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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)).
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202
ABCC7 p.Glu1371Gln
X
ABCC7 p.Glu1371Gln 21594800:202:178
status:
NEW
view ABCC7 p.Glu1371Gln details
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.
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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).
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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
view ABCC7 p.Gly551Asp details
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.
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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.
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271
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:271:127
status:
NEW
view ABCC7 p.Gly551Asp details
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.
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272
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:272:8
status:
NEW
view ABCC7 p.Gly551Asp details
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)).
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273
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:273:44
status:
NEW
view ABCC7 p.Gly551Asp details
To have any possibility of estimating N for
G551D
-CFTR, the experimental conditions should be adjusted to maximize Po.
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276
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:276:61
status:
NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:276:62
status:
NEW
view ABCC7 p.Gly551Asp details
For example, AMP-PNP and pyrophosphate fail to lock-open the
G551D-
CFTR Cl-channel (37, 38).
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277
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:277:88
status:
NEW
view ABCC7 p.Gly551Asp details
A compromise method used by Bompadre et al. (38) offers a rough estimation of the Po of
G551D
-CFTR.
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278
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:278:82
status:
NEW
view ABCC7 p.Gly551Asp details
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).
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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.
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281
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:281:105
status:
NEW
view ABCC7 p.Gly551Asp details
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.
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283
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:283:90
status:
NEW
view ABCC7 p.Gly551Asp details
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.
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284
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:284:96
status:
NEW
view ABCC7 p.Gly551Asp details
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.
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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
view ABCC7 p.Arg347Pro details
As a result, these CF mutants (e.g.
R334W
and
R347P
; 59, 60) diminish single-channel current amplitude (i).
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297
ABCC7 p.Pro99Leu
X
ABCC7 p.Pro99Leu 21594800:297:28
status:
NEW
view ABCC7 p.Pro99Leu details
ABCC7 p.Pro99Leu
X
ABCC7 p.Pro99Leu 21594800:297:170
status:
NEW
view ABCC7 p.Pro99Leu details
ABCC7 p.Pro99Leu
X
ABCC7 p.Pro99Leu 21594800:297:295
status:
NEW
view ABCC7 p.Pro99Leu details
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).
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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.
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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
view ABCC7 p.Arg117His details
ABCC7 p.Pro574His
X
ABCC7 p.Pro574His 21594800:305:359
status:
NEW
view ABCC7 p.Pro574His details
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.
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308
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:308:4
status:
NEW
view ABCC7 p.Gly551Asp details
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).
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312
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 21594800:312:177
status:
NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Arg117His
X
ABCC7 p.Arg117His 21594800:312:170
status:
NEW
view ABCC7 p.Arg117His details
ABCC7 p.Pro574His
X
ABCC7 p.Pro574His 21594800:312:187
status:
NEW
view ABCC7 p.Pro574His details
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).
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