ABCC7 p.Lys892Glu
| Predicted by SNAP2: | A: N (72%), C: D (53%), D: N (61%), E: N (82%), F: D (63%), G: N (66%), H: N (78%), I: N (66%), L: N (72%), M: N (72%), N: N (78%), P: N (53%), Q: N (82%), R: N (82%), S: N (82%), T: N (82%), V: N (66%), W: D (66%), Y: D (59%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, L: N, M: N, N: N, P: N, Q: N, R: 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
61 In contrast, K114E, K329E, K892E, and R899E, like wild type, gave almost linear I-V relationships under these conditions.
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ABCC7 p.Lys892Glu 18449561:61:27
status: NEW78 The mutations K114E, K329E, K892E, R899E, and R1128E did not affect unitary current amplitude at any voltage (Fig. 4b), consistent with these residues not being involved in Cl- permeation.
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ABCC7 p.Lys892Glu 18449561:78:28
status: NEW[hide] Evidence that extracellular anions interact with a... Can J Physiol Pharmacol. 2009 May;87(5):387-95. doi: 10.1139/y09-023. Zhou JJ, Linsdell P
Evidence that extracellular anions interact with a site outside the CFTR chloride channel pore to modify channel properties.
Can J Physiol Pharmacol. 2009 May;87(5):387-95. doi: 10.1139/y09-023., [PMID:19448737]
Abstract [show]
Extracellular anions enter into the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, interacting with binding sites on the pore walls and with other anions inside the pore. There is increasing evidence that extracellular anions may also interact with sites away from the channel pore to influence channel properties. We have used site-directed mutagenesis and patch-clamp recording to identify residues that influence interactions with external anions. Anion interactions were assessed by the ability of extracellular Pt(NO2)42- ions to weaken the pore-blocking effect of intracellular Pt(NO2)42- ions, a long-range ion-ion interaction that does not appear to reflect ion interactions inside the pore. We found that mutations that remove positive charges in the 4th extracellular loop of CFTR (K892Q and R899Q) significantly alter the interaction between extracellular and intracellular Pt(NO2)42- ions. These mutations do not affect unitary Cl- conductance or block of single-channel currents by extracellular Pt(NO2)42- ions, however, suggesting that the mutated residues are not in the channel pore region. These results suggest that extracellular anions can regulate CFTR pore properties by binding to a site outside the pore region, probably by a long-range conformational change. Our findings also point to a novel function of the long 4th extracellular loop of the CFTR protein in sensing and (or) responding to anions in the extracellular solution.
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No. Sentence Comment
74 Moreover, reversal of the charge at these sites in ECL4 (in the K892E and R899E mutants) had no effect on unitary Cl-conductance in the presence of high symmetrical Cl- concentrations (Zhou et al. 2008), again consistent with these ECL4 residues not being involved directly in formation of the pore.
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ABCC7 p.Lys892Glu 19448737:74:64
status: NEW109 Zhou and Linsdell 391 versing mutations K892E and R899E under different ionic conditions (Zhou et al. 2008).
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ABCC7 p.Lys892Glu 19448737:109:41
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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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.Lys892Glu 25024266:117:75
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.Lys892Glu 25024266:224:380
status: NEW301 To test this prediction, we made the single mutation K892E and the double mutation D110R/K892E.
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ABCC7 p.Lys892Glu 25024266:301:53
status: NEWX
ABCC7 p.Lys892Glu 25024266:301:89
status: NEW302 K892E-CFTR open channels behaved similarly to WT-CFTR, including displaying a stable full open state with single-channel amplitude similar to WT (&#e032;0.77 &#b1; 0.02 pA, n = 5; Fig. 10), outward rectification in the I-V relationship, and WT-like reversal potential (Fig. S4 C and Table 1).
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ABCC7 p.Lys892Glu 25024266:302:0
status: NEW304 The double mutant D110R/K892E-CFTR behaved similarly to D110R-CFTR (Fig. 7 A), displaying flickery openings to the s1, s2, and f states with a very brief open burst duration (Fig. 10 A).
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ABCC7 p.Lys892Glu 25024266:304:24
status: NEW335 (A) Representative single-channel current traces of K892E-, D110R/K892E-, and D110C/K892C-CFTR recorded with the same experimental conditions as Fig. 2 (left), their all-points amplitude histograms (middle), and their mean burst durations (right).
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ABCC7 p.Lys892Glu 25024266:335:52
status: NEWX
ABCC7 p.Lys892Glu 25024266:335:66
status: NEW336 *, P < 0.05 indicates a significant difference between D110R- and D110R/K892E-CFTR; #, P < 0.001 for D110C/K892C-CFTR compared with D110R alone.
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ABCC7 p.Lys892Glu 25024266:336:72
status: NEW382 E116R/K892E-CFTR exhibited an I-V relationship similar to that of WT-CFTR.
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ABCC7 p.Lys892Glu 25024266:382:6
status: NEW383 The burst behavior of E116R/K892E-CFTR was similar to that of E116R-CFTR, suggesting that E116 does not form a salt bridge with K892 when the CFTR channel is in the open state (Fig. S5).
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ABCC7 p.Lys892Glu 25024266:383:28
status: NEW437 Top view of the homology model (McCarty laboratory model; Rahman et al., 2013) with salt bridge residues shown as spheres: R117-E1126, red; E116-R104, green; and D110-K892E, magenta.
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ABCC7 p.Lys892Glu 25024266:437:167
status: NEW