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PMID: 9729613
Linsdell P, Zheng SX, Hanrahan JW
Non-pore lining amino acid side chains influence anion selectivity of the human CFTR Cl- channel expressed in mammalian cell lines.
J Physiol. 1998 Oct 1;512 ( Pt 1):1-16., 1998-10-01
[PubMed]
Sentences
No.
Mutations
Sentence
Comment
20
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:20:12
status:
NEW
view ABCC7 p.Thr339Val details
One mutant,
T339V
, was characterized in detail; its permeation properties were significantly altered, although these effects were not as strong as for T338 mutations.
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59
ABCC7 p.Thr338Phe
X
ABCC7 p.Thr338Phe 9729613:59:204
status:
NEW
view ABCC7 p.Thr338Phe details
Stable expression of all six T338 mutants constructed in BHK cells led to the production of both core-glycosylated (band B) and fully glycosylated (band C) CFTR protein, although some mutants (especially
T338F
) P. Linsdell, S.-X. Zheng and J. W. Hanrahan J. Physiol. 512.12 ) at UNIV OF NORTH CAROLINA on August 8, consistently made less band C protein than wild-type (Fig. 1C).
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60
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:60:55
status:
NEW
view ABCC7 p.Thr339Val details
In contrast, of five mutations made at T339, only one (
T339V
) produced detectable levels of CFTR protein in Western blots using cell lysates from one confluent 10 cm culture plate (approximately 2 ² 10É-3 ² 10É cells; Fig. 1D).
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61
ABCC7 p.Thr339Ser
X
ABCC7 p.Thr339Ser 9729613:61:106
status:
NEW
view ABCC7 p.Thr339Ser details
However, following immunoprecipitation of CFTR protein from five confluent plates, a very small amount of
T339S
mutant CFTR protein was detectable by Western blotting (Fig. 1E), indicating that the BHK cells had been successfully transfected with the mutated plasmid, but that most mutations at T339 caused more severe misprocessing andÏor degradation of the CFTR protein.
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63
ABCC7 p.Thr338Phe
X
ABCC7 p.Thr338Phe 9729613:63:125
status:
NEW
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In contrast, no currents were activated under these conditions in large inside-out patches excised from BHK cells expressing
T338F
(n = 8; not shown).
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81
ABCC7 p.Thr339Ser
X
ABCC7 p.Thr339Ser 9729613:81:38
status:
NEW
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E, Western blot of immunoprecipitated
T339S
protein, carried out as described in Methods.
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82
ABCC7 p.Thr339Ala
X
ABCC7 p.Thr339Ala 9729613:82:35
status:
NEW
view ABCC7 p.Thr339Ala details
ABCC7 p.Thr339Phe
X
ABCC7 p.Thr339Phe 9729613:82:52
status:
NEW
view ABCC7 p.Thr339Phe details
ABCC7 p.Thr339Tyr
X
ABCC7 p.Thr339Tyr 9729613:82:42
status:
NEW
view ABCC7 p.Thr339Tyr details
Similar results were observed with
T339A
,
T339Y
and
T339F
(not shown).
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84
ABCC7 p.Thr338Asn
X
ABCC7 p.Thr338Asn 9729613:84:217
status:
NEW
view ABCC7 p.Thr338Asn details
Example macroscopic current-voltage relationships for wild-type and T338 mutant CFTRs, recorded with symmetrical Cl¦-containing solutions Note the inward rectification observed with some T338 mutants, especially
T338N
.
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91
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:91:125
status:
NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:91:126
status:
NEW
view ABCC7 p.Thr338Ala details
Significant rectification resulting in a -I+50ÏI-0 ratio significantly less than one was observed in all mutants except
T338A
(Fig. 3).
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92
ABCC7 p.Thr338Asn
X
ABCC7 p.Thr338Asn 9729613:92:41
status:
NEW
view ABCC7 p.Thr338Asn details
Rectification was particularly strong in
T338N
(-I+ 50ÏI-50 = 0·33 ± 0·05; n = 6).
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93
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:93:103
status:
NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ser
X
ABCC7 p.Thr338Ser 9729613:93:112
status:
NEW
view ABCC7 p.Thr338Ser details
Single CFTR channel currents in inside-out patches excised from CHO cells stably expressing wild-type,
T338A
or
T338S
are shown in Fig. 4A.
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94
ABCC7 p.Thr338Ile
X
ABCC7 p.Thr338Ile 9729613:94:79
status:
NEW
view ABCC7 p.Thr338Ile details
ABCC7 p.Thr338Asn
X
ABCC7 p.Thr338Asn 9729613:94:63
status:
NEW
view ABCC7 p.Thr338Asn details
ABCC7 p.Thr338Val
X
ABCC7 p.Thr338Val 9729613:94:70
status:
NEW
view ABCC7 p.Thr338Val details
However, clear single channel currents were never resolved for
T338N
,
T338V
or
T338I
, either in CHO cell patches or in patches excised from BHK cells selected using a lower concentration of MTX (20 ìÒ), at potentials as hyperpolarized as -100 mV (data not shown).
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97
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:97:137
status:
NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ser
X
ABCC7 p.Thr338Ser 9729613:97:188
status:
NEW
view ABCC7 p.Thr338Ser details
Mean slope conductance was increased from 7·9 ± 0·1 pS (n = 18) for wild-type to 10·4 ± 0·1 pS (n = 9) for
T338A
and 11·3 ± 0·2 pS (n = 5) for
T338S
.
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99
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:99:23
status:
NEW
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ABCC7 p.Thr338Ser
X
ABCC7 p.Thr338Ser 9729613:99:33
status:
NEW
view ABCC7 p.Thr338Ser details
Although the gating of
T338A
and
T338S
channels was not studied in detail, no striking differences from wild-type were noted.
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101
ABCC7 p.Thr338Ile
X
ABCC7 p.Thr338Ile 9729613:101:74
status:
NEW
view ABCC7 p.Thr338Ile details
ABCC7 p.Thr338Asn
X
ABCC7 p.Thr338Asn 9729613:101:57
status:
NEW
view ABCC7 p.Thr338Asn details
ABCC7 p.Thr338Val
X
ABCC7 p.Thr338Val 9729613:101:64
status:
NEW
view ABCC7 p.Thr338Val details
We attempted to make some estimate of the conductance of
T338N
,
T338V
and
T338I
channels by analysing the increase in current noise associated with macroscopic current activation (Fig. 5).
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107
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:107:22
status:
NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:107:97
status:
NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ser
X
ABCC7 p.Thr338Ser 9729613:107:32
status:
NEW
view ABCC7 p.Thr338Ser details
ABCC7 p.Thr338Ser
X
ABCC7 p.Thr338Ser 9729613:107:107
status:
NEW
view ABCC7 p.Thr338Ser details
Unitary properties of
T338A
and
T338S
CFTR A, examples of single channel activity for wild-type,
T338A
and
T338S
, recorded at -50 mV.
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109
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:109:74
status:
NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ser
X
ABCC7 p.Thr338Ser 9729613:109:88
status:
NEW
view ABCC7 p.Thr338Ser details
B and C, mean single channel current-voltage relationships for wild-type,
T338A
(B) and
T338S
(C).
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111
ABCC7 p.Thr338Ile
X
ABCC7 p.Thr338Ile 9729613:111:52
status:
NEW
view ABCC7 p.Thr338Ile details
ABCC7 p.Thr338Asn
X
ABCC7 p.Thr338Asn 9729613:111:78
status:
NEW
view ABCC7 p.Thr338Asn details
ABCC7 p.Thr338Val
X
ABCC7 p.Thr338Val 9729613:111:88
status:
NEW
view ABCC7 p.Thr338Val details
In contrast to wild-type, activation of macroscopic
T338I
(Fig. 5C and D) and
T338N
and
T338V
(data not shown) Cl¦ currents was associated with only a very small increase in noise.
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112
ABCC7 p.Thr338Ile
X
ABCC7 p.Thr338Ile 9729613:112:228
status:
NEW
view ABCC7 p.Thr338Ile details
ABCC7 p.Thr338Asn
X
ABCC7 p.Thr338Asn 9729613:112:126
status:
NEW
view ABCC7 p.Thr338Asn details
ABCC7 p.Thr338Val
X
ABCC7 p.Thr338Val 9729613:112:175
status:
NEW
view ABCC7 p.Thr338Val details
Analysis of current variance (e.g. Fig. 5D) yielded chord conductances at -50 mV of 0·23 ± 0·02 pS (n = 4) for
T338N
, 0·36 ± 0·10 pS (n = 5) for
T338V
, and 0·17 ± 0·04 pS (n = 5) for
T338I
.
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118
ABCC7 p.Thr338Phe
X
ABCC7 p.Thr338Phe 9729613:118:102
status:
NEW
view ABCC7 p.Thr338Phe details
Thus, the lack of macroscopic Cl¦ currents in membrane patches excised from BHK cells expressing
T338F
(see above) may reflect a non-conducting phenotype for this mutant.
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119
ABCC7 p.Thr338Phe
X
ABCC7 p.Thr338Phe 9729613:119:47
status:
NEW
view ABCC7 p.Thr338Phe details
Alternatively, misprocessing or degradation of
T338F
protein (Fig. 1C) may result in expression levels below our threshold of detection using patch clamp recording.
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121
ABCC7 p.Thr338Ile
X
ABCC7 p.Thr338Ile 9729613:121:0
status:
NEW
view ABCC7 p.Thr338Ile details
T338I
gave only very small currents when intracellular Cl¦ was replaced by other anions, such that accurate measurement of Vrev was not possible, and therefore the selectivity of this mutant was not studied further.
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123
ABCC7 p.Thr338Ile
X
ABCC7 p.Thr338Ile 9729613:123:242
status:
NEW
view ABCC7 p.Thr338Ile details
Estimation of unitary current amplitude from macroscopic current noise A and C, activation of macroscopic CFTR current at -50 mV is associated with a large increase in current noise for wild-type (A), but a much smaller increase in noise for
T338I
(C).
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142
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:142:154
status:
NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ser
X
ABCC7 p.Thr338Ser 9729613:142:160
status:
NEW
view ABCC7 p.Thr338Ser details
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:142:178
status:
NEW
view ABCC7 p.Thr339Val details
ABCC7 p.Thr338Asn
X
ABCC7 p.Thr338Asn 9729613:142:166
status:
NEW
view ABCC7 p.Thr338Asn details
ABCC7 p.Thr338Val
X
ABCC7 p.Thr338Val 9729613:142:172
status:
NEW
view ABCC7 p.Thr338Val details
Permeability of intracellular anions in wild-type and mutant CFTR Cl¦ channels ------------------------------------------------------------ Anion WT
T338A
T338S
T338N
T338V
T339V
------------------------------------------------------------ Thiocyanate 2·63 ± 0·13 (6) 5·85 ± 0·27 (4)* 4·80 ± 0·19 (3)* 8·72 ± 1·03 (4)* 1·92 ± 0·35 (4)* 3·28 ± 0·08 (4)* Nitrate 1·53 ± 0·04 (7) 2·04 ± 0·08 (3)* 1·82 ± 0·03 (4)* 4·22 ± 0·22 (3)* 6·84 ± 1·18 (7)* 1·61 ± 0·02 (3) Bromide 1·23 ± 0·03 (5) 1·74 ± 0·04 (3)* 1·47 ± 0·07 (3)* 1·66 ± 0·15 (3)* 1·04 ± 0·09 (5) 1·39 ± 0·06 (4)* Chloride 1·00 ± 0·01 (10) 1·00 ± 0·02 (11) 1·00 ± 0·02 (6) 1·00 ± 0·03 (10) 1·00 ± 0·04 (11) 1·00 ± 0·06 (10) Iodide 0·84 ± 0·03 (5) 2·09 ± 0·16 (5)* 1·76 ± 0·09 (3)* 1·03 ± 0·05 (3)* 0·79 ± 0·11 (3) 0·84 ± 0·02 (3) Perchlorate 0·25 ± 0·02 (6) 1·35 ± 0·08 (3)* 0·66 ± 0·06 (3)* 0·41 ± 0·03 (3)* 0·54 ± 0·00 (3)* 0·24 ± 0·01 (4) Benzoate 0·069 ± 0·006 (6) 0·17 ± 0·03 (4)* 0·091 ± 0·019 (3) 0·089 ± 0·015 (4) 0·15 ± 0·02 (4)* 0·097 ± 0·014 (4) Hexafluorophosphate < 0·019 (4) 0·53 ± 0·01 (3)* 0·31 ± 0·02 (3)* 0·68 ± 0·02 (3)* 0·39 ± 0·05 (3)* 0·051 ± 0·010 (4)* Fluoride 0·11 ± 0·01 (7) 0·12 ± 0·02 (4) 0·095 ± 0·012 (4) 0·11 ± 0·01 (4) 0·093 ± 0·009 (3) 0·17 ± 0·02 (4)* Formate 0·25 ± 0·01 (8) 0·45 ± 0·04 (3)* 0·43 ± 0·03 (3)* 0·35 ± 0·04 (4)* 0·22 ± 0·01 (3) 0·28 ± 0·02 (3) Acetate 0·090 ± 0·004 (8) 0·19 ± 0·01 (3)* 0·18 ± 0·01 (3)* 0·10 ± 0·02 (5) 0·11 ± 0·02 (3) 0·16 ± 0·01 (3)* Propanoate 0·14 ± 0·01 (3) 0·18 ± 0·02 (4) 0·098 ± 0·010 (4)* 0·077 ± 0·013 (3)* 0·13 ± 0·02 (3) - Pyruvate 0·10 ± 0·01 (5) 0·20 ± 0·01 (3)* 0·13 ± 0·02 (3) 0·075 ± 0·015 (3) 0·17 ± 0·03 (3)* - Methane sulphonate 0·077 ± 0·005 (5) 0·14 ± 0·02 (4)* 0·079 ± 0·014 (3) 0·038 ± 0·004 (3)* 0·088 ± 0·007 (3) - Glutamate 0·096 ± 0·008 (4) 0·082 ± 0·009 (3) 0·080 ± 0·008 (3) 0·060 ± 0·012 (5)* 0·11 ± 0·01 (3) - Isethionate 0·13 ± 0·01 (4) 0·11 ± 0·01 (3) 0·086 ± 0·012 (5)* 0·043 ± 0·007 (3)* 0·067 ± 0·005 (3)* - Gluconate 0·068 ± 0·004 (36) 0·10 ± 0·01 (3)* 0·060 ± 0·004 (3) 0·044 ± 0·004 (3) 0·077 ± 0·009 (3) 0·088 ± 0·021 (5) ------------------------------------------------------------ Relative permeabilities of different anions present in the intracellular solution under biionic conditions were calculated from macroscopic current reversal potentials (e.g. Figs 7 and 10) as described in Methods.
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150
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:150:64
status:
NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ser
X
ABCC7 p.Thr338Ser 9729613:150:72
status:
NEW
view ABCC7 p.Thr338Ser details
Note that all lyotropic anions showed the permeability sequence
T338A
>
T338S
> wild-type, again suggesting that the effects of these mutations on pore properties are correlated with the size of the amino acid side chain substituted.
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151
ABCC7 p.Thr338Asn
X
ABCC7 p.Thr338Asn 9729613:151:59
status:
NEW
view ABCC7 p.Thr338Asn details
ABCC7 p.Thr338Val
X
ABCC7 p.Thr338Val 9729613:151:69
status:
NEW
view ABCC7 p.Thr338Val details
However, the permeabilities of the low conductance mutants
T338N
and
T338V
were more difficult to interpret, possibly indicating that substitution of a larger amino acid for T338 causes a more severe disruption of pore function.
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153
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:153:599
status:
NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ser
X
ABCC7 p.Thr338Ser 9729613:153:724
status:
NEW
view ABCC7 p.Thr338Ser details
ABCC7 p.Thr338Asn
X
ABCC7 p.Thr338Asn 9729613:153:849
status:
NEW
view ABCC7 p.Thr338Asn details
ABCC7 p.Thr338Val
X
ABCC7 p.Thr338Val 9729613:153:969
status:
NEW
view ABCC7 p.Thr338Val details
Pore diameter of T338 mutants Previously, we suggested that the double mutant channel, TT338,339AA, had an increased minimum functional pore diameter, based on its increased permeability to extracellular formate, acetate, propanoate and pyruvate ions (Linsdell et P. Linsdell, S.-X. Zheng and J. W. Hanrahan J. Physiol. 512.18 -------------------------------------------------------------------------------------------- Table 2 ---------------------------------------------- Wild-type SCN¦ > NOצ > Br¦ > Cl¦ > I¦ > ClOÚ¦ > formate > F¦ > PFܦ
T338A
SCN¦ > I¦ ü NOצ > Br¦ > ClOÚ¦ > Cl¦ > PFܦ > formate > F¦
T338S
SCN¦ > NOצ ü I¦ > Br¦ > Cl¦ > ClOÚ¦ > formate > PFܦ > F¦
T338N
SCN¦ > NOצ > Br¦ > I¦ = Cl¦ > PFܦ > ClOÚ¦ > formate > F¦
T338V
NOצ > SCN¦ > Br¦ = Cl¦ > I¦ > ClOÚ¦ > PFܦ > formate > F¦ -------------------------------------------------------------------------------------------- Figure 7.
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164
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:164:137
status:
NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ser
X
ABCC7 p.Thr338Ser 9729613:164:160
status:
NEW
view ABCC7 p.Thr338Ser details
ABCC7 p.Thr338Asn
X
ABCC7 p.Thr338Asn 9729613:164:183
status:
NEW
view ABCC7 p.Thr338Asn details
ABCC7 p.Thr338Val
X
ABCC7 p.Thr338Val 9729613:164:209
status:
NEW
view ABCC7 p.Thr338Val details
In each case the data have been fitted by eqn (2), giving minimum functional pore diameters of 0·528 nm (wild-type), 0·576 nm (
T338A
), 0·549 nm (
T338S
), 0·510 nm (
T338N
) and 0·540 nm (
T338V
).
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168
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:168:171
status:
NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ser
X
ABCC7 p.Thr338Ser 9729613:168:196
status:
NEW
view ABCC7 p.Thr338Ser details
ABCC7 p.Thr338Asn
X
ABCC7 p.Thr338Asn 9729613:168:221
status:
NEW
view ABCC7 p.Thr338Asn details
ABCC7 p.Thr338Val
X
ABCC7 p.Thr338Val 9729613:168:249
status:
NEW
view ABCC7 p.Thr338Val details
In each case the data have been fitted using eqn (2) (see Methods), giving estimates of the functional pore diameter (d) of 0·528 nm for wild-type, 0·576 nm for
T338A
, 0·549 nm for
T338S
, 0·510 nm for
T338N
and 0·540 nm for
T338V
. Anions examined (in order of increasing diameter) were: SCN¦, Cl¦, NOצ, Br¦, I¦, ClOÚ¦, benzoate and PFܦ.
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171
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:171:108
status:
NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ser
X
ABCC7 p.Thr338Ser 9729613:171:131
status:
NEW
view ABCC7 p.Thr338Ser details
ABCC7 p.Thr338Asn
X
ABCC7 p.Thr338Asn 9729613:171:154
status:
NEW
view ABCC7 p.Thr338Asn details
ABCC7 p.Thr338Val
X
ABCC7 p.Thr338Val 9729613:171:180
status:
NEW
view ABCC7 p.Thr338Val details
In this case, fits by eqn (2) suggested minimum pore diameters of 0·535 nm (wild-type), 0·615 nm (
T338A
), 0·505 nm (
T338S
), 0·503 nm (
T338N
) and 0·530 nm (
T338V
).
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173
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:173:101
status:
NEW
view ABCC7 p.Thr339Val details
Effects of mutations at T339 Of five amino acid substitutions carried out at position 339, only one (
T339V
) resulted in the production of detectable amounts of CFTR protein (Fig. 1D).
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174
ABCC7 p.Thr339Ala
X
ABCC7 p.Thr339Ala 9729613:174:117
status:
NEW
view ABCC7 p.Thr339Ala details
ABCC7 p.Thr339Ala
X
ABCC7 p.Thr339Ala 9729613:174:119
status:
NEW
view ABCC7 p.Thr339Ala details
ABCC7 p.Thr339Ser
X
ABCC7 p.Thr339Ser 9729613:174:132
status:
NEW
view ABCC7 p.Thr339Ser details
ABCC7 p.Thr339Ser
X
ABCC7 p.Thr339Ser 9729613:174:134
status:
NEW
view ABCC7 p.Thr339Ser details
ABCC7 p.Thr339Phe
X
ABCC7 p.Thr339Phe 9729613:174:164
status:
NEW
view ABCC7 p.Thr339Phe details
ABCC7 p.Thr339Phe
X
ABCC7 p.Thr339Phe 9729613:174:166
status:
NEW
view ABCC7 p.Thr339Phe details
ABCC7 p.Thr339Tyr
X
ABCC7 p.Thr339Tyr 9729613:174:147
status:
NEW
view ABCC7 p.Thr339Tyr details
ABCC7 p.Thr339Tyr
X
ABCC7 p.Thr339Tyr 9729613:174:149
status:
NEW
view ABCC7 p.Thr339Tyr details
Indeed, we found no PKAand ATP-dependent currents in very large inside-out patches excised from BHK cells expressing
T339A (
n = 6),
T339S (
n = 4),
T339Y (
n = 6) or
T339F (
n = 5) using symmetrical 150 mÒ NaCl-containing solutions (not shown).
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175
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:175:76
status:
NEW
view ABCC7 p.Thr339Val details
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:175:78
status:
NEW
view ABCC7 p.Thr339Val details
PKAand ATP-dependent currents were observed, however, in twelve of fourteen
T339V C
FTR patches under the same conditions (Fig. 10A).
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176
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:176:0
status:
NEW
view ABCC7 p.Thr339Val details
T339V
showed slight inward current rectification under these symmetrical ionic conditions (-I+50ÏI-50 = 0·86 ± 0·01; n = 8).
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178
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:178:28
status:
NEW
view ABCC7 p.Thr339Val details
Mean chord conductances for
T339V
were 7·95 ± 0·16 pS (n = 4) at -50 mV and 6·37 ± 0·29 pS (n = 3) at +50 mV, compared with wild-type values of 7·91 ± 0·09 pS (n = 18) at -50 mV and 7·85 ± 0·09 pS (n = 12) at +50 mV.
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179
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:179:8
status:
NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ser
X
ABCC7 p.Thr338Ser 9729613:179:18
status:
NEW
view ABCC7 p.Thr338Ser details
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:179:25
status:
NEW
view ABCC7 p.Thr339Val details
As with
T338A
and
T338S
,
T339V
showed apparently normal channel gating, with open probability being time and P. Linsdell, S.-X. Zheng and J. W. Hanrahan J. Physiol. 512.110 Figure 9.
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181
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:181:171
status:
NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ser
X
ABCC7 p.Thr338Ser 9729613:181:196
status:
NEW
view ABCC7 p.Thr338Ser details
ABCC7 p.Thr338Asn
X
ABCC7 p.Thr338Asn 9729613:181:221
status:
NEW
view ABCC7 p.Thr338Asn details
ABCC7 p.Thr338Val
X
ABCC7 p.Thr338Val 9729613:181:249
status:
NEW
view ABCC7 p.Thr338Val details
In each case the data have been fitted using eqn (2) (see Methods), giving estimates of the functional pore diameter (d) of 0·535 nm for wild type, 0·615 nm for
T338A
, 0·505 nm for
T338S
, 0·503 nm for
T338N
and 0·530 nm for
T338V
. Anions examined (in order of increasing diameter) were: formate, acetate, propanoate, pyruvate, methane sulphonate and gluconate.)
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183
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:183:35
status:
NEW
view ABCC7 p.Thr339Val details
Furthermore, as with T338 mutants,
T339V
had negligible cation permeability (data not shown).
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184
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:184:4
status:
NEW
view ABCC7 p.Thr339Val details
The
T339V
mutant also had significant alterations in ionic permeability (Fig. 10B and Table 1), although in general these were not as strong as those observed for T338 mutants.
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185
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:185:37
status:
NEW
view ABCC7 p.Thr339Val details
Indeed, the selectivity sequence for
T339V
(SCN¦ > NOצ > Br¦ > Cl¦ > I¦ > ClOÚ¦ > formate > F¦ > PFܦ) was the same as for wild-type (see Table 2).
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186
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:186:56
status:
NEW
view ABCC7 p.Thr339Val details
Gluconate permeability was not significantly altered in
T339V
, again suggesting no severe disruption of large organic anion permeability.
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187
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:187:45
status:
NEW
view ABCC7 p.Thr339Val details
All of these effects are consistent with the
T339V
mutant having less severely altered pore properties than any of the T338 mutants studied.
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193
ABCC7 p.Thr338Ile
X
ABCC7 p.Thr338Ile 9729613:193:156
status:
NEW
view ABCC7 p.Thr338Ile details
ABCC7 p.Thr338Asn
X
ABCC7 p.Thr338Asn 9729613:193:139
status:
NEW
view ABCC7 p.Thr338Asn details
ABCC7 p.Thr338Val
X
ABCC7 p.Thr338Val 9729613:193:146
status:
NEW
view ABCC7 p.Thr338Val details
The conductance of different T338 mutants varied over almost two orders of magnitude (Fig. 6), although the low conductances estimated for
T338N
,
T338V
and
T338I
should be considered rough approximations only.
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195
ABCC7 p.Thr338Ile
X
ABCC7 p.Thr338Ile 9729613:195:175
status:
NEW
view ABCC7 p.Thr338Ile details
The central portion of TM6 containing both of these key amino acids (T338 and S341) may therefore be the region where conductance is mainly determined. The low conductance of
T338I
could also explain why this mutation is associated with CF (Saba et al. 1993), although partial misprocessing (Fig. 1C) may also play a role.
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196
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:196:40
status:
NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ser
X
ABCC7 p.Thr338Ser 9729613:196:50
status:
NEW
view ABCC7 p.Thr338Ser details
Conversely, the elevated conductance of
T338A
and
T338S
might be advantageous in gene or protein replacement therapies for Alteration of CFTR anion selectivityJ. Physiol. 512.1 11 Figure 10.
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197
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:197:46
status:
NEW
view ABCC7 p.Thr339Val details
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:197:60
status:
NEW
view ABCC7 p.Thr339Val details
Macroscopic current-voltage relationships for
T339V
CFTR A,
T339V
shows slight inward rectification with symmetrical Cl¦-containing solutions.
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198
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:198:86
status:
NEW
view ABCC7 p.Thr339Val details
B, example current-voltage relationships used to investigate the anion selectivity of
T339V
, measured under biionic conditions with ClOڦ or NOצ in the intracellular solution.
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199
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:199:44
status:
NEW
view ABCC7 p.Thr339Val details
Note that the permeability of these ions in
T339V
is similar to that observed in wild-type (Fig. 7).
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205
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:205:20
status:
NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ser
X
ABCC7 p.Thr338Ser 9729613:205:54
status:
NEW
view ABCC7 p.Thr338Ser details
Interestingly, both
T338A
(10·4 pS; Fig. 4B) and
T338S
(11·3 pS; Fig. 4C) have higher conductances than that we reported previously for TT338,339AA (9·9 pS; Linsdell et al. 1997b).
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206
ABCC7 p.Thr338Ser
X
ABCC7 p.Thr338Ser 9729613:206:5
status:
NEW
view ABCC7 p.Thr338Ser details
Thus
T338S
has the highest conductance of any CFTR variant described to date.
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207
ABCC7 p.Thr338Ile
X
ABCC7 p.Thr338Ile 9729613:207:151
status:
NEW
view ABCC7 p.Thr338Ile details
ABCC7 p.Thr338Asn
X
ABCC7 p.Thr338Asn 9729613:207:134
status:
NEW
view ABCC7 p.Thr338Asn details
ABCC7 p.Thr338Val
X
ABCC7 p.Thr338Val 9729613:207:141
status:
NEW
view ABCC7 p.Thr338Val details
All single channel conductances reported in this paper were measured at hyperpolarized potentials; conductance of the mutant channels
T338N
,
T338V
and
T338I
might be significantly lower at depolarized potentials (Fig. 2).
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209
ABCC7 p.Lys335Glu
X
ABCC7 p.Lys335Glu 9729613:209:96
status:
NEW
view ABCC7 p.Lys335Glu details
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:209:128
status:
NEW
view ABCC7 p.Thr339Val details
ABCC7 p.Ile332Lys
X
ABCC7 p.Ile332Lys 9729613:209:149
status:
NEW
view ABCC7 p.Ile332Lys details
Inward rectification is observed in several other TM6 mutants at the single channel level, e.g.
K335E
(Tabcharani et al. 1993),
T339V
(Fig. 11B) and
I332K
(P. Linsdell, J. A. Tabcharani & J. W. Hanrahan, unpublished observations); however, voltage-dependent gating in T338 mutants cannot be excluded.
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222
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:222:22
status:
NEW
view ABCC7 p.Thr339Val details
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:222:57
status:
NEW
view ABCC7 p.Thr339Val details
Unitary properties of
T339V
CFTR A, activity of a single
T339V
channel at -50 mV (cf.
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224
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:224:93
status:
NEW
view ABCC7 p.Thr339Val details
B, mean single channel current-voltage relationships for wild-type (as in Fig. 4B and C) and
T339V
.
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232
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:232:8
status:
NEW
view ABCC7 p.Thr338Ala details
Thus in
T338A
, for example, the relationship between permeability and ionic hydration energy appeared much more linear than for wild-type (Fig. 12B).
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237
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:237:162
status:
NEW
view ABCC7 p.Thr339Val details
However, it is unclear why mutations at T339 should apparently be more sensitive to misprocessing than those at T338 (Fig. 1C and D), or why the non-conservative
T339V
mutation alone should be appropriately processed (Fig. 1D).
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238
ABCC7 p.Thr339Ala
X
ABCC7 p.Thr339Ala 9729613:238:5
status:
NEW
view ABCC7 p.Thr339Ala details
ABCC7 p.Thr339Cys
X
ABCC7 p.Thr339Cys 9729613:238:39
status:
NEW
view ABCC7 p.Thr339Cys details
Both
T339A
(McDonough et al. 1994) and
T339C
(Cheung & Akabas, 1996) mutant channels can be expressed in Xenopus oocytes following injection of in vitro transcribed cRNA.
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239
ABCC7 p.Thr339Ala
X
ABCC7 p.Thr339Ala 9729613:239:171
status:
NEW
view ABCC7 p.Thr339Ala details
Furthermore, the double mutant TT338,339AA can be functionally expressed in CHO cells (Linsdell et al. 1997b), raising the possibility that the detrimental effects of the
T339A
mutation on processing may be 'rescued` by simultaneous mutation of T338.
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240
ABCC7 p.Thr339Val
X
ABCC7 p.Thr339Val 9729613:240:115
status:
NEW
view ABCC7 p.Thr339Val details
Because of low protein expression, we were only able to characterize the permeation properties of one T339 mutant,
T339V
.
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242
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 9729613:242:124
status:
NEW
view ABCC7 p.Thr338Ala details
Relationship between relative permeability and free energy of hydration for different intracellular anions A, wild-type; B,
T338A
.
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