ABCMdb

PMID: 19420138

Barriere H, Bagdany M, Bossard F, Okiyoneda T, Wojewodka G, Gruenert D, Radzioch D, Lukacs GL
Revisiting the role of cystic fibrosis transmembrane conductance regulator and counterion permeability in the pH regulation of endocytic organelles.
Mol Biol Cell. 2009 Jul;20(13):3125-41. Epub 2009 May 6., [PubMed]

Sentences

No. Mutations Sentence Comment

38 ABCC7 p.Gly551Asp MATERIALS AND METHODS DNA Constructs and Chemicals The extracellular triple hemagglutinin (3HA) tag, consisting of amino acid residues of SLEYPYDVPDY-ASYPYDVPDYAYPYDVPD, was inserted into the fourth extracellular loop after residue 897 in the G551D CFTR, as described for the wild-type (wt) and ⌬F508 CFTR (Sharma et al., 2004; Pedemonte et al., 2005). Login to comment 39 ABCC7 p.Gly551Asp The G551D CFTR cDNA was kindly provided by Dr. J. Rommens (Hospital for Sick Children, Toronto). Login to comment 45 ABCC7 p.Trp1282* The IB3 cells have ⌬F508/W1282X genotype (Zeitlin et al., 1991). Login to comment 52 ABCC7 p.Gly551Asp All cell types used were either transiently or stably transfected with wt or G551D CFTR-3HA harboring triple hemagglutinin tags in the fourth extracellular loop (Sharma et al., 2004). Login to comment 53 ABCC7 p.Gly551Asp BHK cell expressing the wt and G551D CFTR-3HA was generated as previously described and clones were selected in the presence of 500 ␮M methotrexate (Sharma et al., 2001). Login to comment 55 ABCC7 p.Gly551Asp ABCC7 p.Trp1282* IB3 bronchial epithelia, expressing endogenous ⌬F508 and W1282X CFTR at undetectable levels, were stably transfected with the pCEP4 expression plasmid encoding the wt or G551D CFTR-3HA. Login to comment 139 ABCC7 p.Gly551Asp Second, to control CFTR-independent endosomal pH changes provoked by the activation of cAMP-dependent PKA, a direct regulator of the v-ATPase and Naϩ /Hϩ exchanger activity (Marshansky and Futai, 2008), we utilized the G551D CFTR-3HA, a class IV CF mutation with severely impaired channel activity (Becq et al., 1994, 2007). Login to comment 140 ABCC7 p.Gly551Asp In the following experiments, first we validated the use of G551D CFTR-3HA and the pH measurement methodology as well as the counterion and passive proton permeability determination in nonpolarized BHK cells. Login to comment 143 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Comparison of the Biosynthetic and Endocytic Membrane Trafficking of wt and G551D CFTR-3HA To utilize the G551D CFTR-3HA for monitoring the vesicular pH without conferring PKA-dependent chloride permeability to endocytic organelles, first we assessed the membrane trafficking pathway of the G551D CFTR in relation to its wt counterpart. Login to comment 144 ABCC7 p.Gly551Asp BHK cells were stably transfected with G551D and wt CFTR-3HA. Login to comment 150 ABCC7 p.Gly551Asp (B) Immunoblot analysis of wt and G551D CFTR-3HA expression in stably transfected BHK cells. Equal amounts of cell lysates were immunoblotted with anti-HA Ab. Login to comment 152 ABCC7 p.Gly551Asp (C) Plasma membrane halide conductance was measured by the iodide efflux assay in BHK monolayers expressing wt or G551D CFTR. Login to comment 155 ABCC7 p.Gly551Asp (D) Subcellular localization of internalized CFTR in BHK cells. Internalized wt and G551D CFTR-3HA was labeled with anti-HA Ab and visualized by TRITC-conjugated secondary Fab. Login to comment 157 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Single optical sections were obtained by laser confocal fluorescence microscopy. Bar, 10 ␮m. of the core-and complex-glycosylated G551D and wt CFTR was comparable, measured by immunoblotting (Figure 1B). Login to comment 158 ABCC7 p.Gly551Asp Likewise, the cell surface expression of the wt and G551D CFTR-3HA was similar, determined by anti-HA Ab-binding assay (Supplemental Figure S2A). Login to comment 159 ABCC7 p.Gly551Asp PKA activation by forskolin, CPT-cAMP, and IBMX failed to activate G551D CFTR contrary to the wt channel, monitored by the iodide efflux assay (Figure 1C). Login to comment 160 ABCC7 p.Gly551Asp The biosynthetic processing efficiency, metabolic stability, and internalization rates of the complex-glycosylated wt and G551D CFTR, measured by pulse-chase experiments, and anti-HA Ab uptake assay, respectively, were similar (Supplemental Figure S2, B and C, and data not shown). Login to comment 161 ABCC7 p.Gly551Asp To determine the postendocytic fate of the G551D CFTR, internalized channels were colocalized with the Tf receptor (Tf-R), a marker of recycling endosomes (Mukherjee et al., 1997). Login to comment 163 ABCC7 p.Gly551Asp Quantitative colocalization of CFTR, on micrographs obtained by fluorescence laser confocal microscopy (FLCM), revealed that 85 Ϯ 2 and 80 Ϯ 4% (mean Ϯ SEM, n ϭ 4 experiments) of internalized Tf was colocalized with G551D and wt CFTR, respectively. Login to comment 164 ABCC7 p.Gly551Asp Conversely, 52 Ϯ 4% of wt and 62 Ϯ 3% of G551D CFTR were confined to Tf-positive endosomes (Supplemental Figure S3A). Login to comment 165 ABCC7 p.Gly551Asp Confinement of internalized G551D and wt CFTR to early endosomes was confirmed with their colocalization with rab5 and EEA1 (Supplemental Figure S3B and data not shown) and exclusion from FITC-dextran-loaded lysosomes (Figure 1D), an observation confirmed on other cells (see below). Login to comment 166 ABCC7 p.Gly551Asp These results, jointly, indicate that the wt and G551D CFTR have overlapping postendocytic membrane trafficking that was further validated by vesicular pH measurements (see below). Login to comment 167 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Monitoring the Postendocytic Fate of G551D CFTR by Vesicular pH Determination Based on the characteristic pH of the endolysosomal compartment (Mukherjee et al., 1997), the postendocytic sorting of G551D and wt CFTR could be inferred from the luminal pH of internalized CFTR-containing vesicles, as has been shown for a variety of cargo molecules (Barriere et al., 2007; Kumar et al., 2007; Duarri et al., 2008; Varghese et al., 2008). Login to comment 173 ABCC7 p.Gly551Asp Importantly, G551D, similar to the wt CFTR, was targeted to mildly acidic recycling endosomes after 1-h chase (pH ϳ 6.52, Figure 2C). Login to comment 174 ABCC7 p.Gly551Asp Considering that the recycling endosomes mean pH is 6.4-6.5, measured in FITC-Tf-loaded BHK cells (Sharma et al., 2004), these results indicate that G551D like its wt counterpart recycles back to the cell surface and largely avoids lysosomal delivery. Login to comment 175 ABCC7 p.Gly551Asp This conclusion is also in line with the limited (Ͻ8%) colocalization of the wt and G551D CFTR with Lamp2- and dextran-loaded lysosomes, determined by the Volocity program (see Figures 1D and 3C and Supplemental Figure S3D). Login to comment 179 ABCC7 p.Gly551Asp Thus the G551D CFTR could serve as a negative control for evaluating the contribution of wt CFTR activation to the endosomal pH regulation. Login to comment 191 ABCC7 p.Gly551Asp In sharp contrast, the pH dissipation remained unaltered in G551D CFTR-expressing cells (Figures 2, E and F, and 4B). Login to comment 192 ABCC7 p.Gly551Asp These results confirmed that wt, but not the G551D CFTR, is susceptible to PKA activation in endosomes (Becq et al., 1994). Login to comment 194 ABCC7 p.Gly551Asp The endosomal pH of PKA-stimulated cells remained unal- tered, regardless whether wt or G551D CFTR was expressed (Figure 2, F, right panel, and G). Login to comment 200 ABCC7 p.Gly551Asp To this end, wt and G551D CFTR-3HA were stably expressed in IB3 and CFBE cells, widely used models of CF respiratory epithelia lacking functional CFTR (Gruenert et al., 1995; Bruscia et al., 2002). Login to comment 201 ABCC7 p.Trp1282* IB3 and CFBE cells were derived from the bronchial epithelia of CF patients with ⌬F508/W1282X and ⌬F508/⌬F508 CFTR genotypes, respectively (Zeitlin et al., 1991; Cozens et al., 1994) and have no detectable CFTR expression by immunoblotting (Figure 3A). Login to comment 203 ABCC7 p.Gly551Asp Heterologous expression of wt and G551D CFTR-3HA was verified by immunoblotting (Figure 3A) and cell surface anti-HA Ab-binding assay (Supplemental Figure 2A). Login to comment 204 ABCC7 p.Gly551Asp Iodide efflux assay revealed that wt but not G551D CFTR expression conferred PKA-stimulated plasma membrane halide Figure 2. Login to comment 205 ABCC7 p.Gly551Asp Monitoring wt and G551D CFTR-3HA endocytic sorting and pH regulation by FRIA in BHK cells. Login to comment 212 ABCC7 p.Gly551Asp (C) G551D CFTR was labeled as described in B. Login to comment 222 ABCC7 p.Gly551Asp (E) CFTR activation enhances the counterion permeability of wt but not G551D CFTR-expressing endosomes in BHK cells. Login to comment 230 ABCC7 p.Gly551Asp Internalized wt and G551D CFTR were primarily targeted to early endosomes and excluded from lysosomes in IB3, CFBE, and HeLa cells, visualized by colocalization with FITC-Tf, EEA1, or rab5 and exclusion from dextran- or Lamp2-containing lysosome (Figure 3C, Supplemental Figure S3, C and D, and data not shown). Login to comment 231 ABCC7 p.Gly551Asp These results indicate that the recycling propensity of endocytosed G551D CFTR is independent of the cellular expression system, as observed previously for wt CFTR (Sharma et al., 2004; Gentzsch et al., 2007; Varga et al., 2008). Login to comment 233 ABCC7 p.Gly551Asp This conclusion was confirmed by the inability of PKA to activate Hϩ efflux from endosomes of G551D CFTR-expressing cells (Figure 4B). Login to comment 234 ABCC7 p.Gly551Asp Importantly, despite full activation of wt CFTR by the agonist cocktail in 1.5-2 min at room temperature (see Supplemental Figure S4B), no significant change in the steady-state pH of wt CFTR-expressing endosomes, including the CFBE and IB3 respiratory epithelia, was observed relative to that in G551D CFTR-expressing cells (Figure 4C). Login to comment 243 ABCC7 p.Gly551Asp On the basis of the overlapping subcellular distribution of FITC-Tf with wt and G551D CFTR (Figure 1D and Supplemental Figure S3A), we followed the endosomal pH after labeling the recycling endosomes with FITC-Tf. Login to comment 245 ABCC7 p.Gly551Asp Wt and G551D CFTR expression, function, and postendocytic localization in CF respiratory epithelia and HeLa cells. Login to comment 246 ABCC7 p.Gly551Asp (A) CFTR expression was probed by immunoblot analysis in mock, wt, and G551D CFTR-3HA expressing CFBE and IB3 respiratory epithelia, as well as in HeLa cells. Equal amounts of cell lysates were immunoblotted with anti-HA Ab. Login to comment 247 ABCC7 p.Gly551Asp (B) Wt and G551D CFTR activity was measured by the iodide efflux assay as described in Figure 1C. Data are means of triplicate determinations from a representative experiment. Login to comment 248 ABCC7 p.Gly551Asp (C) Localization of internalized wt and G551D CFTR in CFBE and IB3 cells. Internalized CFTR was labeled as described in Figure 1D. Login to comment 252 ABCC7 p.Gly551Asp The endosomal counterion conductance was stimulated by about twofold with PKA agonists in wt, but not in G551D CFTR or parental cells (Supplemental Figure S3E). Login to comment 254 ABCC7 p.Gly551Asp The steady-state endosomal pH was not, or only marginally affected by the wt CFTR activation relative to that of the G551D CFTR (Figure 4D), supporting our conclusion that neither ablation nor overexpression of CFTR influences the endosomal pH regulation, presumably due to the relatively high CFTR-independent counterion conductance. Login to comment 265 ABCC7 p.Gly551Asp (A and B) Measurement of endosomal pH dissipation rates in wt and G551D CFTR-3HA complemented CFBE and IB3 CF respiratory epithelia and overexpressing HeLa and MDCK cells. Login to comment 269 ABCC7 p.Gly551Asp Data were analyzed by two-tailed unpaired t tests and indicated as follows: * p ϭ 0.0347 for CFTR G551D control versus forskolin in HeLa. Login to comment 273 ABCC7 p.Gly551Asp (E) Initial acidification rates of endosomes were measured in wt and G551D CFTR-3HA-expressing HeLa and IB3 cells. Login to comment 427 ABCC7 p.Gly551Asp To compare the CFTR-independent and CFTR-dependent counterion and proton permeabilities, endosomes containing functional (wt) or nonfunctional (G551D) CFTR were labeled with the pH-sensitive fluorophore, restricting the probe to CFTR-expressing vesicles. Login to comment