ABCMdb

ABCC7 p.Asp110Arg

ClinVar:	c.328G>C , p.Asp110His D , Pathogenic c.328G>T , p.Asp110Tyr ? , not provided c.330C>A , p.Asp110Glu ? , not provided
CF databases:	c.328G>C , p.Asp110His D , CF-causing ; CFTR1: c.330C>A , p.Asp110Glu (CFTR1) ? , This mutation was detected by DGGE analysis followed by direct sequencing in two CF infants, a girl carrying [delta]F508 in the other chromosome and a boy carrying G542X in the other chromosome, both of Southern Italian origin (Sicilia region). It was never found in other 800 Italian CF chromosomes and in 100 control chromosomes from Italian population. The girl was diagnosed because of positive neonatal screening (persistent neonatal hypertrypsinemia), sweat chloride was 42, 57, and 68 mEq/l on repeated tests. Delayed meconium emission. No respiratory symptoms, pancreatic sufficiency and normal growth at 6 months. The boy presented at 6 months because of metabolic alkalemic diselectrolitemia and bronchiolitis. Neonatal screening was negative. Sweat chloride was 80, 70, 59 and 88 mEq/l on repeated occasions. At 2.5yrs, he is pancreatic sufficient, his growth is in the normal range and he presents no respiratory problems. This mutation was also reported by Aquino et al. (22/02/2000): It abolishes a Scrf I site. This substitution involves a quite conserved residue among species (N110 in the squale), in an intracellular loop. It doesn't affect the charge of the CFTR protein. It was found in an Italian CF patient with pancreatic sufficiency and bearing [delta]F508 on the other chromosome. No other mutation was found after analysis of 14 exons. The deleterious effect of D110E remains to be demonstrated. c.328G>T , p.Asp110Tyr (CFTR1) D , This mutation was found by SSCA and direct DNA sequencing in a CBAVD patient. (reported in Human Reproduction (2000) 15, 1476-1483). c.328G>A , p.Asp110Asn (CFTR1) ? ,
Predicted by SNAP2:	A: D (91%), C: D (91%), E: D (85%), F: D (95%), G: D (95%), H: D (63%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (85%), P: D (95%), Q: D (95%), R: D (95%), S: D (85%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: N, C: D, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: D, Q: N, R: N, S: N, T: N, V: N, W: D, Y: N,

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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.

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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
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
157 In contrast, neither D110R- nor K114D-CFTR altered Vrev. 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
184 D110E-CFTR exhibited a much more stable full open state with mean burst duration significantly longer than D110R-CFTR (P < 0.001; Fig. 7, A 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
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
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
267 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. 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
301 To test this prediction, we made the single mutation K892E and the double mutation D110R/K892E. Login to comment
304 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). Login to comment
311 Its single-channel behavior was very similar to that of D110R-CFTR except with a longer mean burst duration, probably because the cysteine at position 110 carries a partial negative charge that stabilizes channel open pore architecture in the same manner as D110 does (Fig. 10 A). Login to comment
335 (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). Login to comment
336 *, P < 0.05 indicates a significant difference between D110R- and D110R/K892E-CFTR; #, P < 0.001 for D110C/K892C-CFTR compared with D110R alone. 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