PMC2800016_01
Chest radiographs on admission. (A) Chest plain radiograph shows diffuse reticulonodular densities in both central lung areas symmetrically. (B) On computed tomographic image with lung window setting, diffuse bronchiectasis is seen in both lungs. There are hyperlucent areas in the lung parenchyma due to peripheral bronchial obstruction.
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Chest radiographs and computed tomographic (CT) scans showed bilateral diffuse bronchial wall thickening and symmetric bronchiectasis in both lung fields (Fig. 1).
Denaturing gradient gel electrophoresis (DGGE) and nucleotide sequencing. (A) DGGE on CFTR exon 9 using the DNA sample from patient shows a typical heterozygote banding pattern containing heteroduplex bands (see reference no. 10). (B) Nucleotide sequencing shows that the patient's CFTR gene contains a mutation changed from T nucleotide at 1454 to C (heterozygous for L441P, CTG:Leu   CCG; Pro).
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Interestingly, a non-synonymous L441P mutation of CFTR was identified in one allele by DGGE and consecutive nucleotide sequencings (Fig. 2). However, no CFTR mutations including L441P were found in the blood samples from patient's mother (Fig. 2).
Immunoblotting and surface biotinylation of L441P mutant CFTR protein. HEK 293 cells were transfected with plasmids for wild type CFTR or CFTR carrying the L441P mutation and protein samples were blotted with anti-CFTR M3A7 antibody (Cell Signaling Technology, Danvers, MA). (A) Most of the wild type CFTR protein was detected as the fully glycosylated mature form (band C), whereas virtually all of the L441P mutant protein appeared as the core-glycosylated form of around 150 kDa (band B). Surface proteins were collected using the avidin-biotin interaction. Surface proteins were labeled with EZ-Link biotin-LC-Hydrazide (Pierce, Rockford, IL) and the biotinylated proteins were pelleted with the UltraLink Immobilized NeutrAvidin beads (10%, Pierce). (B) The ER-localized protein calnexin was not detected in the biotinlyated fractions, and demonstrating that biotin conjugates were specifically from the cell-surface proteins.
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In immunoblotting of cell lysates, most of the wild type CFTR protein was detected as the fully glycosylated mature form, whereas virtually all of L441P mutant proteins appeared as the ER core-glycosylated form of about 150 kDa, also known as band B (Fig. 3). CFTR protein has a defect in the ER-to-Golgi trafficking of the secretory pathway of membrane protein. Consequently, the surface biotinylation results revealed that the L441P mutant protein failed to reach the plasma membrane, whereas the fully glycosylated form of wild type CFTR was expressed on the cell surface (Fig. 3).
Immunocytochemistry of L441P mutant CFTR. HEK 293 cells were transfected with plasmids for wild type CFTR or CFTR carrying the L441P mutation, immunostained with anti-CFTR 24-1 antibody (R&D Systems) and fluorescein isothiocyante-conjugated secondary antibodies. The calnexin protein was stained with anti-calexin antibodies (Abcam) and rhodamine-conjugated secondary antibodies. Images were collected with a Zeiss LSM510 confocal microscope.
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As shown in Fig. 4, wild type CFTR was mainly localized on the plasma membrane (arrow heads). On the other hand, L441P mutant protein was found in the ER, which was confirmed by co-localization with calnexin, an ER membrane protein (Fig. 4).
cAMP-activated Cl- channel activity of L441P mutant CFTR. HEK 293 cells were transfected with plasmids for wild type CFTR or CFTR carrying the L441P mutation and the cAMP-activated Cl- channel activity was measured in the whole cell configuration. (A) Cells were stimulated with forskolin (5 microM) and the currents were measured at a -30 mV holding potential. Mean currents were normalized as current densities (pA/pF, n=6 for wild type CFTR and n=10 for L441P mutant CFTR). (B) The I-V relationships were obtained with a step pulse from -120 mV to +120 mV applied at peak current.
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Treatments with the adenylyl cyclase activator forskolin (FSK) produced a large inward current in NMDG-Cl solutions (Fig. 5). The I-V relationship measurements revealed the typical CFTR current of a linear I-V relationship when a ramp pulse from -120 mV to +120 mV was applied at peak current (Fig. 5B). However, the cAMP treatment (5 microM FSK) failed to activate the Cl- currents in cells transfected with L441P mutant CFTR (Fig. 5).