ABCMdb

ABCC7 p.Val510Ala

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PMID: 17911111 [PubMed] Wang Y et al: "Correctors promote maturation of cystic fibrosis transmembrane conductance regulator (CFTR)-processing mutants by binding to the protein."

No. Sentence Comment

3 Although replacement of the 18 endogenous cysteines of CFTR with Ser or Ala yields a Cys-less mutant that does not mature at 37 °C, we found that maturation could be restored if Val510 was changed to Ala, Cys, Ser, Thr, Gly, Ala, or Asp. Login to comment
5 The Cys-less/V510A mutant was used for subsequent cross-linking analysis as it yielded relatively high levels of mature protein that was functional in iodide efflux assays. Login to comment
6 We tested for cross-linking between cysteines introduced into TM6 and TM7 of Cys-less CFTR/V510A because cross-linking between TM6 and TM7 of P-glycoprotein, the sister protein of CFTR, was inhibited with the corrector VRT-325. Login to comment
7 Cys-less CFTR/V510A mutant containing cysteines at I340C(TM6) and S877C(TM7) could be cross-linked with a homobifunctional cross-linker. Login to comment
26 Mutations I340C(TM6) and S877C(TM7) were inserted into Cys-less CFTR/V510A singly or together. Login to comment
28 The ⌬F508 mutation was also introduced into the Cys-less/V510A and I340C(TM6)/S877C(TM7)/Cys-less/ V510A mutants. Login to comment
46 Disulfide Cross-linking Analysis-CFTR mutant I340C(TM6)/ S877C(TM7) Cys-less/V510A CFTR was expressed in HEK 293 cells. Login to comment
68 The mutants were expressed in HEK 293 cells at 37 °C, and whole cell extracts were subjected to immunoblot analysis. Fig. 2B shows that changing Val510 to Thr, Gly, Ala, Ser, or Asp promoted maturation of Cys-less CFTR. Login to comment
70 Stable BHK cell lines expressing mutants V510A, V510C, V510S, V510G, or V510D were generated for use in iodide efflux assays. Login to comment
72 The results of the most active (V510A) and least active (V510D) mutants are shown in Fig. 2C. Login to comment
82 ACCELERATED PUBLICATION: CFTR TM Domain Cross-linking 33248 To test whether the Val510 changes could also promote maturation of ⌬F508 CFTR (in a wild-type background), we introduced the Val510 mutations that promoted maturation of Cys-less CFTR (Val510 to Cys, Gly, Ala, Ser, Asp, or Thr) into ⌬F508 CFTR. Login to comment
84 The Cys-less/V510A CFTR mutant was then used for disulfide cross-linking analysis to test whether correctors directly interacted with the protein. Since corrector VRT-325 inhibited cross-linking of P-gp mutant L339C(TM6)/F728C(TM7), we aligned TM segments (6 and 7) of P-gp and CFTR to identify the equivalent residues in CFTR. Login to comment
87 The 16 double cysteine mutants were initially constructed in a Cys-less CFTR lacking V510A, so they required expression at 27 °C for maturation of CFTR (data not shown). Login to comment
90 The I340C(TM6)/S877C(TM7) mutations were then introduced into the Cys-less/V510A CFTR background. Login to comment
91 The I340C(TM6)/S877C(TM7) Cys-less/ V510A CFTR mutant was expressed in HEK 293 cells, and samples were treated with methanethiosulfonate cross-linkers of various sizes and subjected to immunoblot analysis. Login to comment
93 Fig. 3A shows that mutant I340C(TM6)/ S877C(TM7) Cys-less/V510A CFTR could be cross-linked with various methanethiosulfonate cross-linkers. Login to comment
98 C, iodide efflux assays were performed on stable BHK cell lines expressing Cys-less/V510A CFTR (open circles) or Cys-less/V510D CFTR (closed squares). Login to comment
103 Disulfide cross-linking of mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR. Login to comment
104 A, HEK 293 cells expressing mutant I340C(TM6)/ S877C(TM7) Cys-less/V510A CFTR were treated with (ϩ) or without (-) 0.1 mM of the indicated cross-linkers for 16 min at 22 °C. Login to comment
108 B, immunoblot analysis of HEK 293 cells expressing Cys-less/V510A CFTR, mutants I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR, I340C(TM6) Cys-less/V510A CFTR, or S877C(TM7) Cys-less/V510A CFTR were treated with (ϩ) or without (-) cross-linker M11M. Login to comment
113 The I340C(TM6)/S877C(TM7) Cys-less/ V510A CFTR mutant was used as a reporter molecule to study CFTR interactions with CFTR correctors such as corr-4a (4), VRT-325 (5, 17), and VRT-532 (5, 6). Login to comment
114 We first tested the system by using a compound that binds within the CFTR channel pore (at the interface between the two TMDs) and to see whether it could block cross-linking of mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR. Login to comment
116 Accordingly, HEK 293 cells expressing mutant I340C(TM6)/ S877C(TM7) Cys-less/V510A CFTR were preincubated with various concentrations of benzbromarone and then treated with M11M cross-linker. Login to comment
120 Verapamil (Fig. 4E) and demecolcine (Fig. 4F) did not inhibit cross-linking of mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR. Login to comment
122 We could not test whether correctors blocked cross-linking in the ⌬F508/ I340C(TM6)/S877C(TM7)/Cys-less/V510A mutant because mature CFTR was not detected in mutants ⌬F508/Cys-less/ V510A or ⌬F508/I340C(TM6)/S877C(TM7)/Cys-less/V510A even when expressed at 27 °C (Fig. 4, G and H). Login to comment
131 Although V510D was the most efficient suppressor mutation because it promoted maturation of both Cys-less and ⌬F508 CFTRs, it was less useful than the V510A change in Cys-less CFTR because it showed reduced iodide efflux activity. Login to comment
133 The ability of VRT-325, VRT-532, and corr-4a (Fig. 4) to block cross-linking of I340C(TM6)/S877C(TM7) Cys-less/ V510A CFTR suggests that they interact directly with CFTR. Login to comment
138 Effect of CFTR modulators and correctors on disulfide cross-linking of CFTR mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR. Login to comment
139 HEK 293 cells expressing mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR were incubated at 22 °C for 30 min in the absence (None) or presence of theindicatedcompounds.Thesampleswerethentreatedwith(ϩ)orwithout (-) 0.1 mM M11M at 22 °C for 16 min. Login to comment
141 Immunoblot analysis was also performed on mutants Cys-less/V510A (G) or I340C(TM6)/S877C(TM7)/Cys-less/V510A (H) with (⌬F508) or without (-) the ⌬F508 mutation that were transiently expressed in HEK 293 cells for 24 h at 27 °C. Login to comment

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

No. Sentence Comment

40 The location of the V510A and cross-linkable cysteine residues [(M348C(TM6)/T1142C(TM12) (dashed line), T351C(TM6)/T1142C(TM12) (solid line) and W356C(TM6)/W1145C(TM12) (dotted line) mutants] are indicated. Login to comment
47 The cysteine-less CFTR also contained a V510A mutation (cysteine-less/V510A) that promoted maturation at 37◦ C [17]. Login to comment
48 The double-cysteine mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/ T1142C(TM12) and W356C(TM6)/W1145C(TM12) that were shown to be cross-linkable with M8M (3,6-dioxaoctane-1,8-bismethanethiosulfonate) cross-linker [3] were introduced into the cysteine-less/V510A CFTR. Login to comment
93 The cysteine-less/ V510A CFTR mutant exhibited channel activity at the cell surface [17]. Login to comment
95 To test whether the mature and immature forms of cysteine-less CFTR/V510A still exhibited structural differences, the Figure 2 Disulfide cross-linking of cysteine mutants in wild-type or cysteine-less/V510A CFTR background Wild-type CFTR containing the M348C(TM6)/T1142C(TM12) mutations (A) or cysteine-less/V510A CFTR containing M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutation (B) were expressed in HEK-293 cells. Login to comment
99 M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutations were introduced into cysteine-less/V510A CFTR. Login to comment
103 There was a little aggregation of immature CFTR, however, when cysteine-less/V510A containing the M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/ W1145C(TM12) mutations were treated with M8M cross-linker (Figure 2B). Login to comment
105 In contrast, no cross-linked product was detected when single-cysteine mutants M348C(TM6), T351C(TM6), W356C(TM6), T1142(TM12) and W1145C(TM12) in the cysteine-less/V510A background were each treated with M8M cross-linker (results not shown). Login to comment
106 These results suggest that the M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutations in the cysteine-less/V510A CFTR background could act as reporters for monitoring folding of the TMDs because only mature CFTR shows cross-linking. Login to comment
112 The reactions were Figure 3 Concentration-dependence of M8M cross-linking of cysteine mutants (A) HEK-293 cells expressing M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant in the cysteine-less/V510A background were suspended in PBS. Login to comment
124 Slow-migrating product was not detected when single-cysteine mutants M348C(TM6), T351C(TM6), W356C(TM6), T1142C(TM12) and W1145(TM12) in cysteine-less CFTR/V510A were each treated with M8M (results not shown) We then examined whether correctors, channel blockers or potentiators inhibited cross-linking of M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/ W1145C(TM12) mutants. Login to comment
148 (C) M348C(TM6)/T1142C(TM12)/Y563N, T351C(TM6)/T1142C(TM12)/Y563N or W356C(TM6)/W1145C(TM12)/Y563N mutant in the cysteine-less/V510A CFTR background was expressed in the absence (-) or presence (+) of 15 μM corr-4a. Login to comment
151 (D) Membranes were prepared from HEK-293 cells expressing M348C(TM6)/T1142C(TM12)/Y563N, T351C(TM6)/T1142C(TM12)/Y563N or W356C(TM6)/W1145C(TM12)/Y563N mutant in the cysteine-less/V510A background that were grown in the absence (-) or presence (+) 15 μM corr-4a. Login to comment
166 The Y563N mutation was then introduced into M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant in the cysteine-less/V510A background. Login to comment
170 These results indicate that the COPII exit motif mutations in cysteine-less/V510A CFTR still blocked maturation even when expressed in the presence of a corrector. Login to comment
171 Membranes were then prepared from cells transfected with M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant cDNA in the cysteine-less/V510A/Y563N background and grown in the presence or absence of corr-4a. Login to comment
178 To confirm that the corrector was modulating folding in the ER, we expressed wild-type CFTR and T351C(TM6)/T1142C- (TM12)/Y563N/cysteine-less/V510A mutant in the absence or presence of brefeldin A before cross-linking with M8M cross-linker. Login to comment
182 Cross-linking of T351C(TM6)/ T1142C(TM12)/Y563N/cysteine-less/V510A mutant expressed in the presence of brefeldin A with or without corr-4a showed that there was more cross-linked product when corr-4a was present (Figure 6B). Login to comment
186 To test whether corr-4a promoted folding of the F508 mutant in the ER, the F508 mutation was introduced into Figure 6 Effect of brefeldin A on maturation of wild-type CFTR and cross-linking analysis of T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A mutant HEK-293 cells were transfected with wild-type or T351C(TM6)/T1142C(TM12)/Y563N/cysteineless/V510A mutant CFTR cDNAs. Login to comment
189 (B) After 16 h, the medium in cells expressing T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A mutant was replaced with fresh medium containing 10 μg/ml brefeldin A with (+) or without (-) 15 μM corr-4a. Login to comment
193 the double-cysteine mutants M348C(TM6)/T1142C-(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C- (TM12) in the Y563N/cysteine-less/V510A CFTR background. Login to comment
196 Expression in the presence of corr-4a, however, increased the yield of cross-linked product of F508/M348C(TM6)/T1142C(TM12)/Y563N/cysteine-less/ V510A, F508/T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510Aand F508/W356C(TM6)/W1145C(TM12)/Y563N/ cysteine-less/V510A mutants to 5, 11 and 10% respectively (Figure 7). Login to comment
207 The presence Figure 7 Effects of F508 mutation on cross-linking of COPII cysteine mutants HEK-293 cells expressing CFTR F508/M348C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A, F508/T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A or F508/ W356C(TM6)/W1145C(TM12)/Y563N/cysteine-less/V510A mutant were grown in the absence (-) or presence (+) of 15 μM corr-4a. Membranes were prepared, and cross-linking with M8M cross-linker was performed. Login to comment

PMID: 18708637 [PubMed] Loo TW et al: "Processing mutations disrupt interactions between the nucleotide binding and transmembrane domains of P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR)."

No. Sentence Comment

51 We also constructed the V510C(NBD1)/A1067C(TMD2) CFTR mutant because position V510C or V510A promoted maturation of Cys-less CFTR (22). Login to comment
179 It was previously shown that mutations V510C or V510A promoted maturation of Cys-less CFTR (22). Login to comment

PMID: 19466983 [PubMed] Li MS et al: "Cysteine-independent inhibition of the CFTR chloride channel by the cysteine-reactive reagent sodium (2-sulphonatoethyl) methanethiosulphonate."

No. Sentence Comment

28 To facilitate channel protein maturation and expression in the cell membrane, the V510A mutation (Wang et al., 2007) was introduced into this construct using the QuikChange site directed mutagenesis system as described previously (Gong et al., 2002) and verified by DNA sequencing. Login to comment
29 Baby hamster kidney (BHK) and human embryonic kidney cells were transiently transfected with pIRES2-EGFP-cys-less/V510A CFTR cDNA as described previously (Gong et al., 2002), except that 24 h after transfection, cells were transferred to 27°C to promote mature protein expression (see Supporting Information, Figure S1). Login to comment
32 Once trafficked to the cell membrane (see Supporting Information, Figure S1), the properties of cys-less/V510A CFTR channel currents appeared identical to those of wild-type CFTR studied previously, with the exception that single channel conductance appeared increased (see Figures 2 and 3). Login to comment
55 Results Expression of cys-less/V510A CFTR in BHK cells led to the appearance of PKAand ATP-dependent PPi-stimulated macroscopic currents when the cells were grown at 27°C. Examples of such currents, which had similar properties to those of wild-type CFTR studied many times previously (e.g. Linsdell and Hanrahan, 1998; Gong et al., 2002), are shown in Figure 1. Login to comment
66 (A) An example of the leak-subtracted macroscopic current-voltage relationship for cys-less/V510A-CFTR following maximal current stimulation with protein kinase A catalytic subunit (PKA), adenosine 5'-triphosphate (ATP) and pyrophosphate (PPi). Login to comment

PMID: 20133716 [PubMed] Wang W et al: "ATP-independent CFTR channel gating and allosteric modulation by phosphorylation."

No. Sentence Comment

214 We also introduced the V510A substitution into the cysteine-free mutants, which is known to promote their maturation (35). Login to comment

PMID: 20142516 [PubMed] Zhou JJ et al: "Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore."

No. Sentence Comment

27 Cys-less CFTR also included a mutation in the first nucleotide-binding domain (V510A) to increase protein expression in the cell membrane (Li et al., 2009). 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

30 Subsequently, an additional V510A mutation was introduced to promote expression (Wang et al., 2007). Login to comment
31 A single cysteine was then engineered into each position in TM6 on the cysless/V510A background using the QuikChange XL kit (Agilent Technologies). Login to comment
33 Because all of our studies were performed under this cysless/ V510A background, we will refer to this construct as our wild-type (WT) cysless control. Login to comment

PMID: 20952391 [PubMed] Wang G et al: "State-dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gating by a high affinity Fe3+ bridge between the regulatory domain and cytoplasmic loop 3."

No. Sentence Comment

64 To improve expression of a Cys-free CFTR-based construct, V510A was inserted (31), and cells expressing the construct were grown for 1-2 days at 24 °C and then for 2-5 h at 37 °C before measurements. Login to comment
147 Fig. 5A shows that internal diamide (10 ␮M) suppressed ϳ30% of channel activity of a H950C/S832C/ V510A construct, and suppression was partially reversed by FIGURE 2. Login to comment
227 A-E, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing mutants H950C/S832C/V510A (A), H950C/V510A (B), S832C/V510A (C), H954C (D), and the WT hCFTR construct (E). Login to comment

PMID: 21059651 [PubMed] Wang G et al: "The inhibition mechanism of non-phosphorylated Ser768 in the regulatory domain of cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

69 To improve expression, cells expressing Cys-free CFTR-based constructs inserted with V510A (32) were grown for 1-2 days at 24 °C and then for 2-5 h at 37 °C before measurements. Login to comment

PMID: 21594776 [PubMed] Loo TW et al: "Repair of CFTR folding defects with correctors that function as pharmacological chaperones."

No. Sentence Comment

247 To improve the maturation efficiency of Cys-less CFTR, cysteines 590 and 592 were replaced with leucines (16) and valine 510 was changed to alanine (17). 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

34 As in our recent study on CFTR-TM6 [9], we have used a human CFTR variant ("cys-less" CFTR) in which all endogenous cysteine residues had been substituted by other amino acids [32] and which also includes a mutation in NBD1 (V510A) to increase protein expression in the cell membrane [19]. Login to comment

PMID: 21796426 [PubMed] Holstead RG et al: "Functional Differences in Pore Properties Between Wild-Type and Cysteine-Less Forms of the CFTR Chloride Channel."

No. Sentence Comment

26 Cys-less CFTR also included a mutation in the first NBD (V510A) to increase protein expression in the cell membrane (Li et al. 2009). Login to comment
27 The V510A mutation itself is not expected to affect single-channel conductance, which has previously been reported to be similarly increased in cys-less CFTR without (Cui et al. 2006; Mense et al. 2006) and with (Li et al. 2009; Bai et al. 2010) the V510A mutation. Login to comment

PMID: 23060444 [PubMed] Wang G et al: "Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3."

No. Sentence Comment

124 Fig. 7 demonstrates that 10M diamide greatly suppressed the channel activity of S768C/S832C/V510A mutant but failed to affect the channel activities of both S768C/V510A and S832C/V510A. Login to comment
154 Finally, D979A in CF patients causes misprocessing (33). Login to comment
195 Effects of diamide on CFTR mutants at the R-CL3 interface. A-C, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing CFTR mutants S768C/V510A (A), S832C/V510A (B), and S768C/S832C/V510A (C) by using a ramp protocol (afe;80 mV). Login to comment

PMID: 22303012 [PubMed] Wang W et al: "Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7)."

No. Sentence Comment

47 19), and which includes a mutation in the first NBD (V510A) to increase protein expression in the cell membrane (20). Login to comment
49 19), and which includes a mutation in the first NBD (V510A) to increase protein expression in the cell membrane (20). Login to comment

PMID: 22234285 [PubMed] Wang W et al: "Conformational change opening the CFTR chloride channel pore coupled to ATP-dependent gating."

No. Sentence Comment

58 The cys-less version used in the present study also includes a mutation in NBD1 (V510A) to increase protein expression in the cell membrane [19]. 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

60 As in our recent study on CFTR-TM6 (El Hiani and Linsdell, 2010), we have used a human CFTR variant in which all 18 endogenous cysteine residues had been substituted by other amino acids (as described in Mense et al., 2006), and which also includes a mutation in the first nucleotide-binding domain (V510A) to increase protein cross-linked residues in a current homology model of the CFTR membrane-spanning domain. Login to comment
64 Using site-directed mutagenesis, we substituted cysteines for each of 21 consecutive amino acids, from Y84 near the putative intracellular end of TM1 to R104 near the extracellular end, in a V510A cys-less background (see Materials and methods). Login to comment

PMID: 23709221 [PubMed] Cui G et al: "Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function."

No. Sentence Comment

150 MTS Reagents Exhibit No Effects on Single Channel Amplitude of WT-CFTR and Cys-less V510A-CFTR-Because the Arg352 -Asp993 interaction appeared to make the largest contribution to stabilizing the f state, we asked whether forcing these residues to interact would lead to channels that were latched into the open state. Login to comment
155 We further added a secondary mutation to generate Cys-less V510A-CFTR in order to improve expression (9, 25). Login to comment
156 Because it was reported that Cys-less CFTR showed channel behavior similar to that of WT-CFTR with a few nominal differences (9, 26-28), we first used mutants generated on the WT-CFTR background and then confirmed the results in mutants generated on the Cys-less V510A-CFTR background. Login to comment
157 Representative data indicating that charged monofunctional MTS reagents modified the activity of WT-CFTR or Cys-less V510A-CFTR when applied cytoplasmically in excised, inside-out patches are shown in supplemental Fig. 1. Login to comment
160 Supplemental Fig. 1B shows Cys-less V510A-CFTR in the absence and presence of MTS reagents; not surprisingly, MTS reagents exhibited no effects on either single channel amplitude or NPo in the Cys-less channel. Login to comment
161 Based on these results, we resolved to use both WT-CFTR and Cys-less V510A-CFTR as backgrounds to test the consequences of modification of engineered cysteines. Login to comment
175 This added effect was lost on the Cys-less V510A background. Login to comment
179 We repeated the above experiments in R352C/Cys-less V510A-CFTR and D993C/ Cys-less V510A-CFTR to further rule out the possibility of any endogenous cysteines being involved in the process. Login to comment
181 These effects were removed only upon application of the reducing agent DTT and indicate that the Cys-less V510A-CFTR background was identical to the WT-CFTR background with respect to these experiments. Login to comment
185 Before applying MTS-2-MTS to the R352C/D993C-CFTR double mutant, we first tested the effects of this bifunctional linker on WT-CFTR (Fig. 6A) and Cys-less V510A-CFTR (Fig. 6B). Login to comment
186 MTS-2-MTS did not change the single channel amplitude of either channel but decreased NPo of WT-CFTR without changing NPo of Cys-less V510A-CFTR. Login to comment
201 Effects of 100 òe;M MTS-2-MTS on WT-CFTR (A) and Cys-less V510A-CFTR (B) at VM d1d; d1a;100 mV. A, MTS-2-MTS decreased NPo but had no effect on single channel amplitude of WT-CFTR. Login to comment
202 B, the cross-linker had no effect on either NPo or single channel amplitude of Cys-less V510A-CFTR. Login to comment

PMID: 23955087 [PubMed] Wang W et al: "Relative contribution of different transmembrane segments to the CFTR chloride channel pore."

No. Sentence Comment

38 In this study, we have used two different human CFTR variants as backgrounds for further mutagenesis: wild type CFTR and "cys-less" CFTR in which all cysteines have been removed by mutagenesis [30] and that also includes a mutation in the first NBD (V510A) to increase protein expression in the cell membrane [20]. Login to comment

PMID: 24412276 [PubMed] Loo TW et al: "The cystic fibrosis V232D mutation inhibits CFTR maturation by disrupting a hydrophobic pocket rather than formation of aberrant interhelical hydrogen bonds."

No. Sentence Comment

71 Cys-less CFTR was constructed in which all endogenous cysteines were changed to alanine except that Cys590 and Cys592 were changed to leucine and Val510 was changed to alanine. 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

46 In this study, we have used a human CFTR variant in which all cysteines had been removed by mutagenesis (as described in Ref. 23) and that includes a mutation in NBD1 (V510A) to increase protein expression in the cell membrane (24). Login to comment

PMID: 25944907 [PubMed] El Hiani Y et al: "Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore."

No. Sentence Comment

51 In this study, we used a human CFTR variant in which all cysteines had been removed by mutagenesis (as described in Ref. 21) and that includes a mutation in NBD1 (V510A) to increase protein expression in the cell membrane (22). Login to comment