into JPY201 yeast. Transformants were screened using a qualitative mating assay to identify colonies with an increased mating efficiency relative to H5-Delta F508. Plasmids yielding a revertant phenotype were rescued and reintroduced into JPY201 to confirm the phenotype by a quantitative mating assay. Plasmids associated with improved mating efficiencies were sequenced. Two novel point mutations were isolated in the CFTR sequence that substantially rescued the H5-Delta F508 mating defect (Table I), resulting in the change of Ile-539 of the CFTR sequence to a Thr residue (I539T) and of Gly to Glu at the 550 position (G550E).

                              
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Table I
Yeast mating efficiency mediated by the STE6/CFTR chimeras
Results of quantitative mating are expressed as percentage of H5-wt and represent the mean ± S.E. for n = 3 experiments.

Mutations I539T and G550E Partially Rescue CFTRDelta F508-- The two novel Delta F508 revertant mutations isolated in yeast were located either just upstream (I539T) or within (G550E) the CFTR NBD1 signature motif (Fig. 1). Interestingly, the three Delta F508 revertant mutations previously isolated using the STE6/CFTR system, R553Q, R553M, and R555K, are also located within the NBD1 signature motif (28, 29). To evaluate the effect of the novel revertant mutations on CFTRDelta F508 processing, I539T and G550E mutations were introduced into the full-length CFTRDelta F508 cDNA (Delta F/I539T and Delta F/G550E) for expression in mammalian cells. To test whether the combination of the I539T and G550E mutations would result in an additive or synergistic effect in correcting the Delta F508 phenotype, we also constructed a CFTRDelta F508 allele containing both revertant mutations (Delta F/DB). The CFTR variants were expressed in HeLa cells using a vaccinia virus hybrid expression system, and the steady-state level of CFTR protein in the cell lysates was analyzed by SDS-PAGE followed by Western analysis (Fig. 2A). Only the core-glycosylated endoplasmic reticulum form of CFTR, referred to as "band B," was observed for CFTRDelta F508. CFTR wt was present as both band B and "band C" forms. The latter represents mature CFTR that trafficked to the Golgi, where complex oligosaccharide processing takes place. We observed that I539T and, to a lesser extent, G550E partially rescued the CFTRDelta F508-processing defect. The steady-state level of mature CFTRDelta F/DB protein was higher compared with each revertant alone (Fig. 2A).


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  		Fig. 1.   Schematic representation of CFTR. The positions of Phe-508, Ile-539, and Gly-550 amino acid residues within CFTR NBD1 are indicated. The shadowed boxes show the location of Walker A and Walker B motifs, and the hatched box shows the location of the C motif, also referred to as ABC signature motif.


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  		Fig. 2.   The I539T and G550E mutations partially rescue CFTRDelta F508-processing and functional defects. A, the steady-state level of CFTR protein resulting from transient expression in HeLa cells. HeLa cells were infected with vTF7-3 and transfected with pTM plasmid carrying each CFTR variant cDNA. Cell lysates were obtained 18 h after transfection, and 50-µg protein samples were separated by SDS-PAGE. Western blots were probed with anti-CFTR monoclonal antibody M3A7. HeLa cells infected with vTF7-3 served as the control. Positions of the core glycosylated (band B) and complex glycosylated CFTR (band C) are indicated by the arrows. B, FRT monolayers transiently expressing the CFTRDelta F508 variants were incubated in permeable supports for 4 days at 37 °C. Monolayers were mounted in Ussing chambers, and transepithelial chloride currents were recorded after activation with 10 µM forskolin and 100 µM IBMX. The results are expressed as percentage of chloride current mediated by CFTR wt (26.40 ± 2.79 µA/cm2, n = 20) after subtracting the background current observed for non-transfected FRT monolayers (0.25 ± 0.02 µA/cm2) and represent the mean ± S.E. for the number of experiments shown (n). Non-normalized current values were compared by one-way ANOVA, and the asterisks indicate the CFTR variants presenting a significant increase in chloride current over CFTRDelta F508 (alpha  = 0.05) by Student-Newman-Keuls follow-up test. C, chloride currents mediated by CFTR variants were assayed as in B. Non-normalized current values were compared by one-way ANOVA, and the asterisks indicate the CFTR variants presenting a significant increase in chloride current over CFTR wt (alpha  = 0.05) by Student-Newman-Keuls follow-up test.

Next, we assessed the phosphorylation-activated chloride channel function of the fraction of CFTRDelta F508 revertants present in the mature form at the plasma membrane. Mutant and wild type CFTR alleles were transiently expressed in FRT epithelial cells, which are well suited for this purpose, since they polarize to form monolayers with high transepithelial resistance and do not express a cAMP-activated chloride channel (48, 49). CFTR channels are routinely activated by a cAMP-agonist mixture containing 100 µM IBMX and 10 µM forskolin (48). Four days after transfection, FRT monolayers were mounted in modified Ussing chambers, and the transepithelial short circuit chloride current (Isc) was recorded after activation with 10 µM forskolin and 100 µM IBMX. In the absence of CFTR expression, FRT monolayers remain impermeable to chloride in response to cAMP agonists (48). In contrast, FRT cells transfected with CFTR wt respond to cAMP agonists with a rapid increase in Isc, reflecting increased chloride permeability (48). Transepithelial chloride permeability was markedly decreased in FRT monolayers expressing CFTRDelta F508, to 0.76% of CFTR wt Isc (Fig. 2B). The Delta F508 revertants CFTRDelta F/I539T and CFTRDelta F/G550E exhibited 6- and 12-fold increases in chloride current relative to CFTRDelta F508, respectively (Fig. 2B). The combination of the two revertant mutations (CFTRDelta F/DB) resulted in 29% of CFTR wt chloride currents (Fig. 2B), representing a substantial rescue (38-fold) of the chloride impermeability characteristic of epithelia expressing CFTRDelta F508.

To assess the effect of the Delta F508 revertant mutations on CFTR wt chloride channel function, CFTRG550E, CFTRI539T, and a CFTR allele containing both I539T and G550E mutations (CFTRDB) were transiently expressed in FRT cells, and chloride current was measured after activation with 10 µM forskolin and 100 µM IBMX. The results in Fig. 2C indicate that chloride currents mediated by CFTRI539T and CFTRG550E were not significantly different from CFTR wt. However, CFTRDB produced currents 50% higher than CFTR wt.

The G550E Mutation Increases the Sensitivity of CFTR and CFTRDelta F508 to Activation by cAMP Agonists in FRT Cells Transiently Expressing CFTR-- As shown in Fig. 2B, the G550E mutation was more effective than I539T in restoring the chloride channel function of CFTRDelta F508 (12-fold versus 6-fold increase), yet CFTRDelta F/G550E cell lysates contained lower levels of mature protein relative to CFTRDelta F/I539T (Fig. 2A). Interestingly, the G550E mutation occurs in a conserved residue, changing the core consensus ABC signature motif from LSGGQ to LSEGQ. The functional importance of the NBD1 signature motif is evident from the characterization of the CF, causing mutation G551D, which does not affect processing but results in decreased CFTR chloride channel function (41, 50) (Table II).

                              
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Table II
Effect on CFTR-mediated transepithelial chloride currents of CF-causing mutations and Delta F508 revertant mutations within the LSGGQ motif
FRT cells monolayers transiently expressing the CFTR variants were mounted in Ussing chambers, and chloride current values were measured as described in Fig. 2B. Results are expressed as percentage of CFTR wt chloride and represent the mean ± S.E. for the number of experiments indicated (n). The asterisks indicate the variants, presenting a significant increase in chloride current over CFTRDelta F508 (ANOVA, alpha  = 0.05) by SNK follow-up test.

To better understand the mechanism by which the G550E mutation improves the function of CFTRDelta F508, we tested its effect on activation of CFTRDelta F508 and CFTR wt by suboptimal concentrations of forskolin. CFTR expressed in FRT cells is maximally stimulated with 10 µM forskolin (48), with lower concentrations resulting in decreased channel activity (49). Accordingly, CFTR wt, CFTRDelta F508, CFTRG550E, and CFTRDelta F/G550E were transiently expressed in FRT cells, and the transfected cell monolayers were assayed for transepithelial chloride current in response to activation by sub-optimal concentrations of forskolin (0.5 µM) in the absence of IBMX. The results for each variant were expressed as the percentage of maximum chloride current, with the maximum defined by activation of the channels with 10 µM forskolin and 100 µM IBMX (Fig. 3). The G550E mutation substantially increases the sensitivity of CFTRDelta F508 to PKA activation, increasing the level of chloride current activated by the sub-optimal concentration of forskolin (0.5 µM) from 4.66% of maximum activation for CFTRDelta F508 to 29.25% for CFTR Delta F508/G550E (Fig. 3A). Although the G550E mutation did not increase the chloride channel activity of CFTR wt when the channels were activated with the optimal concentration of cAMP agonists (Fig. 2C), we observed that G550E did increase the sensitivity of CFTR wt to activation by the sub-optimal concentration of forskolin (compare 49.54 versus 77.2% of maximum Isc for CFTR wt and CFTR G550E, respectively) (Fig. 3A).


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  		Fig. 3.   The G550E mutation increases sensitivity of CFTR wt and Delta F508 to activation by forskolin after transient expression in FRT cells. A, FRT monolayers transiently expressing the CFTR variants were incubated in permeable supports for 4 days at 37 °C. Monolayers were mounted in Ussing chambers, and transepithelial chloride currents were recorded after activation with a sub-optimal forskolin concentration (0.5 µM). Results are expressed as the percentage of the maximum current for each variant, achieved with activation by 10 µM forskolin and 100 µM IBMX, and represent mean ± S.E. for n = 6 experiments. Current ratios were compared by one-way ANOVA, and the asterisks indicate a significant difference between the two CFTR variants (alpha  = 0.05) by Student-Newman-Keuls follow-up test. B, representative tracings for no CFTR control, CFTRDelta F508, and CFTRDelta F/G550E. Currents were recorded continuously, and the arrows indicate when forskolin and IBMX were added. The dashed lines represent the base lines. C, representative tracings for CFTR wt and CFTR G550E, as described in B.

Characterization of FRT Cell Lines Stably Expressing the CFTRDelta F508 Revertants-- We obtained FRT cell lines stably expressing CFTRDelta F/I539T, CFTRDelta F/G550E, and CFTRDelta F/DB to further characterize the effect of the revertant mutations on CFTRDelta F508 processing and function. FRT-CFTRDelta F508 and FRT-CFTR wt stable cell lines have been previously described (48). Results from CFTR immunoblotting analysis (Fig. 4A) and functional studies (Fig. 4B) confirmed the suppression of the CFTR Delta F508 processing and chloride impermeability defects by I539T and G550E mutations, as observed for the transient expression experiments (Fig. 2, A and B). Functional assays showed a 13-fold increase in transepithelial chloride current for FRT-CFTRDelta F/I539T in relation to FRT-CFTRDelta F508, which is in agreement with the substantial amount of mature protein observed for this cell line (Fig. 4, A and B). Whereas the Isc was not significantly different between FRT-CFTRDelta F/DB and FRT-CFTRDelta F/I539T, we observed decreased levels of both mature (band C) and immature (band B) protein for FRT-CFTRDelta F/DB. Functional studies showed a 6.3-fold increase in Isc for FRT-CFTRDelta F/G550E relative to FRT-CFTR Delta F508 (Fig. 4B), although the mature band C was barely detectable, and the steady-state levels of band B were decreased for the revertant (Fig. 4A). These results suggest that the G550E mutation increases the channel activity of CFTR variants containing the Delta F508 mutation.


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  		Fig. 4.   Effect of I539T and G550E mutations on CFTR Delta F508 temperature sensitivity and dose response to forskolin activation in FRT stable cell lines. A, Western blot analysis of the steady-state level of CFTRDelta F508, CFTRDelta F/I539T, CFTRDelta F/G550E, and CFTRDelta F/DB stably expressed in FRT cells. A membrane-enriched fraction for each cell line was obtained, and 50-µg protein samples were separated by SDS-PAGE. Western blots were probed with anti-CFTR antibody M3A7, as described in Fig. 2A. B, effect of low temperature on transepithelial chloride current for the cell lines in A. FRT cells stably expressing each CFTR variant were grown on permeable supports for 7 days at 37 °C (solid bar) or for 5 days at 37 °C followed by 48 h at 30 °C (striped bar). The polarized monolayers were mounted on Ussing chambers, and the transepithelial chloride current was measured after activation with 10 µM forskolin and 100 µM IBMX. Results represent mean ± S.E. for n = 6 experiments. The asterisks indicate the variants for which the low temperature treatment resulted on a significant increase in current over the control monolayers (37 °C) (p < 0.05, t test). C, dose-response curve for forskolin activation. Monolayers of FRT stable cell lines described in A and B were mounted on Ussing chambers and activated with 10 µM IBMX and decreasing concentrations of forskolin. FRT-CFTRDelta F508 monolayers were incubated for 5 days at 37 °C followed by 48 h at 30 °C; the other FRT cell lines were incubated for 7 days at 37 °C. Results are expressed as the percentage of maximum activation, as in Fig. 3A. Results represent mean ± S.E. for n = 6 measurements.

Temperature Sensitivity of the Delta F508 Revertants-- The CFTRDelta F508-processing defect can be partially reversed by incubating mammalian cells at 25-30 °C (14) or by expressing the mutant in cells that are usually incubated at lower temperatures, such as insect cells (51) and Xenopus oocytes (52). We also investigated the effect of the revertant mutations I539T and G550E on the temperature sensitivity of CFTR Delta F508. To detect the rescue of CFTRDelta F508 by low temperature treatment and study the effect of the revertant mutations on the temperature sensitivity of CFTRDelta F508, FRT monolayers stably expressing each CFTR variant were incubated at 37 °C for 5 days followed by a 48-h incubation at 30 °C before Isc measurements, which were performed at physiological temperature (37 °C) (Fig. 4B). Under these conditions, low temperature treatment of FRT-CFTR Delta F508 resulted in 4.8-fold increase in Isc (Fig. 4B). Also consistent with the results by others (14, 53), we did not observe an increase in cAMP-activated chloride current for FRT monolayers expressing CFTR wt after low temperature treatment (not shown). The G550E mutation attenuated the temperature sensitivity of CFTRDelta F508, as the low temperature treatment resulted in a 2-fold increase in chloride current for CFTRDelta F/G550E. The I539T mutation rendered CFTRDelta F508 and CFTRDelta F/G550E insensitive to incubation at 30 °C (Fig. 4B). The Isc measured after low temperature treatment of FRT-CFTRDelta F508 (12.27 µA/cm2) was comparable with the Isc of FRT-CFTRDelta F/G550E incubated at physiological temperature (15.57 µA/cm2) (Fig. 4B).

Sensitivity of the CFTR Delta F508 Revertants to cAMP Activation-- The increase in sensitivity to forskolin activation observed for the CFTRDelta F/G550E mutant in transient expression (Fig. 3) we hypothesize to be an intrinsic property of the mutant channel. Alternatively, it is possible that the enhanced sensitivity to activation is secondary to a cooperative interaction among the increased number of mutant channels at the plasma membrane (54). To assess the effect of G550E and I539T on the modulation of sensitivity of CFTRDelta F508 to activation while minimizing the potential effect of different channel levels at the plasma membrane, we compared the sensitivity to forskolin activation of CFTR Delta F508 rescued by incubation for 48 h at 30 °C, with CFTRDelta F/G550E, CFTRDelta F/I539T, and CFTRDelta F/DB incubated at 37 °C. FRT monolayers stably expressing each CFTR variant were mounted in Ussing chambers, and the cAMP-activated chloride current was measured in response to decreasing concentrations of forskolin. The results are expressed as the percentage of maximum Isc (obtained by activation with 10 µM forskolin and 100 µM IBMX) for each variant. The results demonstrate that CFTR variants containing the G550E mutation have increased sensitivity to forskolin activation (Fig. 4C). When CFTRDelta F/G550E, incubated at physiological temperature, was compared with CFTRDelta F508 we observed a significant increase in sensitivity to activation by 0.5 µM forskolin, confirming the results from transient expression (Fig. 3). FRT-CFTRDelta F/DB (containing both I539T and G550E) also exhibited a significant increase in sensitivity to activation relative to FRT-CFTRDelta F508 over the entire range of suboptimal forskolin concentrations tested. However, in contrast to the variants containing G550E, increased sensitivity to activation by suboptimal concentrations of cAMP agonist was not observed for the variant containing I539T alone.

The G550E Mutation Increases the Sensitivity of CFTR wt to Activation by cAMP Agonists in FRT Cells Stably Expressing CFTR-- Next, we investigated the dose-response for forskolin activation of CFTR wt and CFTRG550E stably expressed in FRT cells. Polarized FRT monolayers expressing CFTR wt and CFTRG550E (Fig. 5A) were mounted in Ussing chambers and activated with decreasing amounts of forskolin. The resulting Isc values were expressed as the percentage of maximum Isc, achieved by activation with 10 µM forskolin and 100 µM IBMX. The dose-response curve for activation by forskolin showed a remarkable increase in sensitivity for FRT-CFTRG550E relative to CFTR wt over the entire sub-optimal concentration range tested (0.1-1 µM) (Fig. 5B), confirming the results obtained with the transient expression experiments (Fig. 3).


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  		Fig. 5.   Effect of G550E mutation on CFTR dose response to forskolin activation in FRT stable cell lines. A, Western blot analysis of the steady-state level of protein for CFTR wt and CFTR G550E stably expressed in FRT cells, as described in Fig 2A. Transepithelial chloride currents for each CFTR variant were recorded after activation with 10 µM forskolin and 100 µM IBMX (Isc); results are shown as the mean ± S.E. for n = 6 experiments. B, dose-response curve for forskolin activation. Monolayers of FRT stable cell lines described in A were incubated for 7 days at 37 °C and mounted on Ussing chambers. CFTR channels were activated with decreasing concentrations of forskolin. Results are expressed as the percentage of maximum activation. Results represent the mean ± S.E. for n = 6 experiments.

Additional Delta F508 Revertant Mutations at Position G550-- The significant rescue of the Delta F508 defect by the G550E mutation prompted us to screen for other Delta F508 revertant mutations at this codon using site-directed mutagenesis. Additional revertant of the Delta F508 defect, G550H, was identified by the yeast-mating assay (not shown). Because the G550E mutation replaces a Gly residue with the negatively charged Glu, we also constructed a CFTRDelta F/G550D variant to test whether the negative charge resulting from an aspartate substitution would have a similar effect on CFTR channel activation. The effect of mutations at position G550 on chloride channel function of CFTRDelta F508 was investigated by Isc measurements of FRT monolayers transiently expressing each variant. Results were expressed as the percentage of cAMP-activated Isc measured for monolayers expressing CFTR wt (Table II). CFTR variants bearing CF-causing mutations within the NBD1 signature motif, G551D and S549R, were included in the experiment and produced low Isc, as expected. A significant increase in Isc for monolayers expressing each novel Delta F508 revertant at position G550 relative to CFTRDelta F508 was observed. CFTRDelta F/G550H and CFTRDelta F/G550D displayed 50 and 68% of CFTRDelta F/G550E Isc, respectively, demonstrating that these additional revertants were not as effective as G550E in suppressing the CFTR Delta F508 defect. We tested the CFTRDelta F/G550D response to activation by low concentrations of forskolin (0.5 µM) as described in Fig. 3. However, unlike the results observed for CFTRDelta F/G550E, suboptimal forskolin concentration failed to activate CFTRDelta F/G550D (not shown).

The G550E Mutation Modulates the CFTR Delta F508 Response to Activation by Genistein and Millimolar Concentrations of IBMX-- Several compounds have been isolated that optimize channel activity of phosphorylated CFTR by mechanisms that are independent of increase in cAMP (55, 56). The effect of the G550E mutation to increased sensitivity to cAMP-mediated activation of mutant and wt CFTR led us to ask if this mutation would modulate the response of CFTRDelta F508 channels to optimization by two such compounds. We tested the effect of 2 mM IBMX and 50 µM genistein on the PKA-dependent activity of CFTRDelta F508, CFTRDelta F/I539T, CFTRDelta F/G550E, CFTRDelta F/DB, and CFTR wt. FRT stable cell lines expressing the CFTR variants, including CFTRDelta F508, were incubated at physiological temperatures for these experiments, since genistein and high concentrations of IBMX are reported to enhance the channel activity of even small amounts of mutant CFTR present at the plasma membrane (25). Genistein, a natural isoflavone compound, enhances PKA-dependent activity of wild type and mutant CFTR in several cell types (22, 24, 25, 55, 57). Optimal concentrations of genistein have been reported to be around 50 µM (58, 59). Fifty micromolar genistein was added to the apical side of FRT monolayers mounted in Ussing chambers, and 5 min later, the channels were activated with a cAMP-agonist mixture (10 µM forskolin and 100 µM IBMX). Under these conditions, we did not observe channel activation by genistein alone for any of the FRT cell lines. Genistein significantly increased the PKA-activated Isc for all the cell lines containing the Delta F508 mutation, although it produced a smaller increase for cell lines containing the G550E mutation; compare 58 and 70% increase for CFTRDelta F508 and CFTRDelta F/I539T, respectively, with 45 and 25% for CFTRDelta F/G550E and CFTRDelta F/DB (Fig. 6). We did not observe enhancement of PKA-activated chloride currents for FRT-CFTR wt by genistein under the experimental conditions employed (Fig. 6).


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  		Fig. 6.   Effect of the I539T and G550E mutations on CFTR Delta F508 activation by 2 mM IBMX and 50 µM genistein. FRT monolayers stably expressing each CFTR variant were mounted in Ussing chambers and activated with 10 µM forskolin and 2 mM IBMX (solid bar) or 50 µM genistein followed by 10 µM forskolin and 100 µM IBMX (striped bar). Results are shown as the mean ± S.E. for n = 6 experiments and are expressed as the percentage of control currents for each cell line, achieved by activation with 10 µM forskolin and 100 µM IBMX. The asterisks indicate that the Isc values were significantly different from the control for each CFTR variant (t test, p < 0.05).

When present at millimolar concentrations, the xanthine derivative IBMX has been shown to have an effect on CFTRDelta F508 activity that is independent from its well established activity as a phosphodiesterase inhibitor (23, 24, 55, 60). FRT monolayers stably expressing the CFTR variants were mounted in Ussing chambers and activated with 10 µM forskolin and 2 mM IBMX. Two millimolar IBMX resulted in ~80% increase in Isc for FRT-CFTRDelta F508 and FRT-CFTRDelta F/I539T (Fig. 6). Interestingly, 2 mM IBMX did not affect the PKA-activated Isc of the FRT-CFTR wt, FRT-CFTRDelta F/G550E, or FRT-CFTRDelta F/DB under the experimental conditions employed.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Two novel Delta F508 revertant mutations were identified just upstream (I539T) and within (G550E) the core consensus ABC signature motif LSGGQ in the NBD1 of CFTR. Each mutation partially restored processing of mutant CFTRDelta F508 expressed in HeLa cells, with I539T being the most effective. Increased cAMP-activated chloride permeability was also observed in FRT monolayers expressing CFTRDelta F/I539T and CFTRDelta F/G550E to levels 6- and 12-fold higher than CFTRDelta F508, respectively. The larger fraction of processed CFTRDelta F/I539T and CFTRDelta F/G550E observed relative to CFTRDelta F508, thus, represents functional channels localized at the plasma membrane. Furthermore, functional studies using a double revertant allele (CFTRDelta F/DB) showed that I539T and G550E mutations act synergistically to increase CFTRDelta F508 chloride currents to ~29% of CFTR wt, representing a 38-fold increase over the CF mutant. Processing of CFTRDelta F/DB, as indicated by the fully glycosylated form of CFTR, was correspondingly increased. Therefore, the processing defect of CFTRDelta F508 was substantially rescued by second-site mutations in the region of the LSGGQ motif of NBD1, leading to increased functional activity of mutant channels at the plasma membrane.

The I539T and G550E mutations were identified as revertants of the CF-causing mutation Delta F508. It might, thus, be expected that these revertant mutations, identified by virtue of their effects to reverse the Delta F508 defect, would be specific for suppression of Delta F508. However, results by others suggest that G550E can partially rescue another processing-defective CF mutant, A561E (61). Possibly, the Delta F508 and A561E mutations cause misfolding in a similar manner, allowing each to be partially compensated by G550E. Alternatively, the suppressors may have a general effect to increase CFTR processing. In support of the latter possibility, it was observed that the combination of I539T and G550E within wild type CFTR increased functional activity. Further experiments will be necessary to determine the extent to which Delta F508 revertant mutations I539T and G550E suppress other CF mutations within the NBD1 that are associated with defective protein processing.

To further assess the effects of the revertant mutations on CFTRDelta F508, we compared the functional activity of CFTRDelta F508, CFTRDelta F/G550E, and CFTRDelta F/I539T under various experimental conditions. Because Delta F508 is a temperature-sensitive mutation (14), we determined whether revertant mutations alter the temperature sensitivity of CFTRDelta F508. The I539T mutation, when introduced in either CFTRDelta F508 or CFTRDelta F/G550E, rendered these variants insensitive to low temperature treatment. It is possible that the I539T revertant mutation and the low temperature treatment stabilize the same step in the mutant protein folding pathway; thus, their effects are not additive. The temperature insensitivity of CFTRDelta F508 rendered by suppressor mutations has also been observed by others (62). In contrast to I539T, G550E had little effect on CFTRDelta F508 temperature sensitivity, suggesting that it affects the protein folding pathway differently.

In addition to the processing defect, the Delta F508 mutation also impairs the chloride channel function of CFTR. The channel Po of CFTRDelta F508 is decreased relative to CFTR wt (21, 23) due to prolonged closed times, which are also observed for CFTR wt activated by suboptimal concentrations of forskolin (25). Results from patch-clamp studies using NIH 3T3 cells stably expressing CFTR and CFTR Delta F508 indicate that the Delta F508 mutation affects CFTR activation by attenuating PKA-dependent phosphorylation (22). Our results show that the G550E mutation decreased the concentration of forskolin required for half-maximal stimulation of all the CFTR variants tested, CFTR wt, CFTRDelta F508, and CFTRDelta F/I539T. Furthermore, G550E mutation improved PKA-dependent activity of CFTR variants bearing the Delta F508 mutation under optimal and suboptimal forskolin concentration, and improved the wild type channel activity only under suboptimal concentrations of forskolin, but not when maximal PKA activity was promoted, suggesting that this mutation could specifically increase function of underphosphorylated CFTR. Alternatively, G550E could facilitate CFTR phosphorylation under suboptimal PKA activity. Although the role of G550E in CFTR phosphorylation remains to be determined, the effect of G550E to increase the PKA-dependent activity of CFTRDelta F508 is consistent with the higher levels of function associated with CFTRDelta F/G550E relative to the low levels of processed protein observed.

Because IBMX and genistein are known to enhance the functional activity of CFTRDelta F508 (23-25), we assessed the effect of these molecules on CFTRDelta F/G550E, CFTRDelta F/I539T, and CFTRDelta F/DB. Our results show that 2 mM IBMX caused an increase (1.8-fold) in PKA-stimulated chloride current for CFTRDelta F508. In other studies addressing the effect of IBMX on CFTRDelta F508, it has similarly been shown that 2 mM IBMX increases activity of the CFTRDelta F508 channel activated with 10 µM forskolin without inducing further increase in cAMP (55). We further observed no detectable effect of IBMX on CFTR wt, which is consistent with functional studies in Xenopus oocytes (52) and with the observation that IBMX has a higher affinity for NBD1 Delta F508 relative to NBD1 wt (63). The effect of 2 mM IBMX on the activation of CFTRDelta F/I539T was similar to the effect observed for CFTRDelta F508. However, 2 mM IBMX did not increase the PKA-dependent currents of FRT-CFTRDelta F/G550E or FRT-CFTRDelta F/DB. We speculate that G550E could directly alter the binding of IBMX to CFTRDelta F508, impairing the increase in Po or further contributing to the decrease of current amplitude (24). Genistein significantly enhanced PKA-activated chloride currents of CFTRDelta F508, CFTRDelta F/G550E, CFTRDelta F/I539T, and CFTRDelta F/DB, although the currents mediated by CFTR variants containing the G550E mutation were stimulated to a lesser extent. It has been shown that genistein increased the iodide efflux from NIH 3T3 cells expressing CFTRDelta F508 after activation with a range of forskolin concentrations (0.01-100 µM) but increased CFTR wt-mediated efflux only when the channels were activated by suboptimal concentrations of forskolin (25). Genistein has been shown to activate specifically underphosphorylated CFTR (64) and to cause substantial increase in CFTRDelta F508 channel Po (22). The revertant mutations I539T and G550E did not preclude genistein enhancement of the PKA-dependent activity of CFTRDelta F508 implying that, similarly to the CF mutant, the revertants could be underphosphorylated at maximal PKA activity.

I539T significantly enhanced the processing of CFTRDelta F508. Notably, Thr is the most conserved amino acid residue at position 539 for CFTR of most non-primate species as well as for other ABC transporters, including members of the MRP/CFTR subgroup (ABC C) (Fig. 7). A significant level of CFTRDelta F508 chloride channel activity and a detectable level of mature protein have been observed for the homozygous Delta F508 CF mouse (65-68). Our results suggest that the T539 could contribute to the attenuated defect caused by the Delta F508 mutation in the murine CFTR. Gly-550 is a conserved residue within the ABC signature motif of several ABC transporters, including those with high homology to human CFTR (Fig. 7). The G550E mutation represents a non-conservative introduction of a negatively charged Glu residue, changing the LSGGQ core consensus signature sequence of NBD1 to LSEGQ. Interestingly, LSEGQ is the core signature motif found in few members of the human ABC A subgroup, NBD of ABCA6 (69) and NBD2 of ABCA8 and ABCA9 (accession numbers NP009099 and XP085646, respectively).


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  		Fig. 7.   Alignment of peptide sequences from various ABC transporters to the LSGGQ region of human CFTR NBD1. A multiple alignment sequence of NBD1 of CFTR from several species and other highly homologous ABC transporters was generated by the Pileup program (Wisconsin Package Version 10.2. Genetics Computer Group). The positions of the Delta F508 revertant mutations are indicated. CFTR sequences are from Mus musculus (mouse), Ovis aries (sheep), Bos taurus (bovine), Oryctolagus cuniculus (rabbit), Xenopus laevis (xenla), and Squalus acanthias (squac); sulfonylurea receptor 1 (acc8), from Ratus norvergicus (rat), and Homo sapiens (human); multidrug resistance-associated protein 6 (MRP6) from R. norvergicus (rat) and H. sapiens (human).

The highly conserved signature motif fulfills an essential role in the functioning of ABC transporters. Functions assigned to this motif include coupling of energy to translocation (6, 70), activation of ATP hydrolysis after substrate binding to other components of the transporter (71), and mediation of intra-domain interactions (72, 73). Direct participation of the ABC signature motif in the ATP-dependent dimerization of NBDs has also been suggested (74-76). The CF-causing mutation of the invariant Gly residue in the NBD1 signature motif of CFTR (LSGGQ), G551D, has been shown to reduce ATP binding (41, 77) and hydrolysis (18). Crystallographic structures determined for several ABC cassettes (74, 78) reveal a similar subdomain organization, an ATP binding pocket formed by the Walker A and Walker B motifs and a alpha -helical subdomain containing the ABC signature motif and upstream sequences. Thus, sequences corresponding to the CFTR NBD1 region that includes the Phe-508 residue and the revertant mutations (Ile-539-Gly-550) are localized to the same alpha -helical subdomain. The position of LSGGQ in the ABC cassette structure is in agreement with the proposed function in coupling the activities of the catalytic domains with those involved in regulation or transport (79). We speculate that G550E and I539T mutations could restore the LSGGQ-mediated interactions disrupted by the Delta F508 mutation.

The functional importance of the LSGGQ region of CFTR NBD1 has been supported by previous mutational analysis describing processing and functional defects associated with amino acid substitutions in the conserved motif (37-41). Our results indicate that mutations in this region also enhance CFTR Delta F508 processing and function and highlight the importance of the LSGGQ motif as a focus for understanding the defect associated with Delta F508. Elucidation of the role of the LSGGQ motif to mediate the defects associated with Delta F508 may be instrumental in the design of new therapies for CF.

    ACKNOWLEDGEMENTS

We thank Douglas Spiker for participation in the yeast screen, Scott Olenych for helpful discussions, Dr. Norbert Kartner for providing M3A7 monoclonal antibody, and members of Dr. Michael Welsh laboratory for the FTR cells and CFTR expression plasmids. We thank Drs. Debra Fadool and Ann Morris for comments on the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL61234 and a Program Enhancement Grant from Florida State University Research Foundation (to J. L. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biological Science, Biounit-238, Florida State University, Tallahassee, FL 32306. Tel.: 850-644-5121; Fax: 850-644-0418; E-mail: teem@bio.fsu.edu.

Published, JBC Papers in Press, July 10, 2002, DOI 10.1074/jbc.M205644200

    ABBREVIATIONS

The abbreviations used are: CF, cystic fibrosis; CFTR, CF transmembrane conductance regulator; ABC, ATP binding cassette; NBD, nucleotide binding domain; PKA, CAMP-dependent protein kinase; wt, wild type; FRT, Fischer rat thyroid; IBMX, 3-isobutyl-1-methylxanthine; ANOVA, analysis of variance; MRP, multidrug resistance-related protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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