ABCMdb

PMID: 22014307

Liu X, Dawson DC
Cystic fibrosis transmembrane conductance regulator: temperature-dependent cysteine reactivity suggests different stable conformers of the conduction pathway.
Biochemistry. 2011 Nov 29;50(47):10311-7. Epub 2011 Nov 4., [PubMed]

Sentences

No. Mutations Sentence Comment

12 ABCC7 p.Phe337Cys Cheung and Akabas1,2 reported reactivity of F337C CFTR toward MTSEA+ , MTSET+ , and MTSES- . Login to comment 13 ABCC7 p.Phe337Cys Fatehi et al.5 reported reactivity of F337C CFTR toward MTSES- but not MTSET+ , while Beck et al.6 reported no reactivity toward MTSEA+ at this locus. Login to comment 48 ABCC7 p.Phe337Cys ■ RESULTS The representative experiments compiled in Figure 1 illustrate the dramatic effect of increased temperature on the rate of reaction of MTSES- with F337C CFTR. Login to comment 50 ABCC7 p.Phe337Cys Increased temperature dramatically increased the reactivity of F337C CFTR toward MTSES- . Login to comment 51 ABCC7 p.Phe337Cys Oocytes expressing F337C CFTR were activated using a stimulatory cocktail (10 μM Isop and 1 mM IBMX, hatched bar and cross hairs) and then exposed to 1 mM 2-ME to reverse any spurious reactions of the substituted cysteine.11 (A) Exposure to 1 mM MTSES- (dark gray bar and circles) produced no reaction, but 1 mM [Au(CN)2]- (black bar and circles) produced profound inhibition that was not reversed by 1 mM 2-ME but was reversed by 1 mM KCN (white bar and triangles). Login to comment 58 ABCC7 p.Phe337Cys In many such experiments conducted at room temperature, no change in conductance was detectable upon exposure of an oocyte expressing F337C CFTR to 1 mM MTSES- , even for periods exceeding 10 min. Login to comment 60 ABCC7 p.Phe337Cys In this instance, exposure of the oocyte expressing F337C CFTR to MTSES- at room temperature evoked what at first appeared to be a very slow rate of reaction, but the decline spontaneously reversed upon removal of the reagent from the superfusate. Login to comment 62 ABCC7 p.Phe337Cys The inhibition following the reaction of [Au(CN)2]- was not reversed by exposure to a competing thiol, 2-ME, but was readily reversed by exposure to the high-affinity metal ligand, CN- , as expected from previous studies.10,12 Figure 1B depicts an experiment in which an oocyte expressing F337C CFTR was exposed to 1 mM MTSES- at 22 °C, and the superfusate temperature was then increased to 32 °C in the presence of the mixed disulfide. Login to comment 67 ABCC7 p.Phe337Cys An oocyte expressing F337C CFTR was heated to 37 °C for 10 min and then cooled to 22 °C. Login to comment 71 ABCC7 p.Phe337Cys This result indicates that, although reaction of MTSES- with F337C CFTR was readily detectable at 37 °C, exposure to the elevated temperature did not result in any irreversible change in the channel. Login to comment 73 ABCC7 p.Ile336Cys Increased temperature altered the rate of modification of I336C CFTR by MTSES- . Login to comment 74 ABCC7 p.Ile336Cys Oocytes expressing I336C CFTR were activated using a stimulatory cocktail. Login to comment 81 ABCC7 p.Arg334Cys Increased temperature altered the rate of modification of R334C CFTR by MTSES- . Login to comment 82 ABCC7 p.Arg334Cys An oocyte expressing R334C CFTR was activated using a stimulatory cocktail and then (A) exposed to 1 mM 2-ME (white bar and circles) and 3 μM MTSES- (dark gray bar and circles) at room temperature. Login to comment 85 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys ABCC7 p.Ile336Cys Figures 2-4 contain the results of similar experiments conducted with oocytes expressing R334C, I336C, and T338C CFTR channels. Login to comment 86 ABCC7 p.Phe337Cys In the case of R334 and I336, exposure to MTSES- (3 μM and 1 mM, respectively) produced reactions at 22 °C that were slow but, unlike that of F337C CFTR, readily discernible at room temperature. Login to comment 88 ABCC7 p.Thr338Cys The reaction of MTSES- with T338C CFTR was the most rapid at 22 °C and was nevertheless increased at 37 °C. Login to comment 93 ABCC7 p.Thr338Cys ABCC7 p.Phe337Cys As expected, the apparent activation energies varied widely, ranging from being that expected for disulfide exchange15,16 for T338C CFTR to values in the range of those generally associated with protein conformational change.17-20 Figure 6 summarizes the inhibition by MTSES- of CFTR conductance at 22 °C (27 °C for F337C CFTR) and 37 °C in oocytes expressing CFTR constructs bearing substituted cysteines at positions extracellular to (334 and 336-338) and cytoplasmic to (339-342 and 344) the apparent accessibility cutoff defined by Alexander et al.10 It is apparent that, despite the dramatic increases in the reaction rates of cysteines extracellular to the cutoff, the position of the cutoff was unchanged at 37 °C. Login to comment 97 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys ABCC7 p.Ile336Cys ABCC7 p.Phe337Cys Temperature Dependence of MTSES- Modification kMTSES (M-1 s-1 ) mutant 22 °C 30 °C 32 °C 37 °C Ea (kJ/mol) R334C 2648 ± 259 (n = 3) 9411 ± 1210 (n = 5) 18407 ± 3240 (n = 3) 98 I336C 1.2a 2.3 ± 0.1 (n = 3) 6.9 ± 0.4 (n = 4) 88 F337C 2.6 ± 0.7 (27 °C)b (n = 3) 5.1 ± 1.2 (n = 3) 19.4 ± 4.4 (n = 4) 157 T338C 4067 ± 573 (n = 5) 7192 ± 370 (n = 4) 7972 ± 1019 (n = 6) 35 a Value from ref 10. b The reaction rate was undetectable at 22 °C, so the value determined at 27 °C was used. Login to comment 100 ABCC7 p.Thr338Cys Increased temperature increased the rate of reaction of T338C CFTR with MTSES- . Login to comment 101 ABCC7 p.Thr338Cys An oocyte expressing T338C CFTR was activated using a stimulatory cocktail and then (A) exposed to 100 μM DDTC to reverse spurious thiol reactions (white bar and circles). Login to comment 102 ABCC7 p.Thr338Cys Exposure to 5 μM MTSES- (dark gray bar and circles) profoundly inhibited the T338C CFTR conductance. Login to comment 106 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys ABCC7 p.Ile336Cys ABCC7 p.Phe337Cys Arrhenius plots for (A) R334C, (B) I336C, (C) F337C, and (D) T338C CFTR. Login to comment 124 ABCC7 p.Phe337Cys This finding does not, in and of itself, resolve the discrepancy between our data and those of Cheung and Akabas1,2 and Fatehi et al.,5 who reported reactivity of F337C CFTR toward externally applied MTSES- at room temperature, but the observation of a slow, but discernable reaction rate at 27 °C (Table 1) raises the possibility of some, as yet unidentified, condition of their experiments that allows the CFTR channel to access conformations at temperatures within the 22 °C-27 °C range in which the thiol-disulfide exchange reaction can occur at position 337. Login to comment 129 ABCC7 p.Phe337Cys The percent block of CFTR conductance by MTSES- was defined as the change in conductance induced by MTSES- , ΔgES, divided by the conductance at time zero of exposure, gt=0, at 22 °C (27 °C for F337C, white bars) and 37 °C (dark gray bars). Login to comment 131 ABCC7 p.Val345Cys ABCC7 p.Thr339Cys The values at 37 °C (gray bars) from T339C to V345C reflect small changes in conductance in response to the temperature pulse, rather than reactivity toward MTSES- . Login to comment