ABCC7 p.Arg117Ala
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PMID: 25024266
[PubMed]
Cui G et al: "Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR."
No.
Sentence
Comment
20
In contrast, mutants D110R-, E116R-, and R117A-CFTR exhibited instability of the open state and significantly shortened burst duration compared with WT-CFTR and failed to be locked into the open state by AMP-PNP (adenosine 5&#e039;-(&#e062;,&#e067;-imido) triphosphate); charge-retaining mutants showed mainly the full open state with comparably longer open burst duration.
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ABCC7 p.Arg117Ala 25024266:20:41
status: NEW74 Online supplemental material Fig. S1 illustrates representative single-channel current traces of D110R-, E116R-, and R117A-CFTR with a larger time scale (b) whether and how they contribute to maintaining open pore architecture; and (c) whether ECL1 moves during the CFTR gating cycle.
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ABCC7 p.Arg117Ala 25024266:74:117
status: NEW97 Opening rates for WTand R104C/ E116C-CFTR were measured as previously described except that R117A-CFTR were significantly lower than WT-CFTR (Fig. 2 D).
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ABCC7 p.Arg117Ala 25024266:97:94
status: NEW101 Given the instability of D110R-, E116R-, and R117A-CFTR single-channel openings, we asked whether AMP-PNP would lock these mutants into a stable open state.
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ABCC7 p.Arg117Ala 25024266:101:45
status: NEW103 In contrast, AMP-PNP did not affect mean burst duration or single-channel amplitude of R117A-CFTR but did increase the apparent open probability (P < 0.05; Fig. 3).
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ABCC7 p.Arg117Ala 25024266:103:87
status: NEW115 Fig. S3 shows representative I-V curves of D110R-, E116R-, and R117A-CFTR recorded in symmetrical 150 mM Cl&#e032; solution with the inside-out macropatch technique.
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ABCC7 p.Arg117Ala 25024266:115:63
status: NEW124 To probe the potential mechanisms by which mutation of these charged residues leads to CF, we first recorded the single-channel behavior of a series of CFTR channel mutants bearing a single mutation at one of the six charged sites (D110R, D112R, K114D, E115R, E116R, or R117A).
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ABCC7 p.Arg117Ala 25024266:124:270
status: NEW129 Unlike WT-CFTR, which opens mainly to the full open state(f)withsubconductancestatesasrareevents,D110R-, E116R-, and R117A-CFTR exhibited multiple open states, including subconductance state 1 (s1), subconductance state 2 (s2), and the f state (Fig. S1).
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ABCC7 p.Arg117Ala 25024266:129:117
status: NEW133 The exception was K114D-CFTR, which exhibited mean burst duration significantly shorter than that of WT-CFTR, but much longer than that of D110R-, E116R-, and R117A-CFTR.
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ABCC7 p.Arg117Ala 25024266:133:159
status: NEW145 (A) Representative single-channel current traces and their all-points histograms for WT-, D110R-, D112R-, K114D-, E115R-, E116R-, and R117A-CFTR from inside-out membrane patches excised from Xenopus oocytes, with symmetrical 150 mM Cl&#e032; solution in the presence of 1 mM MgATP and 50 U/ml PKA.
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ABCC7 p.Arg117Ala 25024266:145:134
status: NEW149 (B and C) Single-channel amplitudes of the full open state (B) and mean burst durations (C) of WT-, D110R-, D112R-, K114D-, E115R-, E116R-, and R117A-CFTR.
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ABCC7 p.Arg117Ala 25024266:149:144
status: NEW150 (D) Apparent open probability of WT-, D110R-, E116R-, and R117A-CFTR.
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ABCC7 p.Arg117Ala 25024266:150:58
status: NEW154 ECL1 mutations shift the reversal potential in macroscopic currents To further verify that these ECL1 amino acids do not strongly or directly affect ion conduction and permeation, we compared the reversal potentials (Vrev) of D110R-, K114D-, E116R-, and R117A-CFTR with WT-CFTR and with the R334A mutant, which has been shown to have a profound effect on Vrev compared with WT-CFTR, consistent with the role of R334 in providing charge in the outer mouth of the open channel.
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ABCC7 p.Arg117Ala 25024266:154:254
status: NEW156 E116R- and R117A-CFTR exhibited significantly right-shifted reversal potentials compared with WT-CFTR, but the effects were not as large as for R334A-CFTR.
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ABCC7 p.Arg117Ala 25024266:156:11
status: NEW160 Of the ECL1 mutants we examined, the rectification ratios for D110R-, K114D-, and R117A-CFTR were similar to WT-CFTR (Fig. 6), whereas E116R-CFTR showed significantly reduced outward rectification.
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ABCC7 p.Arg117Ala 25024266:160:82
status: NEW161 We also examined the I-V relationship of D110R-, E116R-, and R117A-CFTR with the inside-out macropatch technique in symmetrical that the charged amino acids in ECL1 might be involved in establishing the appropriate architecture for GlyH-101 binding and function.
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ABCC7 p.Arg117Ala 25024266:161:61
status: NEW172 Figure 3.ߓ R117A-CFTR failed to be locked into a stable open state by AMP-PNP.
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ABCC7 p.Arg117Ala 25024266:172:17
status: NEW173 R117A-CFTR was activated with 1 mM Mg-ATP and PKA and recorded under control conditions with ATP + PKA (&#e032;AMP-PNP), followed by addition of 2.75 mM AMP-PNP (+AMP-PNP).
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ABCC7 p.Arg117Ala 25024266:173:0
status: NEW176 AMP-PNP had no effect on mean burst duration or single-channel amplitude but significantly increased apparent open probability of R117A-CFTR (n = 4).
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ABCC7 p.Arg117Ala 25024266:176:130
status: NEW187 Under these conditions, E116R-CFTR exhibited slight inward rectification, but both D110R- and R117A-CFTR exhibited linear I-V relationships like that of WT-CFTR.
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ABCC7 p.Arg117Ala 25024266:187:94
status: NEW206 All three mutants exhibited very brief openings to the s1, s2, and f states, with mean burst durations significantly lower than that of WT-CFTR (P < 0.001; Fig. 8 B) and R117A-CFTR (Fig. 2 C), but not different from D110R-CFTR and E116R-CFTR (Fig. 2 C).
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ABCC7 p.Arg117Ala 25024266:206:170
status: NEW213 was less marked when R117K- and R117A-CFTR were compared (P < 0.05; Fig. 7, C and E).
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ABCC7 p.Arg117Ala 25024266:213:32
status: NEW224 Tab l e 1 Reversal potentials of WT-CFTR and mutants in ND96 bath solution CFTR n Vrev mV WT 14 &#e032;27.75 &#b1; 0.78 R334A 6 &#e032;12.15 &#b1; 1.64a R117A 6 &#e032;22.51 &#b1; 0.85a E116R 5 &#e032;21.45 &#b1; 1.14a K114D 5 &#e032;24.68 &#b1; 3.22 D110R 5 &#e032;27.64 &#b1; 3.29 R104E 5 &#e032;21.15 &#b1; 1.08a R899C 4 &#e032;25.30 &#b1; 3.94 D891C 6 &#e032;25.81 &#b1; 2.44 K892E 5 &#e032;23.70 &#b1; 3.62 E1124R 5 &#e032;18.32 &#b1; 0.43a E1126R 5 &#e032;20.67 &#b1; 3.16b R117E/E1126R 6 &#e032;23.06 &#b1; 1.37b R104E/E116R 6 &#e032;27.17 &#b1; 1.08 Values are mean &#b1; SEM.
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ABCC7 p.Arg117Ala 25024266:224:153
status: NEW246 (A-C) Representative single-channel currents of D110R- and D110E- (A), E116R- and E116D- (B), and R117A- and R117K-CFTR (C) recorded under the same conditions as Fig. 2 A.
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ABCC7 p.Arg117Ala 25024266:246:98
status: NEW248 (D) Mean single-channel amplitude of WT-, D110R-, D110E-, E116R-, E116D-, R117A-, and R117K-CFTR.
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ABCC7 p.Arg117Ala 25024266:248:74
status: NEW250 (E) Mean burst duration of WT-, D110R-, D110E-, E116R-, E116D-, R117A-, and R117K-CFTR.
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ABCC7 p.Arg117Ala 25024266:250:64
status: NEW251 #, P < 0.001 indicates differences between D110R- and D110E-CFTR or E116R- and E116D-CFTR; *, P < 0.05 indicates differences between R117A- and R117K-CFTR.
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ABCC7 p.Arg117Ala 25024266:251:133
status: NEW406 R117E/E1126R-CFTR opened to a full open state much more often compared with R117A-CFTR (Fig. 2).
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ABCC7 p.Arg117Ala 25024266:406:76
status: NEW408 The mean burst duration of R117E/E1126R-CFTR was significantly longer than that of R117A- and R117C-CFTR.
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ABCC7 p.Arg117Ala 25024266:408:83
status: NEW423 As shown here, mean burst durations of charge-retaining mutants D110E-, E116D-, and R117K-CFTR are significantly longer than their related charge-reversing or charge-destroying mutants D110R-, E116R-, and R117A-CFTR but distinctly shorter than that of WT-CFTR (Fig. 7).
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ABCC7 p.Arg117Ala 25024266:423:205
status: NEW