ABCC7 p.Arg104Glu
| Predicted by SNAP2: | A: D (59%), C: D (71%), D: D (85%), E: D (63%), F: D (80%), G: D (59%), H: N (66%), I: D (75%), K: N (87%), L: D (53%), M: N (53%), N: N (61%), P: D (85%), Q: N (66%), S: N (57%), T: N (57%), V: D (75%), W: D (85%), Y: D (53%), |
| Predicted by PROVEAN: | A: N, C: D, D: D, E: N, F: N, G: D, H: N, I: N, K: N, L: N, M: N, N: N, P: D, Q: N, S: N, T: N, V: N, W: N, Y: N, |
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[hide] Identification of positive charges situated at the... Pflugers Arch. 2008 Nov;457(2):351-60. Epub 2008 May 1. Zhou JJ, Fatehi M, Linsdell P
Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore.
Pflugers Arch. 2008 Nov;457(2):351-60. Epub 2008 May 1., [PMID:18449561]
Abstract [show]
We have used site-directed mutagenesis and functional analysis to identify positively charged amino acid residues in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel that interact with extracellular anions. Mutation of two positively charged arginine residues in the first extracellular loop (ECL) of CFTR, R104, and R117, as well as lysine residue K335 in the sixth transmembrane region, leads to inward rectification of the current-voltage relationship and decreased single channel conductance. These effects are dependent on the charge of the substituted side chain and on the Cl(-) concentration, suggesting that these positive charges normally act to concentrate extracellular Cl(-) ions near the outer mouth of the pore. Side chain charge-dependent effects are mimicked by manipulating charge in situ by mutating these amino acids to cysteine followed by covalent modification with charged cysteine-reactive reagents, confirming the location of these side chains within the pore outer vestibule. State-independent modification of R104C and R117C suggests that these residues are located at the outermost part of the pore. We suggest that ECL1 contributes to the CFTR pore external vestibule and that positively charged amino acid side chains in this region act to attract Cl(-) ions into the pore. In contrast, we find no evidence that fixed positive charges in other ECLs contribute to the permeation properties of the pore.
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No. Sentence Comment
60 As shown in Fig. 2, similar inward rectification is observed in the charge-reversing mutants R104E, R117E, and K335E and to a much lesser extent, R1128E, under symmetrical ionic conditions in excised membrane patches.
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ABCC7 p.Arg104Glu 18449561:60:93
status: NEW67 In contrast to the linear I-V relationship seen in wild-type CFTR with symmetrical 154 mM Cl- solutions, mutants R104E, R117E, and K335E showed clear inward rectification.
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ABCC7 p.Arg104Glu 18449561:67:113
status: NEW73 As shown in Fig. 3b, macroscopic current rectification was indeed sensitive to symmetrical Cl-concentration in R104E, R117E, and K335E, being more pronounced at low Cl-concentration.
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ABCC7 p.Arg104Glu 18449561:73:111
status: NEW79 However, current amplitude was significantly reduced in R104E, R117E, and K335E (Fig. 4a, b).
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ABCC7 p.Arg104Glu 18449561:79:56
status: NEW91 All mutants depicted (R104Q, R117Q, K335A, R1128Q, R104E, R117E, K335E, R1128E) showed rectification ratios significantly different from wild type (asterisk P<0.05).
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ABCC7 p.Arg104Glu 18449561:91:51
status: NEW92 b The degree of rectification in R104E, R117E, and K335E is dependent on the Cl-concentration.
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ABCC7 p.Arg104Glu 18449561:92:33
status: NEW104 In fact, at both sites, pCMBS had a greater effect on the rectification ratio than MTSES (P<0.05), making them closer to results obtained with R104E and R117E.
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ABCC7 p.Arg104Glu 18449561:104:143
status: NEW116 All mutants depicted (R104Q, R117Q, K335A, R104E, R117E, K335E) significantly different from wild type (asterisk P<0.05).
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ABCC7 p.Arg104Glu 18449561:116:43
status: NEW145 The reduction in unitary current for Cl-efflux seen in R104Q, R104E, and K335E (Fig. 4) further suggests that these residues may play an electrostatic role in Fig. 8 Mutation of positively charged residues weakens the apparent inhibitory effect of external Pt(NO2)4 2- ions.
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ABCC7 p.Arg104Glu 18449561:145:62
status: NEW[hide] Three charged amino acids in extracellular loop 1 ... J Gen Physiol. 2014 Aug;144(2):159-79. doi: 10.1085/jgp.201311122. Epub 2014 Jul 14. Cui G, Rahman KS, Infield DT, Kuang C, Prince CZ, McCarty NA
Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR.
J Gen Physiol. 2014 Aug;144(2):159-79. doi: 10.1085/jgp.201311122. Epub 2014 Jul 14., [PMID:25024266]
Abstract [show]
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) bears six extracellular loops (ECL1-6); ECL1 is the site of several mutations associated with CF. Mutation R117H has been reported to reduce current amplitude, whereas D110H, E116K, and R117C/L/P may impair channel stability. We hypothesized that these amino acids might not be directly involved in ion conduction and permeation but may contribute to stabilizing the outer vestibule architecture in CFTR. We used cRNA injected oocytes combined with electrophysiological techniques to test this hypothesis. Mutants bearing cysteine at these sites were not functionally modified by extracellular MTS reagents and were blocked by GlyH-101 similarly to WT-CFTR. These results suggest that these three residues do not contribute directly to permeation in CFTR. 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'-(beta,gamma-imido) triphosphate); charge-retaining mutants showed mainly the full open state with comparably longer open burst duration. These interactions suggest that these ECL1 residues might be involved in maintaining the outer pore architecture of CFTR. A CFTR homology model suggested that E116 interacts with R104 in both the closed and open states, D110 interacts with K892 in the fully closed state, and R117 interacts with E1126 in the open state. These interactions were confirmed experimentally. The results suggest that D110, E116, and R117 may contribute to stabilizing the architecture of the outer pore of CFTR by interactions with other charged residues.
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None has been submitted yet.
No. Sentence Comment
117 Fig. S5 illustrates representative single-channel current traces of E116R/ K892E- and R104E/D110R-CFTR and their mean burst durations.
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ABCC7 p.Arg104Glu 25024266:117:86
status: NEW118 Fig. S6 shows representative I-V plots of double mutants R104E/ E116R- and R117E/E1126R-CFTR and their rectification ratio.
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ABCC7 p.Arg104Glu 25024266:118:57
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.Arg104Glu 25024266:224:283
status: NEWX
ABCC7 p.Arg104Glu 25024266:224:520
status: NEW233 E116 forms a salt bridge with R104 in the open state as well as in the closed state To test the above prediction that R104 is a partner for E116, we studied the single mutant R104E-CFTR and the charge-swap double mutants R104E/E116R- and R104E/D110R-CFTR.
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ABCC7 p.Arg104Glu 25024266:233:175
status: NEWX
ABCC7 p.Arg104Glu 25024266:233:221
status: NEWX
ABCC7 p.Arg104Glu 25024266:233:238
status: NEW235 Both R104E-CFTR and R104E/E116R-CFTR exhibited reduced outward rectification with similar reversal potentials, both significantly different from WT-CFTR (Figs.
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ABCC7 p.Arg104Glu 25024266:235:5
status: NEWX
ABCC7 p.Arg104Glu 25024266:235:20
status: NEW237 Single-channel recording of R104E-CFTR (representative trace in Fig. 9 A) revealed Instead, we found that mutations at E217, D891, R899, or E1124 had no significant effect on single-channel behavior (Fig. S4 A, bottom).
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ABCC7 p.Arg104Glu 25024266:237:28
status: NEW264 Mean burst duration of R104E-CFTR was 325 &#b1; 54.08 ms, significantly shorter than WT but significantly longer than E116R-CFTR (Fig. 9 A, right).
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ABCC7 p.Arg104Glu 25024266:264:23
status: NEW265 Single-channel behavior of the double mutant R104E/ E116R-CFTR was similar to R104E-CFTR, with a long, stable f open state and a mean burst duration significantly longer than that of E116R-CFTR (Fig. 9 A, right).
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ABCC7 p.Arg104Glu 25024266:265:45
status: NEWX
ABCC7 p.Arg104Glu 25024266:265:78
status: NEW267 In contrast, single-channel properties of the R104E/D110R-CFTR double mutant were not significantly different from the D110R-CFTR single mutant (Fig. S5), suggesting no interaction between R104 and D110.
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ABCC7 p.Arg104Glu 25024266:267:46
status: NEW284 (A) Representative single-channel current traces of R104E- and R104E/E116R-CFTR recorded with the same experimental conditions as Fig. 2 (left), their all-points amplitude histograms (middle), and mean burst durations (right).
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ABCC7 p.Arg104Glu 25024266:284:52
status: NEWX
ABCC7 p.Arg104Glu 25024266:284:63
status: NEW285 **, P < 0.01 indicates a significant difference between E116R- and R104E/ E116R-CFTR.
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ABCC7 p.Arg104Glu 25024266:285:67
status: NEW409 However, as noted previously for R104E/E116R-CFTR, the charge swap mutant did not completely recover the behavior of Figure 12.ߓ R117 forms a salt bridge with E1126 in the open state.
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ABCC7 p.Arg104Glu 25024266:409:33
status: NEW