ABCC7 p.Lys190Cys
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PMID: 20133716
[PubMed]
Wang W et al: "ATP-independent CFTR channel gating and allosteric modulation by phosphorylation."
No.
Sentence
Comment
71
(F) Very large ATP-independent current for K190C/K978C-CFTR.
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ABCC7 p.Lys190Cys 20133716:71:43
status: NEW104 We were clued that ADP inhibits the ATP-independent activity of the constitutive mutants (e.g., K190C/K978C-CFTR channels) by the finding that their currents were lowest when hexokinase/glucose was added to induce current deactivation in the presence of 1.5 mM ATP (instead of perfusing ATP from the bath before adding the enzyme).
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ABCC7 p.Lys190Cys 20133716:104:96
status: NEW106 Fig. S1D shows that the currents mediated by the K190C/K978C constitutive mutant increased on subsequent bath perfusion to remove the enzyme and all nucleotides and were reversibly inhibited by about 50% by adding back ADP alone.
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ABCC7 p.Lys190Cys 20133716:106:49
status: NEW108 The constitutive activity of the K190C/K978C mutant was not inhibited by ADP when these loop mutations were introduced into a deletion construct that lacks NBD2 (Fig. S7 and Fig. 3).
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ABCC7 p.Lys190Cys 20133716:108:33
status: NEW125 (C) Titration of ADP inhibition of K190C/K978C-CFTR current in the absence of ATP.
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ABCC7 p.Lys190Cys 20133716:125:35
status: NEW160 We also introduced the double constitutive loop mutant (K190C/K978C) into a construct lacking both the R domain and NBD2 (Fig. 4E).
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ABCC7 p.Lys190Cys 20133716:160:56
status: NEW187 (E) K190C/K978C/ΔR/Δ1198 channels are maximallyactiveunderbaselineconditions.
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ABCC7 p.Lys190Cys 20133716:187:4
status: NEW
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
22
Our recent study also demonstrated that a K190C/S mutation from CL1 enhances ATP-independent channel opening induced by a K978C/P/S mutation from CL3 (17).
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ABCC7 p.Lys190Cys 21059651:22:42
status: NEW
PMID: 21965669
[PubMed]
Wang W et al: "Thermally unstable gating of the most common cystic fibrosis mutant channel (DeltaF508): "rescue" by suppressor mutations in nucleotide binding domain 1 and by constitutive mutations in the cytosolic loops."
No.
Sentence
Comment
7
Here, we determined whether these various rescue protocols induce a ⌬F508-CFTR conformation that is thermally stable in excised membrane patches. We also tested the impact of constitutive cytosolic loop mutations that increase ATP-independent channel activity (K978C and K190C/K978C) on ⌬F508-CFTR function.
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ABCC7 p.Lys190Cys 21965669:7:278
status: NEW52 In a recent study we showed that two cytosolic loop mutations (e.g. K190C and K978C) promote ATP-independent CFTR channel activity, allosterically increase ATP and PKA sensitivity, and also significantly restore the function of mutant CFTR channels that cannot be activated by ATP binding or NBD dimerization (e.g. G551D and ⌬1198, which lacks NBD2) (47).
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ABCC7 p.Lys190Cys 21965669:52:68
status: NEW57 The cytosolic loop mutations (K978C and K190C/K978C) do not improve ⌬F508-CFTR processing but do increase the ATP-independent channel activity of ⌬F508 channels that are delivered to the cell surface by incubation at low temperature.
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ABCC7 p.Lys190Cys 21965669:57:40
status: NEW64 K978C and K190C/K978C mutations, which promote ATP-independent channel activity (47), were introduced into the ⌬F508-CFTR construct.
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ABCC7 p.Lys190Cys 21965669:64:10
status: NEW162 Constitutive Loop Mutations Increase ATP-independent Channel Activity of ⌬F508-CFTR, Which Is More Stable at Physiologic Temperature-Previously, we showed that several mutations in intracellular loop1 and loop3 (e.g. K190C and K978C) increase the ATP-independent channel activities of several CFTR constructs, including mutant forms that cannot be activated by ATP (e.g. G551D and ⌬1198-CFTR, an NBD2 deletion mutant) (47).
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ABCC7 p.Lys190Cys 21965669:162:224
status: NEW164 Fig. 6A shows that neither K978C nor K190C/K978C improved the maturation of ⌬F508-CFTR when cells were grown either at 37 °C or 27 °C, as determined by immunoblotting.
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ABCC7 p.Lys190Cys 21965669:164:37
status: NEW173 To follow up this result we examined the thermal stability of the K190C/K978C/⌬F508 construct that has a greater relative ATP-independent channel activity.
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ABCC7 p.Lys190Cys 21965669:173:66
status: NEW174 Fig. 7B shows that the K190C/K978C double mutation greatly protected the channel activity from thermal inactivation with nearly all of the remaining current following warming the bath being ATP-independent. As a control we also tested the thermal stability of the ATP-independent channel activity of G551D-CFTR channels with the K978C mutation.
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ABCC7 p.Lys190Cys 21965669:174:23
status: NEW185 Error bars, S.E. Thermal Instability of ⌬F508-CFTR Gating DECEMBER 9, 2011•VOLUME 286•NUMBER 49 JOURNAL OF BIOLOGICAL CHEMISTRY 41943 the ATP-independent channel activity of K978C/⌬F508-CFTR and to a greater extent K190C/K978C/⌬F508-CFTR was resistant to temperature-induced inactivation.
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ABCC7 p.Lys190Cys 21965669:185:244
status: NEW195 We found that constitutive mutations in cytosolic loop1 and loop3 (i.e. K978C and K190C/K978C) increased ATP-independent channel activity of low temperature-rescued ⌬F508-CFTR.
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ABCC7 p.Lys190Cys 21965669:195:82
status: NEW199 FIGURE6.Cytosolicloopmutations(K978CandK190C/K978C)increaseATP-independentchannelactivityoflowtemperature-recued⌬F508-CFTR.A, immunoblot showing that loop mutations do not improve maturation of ⌬F508 at 27 °C. B-D, ATP-dependent channel activities of ⌬F508, K978C/⌬F508, and K190C/K978C/⌬F508 revealed by adding hexokinase/glucose to scavenge ATP.
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ABCC7 p.Lys190Cys 21965669:199:308
status: NEW201 E, mean fractional ATP-independent currents for ⌬F508 (n ϭ 6), K978C/⌬F508 (n ϭ 5), and K190C/K978C/⌬F508-CFTR (n ϭ 5).
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ABCC7 p.Lys190Cys 21965669:201:114
status: NEW218 K978C/⌬F508 and K190C/K978C/⌬F508-CFTR are more stable at physiologic temperature.
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ABCC7 p.Lys190Cys 21965669:218:23
status: NEW221 B, effect of raising temperature on K190C/K978C/⌬F508-CFTR, which is more thermally stable.
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ABCC7 p.Lys190Cys 21965669:221:36
status: NEW223 D, mean fractional current decrease for ⌬F508 (data from Fig. 1D), K978C/⌬F508, K190C/K978C/⌬F508, and K978C/ G551D.
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ABCC7 p.Lys190Cys 21965669:223:94
status: NEW237 A and B, effect of raising temperature on ATP-independent currents of K978C/⌬F508- and K190C/K978C/⌬F508-CFTR.
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ABCC7 p.Lys190Cys 21965669:237:94
status: NEW239 C, mean fractional loss of ATP-independent current for K978C/⌬F508 and K190C/ K978C/⌬F508-CFTR (n ϭ 4 for each construct).
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ABCC7 p.Lys190Cys 21965669:239:78
status: NEW
PMID: 23620589
[PubMed]
Okeyo G et al: "Converting nonhydrolyzable nucleotides to strong cystic fibrosis transmembrane conductance regulator (CFTR) agonists by gain of function (GOF) mutations."
No.
Sentence
Comment
104
Accordingly, we compared the activation by AMP-PNP of several previously described GOF CFTR mutants with increasing degrees of unliganded activity (K978P, K978C, and K190C/K978C; see Fig. 3).
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ABCC7 p.Lys190Cys 23620589:104:166
status: NEW105 All three GOF mutants were more strongly activated by AMP-PNP than WT-CFTR, with the lowest and greatest relative activation observed for the weakest (K978P; Fig. 3, A, C, and D) and strongest (K190C/K978C; Fig. 3, B, C, and D) GOF mutant.
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ABCC7 p.Lys190Cys 23620589:105:195
status: NEW169 A and B, representative macropatch records show AMP-PNP activation of K978P-CFTR (A) and K190C/K978C-CFTR (B).
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ABCC7 p.Lys190Cys 23620589:169:89
status: NEW171 Note the previously described voltage-dependent rectification of K190C/K978C-CFTR currents (13).
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ABCC7 p.Lys190Cys 23620589:171:65
status: NEW
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
103
A-D, examples of modification of four different mutants (A, R303C, used in this study as a positive control; B, K190C; C, K370C; D, R1048C) by MTSES (left panels) or MTSET (right panels).
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ABCC7 p.Lys190Cys 25944907:103:112
status: NEW109 Application of MTSES (200 òe;M) following channel activation with PKA and ATP never caused an increase in macroscopic current amplitude but decreased current amplitude in K190C, R248C, R251C, R303C, K370C, K946C, R975C, K1041C, and R1048C (Figs. 2 and 3).
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ABCC7 p.Lys190Cys 25944907:109:175
status: NEW110 The effect of MTSET on these MTSES-sensitive mutants was to increase (K190C and R303C), decrease (R248C, K946C, K1041C, and R1048C), or have no effect (R251C, K370C, and R975C) on macroscopic current amplitude (Figs. 2 and 3).
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ABCC7 p.Lys190Cys 25944907:110:70
status: NEW121 In contrast, the inhibitory effects of MTSES on K190C, R248C, K370C, and R1048C were preserved following PPi treatment (Figs. 4 and 5), suggesting that modification with this negatively charged reagent caused a major inhibition of Clafa; conductance in open channels in each of these mutants.
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ABCC7 p.Lys190Cys 25944907:121:48
status: NEW123 This implies that modification with positively charged MTSET caused an increase in Clafa; conductance in each of K190C, R248C, R303C, K370C, and R1048C, an effect that may potentially be masked by inhibitory effects on channel open probability in normally gating channels.
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ABCC7 p.Lys190Cys 25944907:123:116
status: NEW132 Error bars represent the means afe; S.E. from three to five patches. Cytoplasmic Entrance to the CFTR Channel Pore JUNE 19, 2015ߦVOLUME 290ߦNUMBER 25 JOURNAL OF BIOLOGICAL CHEMISTRY 15859 rent amplitude in each of K190C, R248C, R303C, K370C, K1041C, and R1048C.
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ABCC7 p.Lys190Cys 25944907:132:230
status: NEW139 Following channel activation with PKA and ATP, channels were "locked" in the open statebyapplicationofPPi (2mM;asindicatedbyhatchedbars)priortoadditionofMTSreagent(200òe;M;asindicatedbywhitebars).TheidentityofCFTRcurrents was confirmed by sensitivity to CFTRinh-172 (-172; 5 òe;M; indicated by black bars) at the end of the recording except for very small residual currents for K190C following MTSES modification.
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ABCC7 p.Lys190Cys 25944907:139:386
status: NEW141 Cytoplasmic Entrance to the CFTR Channel Pore 15860 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 25ߦJUNE 19, 2015 at SEMMELWEIS UNIV OF MEDICINE on December 4, amplitudes in unmodified channels was Cys-less b; K946C b03; R975C b0e; K370C b0e; R251C b0e; K1041C b03; R248C b0e; R1048C b0e; R303C b0e; K190C (Fig. 7A).
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ABCC7 p.Lys190Cys 25944907:141:345
status: NEW142 Among channels showing MTSES-sensitive current amplitudes, the order following modification was K1041C b0e; R1048C b0e; K370C b0e; R248C b0e; R303C b0e; K190C (Fig. 6, B and C, and 7B).
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ABCC7 p.Lys190Cys 25944907:142:168
status: NEW155 Single channel recording indicated directly that for each of K190C, R248C, R303C, K370C, K1041C, and R1048C Clafa; conductance was reduced relative to Cys-less (Figs. 6 and 7A), Clafa; conductance was further reduced by modification by negatively charged MTSES (Figs. 6 and 7B), and Clafa; conductance was restored to near wild-type (Cys-less) values by modification by positively charged MTSET (Figs. 6 and 7C).
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ABCC7 p.Lys190Cys 25944907:155:61
status: NEW166 In contrast, the effects of MTS modification of PPi-treated channels in K190C, R248C, R303C, K370C, and R1048C (Fig. 5) closely mirrored the charge-dependent effects of MTS modification on single channel current amplitude (Figs. 6 and 7).
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ABCC7 p.Lys190Cys 25944907:166:72
status: NEW173 We believe that the functional importance of Lys-190, Arg-248, Arg-303, and Lys-370 is likely greater than that of Lys-1041 and Arg-1048 because of the greater reduction in single channel current amplitude following introduction of a negative charge by MTSES modification in K190C, R248C, R303C, and K370C FIGURE 6.
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ABCC7 p.Lys190Cys 25944907:173:275
status: NEW181 Single channel current amplitude was very low in MTSES- modified K190C, R248C, R303C, and K370C channels (b0d;25% of Cys-less current amplitude; Figs. 6, B and C, and 7B), suggesting that most permeating Clafa; ions must pass close to these residues.
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ABCC7 p.Lys190Cys 25944907:181:65
status: NEW