ABCMdb

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 ABCC7 p.Cys524Ser ABCC7 p.Cys276Ser ABCC7 p.Cys491Ser ABCC7 p.Cys225Ser ABCC7 p.Cys1458Ser ABCC7 p.Cys1410Ser ABCC7 p.Cys866Ser ABCC7 p.Cys832Ser ABCC7 p.Cys343Ser ABCC7 p.Cys76Ser ABCC7 p.Cys1400Ser ABCC7 p.Cys1395Ser ABCC7 p.Cys1355Ser ABCC7 p.Cys590Leu ABCC7 p.Cys592Leu 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 ABCC7 p.Arg352Cys ABCC7 p.Gln353Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Thr351Cys ABCC7 p.Met348Cys ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys ABCC7 p.Phe342Cys ABCC7 p.Val350Cys ABCC7 p.Gly330Cys ABCC7 p.Ser341Cys ABCC7 p.Val345Cys ABCC7 p.Ile332Cys ABCC7 p.Ile336Cys ABCC7 p.Ile340Cys ABCC7 p.Ala349Cys ABCC7 p.Ile344Cys ABCC7 p.Phe337Cys ABCC7 p.Thr339Cys ABCC7 p.Leu346Cys 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 ABCC7 p.Leu333Cys Figure 2 contains an example of results obtained with L333C CFTR on a wt background (L333C/wt). Login to comment 136 ABCC7 p.Leu333Cys FIGURE 2: Functional impact of covalent labeling of L333C CFTR was charge-independent. Login to comment 140 ABCC7 p.Leu333Cys 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 Lack of Reactivity of F337C CFTR toward MTSETþ and MTSES- . Login to comment 149 ABCC7 p.Phe337Cys 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 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 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 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 ABCC7 p.Phe337Cys 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 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 ABCC7 p.Phe337Cys 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 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 F337C/Cys-less CFTR was also reactive toward the second, channel-permeant probe, [Ag(CN)2]- . Login to comment 165 ABCC7 p.Ser341Cys ABCC7 p.Phe337Cys 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 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 The inhibition was readily and FIGURE 3: Selective reactivity of F337C CFTR. Login to comment 176 ABCC7 p.Ser341Cys 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 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 We also reported, however, that R352C/wt CFTR displayed an unusual, spontaneously reversible reaction with MTSEAþ (7). Login to comment 182 ABCC7 p.Arg352Gln ABCC7 p.Arg352His ABCC7 p.Arg352Cys 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 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 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 ABCC7 p.Arg352Cys 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 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 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 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 ABCC7 p.Ile331Cys 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 ABCC7 p.Arg352Cys ABCC7 p.Gln353Cys ABCC7 p.Thr351Cys ABCC7 p.Ser341Cys ABCC7 p.Ile344Cys ABCC7 p.Phe337Cys Note the lack of consistent results reported for F337C, S341C, I344C, R347C, T351C, R352C, and Q353C (shaded). Login to comment 299 ABCC7 p.Arg352Cys 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 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