ABCMdb

ABCC7 p.Glu116Arg

ClinVar:	c.346G>A , p.Glu116Lys ? , not provided c.346G>C , p.Glu116Gln ? , not provided
CF databases:	c.346G>A , p.Glu116Lys (CFTR1) D , A missense mutation in exon 4 of the CFTR gene was detected by DGGE and identified by direct sequencing. The nucleotide position 478 is chnaged from G to A, leading to a subsitution of glutamic acid for lysine at position 116. This mutation abolishes a MnI restriction site. The mutation on the other chromosome is the deletion of [delta]F508 c.346G>C , p.Glu116Gln (CFTR1) ? , This mutation was detected in a thirty year old male with UAVD by multiplex heteroduplex analysis on the MDE gel matrix and direct sequencing. The patient was also heterozygous for the [delta]F508 mutation. Clinical Data: UAVD, borderline sweat test, chronic sinusitis
Predicted by SNAP2:	A: N (72%), C: D (59%), D: N (87%), F: D (71%), G: N (61%), H: D (63%), I: N (61%), K: N (61%), L: N (57%), M: D (63%), N: N (78%), P: N (57%), Q: N (82%), R: N (57%), S: N (78%), T: N (78%), V: N (66%), W: D (75%), Y: D (66%),
Predicted by PROVEAN:	A: N, C: D, D: N, F: D, G: N, H: N, I: N, K: N, L: D, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: D,

[switch to compact view]
Comments [show]

None has been submitted yet.

Publications
[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.

Comments [show]

None has been submitted yet.

Sentences [show]
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. Login to comment
74 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. Login to comment
101 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. Login to comment
104 Similar results were seen in D110R- and E116R-CFTR (mean burst duration for E116R: &#e032;AMP-PNP, 37.72 &#b1; 3.07 ms; +AMP-PNP, 36.16 &#b1; 5.73 ms, n = 3; and for D110R-CFTR: &#e032;AMP-PNP, 22.24 &#b1; 1.8 ms; +AMP-PNP, 19.74 &#b1; 0.69 ms, n = 4). Login to comment
115 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. Login to comment
117 Fig. S5 illustrates representative single-channel current traces of E116R/ K892E- and R104E/D110R-CFTR and their mean burst durations. Login to comment
118 Fig. S6 shows representative I-V plots of double mutants R104E/ E116R- and R117E/E1126R-CFTR and their rectification ratio. Login to comment
124 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). Login to comment
129 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). Login to comment
133 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. Login to comment
134 The apparent open probabilities of D110R-, E116R-, and in these positions were modified by ET+ and ES&#e032; , but their modification failed to affect ion conduction because these amino acids are located too far away from the Cl&#e032; conduction pathway. Login to comment
145 (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. Login to comment
149 (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. Login to comment
150 (D) Apparent open probability of WT-, D110R-, E116R-, and R117A-CFTR. Login to comment
154 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. Login to comment
156 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. Login to comment
160 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. Login to comment
161 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. Login to comment
185 Similar results were observed when the burst durations of E116D-CFTR and E116R-CFTR were compared (P < 0.001; Fig. 7, B and E). Login to comment
187 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. Login to comment
204 To test this hypothesis, we first made the mutants E116R/R117E-, D110R/R117E-, and D110R/E116R/R117E-CFTR and studied their single-channel properties. Login to comment
206 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). Login to comment
219 WT-CFTR, K114D-CFTR, and E116R-CFTR currents were generated under a voltage protocol wherein membrane potential was held at 0 mV for 50 ms then ramped from &#e032;100 mV to 100 mV over 300 ms with the TEVC technique. Login to comment
224 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. Login to comment
233 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. Login to comment
235 Both R104E-CFTR and R104E/E116R-CFTR exhibited reduced outward rectification with similar reversal potentials, both significantly different from WT-CFTR (Figs. Login to comment
246 (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. Login to comment
248 (D) Mean single-channel amplitude of WT-, D110R-, D110E-, E116R-, E116D-, R117A-, and R117K-CFTR. Login to comment
250 (E) Mean burst duration of WT-, D110R-, D110E-, E116R-, E116D-, R117A-, and R117K-CFTR. Login to comment
251 #, 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. Login to comment
264 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). Login to comment
265 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). Login to comment
266 Although the charge swap mutant did not fully recover WT-CFTR behavior, its recovery compared with E116R-CFTR suggests that E116 may form a salt bridge with R104 when CFTR channels are in the open state. Login to comment
271 (A) Representative single-channel currents of R117E/ E116R-, R117E/D110R-, and R117E/E116R/ D110R-CFTR and corresponding all-points amplitude histograms recorded under the same conditions as Fig. 2 A. Login to comment
274 (C) Mean single-channel amplitudes of WT-, R117E/ E116R-, R117E/D110R-, and R117E/E116R/ D110R-CFTR. Login to comment
284 (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). Login to comment
285 **, P < 0.01 indicates a significant difference between E116R- and R104E/ E116R-CFTR. Login to comment
382 E116R/K892E-CFTR exhibited an I-V relationship similar to that of WT-CFTR. Login to comment
383 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). Login to comment
409 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. Login to comment
423 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). Login to comment