PMID: 11927667

Gong X, Burbridge SM, Cowley EA, Linsdell P
Molecular determinants of Au(CN)(2)(-) binding and permeability within the cystic fibrosis transmembrane conductance regulator Cl(-) channel pore.
J Physiol. 2002 Apr 1;540(Pt 1):39-47., 2002-04-01 [PubMed]
Sentences
No. Mutations Sentence Comment
12 ABCC7 p.Arg352Gln
X
ABCC7 p.Arg352Gln 11927667:12:201
status: NEW
view ABCC7 p.Arg352Gln details
ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 11927667:12:191
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 11927667:12:144
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 11927667:12:137
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 11927667:12:130
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Ile344Ala
X
ABCC7 p.Ile344Ala 11927667:12:154
status: NEW
view ABCC7 p.Ile344Ala details
ABCC7 p.Lys329Ala
X
ABCC7 p.Lys329Ala 11927667:12:224
status: NEW
view ABCC7 p.Lys329Ala details
Channel block by 100 mM Au(CN)2 _ , a measure of intrapore anion binding affinity, was significantly weakened in the CFTR mutants K335A, F337S, T338A and I344A, significantly strengthened in S341A and R352Q and unaltered in K329A. Login to comment
13 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 11927667:13:73
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 11927667:13:63
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 11927667:13:107
status: NEW
view ABCC7 p.Phe337Ser details
Relative Au(CN)2 _ permeability was significantly increased in T338A and S341A, significantly decreased in F337S and unaffected in all other mutants studied. Login to comment
42 ABCC7 p.Arg352Gln
X
ABCC7 p.Arg352Gln 11927667:42:147
status: NEW
view ABCC7 p.Arg352Gln details
ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 11927667:42:224
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 11927667:42:85
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 11927667:42:78
status: NEW
view ABCC7 p.Phe337Ser details
Some of these have previously been associated with altered anion selectivity (F337S, T338A; Linsdelletal.1998,2000),alteredanion:cationselectivity(R352Q; Guinamard & Akabas, 1999), or disrupted open channel blocker binding (S341A; McDonough et al. 1994). Login to comment
70 ABCC7 p.Arg352Gln
X
ABCC7 p.Arg352Gln 11927667:70:204
status: NEW
view ABCC7 p.Arg352Gln details
Block of PPi-stimulated wild-type and mutant CFTR Cl_ currents by Au(CN)2 _ A, example CFTR I-V relationships recorded from inside-out membrane patches from BHK cells transfected with wild-type (left) or R352Q-CFTR (right). Login to comment
72 ABCC7 p.Arg352Gln
X
ABCC7 p.Arg352Gln 11927667:72:97
status: NEW
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The PPi-induced increase in current amplitude was significantly different from wild-type only in R352Q (* P < 0.001, Student`s two-tailed t test). Login to comment
78 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 11927667:78:58
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 11927667:78:51
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 11927667:78:44
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Ile344Ala
X
ABCC7 p.Ile344Ala 11927667:78:68
status: NEW
view ABCC7 p.Ile344Ala details
ABCC7 p.Lys329Ala
X
ABCC7 p.Lys329Ala 11927667:78:37
status: NEW
view ABCC7 p.Lys329Ala details
Currents carried by the CFTR mutants K329A, K335A, T338A, S341A and I344A were also stimulated an average of 2_3-fold by PPi (Fig. 2B). Login to comment
79 ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 11927667:79:19
status: NEW
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The stimulation of F337S appeared somewhat less (1.46 ± 0.24-fold, n = 5), although this was not significantly different from wild-type (P > 0.1, Student`s two-tailed t test). Login to comment
80 ABCC7 p.Arg352Gln
X
ABCC7 p.Arg352Gln 11927667:80:35
status: NEW
view ABCC7 p.Arg352Gln details
In contrast, while only very small R352Q currents were recorded in the presence of PKA and ATP, currents were massively stimulated on addition of PPi (by an average of 26.3 ± 5.7-fold; Fig. 2A, B). Login to comment
81 ABCC7 p.Arg352Gln
X
ABCC7 p.Arg352Gln 11927667:81:108
status: NEW
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Although single channel recordings were not carried out, this implies that the channel open probability for R352Q in the absence of PPi is very low (< 0.04). Login to comment
82 ABCC7 p.Arg334Ala
X
ABCC7 p.Arg334Ala 11927667:82:114
status: NEW
view ABCC7 p.Arg334Ala details
ABCC7 p.Thr351Ala
X
ABCC7 p.Thr351Ala 11927667:82:124
status: NEW
view ABCC7 p.Thr351Ala details
Two other mutants were also transfected into BHK cells; however, even in the presence of PPi, currents carried by R334A and T351A were too small for proper analysis (not shown). Login to comment
86 ABCC7 p.Lys329Ala
X
ABCC7 p.Lys329Ala 11927667:86:40
status: NEW
view ABCC7 p.Lys329Ala details
However, for all mutants studied except K329A, the blocking effects of 100 mM Au(CN)2 _ at _100 mV were significantly altered relative to wild-type (P < 0.05 in each case, Student`s two-tailed t test). Login to comment
87 ABCC7 p.Arg352Gln
X
ABCC7 p.Arg352Gln 11927667:87:119
status: NEW
view ABCC7 p.Arg352Gln details
ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 11927667:87:127
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 11927667:87:178
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 11927667:87:170
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 11927667:87:162
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Ile344Ala
X
ABCC7 p.Ile344Ala 11927667:87:154
status: NEW
view ABCC7 p.Ile344Ala details
ABCC7 p.Lys329Ala
X
ABCC7 p.Lys329Ala 11927667:87:146
status: NEW
view ABCC7 p.Lys329Ala details
Comparison between different channel variants at _100 mV reveals the sensitivity to this concentration of Au(CN)2 _ is R352Q > S341A > wild-type, K329A > I344A > K335A = F337S > T338A. Login to comment
88 ABCC7 p.Arg352Gln
X
ABCC7 p.Arg352Gln 11927667:88:213
status: NEW
view ABCC7 p.Arg352Gln details
ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 11927667:88:221
status: NEW
view ABCC7 p.Ser341Ala details
At depolarised voltages, where the blocking effects of 100 mM Au(CN)2 _ are weak, block of most mutants was not significantly different from wild-type; the only differences in Au(CN)2 _ sensitivity at +60 mV were R352Q > S341A > wild-type. Login to comment
89 ABCC7 p.Arg352Gln
X
ABCC7 p.Arg352Gln 11927667:89:236
status: NEW
view ABCC7 p.Arg352Gln details
Although block of PPi-stimulated channels by 100 mM Au(CN)2 _ was strongly voltage dependent in wild-type and most mutants, consistent with the 'conduction effect` of this anion (Linsdell & Gong, 2002), the strong inhibitory effects on R352Q were practically voltage independent. Login to comment
91 ABCC7 p.Arg352Gln
X
ABCC7 p.Arg352Gln 11927667:91:41
status: NEW
view ABCC7 p.Arg352Gln details
ABCC7 p.Arg352Gln
X
ABCC7 p.Arg352Gln 11927667:91:206
status: NEW
view ABCC7 p.Arg352Gln details
Together with the extreme stimulation of R352Q current by PPi (Fig. 2A and B), this suggests that we are unable to separate permeation from gating effects in this mutant and, as such, the potential role of R352Q in contributing to intrapore anion binding cannot be addressed in the present study. Login to comment
95 ABCC7 p.Lys329Ala
X
ABCC7 p.Lys329Ala 11927667:95:12
status: NEW
view ABCC7 p.Lys329Ala details
Results for K329A were indistinguishable from wild-type (not shown). Login to comment
100 ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 11927667:100:100
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 11927667:100:63
status: NEW
view ABCC7 p.Phe337Ser details
Relative Au(CN)2 _ permeability was significantly decreased in F337S and significantly increased in T338A (Fig. 4), consistent with the previously described opposite effects of these mutants on CFTR lyotropic anion selectivity (Linsdell et al. 1998, 2000). Login to comment
101 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 11927667:101:15
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 11927667:101:106
status: NEW
view ABCC7 p.Thr338Ala details
Interestingly, S341A also significantly increased PAu(CN)2/PCl (Fig. 4), although to a lesser extent than T338A. Login to comment
116 ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 11927667:116:229
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 11927667:116:219
status: NEW
view ABCC7 p.Phe337Ser details
Au(CN)2 _ permeability of different CFTR variants A, example CFTR I-V relationships recorded with 150 m KAu(CN)2 in the extracellular solution and 150 m KCl in the intracellular solution, for wild-type, F337S and T338A. Login to comment
123 ABCC7 p.Arg352Gln
X
ABCC7 p.Arg352Gln 11927667:123:144
status: NEW
view ABCC7 p.Arg352Gln details
ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 11927667:123:134
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 11927667:123:87
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 11927667:123:80
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 11927667:123:73
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Lys329Ala
X
ABCC7 p.Lys329Ala 11927667:123:168
status: NEW
view ABCC7 p.Lys329Ala details
At this voltage, block by 100 mM Au(CN)2 _ was significantly weakened in K335A, F337S, T338A and I334A, significantly strengthened in S341A and R352Q and unaffected in K329A (Fig. 3). Login to comment
124 ABCC7 p.Arg352Gln
X
ABCC7 p.Arg352Gln 11927667:124:78
status: NEW
view ABCC7 p.Arg352Gln details
ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 11927667:124:86
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 11927667:124:137
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 11927667:124:129
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Lys335Ala
X
ABCC7 p.Lys335Ala 11927667:124:121
status: NEW
view ABCC7 p.Lys335Ala details
ABCC7 p.Ile344Ala
X
ABCC7 p.Ile344Ala 11927667:124:113
status: NEW
view ABCC7 p.Ile344Ala details
ABCC7 p.Lys329Ala
X
ABCC7 p.Lys329Ala 11927667:124:105
status: NEW
view ABCC7 p.Lys329Ala details
The sequence of relative sensitivity to block by 100 mM Au(CN)2 _ at _100 mV (R352Q > S341A > wild-type, K329A > I344A > K335A = F337S > T338A) suggests that T338 normally makes the strongest contribution to Au(CN)2 _ binding within the pore, with nearby residues K335 and F337 also making large contributions. Login to comment
126 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 11927667:126:141
status: NEW
view ABCC7 p.Ser341Ala details
Interestingly, in spite of its previous association with disrupted anion binding within the pore (McDonough et al. 1994; Zhang et al. 2000), S341A showed significantly strengthened Au(CN)2 _ block at all potentials (Fig. 3), suggesting that the polar hydroxyl side chain of S341 does not contribute to lyotropic anion binding. Login to comment
128 ABCC7 p.Arg352Gln
X
ABCC7 p.Arg352Gln 11927667:128:41
status: NEW
view ABCC7 p.Arg352Gln details
As discussed in the text, the effects of R352Q cannot be confidently ascribed to open channel block and, as such, we are unable to determine the role of R352 in anion binding in the pore. Login to comment
139 ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 11927667:139:25
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 11927667:139:131
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 11927667:139:15
status: NEW
view ABCC7 p.Phe337Ser details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 11927667:139:83
status: NEW
view ABCC7 p.Phe337Ser details
The effects of F337S and T338A on PAu(CN)2/PCl are consistent with the disruption (F337S; Linsdell et al. 2000) and strengthening (T338A; Linsdell et al. 1998) of lyotropic anion selectivity previously described in these two mutants. Login to comment
140 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 11927667:140:59
status: NEW
view ABCC7 p.Ser341Ala details
However, the dramatic increase in PAu(CN)2/PCl observed in S341A (Fig. 4) suggests that the region of the pore which predominantly controls selectivity between different anions may extend further towards the intracellular end of TM6 than previously appreciated. Login to comment
142 ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 11927667:142:148
status: NEW
view ABCC7 p.Thr338Ala details
Multiple TM6 residues contribute to anion binding, as determined by Au(CN)2 _ block of Cl_ permeation; the dramatic weakening of Au(CN)2 _ block in T338A suggests a particularly strong role of this residue in binding. Login to comment
147 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 11927667:147:60
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 11927667:147:53
status: NEW
view ABCC7 p.Thr338Ala details
ABCC7 p.Phe337Ser
X
ABCC7 p.Phe337Ser 11927667:147:46
status: NEW
view ABCC7 p.Phe337Ser details
Only mutations in the central portion of TM6 (F337S, T338A, S341A) affected both Au(CN)2 _ binding and Au(CN)2 _ permeability (Figs 3_5). Login to comment
154 ABCC7 p.Arg352Gln
X
ABCC7 p.Arg352Gln 11927667:154:285
status: NEW
view ABCC7 p.Arg352Gln details
ABCC7 p.Thr351Ala
X
ABCC7 p.Thr351Ala 11927667:154:202
status: NEW
view ABCC7 p.Thr351Ala details
Unfortunately, we were unable to test the role of TM6 residues more intracellular than I344 on anion binding; R347 was not studied because of its association with pore stability (Cotten & Welsh, 1999), T351A was not well expressed in BHK cells and the blocking effects of Au(CN)2 _ on R352Q most likely do not reflect open channel block. Login to comment
157 ABCC7 p.Ser341Ala
X
ABCC7 p.Ser341Ala 11927667:157:363
status: NEW
view ABCC7 p.Ser341Ala details
ABCC7 p.Thr338Ala
X
ABCC7 p.Thr338Ala 11927667:157:284
status: NEW
view ABCC7 p.Thr338Ala details
Nevertheless, there does not seem to be a strong correlation between these two aspects of pore function, such that they may be controlled independently by the same structural featuresofthepore.Thus,F337Sisassociatedwithweakened Au(CN)2 _ binding and decreased Au(CN)2 _ permeability, T338A with weakened Au(CN)2 _ binding and increased Au(CN)2 _ permeability and S341A with strengthened Au(CN)2 _ bindingandincreasedpermeability(Figs3and4). Login to comment