ABCMdb

ABCC7 p.Met348Cys

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PMID: 15272010 [PubMed] Chen EY et al: "The DeltaF508 mutation disrupts packing of the transmembrane segments of the cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

56 TM6 point mutations (M348C, T351C, and W356C) were generated in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; TM12 point mutations (T1142C and W1145C) were generated in the EcoRV (bp 2996) 3 EcoRI (bp 3643) fragment; the ⌬F508 mutation was generated in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment. Login to comment
146 Three positive cross-linking mutants, M348C/T1142C, T351C/T1142C, and W356C/W1145C were identified (see Fig. 3B, band X) and selected for further study. Login to comment
148 Fig. 2B shows the expression of WT CFTR, the single cysteine mutants M348C, T351C, W356C, T1142C, and W1145C, and the double cysteine mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C. Login to comment
150 The cross-linking patterns of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C showed differences when treated with different cross-linkers. Login to comment
151 Mutant M348C/T1142C, for example, showed cross-linking with M5M and M8M but not with M17M. Login to comment
153 It is interesting to note that both M348C and T351C in TM6 showed cross-linking to T1142C in TM12. Login to comment
159 Because the cross-linkable mutants M348C/T1142C, T351C/ T1142C, and W356C/W1145C also contained the 18 endogenous cysteines, it was important to test whether any of the single M348C, T351C, W356C, T1142C, or W1145C mutants showed evidence of cross-linking with endogenous cysteines. Login to comment
182 Despite the problems with aggregation, cross-linking analysis still appeared to be a useful assay because the putative cross-linked products were specific to the double cysteine mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C (Fig. 3B, band X). Login to comment
183 To ensure that band X was indeed the product of disulfide cross-linking between the introduced cysteines of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C, we added DTT after cross-linking. Login to comment
187 Each cDNA contained one of the cysteine mutations M348C, T351C, W356C, T1142C, or W1145C. Login to comment
188 It was found that co-expression of the single cysteine mutants M348C plus T1142C, T351C plus T1142C or W356C plus W1145C followed by treatment with the cross-linkers M5M, M8M, or M17M did not lead to cross-linking (formation of band X) (data not shown). Login to comment
189 This indicates that cross-linking occurs intramolecularly and not intermolecularly. To compare the inter-TMD interactions between WT and misprocessed CFTRs, the ⌬F508 mutation was introduced into the positive cross-linking double cysteine constructs M348C/ T1142C, T351C/T1142C, and W356C/W1145C. Login to comment
191 As shown in Fig. 6A, incorporation of the ⌬F508 mutation into mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C abolished cross-linking. Login to comment
196 To test whether the lack of cross-linking in the ⌬F508 series of double cysteine mutants was due to inaccessibility of thiol-reactive cross-linkers to the ER membrane, we tested whether mutants M348C/T1142C, T351C/ T1142C, and W356C/W1145C (lacking ⌬F508 mutation) would still show cross-linking then they were located in an intracellular membrane. Login to comment
197 To block trafficking of the mutants to the cell surface, we pretreated cells expressing mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C with 10 ␮g/ml brefeldin A. Login to comment
212 As shown in Fig. 6B, brefeldin A blocked processing of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C. Login to comment
215 Because the mature form of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C but not WT CFTR showed cross-linking, it was important to determine whether the mutants were still active. Login to comment
235 The M348C/T1142C mutant showed a similar level of activity as WT CFTR. Login to comment
248 Iodide efflux assays were performed on stable CHO cell lines expressing WT or one of the positive cross-linking double cysteine mutants (M348C/T1142C, T351C/ T1142C, and W356C/W1145C) as described under "Experimental Procedures." Login to comment
262 We were able to identify three mutants, M348C/T1142C, T351C/T1142C, and W356C/W1145C, that showed disulfide cross-linking in the mature WT background but not in the ⌬F508 background. Login to comment
263 Various control experiments were done to confirm that the mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C were indeed cross-linked through the introduced cysteines via the disulfide cross-linker. Login to comment
266 Finally, cross-linking was not observed when the cysteines in mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C were co-expressed on separate CFTR molecules. Login to comment
268 The ability to detect cross-linked products between TMD1 and TMD2 such as observed with mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C could be particularly useful in future studies to monitor dynamic changes in the molecule associated with phosphorylation or ATP binding/ hydrolysis. Login to comment

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

PMID: 18361776 [PubMed] Loo TW et al: "Correctors promote folding of the CFTR in the endoplasmic reticulum."

No. Sentence Comment

230 An example is the channel blocker, benzbromarone, which also inhibited cross-linking between cysteine residues in TM6 and TM7 [I340C(TM6)/S877C(TM7) mutant] [17], as well as between cysteine residues in TM6 and TM12 [M348C- (TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutants] (Figure 4). Login to comment

PMID: 18597042 [PubMed] Mornon JP et al: "Atomic model of human cystic fibrosis transmembrane conductance regulator: membrane-spanning domains and coupling interfaces."

No. Sentence Comment

153 Interestingly, it appears that all the CFTR mutants for which disulfide cross-linking was detected (M348C in TM6 and T1142C in TM12; T351C in TM6 and T1142C in TM12; W356C in TM6 and W1145C in TM12) line the chloride channel pore and face each other (Fig. 3A). 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

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

17 For I344C and M348C, the open time was prolonged and the closed time was shortened after modification, suggesting that depositions of positive charges at these positions stabilize the open state but destabilize the closed state. Login to comment
107 Spontaneous ATP-independent gating of cysless/I344C and cysless/M348C was also increased by MTSET because after the removal of ATP, there remained a substantial amount of current, which can be inhibited by CFTR-specific inhibitor, K335C, F337, and T338C at 50 mV membrane potential (0.46 pA for cysless/WT). Login to comment
120 In addition, MTSET modification can more than double the current in M348C channels. Login to comment
123 A representative result of the experiments with the cysless/ M348C construct is shown in Fig. 5. Login to comment
135 (D) MTSET increased ATP-dependent current in cysless/M348C channels. Login to comment
146 Interestingly, MTSET modification of M348C also slightly but significantly increased (12 ± 1%; n = 5) the single-channel amplitude (Fig. 5, A and C). Login to comment
154 Again, gating Figure 5. Effects of MTSET on a single cysless/M348C channel. Login to comment
156 (B) Gating parameters of the cysless/M348C channel before (black) and after (blue) modification, as extracted from the traces in A. Those of the cysless/ WT (gray) in the presence of 2 mM ATP are also included for comparison (traces not depicted). Login to comment
157 n = 5 for cysless/M348C. Login to comment
159 (C) Single-channel amplitude of the cysless/M348C channel before and after modification in the same patch. Login to comment
186 Instead, we will focus on the four other positive hits (i.e., I344C, V345C, M348C, and Q353C). Login to comment
201 (B) Single-channel amplitude, Po, open time and closed time of MTSET- (blue) and MTSEA-modified (green) cysless/R352C channel, as determined by Gaussian fitting and kinetics analysis; n = 6. inhibition of the macroscopic mean current (Fig. 4 C) and the single-channel current in the case of the cysless/ M348C channel might be due to oxidation of the introduced cysteine to a state not reactive toward either DTT or MTS reagents. Login to comment
212 Similar results were obtained with the cysless/V345C and cysless/ M348C channels. Login to comment
256 (A and B) Macroscopic recordings of cysless/ V345C and cysless/M348C showing modification by 1 mM MTSES when the membrane potential is held at 50 mV (left) and 100 mV (right). Login to comment
279 Similar results were obtained for cysless/M348C: 0.36 ± 0.03 s (n = 3) before and 0.55 ± 0.03 s (n = 3) after modification. Login to comment
290 For example, biochemical studies demonstrated that both M348C and T351C can be cross-linked to T1142C in TM12 (Chen et al. 2004). Login to comment
316 In fact, our data show that the Po of spontaneous gating in the absence of ATP (Bompadre et al., 2007; Wang et al., 2010) is visibly increased by MTSET modification (Figs. 4 D and 12 B; Po is 0.19 ± 0.04, n = 5 for cysless/M348C, and 0.63 ± 0.03, n = 4 for cysless/ I344C). Login to comment

PMID: 21520952 [PubMed] Loo TW et al: "Benzbromarone stabilizes DeltaF508 CFTR at the cell surface."

No. Sentence Comment

28 It was shown that benzbromarone appeared to interact with the CFTR TMDs because 200 μM benzbromarone blocked cross-linking between cysteines introduced into TM segments 6 and 12 (M348C/T1142C).23 This concentration of benzbromarone is now shown to inhibit maturation of CFTR (Figure 1A). Login to comment
50 (C) Effect of benzbromarone on cross-linking (X-link) between cysteines in TMD1 and TMD2 (M348C/T1142C) or NBD1 and TMD2 (V510C/A1067C).7 (D) Immunoblot of cells expressing CFTR TMD1þ2 in the absence (À) or presence (þ) of 0.05 mM benzbromarone. Login to comment

PMID: 21796338 [PubMed] Qian F et al: "Functional arrangement of the 12th transmembrane region in the CFTR chloride channel pore based on functional investigation of a cysteine-less CFTR variant."

No. Sentence Comment

140 In this respect, the slow rate of modification observed in N1138C (Fig. 3b) is similar to that we reported for P99C and L102C in TM1 [41] and T338C and S341C in TM6 [9], and the much higher modification rate constant for T1142C, S1141C, and (to a lesser extent) M1140C is closer to that reported for K95C in TM1 [41] and I344C, V345C, and M348C in TM6 [9]. Login to comment
207 However, charge-conservative mutations in the analgous part of TM6-for example, in I344C, V345C, M348C, and A349C-also failed to significantly alter Cl-conductance [4]. 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

259 Chen et al.41 observed cross-linking of M348C and T1142C by M5M and M8M but not M17M. Login to comment
262 The two ends of M17M are predicted by the MD simulation to come close to each other in free solution, forming a folded structure, theoretically allowing cross-linking of engineered cysteines such as M348C and T1142C. Login to comment

PMID: 22042986 [PubMed] Bai Y et al: "Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7)."

No. Sentence Comment

198 (C) Second-order rate constants (MTSES  ) of Texas red MTSEA+ modification for cysless/ S1141C-, cysless/N1148C-, cysless/ I344C-, and cysless/M348C-CFTR channels. 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

139 Our work concerning intracellular MTS reagent modification in TM6 also identified some cysteines that could be modified in both activated and nonactivated channels (e.g., V345C and M348C), and others that could apparently be modified only after channel activation (e.g., T338C, S341C, and I344C), suggesting a state-dependent conformational change that alters access of internally applied MTS reagents into the pore (El Hiani and Linsdell, 2010). Login to comment
162 (A-C) Example leak-subtracted I-V relationships for K95C/I344C (A), Q98C/I344C (B), and Q98C/M348C (C) after channel activation with 20 nM PKA and 1 mM ATP. Login to comment
166 Note that cys-less CFTR, the single mutants K95C, Q98C, or I344C, and the double mutant Q98C/M348C were all insensitive to CuPhe under these conditions. Login to comment
182 However, K95C/ I344C, Q98C/I344C, and Q98C/M348C did generate macroscopic PKA- and ATP-dependent currents in inside-out patches. Login to comment
186 Furthermore, the lack of effect of CuPhe on Q98C/M348C indicated that not all double-cysteine mutants were CuPhe sensitive, which we take to indicate that only nearby cysteine side chains can be cross-linked by this reagent. Login to comment
244 In contrast, we found no evidence for disulfide bond formation between a pair of introduced cysteine side chains that would be predicted (based on Fig. 9) to be further apart, Q98C in TM1 and M348C in TM6 (Fig. 6, C and D). Login to comment
256 For comparison, the MTSES modification rate constant for P99C and L102C (Fig. 3) was similar to that of T338C and S341C in TM6 (El Hiani and Linsdell, 2010) (all between 100 and 150 M1 s1 ), and the modification rate constant for K95C was comparable to, or slightly greater than, that of I344C, V345C, and M348C (El Hiani and Linsdell, 2010) (all between 2,000 and 4,000 M1 s1 ). 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
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

PMID: 23223629 [PubMed] Jih KY et al: "Nonequilibrium gating of CFTR on an equilibrium theme."

No. Sentence Comment

185 Likewise in Bai et al. (8), chemical modifications of an engineered cysteine (I344C or M348C) in TM6 drastically increase ATP-independent activity to the level of ATP-dependent activity before modifications. Login to comment

PMID: 23442957 [PubMed] Gao X et al: "Cysteine scanning of CFTR's first transmembrane segment reveals its plausible roles in gating and permeation."

No. Sentence Comment

164 Similar to what we observed for I344C- and M348C-CFTR (16), this robust ATP-independent gating was seen following modification by MTSET but not by MTS-ethylammonium (MTSEA) (Fig. S4). Login to comment

PMID: 25143385 [PubMed] El Hiani Y et al: "Metal bridges illuminate transmembrane domain movements during gating of the cystic fibrosis transmembrane conductance regulator chloride channel."

No. Sentence Comment

51 To investigate potential Cd2af9; bridges formed between pore-lining cysteine side chains exposed in the inner vestibule of the CFTR pore, we combined individual cysteines that we previously found to be accessible to cytoplasmically applied methanethiosulfonate reagents in three important pore-lining TMs: TM1 (K95C, Q98C) (13), TM6 (I344C, V345C, M348C, A349C) (15), and TM12 (M1140C, S1141C, T1142C, Q1144C, W1145C, V1147C, N1148C) (16), to generate a total of 50 double cysteine mutants (8 TM1:TM6; 14 TM1:TM12; 28 TM6:TM12). Login to comment
71 In contrast, the remaining seven double cysteine mutants, namely I344C/S1141C (Fig. 2, C and D), V345C/S1141C, M348C/ S1141C (Fig. 2, C and E), M348C/V1144C, M348C/W1145C, M348C/V1147C, and M348C/N1148C, all showed increased sensitivity to Cd2af9; , leading to a significant decrease in Ki as compared with either of the single cysteine mutants from which they were derived (estimated Ki values b0d; 50 òe;M; Fig. 3). Login to comment
77 In particular, M348C/S1141C was associated with a dramatic increase in sensitivity to Cd2af9; (Fig. 2, C and E, and Fig. 3) with an estimated Ki of 0.14 afe; 0.02 òe;M (n afd; 5). Login to comment
80 In each case, PPi treatment resulted in a weakening of Cd2af9; inhibition (Fig. 4A) and a significant increase in Ki (Fig. 4B) of between 2.3-fold (in I344C/S1141C) and 97-fold (in M348C/ S1141C). Login to comment
83 As shown in Fig. 5, all E1371Q-containing channels tested were only weakly sensitive to inhibition by Cd2af9; , resulting in a significant increase in Ki both in single cysteine (I344C, M348C, S1141C) and in double cysteine (I344C/S1141C, Fig. 5, A-C; M348C/S1141C, Fig. 5, A, D, and E) mutant channels. Login to comment
84 However, the effect of the E1371Q mutation was greater in the double cysteine mutants; this gating mutation increased Ki 30-fold in I344C/S1141C (Fig. 5C) and 2500-fold in M348C/S1141C (Fig. 5E). Login to comment
98 C, sample time courses (upper panels) and I-V curves (lower panels) recorded from similar experiments for the double cysteine mutants I344C/S1141C (left) and M348C/S1141C (right). Login to comment
127 Note that the Ki for M348C/S1141C (0.14 afe; 0.02 òe;M, n afd; 5) is too small to be visible on this scale, but was significantly different from either M348C or S1141C alone (p b0d; 0.0005; see also Fig. 2E). Login to comment
137 Thus, M348C is able to form Cd2af9; bridges with cysteines at multiple positions in TM12 (S1141C, Q1144C, W1145C, V1147C, N1148C) (Fig. 8B), and S1141C is able to form Cd2af9; bridges with cysteines both in TM1 (K95C) and in TM6 (I344C, V345C, M348C) (Fig. 8C). Login to comment
139 For example, M348C is able to form Cd2af9; bridges with TM12 sites on a broad face of the TM12 helical face (Fig. 8B, top panel), suggesting some degree of relative rotational flexibility or movement, and also across a distance spanning two helical turns (from Ser-1141 to Asn-1148) (Fig. 8B, bottom panel), suggesting also some translational flexibility or movement. Login to comment
146 Most striking in this respect was the M348C/S1141C mutant FIGURE 4. Login to comment
148 A, mean fractional current remaining following the addition of different concentrations of Cd2af9; in M348C/S1141C channels in the presence of PKA and ATP (F) or following activation by PKA and ATP followed by treatment with 2 mM PPi to maximize channel open probability (E). Login to comment
152 The unusually high apparent Cd2af9; binding affinity of this double cysteine mutant (Figs. 3B and 4B), more than 700-fold lower Ki than the corresponding single cysteine mutants (Figs. 2E and 3), suggests that M348C and S1141C are uniquely well positioned to coordinate tight Cd2af9; binding in closed channels. Login to comment
153 However, when the channel is open, these two cysteines do not appear to coordinate Cd2af9; at all because the apparent Cd2af9; affinity in M348C/S1141C/ E1371Q channels appears the same as in M348C/E1371Q or S1141C/E1371Q (Fig. 5, D and E). Login to comment
164 A, sample time courses and I-V curves illustrating the low Cd2af9; sensitivity of constitutively active I344C/S1141C/E1371Q (left panels) and M348C/S1141C/E1371Q (right panels) channels in inside-out patches. Experiments were performed as described in the legend for Fig. 2. Login to comment
175 As described above, several different Cd2af9; bridges can form between M348C (TM6) and TM12 (Fig. 8, A and B), as well as between S1141C (TM12) and TM6; all appear to stabilize the closed state (Figs. 3 and 4). Login to comment
195 )Residuesinredarethosewithpore-liningsidechainsmutatedinthepresentstudy;thoseinblackarenon-pore-lining.Redlinesconnectresiduesthat canformCd2af9; bridgesfollowingmutationtocysteine.B,relativelocationandorientationofMet-348(TM6)withthoseresiduesinTM12,whichcanformCd2af9; bridges with M348C following mutation to cysteine. Login to comment

PMID: 26496611 [PubMed] Sorum B et al: "Timing of CFTR Pore Opening and Structure of Its Transition State."

No. Sentence Comment

74 Timing of Motion at Position 348 in the Pore Region (A) Inward single-channel currents of the cut-DR(D1370N) CFTR background construct (top trace) and of channels bearing mutations M348I, M348K, M348C, M348N, and M348A, respectively, in the same background. Currents were recorded at 80 mV, in symmetrical 140 mM Cl ; dashes on the left mark zero-current level. Login to comment