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PMID: 10516113
Illek B, Zhang L, Lewis NC, Moss RB, Dong JY, Fischer H
Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein.
Am J Physiol. 1999 Oct;277(4 Pt 1):C833-9.,
[PubMed]
Sentences
No.
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Sentence
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6
ABCC7 p.Gly551Asp
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ABCC7 p.Gly551Asp 10516113:6:137
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It is published 12 times aAJP - Cell Physiology rapid communication Defective function of the cystic fibrosis-causing missense mutation
G551D
is recovered by genistein BEATE ILLEK,1 LEI ZHANG,2 NANCY C. LEWIS,1 RICHARD B. MOSS,3 JIAN-YUN DONG,2 AND HORST FISCHER1 1Research Institute and Pulmonary Center, Children`s Hospital Oakland, Oakland 94609; 2Laboratory Medicine, University of California at San Francisco, San Francisco 94143; and 3Pediatric Pulmonary Medicine, Stanford University Medical Center, Stanford, California 94305 Illek, Beate, Lei Zhang, Nancy C. Lewis, Richard B. Moss, Jian-Yun Dong, and Horst Fischer.
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7
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:7:68
status:
NEW
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Defective function of the cystic fibrosis-causing missense mutation
G551D
is recovered by genistein.
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8
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:8:177
status:
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Am. J. Physiol. 277 (Cell Physiol. 46): C833-C839, 1999.-The patch-clamp technique was used to investigate the effects of the isoflavone genistein on disease-causing mutations (
G551D
and ⌬F508) of the cystic fibrosis transmembrane conductance regulator (CFTR).
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9
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:9:65
status:
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ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:9:158
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In HeLa cells recombinantly expressing the trafficking-competent
G551D
-CFTR, the forskolin-stimulated Cl currents were small, and average open probability of
G551D
-CFTR was Po ϭ 0.047 Ϯ 0.019.
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10
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:10:74
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view ABCC7 p.Gly551Asp details
Addition of genistein activated Cl currents ϳ10-fold, and the Po of
G551D
-CFTR increased to 0.49 Ϯ 0.12, which is a Po similar to wild-type CFTR.
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13
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:13:70
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Therefore, we tested the effects of genistein in CF patients with the
G551D
mutation in nasal potential difference (PD) measurements in vivo.
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14
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:14:37
status:
NEW
view ABCC7 p.Gly551Asp details
The perfusion of the nasal mucosa of
G551D
CF patients with isoproterenol had no effect; however, genistein stimulated Cl-dependent nasal PD by, on average, -2.4 Ϯ 0.6 mV, which corresponds to 16.9% of the responses (to beta-adrenergic stimulation) found in healthy subjects.
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18
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:18:62
status:
NEW
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The glycine-to- aspartic acid missense mutation at codon 551 (
G551D
) is the third commonest CF mutation, with a worldwide frequency of ϳ2% on CF chromosomes (25, 27).
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19
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:19:22
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Higher frequencies of
G551D
are found in populations of Celtic descent, with incidences of 6.9% and 5.5% in the Irish and Australian CF populations, respectively (3, 12, 25).
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22
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:22:4
status:
NEW
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The
G551D
-CFTR protein traffics to the apical membrane, but Cl channel function is markedly reduced (class III mutation) (27).
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23
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:23:90
status:
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Compared with CF patients homozygous for the ⌬F508 mutation, patients carrying the
G551D
mutation show a threefold reduction in the incidence of meconium ileus (neonatal intestinal blockage), a trend toward later age at diagnosis of pancreatic insufficiency (12), and milder disease in homozygotes (22).
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24
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:24:54
status:
NEW
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ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:24:197
status:
NEW
view ABCC7 p.Gly551Asp details
Interestingly, both the ⌬F508 mutation and the
G551D
mutation are in NBD1 of CFTR; however, ATP binding of NBD1 is not affected by the ⌬F508 mutation but is significantly impaired in
G551D
-CFTR (21).
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28
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:28:35
status:
NEW
view ABCC7 p.Gly551Asp details
We therefore tested its effects on
G551D
-CFTR, which is present in the cell membrane, and on ⌬F508-CFTR, once it was translocated in sufficient quantity to the membrane after 4-PB treatment.
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29
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:29:72
status:
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MATERIALS AND METHODS Generation of recombinant adenovirus carrying the
G551D
gene.
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30
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:30:41
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Recombinant adenovirus carrying the CFTR
G551D
mutation was generated by a modified ligation procedure.
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31
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:31:45
status:
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With the use of PCR-mediated point mutation,
G551D
was generated by converting the codon for glycine (GGT) at position 551 to an aspartic acid codon (GAT).
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35
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:35:4
status:
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The
G551D
cDNA was inserted into a shuttle vector, pLadC, which contains the left-hand terminal of adenovirus sequence and a cytomegalovirus promoter.
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36
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:36:88
status:
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The recombinant adenovirus, AdcG551D, was generated by ligation of the linearized pLadC-
G551D
to the right-hand portion of the adenovirus sub360, which contains the deletion in the E1a and E3 regions.
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40
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:40:13
status:
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The inserted
G551D
cDNA was confirmed using PCR protocols.
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45
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:45:80
status:
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Twenty-four hours before use, cells were infected with viral vectors containing
G551D
-CFTR, wtCFTR, or LacZ as control at a multiplicity of infection of ϳ100.
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63
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:63:27
status:
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Patients with at least one
G551D
-CFTR allele were recruited from Children`s Hospital Oakland and from the Stanford CF Center at Lucile Salter Packard Children`s Hospital.
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70
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:70:0
status:
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G551D
-CFTR-mediated current is stimulated by genistein.
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71
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:71:52
status:
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A: whole cell recording from a HeLa cell expressing
G551D
-CFTR.
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89
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:89:162
status:
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F: average calculated Po values during forskolin treatment (fsk, open bars) and forskolin ϩ genistein treatment (geni, solid bars) in HeLa cells expressing
G551D
or wild-type (wt) CFTR.
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90
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:90:40
status:
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view ABCC7 p.Gly551Asp details
Genistein significantly increased Po of
G551D
-CFTR (P ϭ 0.01, n ϭ 4) and wtCFTR (P ϭ 0.02, n ϭ 4, paired t-tests).
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94
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:94:32
status:
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RESULTS Effects of genistein on
G551D
-CFTR in patch-clamp recordings.
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95
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:95:29
status:
NEW
view ABCC7 p.Gly551Asp details
HeLa cells infected with the
G551D
-CFTR-containing adenovirus were patch clamped in the whole cell and the cell-attached mode.
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96
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:96:45
status:
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view ABCC7 p.Gly551Asp details
Figure 1 shows a whole cell recording from a
G551D
-CFTR-expressing cell.
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101
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:101:79
status:
NEW
view ABCC7 p.Gly551Asp details
Current variance to mean current plots was used to calculate the average Po of
G551D
-CFTR-mediated whole cell current.
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106
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:106:37
status:
NEW
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Figure 1F shows average Po values of
G551D
-CFTR compared with wtCFTR.
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107
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:107:19
status:
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view ABCC7 p.Gly551Asp details
Note the low Po of
G551D
-CFTR during forskolin stimulation (0.047 Ϯ 0.19, n ϭ 4), which was increased by genistein to 0.49 Ϯ 0.12, a value not different from wtCFTR.
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109
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:109:47
status:
NEW
view ABCC7 p.Gly551Asp details
Figure 2 shows the effects of genistein on the
G551D
-CFTR channel in a cell-attached patch-clamp recording.
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114
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:114:60
status:
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view ABCC7 p.Gly551Asp details
Figure 2B shows the average single channel I-V relation for
G551D
-CFTR recorded in the cell-attached mode. Single channel I-V relations were typically outwardly rectifying in the cell-attached mode (7), with a conductance in the positive voltage range of g ϭ 8.7 Ϯ 1.0 pS (n ϭ 4, forskolin) and 9.6 Ϯ 0.8 pS (forskolin ϩ genistein), which were similar to wtCFTR (g ϭ 8.6 Ϯ 0.91 pS, n ϭ 3) recorded under the same conditions.
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116
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:116:143
status:
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view ABCC7 p.Gly551Asp details
Genistein showed no effects on Cl current, indicating that 1) HeLa cells had no endogenous genistein-activated Cl current, and 2) unstimulated
G551D
-CFTR could not be activated by genistein alone.
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118
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:118:65
status:
NEW
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ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:118:153
status:
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view ABCC7 p.Gly551Asp details
This indicated that genistein was not a direct channel opener of
G551D
-CFTR but relied on regulation by the cAMP/protein kinase A (PKA) pathway and that
G551D
-CFTR was only active when both genistein was present and PKA was active.
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119
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:119:45
status:
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view ABCC7 p.Gly551Asp details
In summary, these patch-clamp data show that
G551D
-CFTR is a channel with markedly reduced open probability.
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120
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:120:13
status:
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Treatment of
G551D
-CFTR with genistein effectively restored regulation of Po by the cAMP pathway similar to wtCFTR.
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127
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:127:27
status:
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Cell-attached recording of
G551D
-CFTR.
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138
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:138:85
status:
NEW
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We performed nasal potential difference (PD) measurements in CF patients bearing the
G551D
mutation on at least one allele and in healthy human subjects.
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141
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:141:81
status:
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Figure 5 shows measurements of nasal PD from a healthy volunteer (Fig. 5A) and a
G551D
CF patient (Fig. 5B).
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143
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:143:154
status:
NEW
view ABCC7 p.Gly551Asp details
Perfusion with Cl-free solution and subsequent addition of isoproterenol hyperpolarized nasal PD in healthy subjects, whereas nasal PD was depolarized in
G551D
CF patients, indicating a lack of a beta-adrenergic- stimulated Cl conductance in CF patients.
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144
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:144:100
status:
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view ABCC7 p.Gly551Asp details
However, perfusion of the nasal mucosa with genistein significantly hyperpolarized nasal PD both in
G551D
CF patients and in healthy subjects, indicative of a genistein-induced Cl conductance in both groups.
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146
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:146:3
status:
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In
G551D
CF patients, genistein caused a hyperpolarization of, on average, -2.4 Ϯ 0.6 mV (n ϭ 5), which corresponds to 16.9% of the average response of normal subjects to treatment with Cl-free solution and isoproterenol (-14.0 Ϯ 2.1 mV, n ϭ 8).
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147
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:147:111
status:
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Another tested flavonoid, quercetin, was also active but less potent than genistein in stimulating nasal PD in
G551D
CF patients (by -0.9 Ϯ 0.3 mV, n ϭ 4, significantly different from 0, one sample t-test).
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148
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:148:45
status:
NEW
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DISCUSSION Genistein restores cAMP-dependent
G551D
-CFTR activity.
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149
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:149:31
status:
NEW
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ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:149:58
status:
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view ABCC7 p.Gly551Asp details
For this study we selected the
G551D
mutation because the
G551D
-CFTR protein was described as a trafficking-competent mutant (23).
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150
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:150:95
status:
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view ABCC7 p.Gly551Asp details
We found that acute treatment of cells with genistein in the presence of forskolin resulted in
G551D
-CFTR activity that was functionally similar to wtCFTR during stimulation with forskolin.
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151
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:151:25
status:
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view ABCC7 p.Gly551Asp details
The genistein-stimulated
G551D
-CFTR had a single channel conductance of 9.6 Ϯ 0.8 pS, showed time-and voltage-independent currents, and Po was 0.49 Ϯ 0.12, which are typical biophysical characteristics of wtCFTR.
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152
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:152:28
status:
NEW
view ABCC7 p.Gly551Asp details
In addition, stimulation of
G551D
-CFTR by genistein required intrinsic regulation of the channel by the cAMP/PKA system.
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155
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:155:27
status:
NEW
view ABCC7 p.Gly551Asp details
Genistein has no effect on
G551D
-CFTR-mediated Cl currents in unstimulated cells.
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156
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:156:47
status:
NEW
view ABCC7 p.Gly551Asp details
Whole cell recording of a HeLa cell expressing
G551D
-CFTR.
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171
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:171:24
status:
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Effects of genistein in
G551D
CF patients.
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172
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:172:29
status:
NEW
view ABCC7 p.Gly551Asp details
Our nasal PD measurements in
G551D
CF patients showed the typical abnormalities present in other CF patients, which are: a hyperpolarized basal nasal PD, an increased amiloride-sensitive PD, a depolarization of nasal PD with Cl-free solutions, and a lack of response to the beta-adrenergic agonist isoproterenol (20).
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173
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:173:50
status:
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view ABCC7 p.Gly551Asp details
In contrast, perfusion of the nasal epithelium of
G551D
CF patients with genistein resulted in a hyperpolarization of nasal PD in all patients, which under the experimental conditions corresponds to an activation of a Cl conductance.
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183
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:183:47
status:
NEW
view ABCC7 p.Gly551Asp details
Thus the responses to genistein of nasal PD in
G551D
CF patients correspond to 13.6% of normal in terms of Cl current, which may have therapeutic significance.
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189
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:189:64
status:
NEW
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Genistein activates Cl-selective potentials in CF patients with
G551D
mutation.
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194
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:194:32
status:
NEW
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B: measurement of nasal PD in a
G551D
CF patient.
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195
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:195:57
status:
NEW
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Patient was a 14-year-old Caucasian female with genotype
G551D
/⌬F508.
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200
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:200:24
status:
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C: average responses of
G551D
CF patients (n ϭ 5) to treatment with amiloride (amil, 50 µM), Cl-free solution (exchanged for gluconate) and 10 µM isoproterenol (Cl free/iso), and 30 µM genistein (geni), in comparison to measurements in healthy subjects (n ϭ 8).
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202
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:202:69
status:
NEW
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ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:202:119
status:
NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:202:125
status:
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ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:202:150
status:
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Genotypes (and responses of PD to genistein) of patients were: three
G551D
/⌬F508 (-0.8, -2.4, and -2.3 mV), one
G551D
/
G551D
(-2.0 mV), and one
G551D
/unknown (-4.3 mV).
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205
ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:205:3
status:
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view ABCC7 p.Gly551Asp details
In
G551D
CF patients average nasal PD values were: NaCl, -33.8 Ϯ 3.2 mV; amiloride, -11.7 Ϯ 0.9 mV; isoproterenol/Cl free, -6.4 Ϯ 1.7 mV; and genistein, -8.7 Ϯ 1.6 mV.
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