ABCMdb

PMID: 7540133

Champigny G, Imler JL, Puchelle E, Dalemans W, Gribkoff V, Hinnrasky J, Dott K, Barbry P, Pavirani A, Lazdunski M
A change in gating mode leading to increased intrinsic Cl- channel activity compensates for defective processing in a cystic fibrosis mutant corresponding to a mild form of the disease.
EMBO J. 1995 Jun 1;14(11):2417-23., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Pro574His ABCC7 p.Arg560Thr Immunochemical and functional analyses indicate that the rank order of CFTR expression at the cell surface is: wild type CFTR > P574H > > AF508 > > R560T - 0. Login to comment 2 ABCC7 p.Pro574His Patch-clamp analysis indicates that the open probability of P574H Cl- channels is almost twice as high as that ofthe wild type CFTR-CI- channel. Login to comment 3 ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Pro574His This increased intrinsic activity of individual P574H CFTR-Cl- channels compensates for the lower number of P574H CFTR-Cl- channels reaching the cell surface, and probably explains the milder form of CF associated with the P574H mutation. Login to comment 4 ABCC7 p.Pro574His NS004, a recently described activator, restores near normal CFTR activity in cells expressing the P574H-CFTR channel. Login to comment 5 ABCC7 p.Pro574His The P574H mutation modifies the gating mode of the channel with a large increase (.X7) in the mean channel open time. Login to comment 19 ABCC7 p.Pro574His This work analyses the molecular properties of a CFTR protein expressed with a recombinant vaccinia virus, which carries the mutation P574H, previously shown to be associated with a mild form of the disease (Kristidis et al., 1992). Login to comment 21 ABCC7 p.Arg560Thr The properties of this mutation are compared with those of three severe mutations A1507, AF508 and R560T, also affecting the first nucleotide binding domain. Login to comment 22 ABCC7 p.Pro574His Results Immunolocalization of P574H-CFTR and other CFTR mutant proteins Recombinant vaccinia viruses expressing wild type or mutant CFTR were constructed to study the effects of al. P 574 H AF 508 R 560 T Fig. 1. Login to comment 23 ABCC7 p.Pro574His ABCC7 p.Arg560Thr Immunodetection of wild type, AF508-, R560T- and P574H-CFTRs in Vero cells. Login to comment 24 ABCC7 p.Pro574His ABCC7 p.Arg560Thr Standard fluorescence microscopic photomicrographs (A, C, E and G) and XY and XZ confocal optical section (B, D, F and H) of Vero cells expressing wild type (A and B), P574H- (C and D), AF508- (E and F) and R560T-CFTRs (G and H). Login to comment 27 ABCC7 p.Pro574His ABCC7 p.Arg560Thr A1507, AF508, R560T and to compare them with the P574H mutation. Login to comment 32 ABCC7 p.Arg560Thr ABCC7 p.Arg560Thr The R560T mutation resulted in very low immunolabelling of R560T-CFTR expressing cells, which was strictly located within the cytosol. Login to comment 34 ABCC7 p.Pro574His In cells expressing P574H-CFTR, the immunolabelling was mainly intracellular, and preferentially localized in the Golgi region, although some cells also exhibited focal CFTR labelling on the plasma membrane (Figure 1C and D). Login to comment 38 ABCC7 p.Pro574His ABCC7 p.Arg560Thr Figure 2 shows the percentage of cells that were positive for membrane immunolabelling after incubation with the 80 c ._ CD 60 m 40 E c 20 0 _ Vero M Chang I -E _m7A - WT P574H AF508 R560T Fig. 2. Login to comment 39 ABCC7 p.Pro574His ABCC7 p.Arg560Thr Analysis of the cell surface expression of wild type, AF508-, R560T- and P574H-CFTRs. Login to comment 40 ABCC7 p.Pro574His ABCC7 p.Arg560Thr The number of Vero and Chang cells used for the analysis was equal to 125 and 223 for wild type CFTR, 59 and 288 for AF508, 169 and 317 for R560T, 88 and 479 for P574H respectively. Login to comment 45 ABCC7 p.Pro574His ABCC7 p.Pro574His The percentage of cells with positive plasma-membrane labelling was lower for P574H and AF508, but remained significantly higher than zero (20-30% for P574H and 2-6% for AF508). Login to comment 46 ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Arg560Thr No R560T-expressing cells exhibited plasma-membrane immunolabelling. Login to comment 47 ABCC7 p.Arg560Thr No R560T-expressing cells exhibited plasmamembrane immunolabelling. Login to comment 50 ABCC7 p.Pro574His A diffuse band of 160-170 kDa corresponding to the fully glycosylated CFTR was only detected in cells infected with the recombinant virus expressing wild type CFTR, but in some experiments small amounts of 160-170 kDa glycosylated protein could be observed in cells expressing AF508 or P574H (not shown). Login to comment 51 ABCC7 p.Pro574His A diffuse band of 160-170 kDa corresponding to the fully glycosylated CFTR was only detected in cells infected with the recombinant virus expressing wild type CFTR, but in some experiments small amounts of 160-170 kDa glycosylated protein could be observed in cells expressing AF508 or P574H (not shown). Login to comment 52 ABCC7 p.Pro574His ABCC7 p.Arg560Thr In addition, the absence of fully glycosylated forms of CFTR in cells expressing A1507, AF508, R560T and P574H mutant proteins confirms that these mutations are associated with defective processing. Login to comment 53 ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Arg560Thr Functional characterization of P574H-CFTR as compared with other CFTR mutant proteins The functional activity of CFTR mutant proteins was measured in two different experimental conditions that permit activation of the CFTR-CI- channels. Login to comment 54 ABCC7 p.Pro574His ABCC7 p.Pro574His In the first 'Mock WT P574H R560TAF508 A1507 170kDam F 140kDamp _ mm ----MD Fig. 3. Login to comment 55 ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Arg560Thr Western blotting analysis of the 'mock'-infected, wild type, AF508-, R560T- and P574H-CFTRs. Login to comment 56 ABCC7 p.Pro574His ABCC7 p.Arg560Thr Western blotting analysis of the 'mock'-infected, wild type, AF508-, R560T- and P574H-CFTRs. Login to comment 66 ABCC7 p.Arg560Thr When 1251- efflux were performed on Vero cells expressing A1507 and R560T CFTRs, no significant stimulation of the efflux was observed, even in conditions permitting a maximal activation of wild type CFTR or of AF508 mutant protein, i.e. 10 ,uM forskolin + 20 jM NS004 (Gribkoff et al., 1994). Login to comment 67 ABCC7 p.Arg560Thr When 1251efflux were performed on Vero cells expressing A1507 and R560T CFTRs, no significant stimulation of the efflux was observed, even in conditions permitting a maximal activation of wild type CFTR or of AF508 mutant protein, i.e. 10 ,uM forskolin + 20 jM NS004 (Gribkoff et al., 1994). Login to comment 68 ABCC7 p.Pro574His Very different results were observed for P574H (Figure 4C-E). Login to comment 69 ABCC7 p.Pro574His Very different results were observed for P574H (Figure 4C-E). Login to comment 71 ABCC7 p.Pro574His ABCC7 p.Arg560Thr 1050 Istim-Ictrl 700 (pA) 350 P574H R560T A1507 urIC) 0L It) C~ a ° Fig. 4. Login to comment 72 ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Arg560Thr ABCC7 p.Arg560Thr Functional analysis of the A1507-, R560T- and P574H-CFTRs. Login to comment 73 ABCC7 p.Pro574His ABCC7 p.Arg560Thr Functional analysis of the A1507-, R560T- and P574H-CFTRs. Login to comment 74 ABCC7 p.Arg560Thr (B) Time-course of the activation of the R560T-CFTR mediated 1251- efflux by 10 ,uM forskolin (EO1) and 10 jM forskolin + 20 jM NS004 (A). Login to comment 75 ABCC7 p.Pro574His ABCC7 p.Arg560Thr (C) Time-course of the activation of the P574H-CFTR mediated 125p- efflux by 10 ,uM forskolin (O), 20 ,uM NS004 (M) and 10 jM forskolin + 20 ,uM NS004 (A). Login to comment 76 ABCC7 p.Pro574His (C) Time-course of the activation of the P574H-CFTR mediated 125p- efflux by 10 ,uM forskolin (O), 20 ,uM NS004 (M) and 10 jM forskolin + 20 ,uM NS004 (A). Login to comment 77 ABCC7 p.Pro574His ABCC7 p.Arg560Thr Results are shown as mean SD (D) Whole cell patch-clamp studies of wild type, A1507, AF508, R560T and P574H after stimulation by 10 jM forskolin. Login to comment 78 ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Arg560Thr ABCC7 p.Arg560Thr (E) Activation of the A1507, R560T and P574H -C1- currents (Istim) by 20 jM NS004 and/or forskolin 10 jiM. Login to comment 79 ABCC7 p.Pro574His ABCC7 p.Arg560Thr (E) Activation of the A1507, R560T and P574H -C1- currents (Istim) by 20 jM NS004 and/or forskolin 10 jiM. Login to comment 81 ABCC7 p.Pro574His wild type, P574H and AF508 respectively). Login to comment 82 ABCC7 p.Pro574His wild type, P574H and AF508 respectively). Login to comment 84 ABCC7 p.Pro574His Values of the Cl- current did not differ very significantly between P574H and the wild type proteins, suggesting that in that configuration there might be no significant difference in activity between the two different channel proteins. Login to comment 85 ABCC7 p.Pro574His ABCC7 p.Pro574His Figure 4E shows that NS004 can stimulate the CFTR-C1- current in P574H-CFTR expressing cells, either when used alone or in combination with forskolin. Login to comment 86 ABCC7 p.Pro574His ABCC7 p.Arg560Thr ABCC7 p.Arg560Thr A Cl- current in R560T and A1507 expressing cells was stimulated by forskolin (Figure 4D), but its amplitude remained always very small in comparison with the Cl- current generated by the expression of wild type CFTR (2.6 and 3.7% of the wild type, for A1507 and R560T respectively), and the stimulating effect of NS004 remained modest (Figure 4E). Login to comment 87 ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Arg560Thr ABCC7 p.Arg560Thr The mutant protein P574H has a higher intrinsic Ct channel activity than wild type In order to understand why the P574H mutant protein displayed a high residual C1- channel activity, despite its defective cell-surface expression, the properties of the P574H-Cl- channel were compared with those of the wild type CFTR using the cell-attached patch-clamp technique. Login to comment 88 ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Pro574His ABCC7 p.Pro574His When cells expressing wild type or P574H Cl- channels were exposed to NS004, an increase in the open probability was noticed in both cases (not shown), but the comparison shown here was performed after stimulation by a 'cocktail' of forskolin (5 ,uM), 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (cpt-cAMP, 100 ,uM) and isobutylmethylxanthine (IBMX, 100 gM) and in the absence of NS004. Login to comment 89 ABCC7 p.Pro574His ABCC7 p.Pro574His Long lasting recordings of P574H mutant CFTR and wild type CFTR show the typical difference observed between the activity of the two C1- channels (Figure 5A). Login to comment 90 ABCC7 p.Pro574His ABCC7 p.Pro574His Although the unitary current amplitude was not modified by the P574H mutation (Figure 5B), the mean number of active Cl- channels was reduced by a factor of -2 (i.e. 46% of the wild type CFTR) (Figure 5C) and the open probability of the channel was increased by 59% (Figure SD). Login to comment 91 ABCC7 p.Pro574His ABCC7 p.Pro574His The conductance of the P574H C1- channel is 5.1 pS, very similar if not identical to that found for the normal CFTR channel (5.3 pS) under the same conditions. Login to comment 92 ABCC7 p.Pro574His ABCC7 p.Pro574His Figure 5E presents a comparative analysis of the mean open and mean closed times of P574H CFTR and normal CFTR after stimulation by the cocktail of forskolin, cpt-cAMP and IBMX. Login to comment 93 ABCC7 p.Pro574His ABCC7 p.Pro574His The P574H mutation led to a large increase (a factor of 6.8) in the mean open time of the Cl- channel to a value of 10.4 s. Login to comment 94 ABCC7 p.Pro574His The P574H mutation led to a large increase (a factor of 6.8) in the mean open time of the Cl-channel to a value of 10.4 s. Login to comment 96 ABCC7 p.Arg560Thr All mutations affecting the first nucleotide binding domain that have been described so far lead to a 2420 5 rstim rbas 3 B R560T 5 - on=4 A n=8 3 1 ' . Login to comment 97 ABCC7 p.Pro574His ABCC7 p.Arg560Thr .-.. 2 4 time(minutes) 6 Istim-lctd (pA) I J * NS004 1OgM U ForskliO±M O NS004 + Forsk 1 E WT P574H N-4. Login to comment 98 ABCC7 p.Pro574His ABCC7 p.Pro574His Po-0.72 o.4pAIC _L|j C_. -C ~- - Al - - --- --- -- N-4, Po-0.6 N-8, Po.0.31 cJ/\S71q2 i(pA) 8 4How P574H A _._ E 1.___ *FfoP574H2~~ III , * I I~~~~~~~~- 0 35I~~~~I~~II' InLLLEiJ~~~~+60 mV 60 mV ,. Login to comment 99 ABCC7 p.Pro574His : wr(rw o P574H (r-7) Mean open Mean doe tl -e Fig. 5. Login to comment 100 ABCC7 p.Pro574His ABCC7 p.Pro574His Single channel comparison between wild type and P574H mutant Cl- channels. Login to comment 101 ABCC7 p.Pro574His (A) Typical cell-attached recordings on forskolin-stimulated WT-CFTR-infected cell (left) and P574H-CFTR infected cell (right); c = closed state of the channels. Login to comment 102 ABCC7 p.Pro574His ABCC7 p.Pro574His (B) Mean I-V relationships for wild type and P574H mutant CFTR. Login to comment 103 ABCC7 p.Pro574His ABCC7 p.Pro574His (C) Histograms showing the mean number of active CFTR-Cl- channels in wild type and P574H CFTR expressing cells. Login to comment 104 ABCC7 p.Pro574His (D) Histograms showing the mean open probability of active CFTR-Cl- channels in wild type and P574H CFTR expressing cells at ±60 mV. Login to comment 105 ABCC7 p.Pro574His (B) Mean I-V relationships for wild type and P574H mutant CFTR. Login to comment 106 ABCC7 p.Pro574His ABCC7 p.Pro574His (E) Histograms showing the mean open and closed time for wild type and P574H Cl- channels at -60 mV. Login to comment 107 ABCC7 p.Pro574His (D) Histograms showing the mean open probability of active CFTR-Cl-channels in wild type and P574H CFTR expressing cells at &#b1;60 mV. Login to comment 109 ABCC7 p.Pro574His ABCC7 p.Arg560Thr No significant Cl- channel activity was observed in cells expressing the two mutant proteins A1507 and R560T-CFTR corresponding to severe mutations. Login to comment 112 ABCC7 p.Arg560Thr No significant Cl-channel activity was observed in cells expressing the two mutant proteins A1507 and R560T-CFTR corresponding to severe mutations. Login to comment 114 ABCC7 p.Arg560Thr Since no A1507 or R560T proteins could be detected at the plasma membrane, and since their expression generated <4% of the whole-cell Cl- current generated by the wild type (Figure 4D), these mutant channels can be considered as members of class II. Login to comment 115 ABCC7 p.Arg560Thr It may be that in addition to their severe sorting defect, A1507-CFTR and R560T-CFTR also have altered Cl- channel properties, as has been reported for the AF508 mutant protein (Dalemans et al., 1991). Login to comment 117 ABCC7 p.Pro574His ABCC7 p.Arg560Thr A different situation was observed with the P574H mutation. Login to comment 118 ABCC7 p.Arg560Thr It may be that in addition to their severe sorting defect, A1507-CFTR and R560T-CFTR also have altered Cl-channel properties, as has been reported for the AF508 mutant protein (Dalemans et al., 1991). Login to comment 119 ABCC7 p.Pro574His In this case the anti-CFTR monoclonal antibody labelled the cell surface of 20-30% of P574H-CFTR expressing cells (Figures 1 and 2). Login to comment 120 ABCC7 p.Pro574His A different situation was observed with the P574H mutation. Login to comment 121 ABCC7 p.Pro574His This alteration in the traffic toward the cell surface of the P574H mutant protein was confirmed by functional studies. Login to comment 122 ABCC7 p.Pro574His ABCC7 p.Pro574His The number of active channels per patch (estimated by assuming independence between the channels present in a patch) was about half for P574H as compared with wild type (Figure 5C and D). Login to comment 123 ABCC7 p.Pro574His Immature P574H-CFTR accumulated mainly in the Golgi apparatus and in the cytoplasm, whereas diffuse labelling restricted to the cytoplasm was observed for AF508-CFTR. Login to comment 124 ABCC7 p.Pro574His This alteration in the traffic toward the cell surface of the P574H mutant protein was confirmed by functional studies. Login to comment 125 ABCC7 p.Pro574His ABCC7 p.Pro574His An interesting property of the P574H mutant protein is that it is associated with defective sorting and increased intrinsic activity of the Cl- channel (Figures 4 and 5). Login to comment 126 ABCC7 p.Pro574His ABCC7 p.Pro574His This increased Cl- channel activity is not due to a change of the channel conductance (5.1 pS and 5.3 pS, for P574H- and WT-CFTR, respectively) but is associated with an increase in the time during which the channels remain open. Login to comment 128 ABCC7 p.Pro574His ABCC7 p.Pro574His The open probability of the P574H-CFTR Cl- channel is nearly 60% higher than normal. Login to comment 129 ABCC7 p.Pro574His ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro An initial conclusion from these observations is that mutations associated with mild CF: (i) can lead to partial defective processing, less severe than previously observed for severe mutations, and (ii) are not necessarily associated with a decrease in intrinsic channel activity as previously reported for R 117H, R334W and R347P. Login to comment 130 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro These three mutations, identified in mildly affected patients, are located at the extemal end of the second (R 117H) and the sixth (R334W, R347P) putative membrane-spanning sequences. Login to comment 131 ABCC7 p.Pro574His The open probability of the P574H-CFTR Cl-channel is nearly 60% higher than normal. Login to comment 132 ABCC7 p.Pro574His ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro A second conclusion is that the 'quality control' which prevents the trafficking of mutated CFTR-Cl- channels to the plasma membrane does not only eliminate mutants with reduced or abolished Cl- channel activity, since the intrinsic activity of the P574H mutant protein is higher than normal. Login to comment 133 ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro These three mutations, identified in mildly affected patients, are located at the extemal end of the second (R 117H) and the sixth (R334W, R347P) putative membrane-spanning sequences. Login to comment 134 ABCC7 p.Pro574His 2421 A E I aL The P574H mutation has been identified in compound heterozygotes where it is associated with other mutations such as AF508 (Kristidis et al., 1992). Login to comment 135 ABCC7 p.Pro574His A second conclusion is that the 'quality control' which prevents the trafficking of mutated CFTR-Cl-channels to the plasma membrane does not only eliminate mutants with reduced or abolished Cl-channel activity, since the intrinsic activity of the P574H mutant protein is higher than normal. Login to comment 136 ABCC7 p.Pro574His Results from 125v efflux measurements (Figure 4) indicate that the P574H- Cl- channel activity is -70% of the activity measured with wild type CFTR-expressing Vero cells (as measured by the ratios of the maximal stimulation factors). Login to comment 137 ABCC7 p.Pro574His ABCC7 p.Pro574His This would mean, assuming that the activity observed in Vero cells can be extrapolated to polarized epithelial cells, that residual in vivo Cl- transport activity for patients bearing P574H mutation would be at most 35% of the normal value. Login to comment 139 ABCC7 p.Pro574His Results from 125v efflux measurements (Figure 4) indicate that the P574H- Cl- channel activity is -70% of the activity measured with wild type CFTR-expressing Vero cells (as measured by the ratios of the maximal stimulation factors). Login to comment 140 ABCC7 p.Pro574His This would mean, assuming that the activity observed in Vero cells can be extrapolated to polarized epithelial cells, that residual in vivo Cl-transport activity for patients bearing P574H mutation would be at most 35% of the normal value. Login to comment 142 ABCC7 p.Pro574His The association of the P574H mutation with a mild clinical status of the disease is certainly explained by the relatively high intrinsic activity of the corresponding CFTR-C1- channel. Login to comment 145 ABCC7 p.Pro574His The association of the P574H mutation with a mild clinical status of the disease is certainly explained by the relatively high intrinsic activity of the corresponding CFTR-C1-channel. Login to comment 150 ABCC7 p.Pro574His The P574H mutation also increases the mean closed times at -60 mV but only by a factor of 2.6. Login to comment 153 ABCC7 p.Pro574His The P574H mutation also increases the mean closed times at -60 mV but only by a factor of 2.6. Login to comment 155 ABCC7 p.Pro574His The P574H mutation described in this work favours the long open state situation, and as such might become useful for further mechanistic studies of the Cl- channel activity as have been recently carried out with the normal CFTR protein (Fisher and Machen, 1994). Login to comment 158 ABCC7 p.Pro574His The P574H mutation described in this work favours the long open state situation, and as such might become useful for further mechanistic studies of the Cl-channel activity as have been recently carried out with the normal CFTR protein (Fisher and Machen, 1994). Login to comment 164 ABCC7 p.Pro574His ABCC7 p.Arg560Thr Light fluorescence microscopy Vero and Chang cells were grown on Lab Tek chamber slides and were infected using a recombinant vaccinia virus expression system carrying either the wild type or the mutant (AF508, R560T or P574H CFTR) cDNAs under the control of the T7 promoter. Login to comment 167 ABCC7 p.Pro574His ABCC7 p.Arg560Thr Light fluorescence microscopy Vero and Chang cells were grown on Lab Tek chamber slides and were infected using a recombinant vaccinia virus expression system carrying either the wild type or the mutant (AF508, R560T or P574H CFTR) cDNAs under the control of the T7 promoter. Login to comment