PMID: 19754156

Alexander C, Ivetac A, Liu X, Norimatsu Y, Serrano JR, Landstrom A, Sansom M, Dawson DC
Cystic fibrosis transmembrane conductance regulator: using differential reactivity toward channel-permeant and channel-impermeant thiol-reactive probes to test a molecular model for the pore.
Biochemistry. 2009 Oct 27;48(42):10078-88., 2009-10-27 [PubMed]
Sentences
No. Mutations Sentence Comment
42 ABCC7 p.Cys1344Ser
X
ABCC7 p.Cys1344Ser 19754156:42:112
status: NEW
view ABCC7 p.Cys1344Ser details
ABCC7 p.Cys524Ser
X
ABCC7 p.Cys524Ser 19754156:42:70
status: NEW
view ABCC7 p.Cys524Ser details
ABCC7 p.Cys276Ser
X
ABCC7 p.Cys276Ser 19754156:42:49
status: NEW
view ABCC7 p.Cys276Ser details
ABCC7 p.Cys491Ser
X
ABCC7 p.Cys491Ser 19754156:42:63
status: NEW
view ABCC7 p.Cys491Ser details
ABCC7 p.Cys225Ser
X
ABCC7 p.Cys225Ser 19754156:42:42
status: NEW
view ABCC7 p.Cys225Ser details
ABCC7 p.Cys1458Ser
X
ABCC7 p.Cys1458Ser 19754156:42:152
status: NEW
view ABCC7 p.Cys1458Ser details
ABCC7 p.Cys1410Ser
X
ABCC7 p.Cys1410Ser 19754156:42:144
status: NEW
view ABCC7 p.Cys1410Ser details
ABCC7 p.Cys866Ser
X
ABCC7 p.Cys866Ser 19754156:42:105
status: NEW
view ABCC7 p.Cys866Ser details
ABCC7 p.Cys832Ser
X
ABCC7 p.Cys832Ser 19754156:42:98
status: NEW
view ABCC7 p.Cys832Ser details
ABCC7 p.Cys343Ser
X
ABCC7 p.Cys343Ser 19754156:42:56
status: NEW
view ABCC7 p.Cys343Ser details
ABCC7 p.Cys76Ser
X
ABCC7 p.Cys76Ser 19754156:42:29
status: NEW
view ABCC7 p.Cys76Ser details
ABCC7 p.Cys1400Ser
X
ABCC7 p.Cys1400Ser 19754156:42:136
status: NEW
view ABCC7 p.Cys1400Ser details
ABCC7 p.Cys1395Ser
X
ABCC7 p.Cys1395Ser 19754156:42:128
status: NEW
view ABCC7 p.Cys1395Ser details
ABCC7 p.Cys1355Ser
X
ABCC7 p.Cys1355Ser 19754156:42:120
status: NEW
view ABCC7 p.Cys1355Ser details
ABCC7 p.Cys590Leu
X
ABCC7 p.Cys590Leu 19754156:42:77
status: NEW
view ABCC7 p.Cys590Leu details
ABCC7 p.Cys592Leu
X
ABCC7 p.Cys592Leu 19754156:42:84
status: NEW
view ABCC7 p.Cys592Leu details
The Cys-less CFTR construct (C76S, C126S, C225S, C276S, C343S, C491S, C524S, C590L, C592L, C657S, C832S, C866S, C1344S, C1355S, C1395S, C1400S, C1410S, C1458S) was a gift from Drs. Martin Mense and David Gadsby and was used in their pGEMHE vector previously described (13). Login to comment
52 ABCC7 p.Arg347Cys
X
ABCC7 p.Arg347Cys 19754156:52:792
status: NEW
view ABCC7 p.Arg347Cys details
ABCC7 p.Arg352Cys
X
ABCC7 p.Arg352Cys 19754156:52:892
status: NEW
view ABCC7 p.Arg352Cys details
ABCC7 p.Gln353Cys
X
ABCC7 p.Gln353Cys 19754156:52:916
status: NEW
view ABCC7 p.Gln353Cys details
ABCC7 p.Arg334Cys
X
ABCC7 p.Arg334Cys 19754156:52:359
status: NEW
view ABCC7 p.Arg334Cys details
ABCC7 p.Lys335Cys
X
ABCC7 p.Lys335Cys 19754156:52:419
status: NEW
view ABCC7 p.Lys335Cys details
ABCC7 p.Thr338Cys
X
ABCC7 p.Thr338Cys 19754156:52:557
status: NEW
view ABCC7 p.Thr338Cys details
ABCC7 p.Thr351Cys
X
ABCC7 p.Thr351Cys 19754156:52:878
status: NEW
view ABCC7 p.Thr351Cys details
ABCC7 p.Met348Cys
X
ABCC7 p.Met348Cys 19754156:52:806
status: NEW
view ABCC7 p.Met348Cys details
ABCC7 p.Leu333Cys
X
ABCC7 p.Leu333Cys 19754156:52:315
status: NEW
view ABCC7 p.Leu333Cys details
ABCC7 p.Ile331Cys
X
ABCC7 p.Ile331Cys 19754156:52:257
status: NEW
view ABCC7 p.Ile331Cys details
ABCC7 p.Phe342Cys
X
ABCC7 p.Phe342Cys 19754156:52:683
status: NEW
view ABCC7 p.Phe342Cys details
ABCC7 p.Val350Cys
X
ABCC7 p.Val350Cys 19754156:52:864
status: NEW
view ABCC7 p.Val350Cys details
ABCC7 p.Gly330Cys
X
ABCC7 p.Gly330Cys 19754156:52:243
status: NEW
view ABCC7 p.Gly330Cys details
ABCC7 p.Ser341Cys
X
ABCC7 p.Ser341Cys 19754156:52:659
status: NEW
view ABCC7 p.Ser341Cys details
ABCC7 p.Val345Cys
X
ABCC7 p.Val345Cys 19754156:52:754
status: NEW
view ABCC7 p.Val345Cys details
ABCC7 p.Ile332Cys
X
ABCC7 p.Ile332Cys 19754156:52:301
status: NEW
view ABCC7 p.Ile332Cys details
ABCC7 p.Ile336Cys
X
ABCC7 p.Ile336Cys 19754156:52:478
status: NEW
view ABCC7 p.Ile336Cys details
ABCC7 p.Ile340Cys
X
ABCC7 p.Ile340Cys 19754156:52:635
status: NEW
view ABCC7 p.Ile340Cys details
ABCC7 p.Ala349Cys
X
ABCC7 p.Ala349Cys 19754156:52:840
status: NEW
view ABCC7 p.Ala349Cys details
ABCC7 p.Ile344Cys
X
ABCC7 p.Ile344Cys 19754156:52:720
status: NEW
view ABCC7 p.Ile344Cys details
ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 19754156:52:523
status: NEW
view ABCC7 p.Phe337Cys details
ABCC7 p.Thr339Cys
X
ABCC7 p.Thr339Cys 19754156:52:611
status: NEW
view ABCC7 p.Thr339Cys details
ABCC7 p.Leu346Cys
X
ABCC7 p.Leu346Cys 19754156:52:778
status: NEW
view ABCC7 p.Leu346Cys details
We proposed that these spontaneous changes, that are not seen in either wt or Cys-less CFTR, reflect the coordination of trace Table 1: Percent Change in Oocyte Conductance in the Presence of Compounda MTSETþ MTSES- [Ag(CN)2]- [Au(CN)2]- G330C O O O O I331C -51.6 ( 6.3 -28.9 ( 2.1 -63.1 ( 8.8 O I332C O O O O L333C -58.5 ( 4.8 -47.5 ( 7.6 -83.1 ( 2.2 O R334C þ76.9 ( 11.3 -84.4 ( 1.5 -67.4 ( 7.4 -41.4 ( 3.1 K335C þ10.7 ( 2.4 -37.3 ( 1.5 -29.1 ( 6.4 -54.6 ( 4.7 I336C -54.4 ( 7.9 -75.0 ( 0.6 -81.2 ( 10.5 O F337C O O -89.6 ( 1.9 -90.1 ( 1.3 T338C -37.1 ( 3.3 -85.4 ( 2.5 -75.0 ( 5.2 -88.3 ( 1.6 T339C O O -24.5 ( 7.2 O I340C O O -93.8 ( 1.0 O S341C O O -49.3 ( 4.8 O F342C O O -84.7 ( 1.8 O C343 O O O O I344C O O -66.9 ( 9.3 -77.9 ( 2.1 V345C O O -49.1 ( 9.3 O L346C O O O O R347C O O O O M348C O O -47.9 ( 8.8 -50.1 ( 3.3 A349C O O -19.0 ( 2.0 O V350C O O O O T351C O O O O R352C O O -77.5 ( 1.3 O Q353C O O -72.6 ( 4.5 -76.7 ( 2.8 a Values are means ( SE of three or more oocytes. Login to comment
130 ABCC7 p.Leu333Cys
X
ABCC7 p.Leu333Cys 19754156:130:54
status: NEW
view ABCC7 p.Leu333Cys details
ABCC7 p.Leu333Cys
X
ABCC7 p.Leu333Cys 19754156:130:85
status: NEW
view ABCC7 p.Leu333Cys details
Figure 2 contains an example of results obtained with L333C CFTR on a wt background (L333C/wt). Login to comment
136 ABCC7 p.Leu333Cys
X
ABCC7 p.Leu333Cys 19754156:136:52
status: NEW
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FIGURE 2: Functional impact of covalent labeling of L333C CFTR was charge-independent. Login to comment
140 ABCC7 p.Leu333Cys
X
ABCC7 p.Leu333Cys 19754156:140:41
status: NEW
view ABCC7 p.Leu333Cys details
Reactions of MTSETþ and MTSES- with L333C/wt CFTR channels were reversed by exposure to DTT, but the rate of reversal was slower in the case of MTSES- -modified channels, as expected due to the unfavorable electrostatic interaction between the negatively charged adduct and the anionic DTT. Login to comment
147 ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 19754156:147:22
status: NEW
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Lack of Reactivity of F337C CFTR toward MTSETþ and MTSES- . Login to comment
149 ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 19754156:149:42
status: NEW
view ABCC7 p.Phe337Cys details
Beck et al. (9) reported no reactivity of F337C on a wt CFTR background (F337/wt CFTR) toward MTSEAþ or MTSES- , but Cheung and Akabas (5, 6) and Fatehi and Linsdell (10) reported reactivity toward both MTSETþ and MTSES- . Login to comment
150 ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 19754156:150:63
status: NEW
view ABCC7 p.Phe337Cys details
Accordingly, we examined the reactivity toward MTS reagents of F337C CFTR (wt and Cys-less backgrounds) carefully to ensure that any change in conductance observed in the presence of an MTS reagent met our criteria for a thiol-disulfide exchange reaction. Login to comment
151 ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 19754156:151:65
status: NEW
view ABCC7 p.Phe337Cys details
In some experiments weobserved that exposureofoocytes expressing F337C/wt CFTR to MTSETþ or MTSES- produced decreases in conductance.Someofthesechangeswerereversed by simplywashing off the compound while others persisted to a variable extent after washing. Login to comment
152 ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 19754156:152:151
status: NEW
view ABCC7 p.Phe337Cys details
We determined, however, that these variable effects of exposure to MTSET- or MTSES- were not due to thiol-disulfide exchange reactions.3 Reactivity of F337C CFTR toward Channel-Permeant Probes. Login to comment
153 ABCC7 p.Thr338Cys
X
ABCC7 p.Thr338Cys 19754156:153:114
status: NEW
view ABCC7 p.Thr338Cys details
ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 19754156:153:18
status: NEW
view ABCC7 p.Phe337Cys details
The reactivity of F337C/wt CFTR toward the channel-permeant probes, although similar to that seen previously with T338C/wt CFTR (12), differed significantly in detail. Login to comment
154 ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 19754156:154:31
status: NEW
view ABCC7 p.Phe337Cys details
Exposure of oocytes expressing F337C/wt CFTR to 1 mM [Au(CN)2]- produced a profound inhibition that was not reversed by superfusing the oocytes with a [Au(CN)2]- -free solution (Figure 3). Login to comment
155 ABCC7 p.Thr338Cys
X
ABCC7 p.Thr338Cys 19754156:155:174
status: NEW
view ABCC7 p.Thr338Cys details
ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 19754156:155:20
status: NEW
view ABCC7 p.Phe337Cys details
After inhibition of F337C conductance by [Au- (CN)2]- , exposure of oocytes to a competing thiol, 2-ME, did not reverse the inhibition of conductance as previously seen with T338C/wtCFTR(12), butthe inhibition wasrelievedbyexposing the oocyte to a solution containing 1 mM KCN as expected from the high-affinity liganding of Au(1) by the cyanide anion (12). Login to comment
156 ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 19754156:156:54
status: NEW
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Similar results were obtained with oocytes expressing F337C/ Cys-less CFTR, confirming that the cysteine at 337 is the site of the reaction with [Au(CN)2]- (Supporting Information Figure S3). Login to comment
157 ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 19754156:157:0
status: NEW
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F337C/Cys-less CFTR was also reactive toward the second, channel-permeant probe, [Ag(CN)2]- . Login to comment
165 ABCC7 p.Ser341Cys
X
ABCC7 p.Ser341Cys 19754156:165:109
status: NEW
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ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 19754156:165:121
status: NEW
view ABCC7 p.Phe337Cys details
That this could reflect the size and/or polarity of the latter reagents is suggested by the observation that S341C, like F337C CFTR, clearly reacts with a smaller MTS compound, MMTS (not shown). Login to comment
166 ABCC7 p.Ser341Cys
X
ABCC7 p.Ser341Cys 19754156:166:55
status: NEW
view ABCC7 p.Ser341Cys details
The results depicted in Figure 4 show that exposure of S341C/ Cys-less CFTR to 100 μM [Ag(CN)2]- produced about 70% inhibition of conductance (gCl), substantially greater than that seen in wt or Cys-less CFTR. Login to comment
167 ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 19754156:167:65
status: NEW
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The inhibition was readily and FIGURE 3: Selective reactivity of F337C CFTR. Login to comment
176 ABCC7 p.Ser341Cys
X
ABCC7 p.Ser341Cys 19754156:176:25
status: NEW
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In contrast, exposure of S341C CFTR to [Au(CN)2]- produced inhibition that was similar to that seen in the wt or Cys-less CFTR and was not altered by KCN, indicating the absence of a ligand exchange reaction. Login to comment
180 ABCC7 p.Arg352Cys
X
ABCC7 p.Arg352Cys 19754156:180:32
status: NEW
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We had previously reported that R352C/wt CFTR was not reactive toward MTSETþ and MTSES- (7), a result consistent with the findings of Beck et al. (9). Login to comment
181 ABCC7 p.Arg352Cys
X
ABCC7 p.Arg352Cys 19754156:181:32
status: NEW
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We also reported, however, that R352C/wt CFTR displayed an unusual, spontaneously reversible reaction with MTSEAþ (7). Login to comment
182 ABCC7 p.Arg352Gln
X
ABCC7 p.Arg352Gln 19754156:182:124
status: NEW
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ABCC7 p.Arg352His
X
ABCC7 p.Arg352His 19754156:182:136
status: NEW
view ABCC7 p.Arg352His details
ABCC7 p.Arg352Cys
X
ABCC7 p.Arg352Cys 19754156:182:248
status: NEW
view ABCC7 p.Arg352Cys details
In those experiments, however, qualitatively identical, reversible reactivity toward MTSEAþ was also seen using either R352Q/wt or R352H/wt CFTR constructs, suggesting that the target of MTSEAþ was 1 of the 18 endogenous cysteines in the R352C/wt protein (7). Login to comment
184 ABCC7 p.Arg352Cys
X
ABCC7 p.Arg352Cys 19754156:184:54
status: NEW
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The experiment depicted in Figure 5 demonstrates that R352C/Cys-less CFTR exhibited reactivity toward [Ag(CN)2]- . Login to comment
185 ABCC7 p.Arg352Cys
X
ABCC7 p.Arg352Cys 19754156:185:64
status: NEW
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Reactivity toward [Au(CN)2]- was not seen, and experiments with R352C/ wt CFTR yielded identical results (not shown). Login to comment
186 ABCC7 p.Arg352Cys
X
ABCC7 p.Arg352Cys 19754156:186:31
status: NEW
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ABCC7 p.Arg352Cys
X
ABCC7 p.Arg352Cys 19754156:186:44
status: NEW
view ABCC7 p.Arg352Cys details
We also confirmed that neither R352C/wt nor R352C/Cys-less exhibits reactivity toward either MTSETþ or MTSES- (not shown), contrary to the reports of Chueng and Akabas (5, 6). Login to comment
209 ABCC7 p.Ser341Cys
X
ABCC7 p.Ser341Cys 19754156:209:78
status: NEW
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Position 332, predicted to reside in the FIGURE 4: Differential reactivity of S341C/Cys-less CFTR toward [Ag(CN)2]- and [Au(CN)2]- . Login to comment
212 ABCC7 p.Arg352Cys
X
ABCC7 p.Arg352Cys 19754156:212:10
status: NEW
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FIGURE 5: R352C/Cys-less CFTR was reactive toward [Ag(CN)2]- (1 mM) as judged by reversal of inhibition by adding KCN (1 mM) in the continued presence of [Ag(CN)2]- . Login to comment
241 ABCC7 p.Arg352Cys
X
ABCC7 p.Arg352Cys 19754156:241:0
status: NEW
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R352C/wt CFTR is not reactive toward MTSETþ and MTSES- , but St. Aubin and Linsdell (40), Cui et al. (41), and Jordan et al. (3) identified R352 as critical for the maintenance of normal anion conduction and gating in CFTR channels. Login to comment
271 ABCC7 p.Leu333Cys
X
ABCC7 p.Leu333Cys 19754156:271:55
status: NEW
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ABCC7 p.Ile331Cys
X
ABCC7 p.Ile331Cys 19754156:271:46
status: NEW
view ABCC7 p.Ile331Cys details
Beck et al. (9) reported that modification of I331C or L333C CFTR channels results in a profound reduction in open probability, and the authors suggested that these sites may experience significant movement during the gating cycle. Login to comment
281 ABCC7 p.Arg347Cys
X
ABCC7 p.Arg347Cys 19754156:281:70
status: NEW
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ABCC7 p.Arg352Cys
X
ABCC7 p.Arg352Cys 19754156:281:84
status: NEW
view ABCC7 p.Arg352Cys details
ABCC7 p.Gln353Cys
X
ABCC7 p.Gln353Cys 19754156:281:95
status: NEW
view ABCC7 p.Gln353Cys details
ABCC7 p.Thr351Cys
X
ABCC7 p.Thr351Cys 19754156:281:77
status: NEW
view ABCC7 p.Thr351Cys details
ABCC7 p.Ser341Cys
X
ABCC7 p.Ser341Cys 19754156:281:56
status: NEW
view ABCC7 p.Ser341Cys details
ABCC7 p.Ile344Cys
X
ABCC7 p.Ile344Cys 19754156:281:63
status: NEW
view ABCC7 p.Ile344Cys details
ABCC7 p.Phe337Cys
X
ABCC7 p.Phe337Cys 19754156:281:49
status: NEW
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Note the lack of consistent results reported for F337C, S341C, I344C, R347C, T351C, R352C, and Q353C (shaded). Login to comment
299 ABCC7 p.Arg352Cys
X
ABCC7 p.Arg352Cys 19754156:299:18
status: NEW
view ABCC7 p.Arg352Cys details
The reactivity of R352C CFTR toward the channel-permeant probe, [Ag(CN)2]- , clearly implicates R352 as a "pore-lining" residue, as suggested by the effect of amino acid substitutions at this position on single-channel conductance, envisioned by St. Aubin and Linsdell (40) as contributing a positive electrostatic potential to a cytoplasmic vestibule for the pore. Login to comment
306 ABCC7 p.Asp993Arg
X
ABCC7 p.Asp993Arg 19754156:306:93
status: NEW
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Conversely, Cui et al. (41) reported that substitution of a positive charge at position 993 (D993R) increased single-channel conductance for inward current above that of wt CFTR. Login to comment