PMID: 21594800

Cai Z, Sohma Y, Bompadre SG, Sheppard DN, Hwang TC
Application of high-resolution single-channel recording to functional studies of cystic fibrosis mutants.
Methods Mol Biol. 2011;741:419-41., [PubMed]
Sentences
No. Mutations Sentence Comment
156 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:156:125
status: NEW
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This technical issue is especially relevant when dealing with CF mutants that profoundly disrupt CFTR channel gating (e.g. G551D-CFTR (37, 38)). Login to comment
202 ABCC7 p.Glu1371Gln
X
ABCC7 p.Glu1371Gln 21594800:202:178
status: NEW
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Furthermore, relaxation analysis of macroscopic currents is the method of choice to analyse the burst duration of CFTR constructs that open for tens of hundreds of seconds (e.g. E1371Q, (47)), because it can be very difficult to collect sufficient gating transitions for microscopic kinetic analysis of channel gating for these CFTR constructs. Login to comment
267 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:267:34
status: NEW
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The third most common CF mutation G551D is a classic example of a class III mutation because it completely abrogates the ATP-dependence of CFTR channel gating (38). Login to comment
268 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:268:38
status: NEW
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ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:268:177
status: NEW
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Because of the extremely low Po of G551D-CFTR, the methods used to study the single-channel behaviour of wild-type CFTR require some refinement before they can be applied to G551D-CFTR. Login to comment
270 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:270:140
status: NEW
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The issue resides in the difficulty of properly assessing the number of channels (N) present in the membrane patch given the very low Po of G551D-CFTR. Login to comment
271 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:271:127
status: NEW
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Although it is tempting to assume that N is equivalent to the number of simultaneous channel openings observed, in the case of G551D-CFTR, this can lead to a gross underestimation of N. Login to comment
272 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:272:8
status: NEW
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Because G551D-CFTR Cl-channels remain closed most of the time, different channel openings will likely originate from different channels present in the same membrane patch (for a discussion of this problem, see (51)). Login to comment
273 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:273:44
status: NEW
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To have any possibility of estimating N for G551D-CFTR, the experimental conditions should be adjusted to maximize Po. Login to comment
276 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:276:61
status: NEW
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ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:276:62
status: NEW
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For example, AMP-PNP and pyrophosphate fail to lock-open the G551D-CFTR Cl-channel (37, 38). Login to comment
277 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:277:88
status: NEW
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A compromise method used by Bompadre et al. (38) offers a rough estimation of the Po of G551D-CFTR. Login to comment
278 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:278:82
status: NEW
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When CHO cells are transfected with the same amount of cDNA encoding wild-type or G551D-CFTR, Western blot analysis demonstrates that the cells produce similar amounts of band C (mature protein) for the two constructs (38). Login to comment
279 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:279:53
status: NEW
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This indicates that similar numbers of wild-type and G551D-CFTR Cl-channels are present at the cell surface. Login to comment
281 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:281:105
status: NEW
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Because neither the number of active channels nor the single-channel current amplitude is altered by the G551D mutation, the mean current amplitude directly reflects Po. Login to comment
283 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:283:90
status: NEW
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One potential solution to the technical difficulty of measuring the Po of CF mutants like G551D is to use CFTR potentiators or ATP analogues to augment robustly channel gating and hence Po. Login to comment
284 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:284:96
status: NEW
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However, so far in the literature, the most efficacious small molecules only increase the Po of G551D-CFTR by ~ 5-fold, far lower than the ~100-fold decrease of Po caused by this mutation. Login to comment
287 ABCC7 p.Arg334Trp
X
ABCC7 p.Arg334Trp 21594800:287:36
status: NEW
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ABCC7 p.Arg347Pro
X
ABCC7 p.Arg347Pro 21594800:287:46
status: NEW
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As a result, these CF mutants (e.g. R334W and R347P; 59, 60) diminish single-channel current amplitude (i). Login to comment
297 ABCC7 p.Pro99Leu
X
ABCC7 p.Pro99Leu 21594800:297:28
status: NEW
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ABCC7 p.Pro99Leu
X
ABCC7 p.Pro99Leu 21594800:297:170
status: NEW
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ABCC7 p.Pro99Leu
X
ABCC7 p.Pro99Leu 21594800:297:295
status: NEW
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For example, the CF mutant, P99L, in the first transmembrane segment attenuated the single-channel conductance (wild-type, 7.72 ± 0.22 pS (means ± SEM; n = 4); P99L, 4.97 ± 0.24 pS (n = 5, p < 0.0001)) and altered the anion selectivity sequence (wild-type, Br- ≥ Cl- > I-; P99L, Br- ≥ Cl- = I-) (62). Login to comment
298 ABCC7 p.Pro99Cys
X
ABCC7 p.Pro99Cys 21594800:298:108
status: NEW
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However, Akabas et al. (63) concluded that P99 does not line the CFTR pore because the site-directed mutant P99C did not react with methanethiosulfonate reagents. Login to comment
305 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:305:311
status: NEW
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ABCC7 p.Arg117His
X
ABCC7 p.Arg117His 21594800:305:337
status: NEW
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ABCC7 p.Pro574His
X
ABCC7 p.Pro574His 21594800:305:359
status: NEW
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Using biochemical (N) and functional Table 27.1 Comparison of predicted apical membrane Cl- current and measured cAMP-activated apical membrane Cl- current for wild-type and mutant CFTRs CFTR N (%) i (%) Po (%) N × i × Po (%) ICFTR (apical) (%) Wild-type 100 100 100 100 100 F508del 4 100 30 1.2 0 G551D 100 100 2.5 2.5 1.5 R117H 100 86 28 24 15 P574H 15 100 139 21.1 17 N, the number of Cl-channels in the apical membrane; i, single-channel current amplitude; Po, open probability; N × i × Po, the predicted apical membrane Cl- current; ICFTR (apical), measured cAMP-activated apical membrane Cl- current. Login to comment
308 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:308:4
status: NEW
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For G551D data, N and ICFTR values are from Bompadre et al. (38) and Zegarra-Moran et al. (64), while i and Po data are from Cai et al. (37). Login to comment
312 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:312:177
status: NEW
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ABCC7 p.Arg117His
X
ABCC7 p.Arg117His 21594800:312:170
status: NEW
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ABCC7 p.Pro574His
X
ABCC7 p.Pro574His 21594800:312:187
status: NEW
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Table 27.1 compares the predicted values of N × i × Po with the observed values of ICFTR(apical) measured in FRT epithelia for F508del-CFTR and the CF mutants, R117H, G551D and P574H, which disrupt CFTR function by different mechanisms (33, 37, 59, 64, 65). Login to comment