ABCMdb

ABCC7 p.Phe337Cys

None has been submitted yet.

Publications

PMID: 18056267 [PubMed] Beck EJ et al: "Conformational changes in a pore-lining helix coupled to cystic fibrosis transmembrane conductance regulator channel gating."

No. Sentence Comment

100 The oocytes 750 500 250 0 µS 180012006000 s IBMX MTSEA Cd 2+ DTT 200 100 0 µS 180012006000 s IBMX DTT Cd 2+ MTSEA A B C -100 -80 -60 -40 -20 0 20 40 % Change in conductance Y325C A326C L327C I328C K329C G330C I331C I332C L333C R334C K335C I336C F337C T338C T339C I340C S341C F342C WT I344C V345C R347C M348C A349C V350C T351C Q353C * * * * * Cd 2+ 1mM MTSEA 1mM D FIGURE 1. Login to comment
218 Finally, the MTSEA reactivity was restricted to only five of twenty-six residues in and flanking TM6 in our study, whereas in the earlier study, residues F337C, S341C, I344C, R347C, T351C, R352C, and Q353C were also shown to be accessible to MTS reagents. Login to comment

PMID: 18167343 [PubMed] Fatehi M et al: "State-dependent access of anions to the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

75 Another mutant, F337C, became significantly more inwardly rectified in the presence of MTSES but was apparently not affected by inclusion of MTSET in the pipette solution (Fig. 3). Login to comment
90 Conformational Change in the Pore on Activation of CFTR 6104 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 10•MARCH , shape when included in the pipette solution or when preincubated with cells (or, in the case of F337C, the same lack of effect) (Fig. 4A). Login to comment
114 F, wild type (both panels); E, R334C (left); Ⅺ, K335C (left); ‚, F337C (right); ƒ, T338C (right); छ, S341C (right) (mean of data from 3-9 patches). Login to comment
115 All MTS treatments led to significant changes in rectification ratio (p Ͻ 0.05) except MTSET-wild type, MTSES-wild type, and MTSET-F337C. Login to comment
142 In contrast, F337C was only very weakly inhibited by Au(CN)2 - either with or without cAMP prestimulation. Login to comment
143 Alteration of I-V shape in F337C by MTSES (Figs. Login to comment
147 Whatever the explanation, this lack of effect appears to be specific for F337C because the four other reactive cysteine mutants studied could be modified functionally by all reagents tested. Login to comment
189 E, the mean change in CFTR macroscopic conductance for R334C, K335C, F337C, and S341C following addition of KCN without (white bars) or with (black bars) cAMP pretreatment is shown (mean of data from 4-5 patches). Login to comment

PMID: 19754156 [PubMed] Alexander C et al: "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."

No. Sentence Comment

52 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
147 Lack of Reactivity of F337C CFTR toward MTSETþ and MTSES- . Login to comment
149 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 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 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 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 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 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 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 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 F337C/Cys-less CFTR was also reactive toward the second, channel-permeant probe, [Ag(CN)2]- . Login to comment
165 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
167 The inhibition was readily and FIGURE 3: Selective reactivity of F337C CFTR. Login to comment
281 Note the lack of consistent results reported for F337C, S341C, I344C, R347C, T351C, R352C, and Q353C (shaded). Login to comment

PMID: 20805575 [PubMed] Bai Y et al: "Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation."

No. Sentence Comment

82 7 out of the 25 mutant channels exhibited a reduced single-channel current amplitude, including, from extracellular to intracellular, R334C, K335C, F337C, T338C, S341C, R347C, and R352C (Fig. 2). Login to comment
95 Single-channel amplitude: cysless/WT,0.46±0.005pA(n=5); cysless/K355C, 0.28 ± 0.011 pA (n = 4); cysless/F337C, 0.19 ± 0.008 pA (n = 3). Login to comment

PMID: 22352759 [PubMed] Norimatsu Y et al: "Cystic fibrosis transmembrane conductance regulator: a molecular model defines the architecture of the anion conduction path and locates a "bottleneck" in the pore."

No. Sentence Comment

218 The dagger denotes that F337C was previously reported by our group to be unreactive toward MTSET+ and MTSES- . Login to comment
338 Recently, however, we used temperature increases from 22 to 37 °C to effect substantial changes in the conformation of the outer vestibule of the CFTR pore that were evident from markedly increased rates of reaction of MTSES- with cysteines substituted at positions 334 and 336-338 at 37 °C.62 In the case of F337C CFTR, a cysteine that was unreactive toward externally applied MTSES- at 22 °C was highly reactive at 37 °C. Despite these substantial changes in conformation, however, the position of the size selectivity barrier was unchanged. Login to comment

PMID: 22923500 [PubMed] Norimatsu Y et al: "Locating a Plausible Binding Site for an Open Channel Blocker, GlyH-101, in the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator."

No. Sentence Comment

108 In this example, externally-applied [Au(CN)2]- was reacted with F337C CFTR. Login to comment
115 Figure 3C and 3D contain the time courses for the reactions of [Au(CN)2]- with F337C and T338C CFTR pre-and post-activation. Login to comment
141 Figure 5C and 5D summarize the inhibition of F337C CFTR and T338C CFTR by [Au(CN)2]- in the presence and in the absence of GlyH-101. Login to comment
144 In Figure 5E and 5F the measured second order rate constants for covalent modification of F337C and T338C CFTR are plotted versus GlyH-101 concentration. Login to comment
151 We also studied the reaction of I1131C CFTR with MTSES- . Login to comment
159 The presence of negative charges near I1131 is consistent with the observed slow reaction of I1131C CFTR with MTSES- (20 M-1 sec-1 ; Figure 7) which is more than 100-fold less than that seen for a Cys at 338 (3.3 x 103 M-1 sec-1 ; Figure 6). Login to comment
160 As reported previously (Norimatsu et al., 2012) the macroscopic conductance of I1131C CFTR was increased by depositing the negatively charged sulfonic acid group via reaction with MTSES- . Login to comment
209 Second, alkylation of T338C CFTR with IAM, which results in covalent addition of an acetamide moiety predicted by the model to create a steric clash with GlyH-101, significantly reduced the apparent binding affinity of GlyH-101. Login to comment
210 In contrast, alkylation of F337C CFTR with IAM is not predicted by the molecular model to cause a steric clash and does not markedly alter GlyH-101 block. Login to comment
220 The state-dependent reactivity of T338C CFTR observed in the current study is consistent with the finding of Beck et al., (2008) that MTSES- reacts slightly faster with a high open probability mutant T338C/E1371Q CFTR than with T338C/wt CFTR.2 Mornon et al., (2009) created a homology model of CFTR based on the inward-facing conformation of a prokaryotic transporter, MsbA (Ward et al., 2007) (PDB code: 3B5X). Login to comment
224 A conformational change of this sort would be consistent with the state-dependent reactivity of F337C and T338C CFTR observed in the current study. Login to comment
225 The MsbA-based model of Mornon et al., (2009) also predicts that the side chain of R334 protrudes into the external aqueous environment, and when R334 is mutated to a cysteine in the MsbA-based model of Mornon et al., (2009) using Maestro (version 9.1, Schrödinger LLC), the reactive thiolate is clearly accessible from the extracellular solution (Figure 9C), consistent with the closed state reactivity of R334C observed in the current study and by Zhang et al., (2005). Login to comment
226 On the other hand, the mechanism that renders R334C CFTR unreactive in the conducting state of CFTR is not clear. Login to comment
158 Figure 3, C and D, contains the time courses for the reactions of [Au(CN)2]afa; with the F337C and T338C CFTRs before TABLE 1 EC50 at 0 mV (mean afe; S.E.M.) for GlyH-101 for wt and mutant CFTRs, with and without modification with iodoacetamide CFTR EC50 at 0 mV òe;M wt 1.1 afe; 0.11 (n afd; 4) K95C 1.4 afe; 0.35 (n afd; 4) F337C 1.8 afe; 0.06 (n afd; 3) F337C af9; iodoacetamide 2.4 afe; 0.29 (n afd; 3) T338C 3.7 afe; 0.27 (n afd; 3) T338C af9; iodoacetamide 24 afe; 2.6 (n afd; 3) Fig. 3. Login to comment
162 The subsequent application of [Au(CN)2]afa; almost completely abolished F337C CFTR conductance. Login to comment
163 C and D, time courses of the decreases in normalized conductance as a result of F337C (C) and T338C (D) modifications with [Au(CN)2]afa; . Login to comment
165 For the F337C CFTR, the abscissa represents cumulative [Au(CN)2]afa; exposure time. Login to comment
167 The reaction rate for the F337C CFTR before activation of the channels was b03;20 times slower than the rate after activation. Login to comment
171 The second-order reaction rate constants for the F337C CFTR before and after activation were 1.5 and 26 Mafa;1 safa;1 , respectively. Login to comment
206 Figure 5, C and D, summarizes the inhibition of the F337C and T338C CFTRs by [Au(CN)2]afa; in the presence and absence of GlyH-101. Login to comment
215 C and D, F337C (C) and T338C (D) CFTR channels were protected by 10 òe;M GlyH-101 from reactions with [Au(CN)2]afa; . Login to comment
217 The F337C CFTR was reacted with 600 òe;M [Au(CN)2]afa; and the T338C CFTR was reacted with 5 òe;M [Au(CN)2]afa; in the presence and absence of 10 òe;M GlyH-101. Login to comment
318 In contrast, alkylation of the F337C CFTR with iodoacetamide was not predicted by the molecular model to cause a steric clash and did not alter GlyH-101 blockade markedly. Login to comment
350 A conformational change of this sort would be consistent with the state-dependent reactivity of the F337C and T338C CFTRs observed in the current study. Login to comment

PMID: 22160394 [PubMed] Cui G et al: "Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers."

No. Sentence Comment

183 F337C-and F337E-CFTR exhibited significantly altered reversal potential, relative permeability, and relative conductance compared to WT-CFTR (Supplementary Tables 1, 2, 3), as did F337A-, S-, Y-, and L-CFTR [24]. Login to comment

PMID: 22014307 [PubMed] Liu X et al: "Cystic fibrosis transmembrane conductance regulator: temperature-dependent cysteine reactivity suggests different stable conformers of the conduction pathway."

No. Sentence Comment

12 Cheung and Akabas1,2 reported reactivity of F337C CFTR toward MTSEA+ , MTSET+ , and MTSES- . Login to comment
13 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 ■ 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 Increased temperature dramatically increased the reactivity of F337C CFTR toward MTSES- . Login to comment
51 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 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 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 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 An oocyte expressing F337C CFTR was heated to 37 °C for 10 min and then cooled to 22 °C. Login to comment
71 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
86 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
93 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 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
106 Arrhenius plots for (A) R334C, (B) I336C, (C) F337C, and (D) T338C CFTR. Login to comment
124 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 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

PMID: 21746847 [PubMed] Wang W et al: "Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

214 In open CFTR channels, internally applied MTS reagents can penetrate far enough into the pore as to modify L102C in TM1 and F337C in TM6. Login to comment
227 Second, whereas cysteines substituted for TM6 residues in the putative narrow pore region-F337C, T338C, and S341C-could be modified by both intracellular and extracellular MTS reagents (El Hiani and Linsdell, 2010), no residues that could be modified from both sides of the membrane were identified in TM1. Login to comment

PMID: 9089437 [PubMed] Cheung M et al: "Locating the anion-selectivity filter of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel."

No. Sentence Comment

107 We did not measure the reaction rate constants for the most extracellular residue, I331C, because we thought that it was unlikely that the reaction rates would be voltage dependent given the absence of voltage dependence at the adjacent, more cytoplasmic residues. We also did not measure the reaction rate constants for the mutants I344C and R347C because, although MTSEAϩ reacted with these residues, MTSES- and MTSETϩ did not react with these k ψ( )( )ln k Ψ 0=( )( ) zFδ RT/( )-ln ψ= t a b l e i Second-order Rate Constants for the Reaction of the MTS Reagents with the Water-exposed Cysteine Mutants k ES (M-1s-1) k EA (M-1s-1) k ET (M-1s-1) mutant -25 mV -50 mV -75 mV -25 mV -50 mV -75 mV -25 mV -50 mV -75 mV L333C 71 Ϯ 3(3) 71 Ϯ 20(2) 71 Ϯ 23(3) 320 Ϯ 89(2) 320 Ϯ 128(2) 333 Ϯ 139(3) 952 Ϯ 136(2) 1,000 Ϯ 350(2) 1,053 Ϯ 443(2) R334C 48 Ϯ 14(2) 48 Ϯ 6(3) 44 Ϯ 8(4) 145 Ϯ 32(2) 163 Ϯ 7(2) 182 Ϯ 21(3) 444 Ϯ 49(2) 454 Ϯ 124(2) 588 Ϯ 95(3) K335C 36 Ϯ 20(3) 23 Ϯ 11(3) 27 Ϯ 16(3) 222 Ϯ 80(3) 121 Ϯ 51(4) 107 Ϯ 30(3) 217 Ϯ 111(3) 235 Ϯ 28(3) 217 Ϯ 95(4) F337C 91 Ϯ 17(2) 80 Ϯ 22(3) 71 Ϯ 20(4) 222 Ϯ 74(2) 222 Ϯ 86(3) 285 Ϯ 81(3) 740 Ϯ 246(3) 740 Ϯ 82(2) 714 Ϯ 51(2) S341C 56 Ϯ 18(3) 56 Ϯ 40(2) 43 Ϯ 12(3) 93 Ϯ 6(3) 110 Ϯ 22(3) 138 Ϯ 34(3) 690 Ϯ 356(3) 556 Ϯ 246(3) 800 Ϯ 224(4) T351C 100 Ϯ 25(5) 57 Ϯ 6(3) 26 Ϯ 9(6) 146 Ϯ 30(4) 195 Ϯ 42(4) 296 Ϯ 18(3) 308 Ϯ 47(10) 392 Ϯ 78(6) 769 Ϯ 89(5) R352C 42 Ϯ 4(3) 26 Ϯ 4(5) 21 Ϯ 6(4) 105 Ϯ 76(3) 137 Ϯ 46(3) 205 Ϯ 58(2) 417 Ϯ 138(4) 800 Ϯ 128(2) 952 Ϯ 408(2) Q353C 125 Ϯ 23(4) 51 Ϯ 12(4) 42 Ϯ 8(4) 83 Ϯ 24(4) 116 Ϯ 42(4) 160 Ϯ 92(3) 189 Ϯ 48(6) 220 Ϯ 48(3) 625 Ϯ 273(4) residues and therefore we could not determine the charge selectivity at these positions.2 The reaction rate constants that we have measured are between 10-and 500-fold slower than the rates of reaction with sulfhydryls in free solution (Table II) (Stauffer and Karlin, 1994). Login to comment

PMID: 8744306 [PubMed] Cheung M et al: "Identification of cystic fibrosis transmembrane conductance regulator channel-lining residues in and flanking the M6 membrane-spanning segment."

No. Sentence Comment

91 Effects of MTS reagents on wild-type cysteines RESULTS in CFTR To identify the residues in and flanking the M6 membrane-spanning segment that are on the water-exposed surface of As reported previously (Akabas et al., 1994b), extracellular applications of the MTS reagents to Xenopus oocytes ex- L2j K329C L. _J *G330C 1331C 1332C L333C R334C K335C 1336C F337C T338C T339C 1340C S341C T342C C343,WT 1344C V345C L346C R347C M348C A349C V350C T351C R352C Q353C 0 2000 4000 6000 8000 0 25 50 PEAK CURRENTS (nA) TIME TO REACH PLATEAU (min) FIGURE 2 Peak CFTR-induced currents and time to reach the plateau current after stimulation with cAMP-activating reagents for 24 cysteine-substitution mutants and wild-type CFTR. Login to comment
109 Accessibility of substituted cysteines to MTSES- A 1-min application of 10 mM MTSES- significantly inhibited the CFIR-induced currents of 9 of the 24 cysteine-substituted mutants (Fig. 4 A), L333C, R334C, K335C, F337C, S341C, R347C, T351C, R352C, and Q353C. Login to comment
90 Effects of MTS reagents on wild-type cysteines RESULTS in CFTR To identify the residues in and flanking the M6 membrane-spanning segment that are on the water-exposed surface of As reported previously (Akabas et al., 1994b), extracellular applications of the MTS reagents to Xenopus oocytes ex- L2j K329C L. _J *G330C 1331C 1332C L333C R334C K335C 1336C F337C T338C T339C 1340C S341C T342C C343,WT 1344C V345C L346C R347C M348C A349C V350C T351C R352C Q353C 0 2000 4000 6000 8000 0 25 50 PEAK CURRENTS (nA) TIME TO REACH PLATEAU (min) FIGURE 2 Peak CFTR-induced currents and time to reach the plateau current after stimulation with cAMP-activating reagents for 24 cysteine-substitution mutants and wild-type CFTR. Login to comment
108 Accessibility of substituted cysteines to MTSES- A 1-min application of 10 mM MTSES- significantly inhibited the CFIR-induced currents of 9 of the 24 cysteine-substituted mutants (Fig. 4 A), L333C, R334C, K335C, F337C, S341C, R347C, T351C, R352C, and Q353C. Login to comment

PMID: 26606940 [PubMed] Wei S et al: "Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels."

No. Sentence Comment

70 Primer sequences for cloning and site-directed mutagenesis Ycf1p Forward cloning primer: CAACACAGGCATGTATATTA- AGAGC Reverse cloning primer: TTAAACTTATGGCGTCAGAG- TTGCC F565A: CATTGACTACTGACTTAGTTGCCCCTGCTTTG- ACTCTGTTC F565S: CATTGACTACTGACTTAGTTTCCCCTGCTTTGA- CTCTGTTC F565L: CATTGACTACTGACTTAGTTTTACCTGCTTTG- ACTCTGTTC G756D: AAGACAAACGAGCTTTTTGATCTCCAGATAAG- GAGATCCC D777N: ACAGCTGGCAAAGGATCATTAAGTAAATAAG- TGTCAGCTC Y1281G: GATCAAGCTCCGGCCTACCACGAGTGGAATA- ATTATTAAAC Yor1p Forward cloning primer: CTAATTGTACATCCGGTTTT- AACC Reverse cloning primer: TTGAGTCATTGCCCTTAA- AATGG F468S: AGGCAACCTGGTAATATTTCTGCCTCTTTATC- TTTATTTC F468A: AGGCAACCTGGTAATATTGCTGCCTCTTTATC- TTTATTTC F468L: AGGCAACCTGGTAATATTCTTGCCTCTTTATC- TTTATTTC G713D: GTGGTATTACTTTATCTGGTGATCAAAAGGCA- CGTATCAATTT Y1222G: ATAGGTAAACCAGGTCTACCGGCAAAATCAA- CATTTTCAA CFTR Forward cloning primer: GAAGAAGCAATGGAAAAA- ATGATTG Reverse cloning primer: TCGGTGAATGTTCTGACCT- TGG F337S: TCATCCTCCGGAAAATATCCACCACCATCTCA- TTCTGC F337A: TCATCCTCCGGAAAATAGCCACCACCATCTCA- TTCTGC F337L: TCATCCTCCGGAAAATATTAACCACCATCTCA- TTCTGC F337C: TCATCCTCCGGAAAATATGCACCACCATCTC- ATTCTGC Immunoblot analysis of CFTR protein expression Expression of the CFTR F337 mutants was verified by immunoblotting as described elsewhere (15). Login to comment
152 Mean percent ATP-free currents 6 SEMs were as follows: WT (0.5 6 0.2%; n = 5); F337L (0.6 6 0.3%; n = 5); F337C (2.5 6 1.4%; n = 5), F337A (9.6 6 1.4%; n = 5), and F337S (15.8 6 4.5%; n = 10). Login to comment
69 Primer sequences for cloning and site-directed mutagenesis Ycf1p Forward cloning primer: CAACACAGGCATGTATATTA- AGAGC Reverse cloning primer: TTAAACTTATGGCGTCAGAG- TTGCC F565A: CATTGACTACTGACTTAGTTGCCCCTGCTTTG- ACTCTGTTC F565S: CATTGACTACTGACTTAGTTTCCCCTGCTTTGA- CTCTGTTC F565L: CATTGACTACTGACTTAGTTTTACCTGCTTTG- ACTCTGTTC G756D: AAGACAAACGAGCTTTTTGATCTCCAGATAAG- GAGATCCC D777N: ACAGCTGGCAAAGGATCATTAAGTAAATAAG- TGTCAGCTC Y1281G: GATCAAGCTCCGGCCTACCACGAGTGGAATA- ATTATTAAAC Yor1p Forward cloning primer: CTAATTGTACATCCGGTTTT- AACC Reverse cloning primer: TTGAGTCATTGCCCTTAA- AATGG F468S: AGGCAACCTGGTAATATTTCTGCCTCTTTATC- TTTATTTC F468A: AGGCAACCTGGTAATATTGCTGCCTCTTTATC- TTTATTTC F468L: AGGCAACCTGGTAATATTCTTGCCTCTTTATC- TTTATTTC G713D: GTGGTATTACTTTATCTGGTGATCAAAAGGCA- CGTATCAATTT Y1222G: ATAGGTAAACCAGGTCTACCGGCAAAATCAA- CATTTTCAA CFTR Forward cloning primer: GAAGAAGCAATGGAAAAA- ATGATTG Reverse cloning primer: TCGGTGAATGTTCTGACCT- TGG F337S: TCATCCTCCGGAAAATATCCACCACCATCTCA- TTCTGC F337A: TCATCCTCCGGAAAATAGCCACCACCATCTCA- TTCTGC F337L: TCATCCTCCGGAAAATATTAACCACCATCTCA- TTCTGC F337C: TCATCCTCCGGAAAATATGCACCACCATCTC- ATTCTGC Immunoblot analysis of CFTR protein expression Expression of the CFTR F337 mutants was verified by immunoblotting as described elsewhere (15). Login to comment
151 Mean percent ATP-free currents 6 SEMs were as follows: WT (0.5 6 0.2%; n = 5); F337L (0.6 6 0.3%; n = 5); F337C (2.5 6 1.4%; n = 5), F337A (9.6 6 1.4%; n = 5), and F337S (15.8 6 4.5%; n = 10). Login to comment

PMID: 25675504 [PubMed] Gao X et al: "Localizing a gate in CFTR."

No. Sentence Comment

78 State-Dependent Reactivity of T338C, F337C, and R334C Implicates the Location of a Gate for CFTR. Login to comment
88 Similar observations were made for F337C-CFTR and R334C-CFTR (Fig. S4 A-D). Login to comment
96 Our previous studies demonstrated that a disease-associated mutation G1349D could decrease the Po of CFTR by ~10-fold (34) without affecting trafficking of the channel (34, 35); we thus engineered this mutation into R334C, K335C, F337C, and T338C backgrounds. Login to comment
192 [Au(CN)2]- , forskolin, with G1349D, /M/s Outside R334C 189 &#b1; 39 - 403 &#b1; 20 537 &#b1; 56 K335C - - 56 &#b1; 9 1,809 &#b1; 201 F337C 437 &#b1; 49 - 20 &#b1; 3 32 &#b1; 6 T338C 752 &#b1; 59 - 1,135 &#b1; 166 118 &#b1; 18 Inside I344C 32 &#b1; 5 37 &#b1; 4 - - N1148C 437 &#b1; 66 2,089 &#b1; 130 - - Residues located extracellularly (extra.) Login to comment