ABCMdb

ABCC7 p.Asp110Cys

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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[hide] Cysteine scanning of CFTR's first transmembrane se... Biophys J. 2013 Feb 19;104(4):786-97. doi: 10.1016/j.bpj.2012.12.048. Gao X, Bai Y, Hwang TC
Cysteine scanning of CFTR's first transmembrane segment reveals its plausible roles in gating and permeation.
Biophys J. 2013 Feb 19;104(4):786-97. doi: 10.1016/j.bpj.2012.12.048., [PMID:23442957]

Abstract [show]
Previous cysteine scanning studies of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have identified several transmembrane segments (TMs), including TM1, 3, 6, 9, and 12, as structural components of the pore. Some of these TMs such as TM6 and 12 may also be involved in gating conformational changes. However, recent results on TM1 seem puzzling in that the observed reactive pattern was quite different from those seen with TM6 and 12. In addition, whether TM1 also plays a role in gating motions remains largely unknown. Here, we investigated CFTR's TM1 by applying methanethiosulfonate (MTS) reagents from both cytoplasmic and extracellular sides of the membrane. Our experiments identified four positive positions, E92, K95, Q98, and L102, when the negatively charged MTSES was applied from the cytoplasmic side. Intriguingly, these four residues reside in the extracellular half of TM1 in previously defined CFTR topology; we thus extended our scanning to residues located extracellularly to L102. We found that cysteines introduced into positions 106, 107, and 109 indeed react with extracellularly applied MTS probes, but not to intracellularly applied reagents. Interestingly, whole-cell A107C-CFTR currents were very sensitive to changes of bath pH as if the introduced cysteine assumes an altered pKa-like T338C in TM6. These findings lead us to propose a revised topology for CFTR's TM1 that spans at least from E92 to Y109. Additionally, side-dependent modifications of these positions indicate a narrow region (L102-I106) that prevents MTS reagents from penetrating the pore, a picture similar to what has been reported for TM6. Moreover, modifications of K95C, Q98C, and L102C exhibit strong state dependency with negligible modification when the channel is closed, suggesting a significant rearrangement of TM1 during CFTR's gating cycle. The structural implications of these findings are discussed in light of the crystal structures of ABC transporters and homology models of CFTR.

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139 (C) Summary of whole-cell SCAM results on the eight residues, G103C-D110C. Login to comment

[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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93 Window current (calculated as the rolling mean of current in a 1-min window, in pA) over each minute for WTand D110C/K892C-CFTR records was measured sequentially for 21 min with Clampfit 10.2. Login to comment
109 Therefore, we performed experiments to investigate the modification of WT-, D110C-, E116C-, and R117C-CFTR by MTSET (ET+ ) and MTSES (ES&#e032; ) with the TEVC technique; R334C-CFTR was used as a positive control (Zhang et al., 2005b). Login to comment
119 Fig. S7 shows representative TEVC current traces of WTand D110C/ K892C-CFTR with DTT pretreatment. Login to comment
140 However, under the same conditions, no functional modifications were observed for any of the three ECL1 cysteine mutants studied (D110C-, E116C-, and R117C-CFTR). Login to comment
144 Our data could be interpreted in two ways: (1) the thiol groups of the engineered cysteines in D110C-, E116C-, and R117C-CFTR were not exposed and therefore unable to be modified by ET+ or ES&#e032; , or (2) the three cysteines Figure 2.ߓ Some ECL1 mutants exhibited decreased burst duration. Login to comment
170 GlyH-101 blocked D110C- and R117C-CFTR similarly to WT-CFTR, whereas E116C-CFTR was also blocked significantly by GlyH-101 (P < 0.01), but less efficaciously than the other two mutants or the WT. Login to comment
189 The data presented so far resolve our first two questions in this paper: (1) Charge-swapping mutations of D110, E116, and R117 of ECL1 destabilize the open state, indicating that these residues contribute to maintaining the outer mouth open pore architecture of CFTR; (2) based Figure 4.ߓ Effects of 1 mM MTSET+ (ET+ ) and MTSES&#e032; (ES&#e032; ) on WT-, D110C-, E116C-, R117C-, and R334C-CFTR. Login to comment
196 Figure 5.ߓ Effects of 2.5 &#b5;M GlyH-101 on WT-, D110C-, E116C-, and R117C-CFTR. Login to comment
310 To test this, we made the double cysteine mutant D110C/K892C-CFTR. Login to comment
312 The data also suggested that at least some of the double mutant D110C/K892C-CFTR channels could be activated by ATP and PKA in the absence of DTT, which would not support the formation of a stable spontaneous disulfide bond. Login to comment
313 To resolve this, we backfilled 1 mM DTT in the pipette solution and recorded single-channel current of WTand D110C/K892C-CFTR from inside-out patches in the presence of cytosolic ATP and PKA, whereas DTT diffused to the tip. Login to comment
329 D110C/K892C-CFTR patch current remained low in the first &#e07a;5 min, then slowly increased over the next 10-15 min as more channels were activated and the number of apparent channels in the patch increased (Fig. 10 C). Login to comment
332 However, patches from oocytes expressing D110C/K892C-CFTR exhibited a very large increase in apparent channel number after exposure to extracellular DTT, consistent with channels being released from the spontaneous disulfide bond and therefore able to open. 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
342 (C) Exposure to 1 mM DTT, backfilled into the pipette, increased the number of active channels in D110C/K892C-CFTR. Login to comment
347 for several minutes, the question remained as to whether D110C/K892C-CFTR channels form a spontaneous disulfide bond in intact cells where CFTR is in the constant presence of ATP. Login to comment
350 In contrast, in D110C/K892C-CFTR, activation by ISO plus DTT (ISO2 + DTT) led to significantly higher current than ISO alone (ISO1). Login to comment
351 These data suggest that in whole oocytes, a fraction of D110C/ K892C channels formed disulfide bonds under resting conditions and were locked into the closed state (C0). Login to comment
353 We then asked whether DTT could further activate D110C/K892C-CFTR current if we used DTT before ISO. Login to comment
354 We used 1 mM DTT alone for 3 min followed by ISO alone (ISO1), which fully activated D110C/K892C-CFTR channels to plateau. Login to comment
357 These data suggest that with prior DTT treatment, disulfide bonds formed during the closed state in D110C/K892C-CFTR were broken by DTT and ISO1 was able to activate all channels to reach maximum current. Login to comment
361 In the absence of 1 mM DTT, D110C/K892C-CFTR channels were activated by ISO1 and ISO2 to a similar level (Fig. S7 C), suggesting that ISO alone was not able to break the spontaneous disulfide bond between K892C and D110C in channels that had formed this bond. Login to comment
362 In summary, the above data, combined with molecular modeling, suggest three important findings: (1) D110 forms a salt bridge with K892 in the closed state; (2) D110C/K892C-CFTR forms a spontaneous disulfide bond when the channel is in the closed state and the energy of CFTR channel gating is not strong enough to break it in the absence of the reducing agent DTT; (3) CFTR may transition to a state where the NBDs are fully dedimerized (C0 closed state), as our simulations suggest that C0 is the only state where these residues approach each other closely enough for a spontaneous disulfide to form. Login to comment
363 unlike WT-CFTR, D110C/K892C-CFTR was modified by DTT when it perfused to the pipette tip over 5-10 min during channel phosphorylation; DTT probably broke the disulfide bond between D110C and K892C, allowing more channels to be activated by ATP and PKA. Login to comment
364 The single-channel current amplitude of D110C/K892C-CFTR remained unchanged in the absence and presence of DTT; individual single-channel openings in the presence of DTT could not be distinguished from those in channels that were able to open before DTT. Login to comment
367 Collectively, the data suggest that D110C/ K892C-CFTR forms a spontaneous disulfide bond when the channel is in the closed state (C0), and this locks the channel closed. Login to comment
368 We further tested the spontaneous disulfide bond in D110C/K892C-CFTR with the macropatch technique. Login to comment
369 We pulled inside-out macropatches from oocytes expressing WT- or D110C/K892C-CFTR and recorded the current in real time during exposure to 1 mM DTT backfilled into the pipette. Login to comment
372 For D110C/ K892C-CFTR ATPand PKA-activated channels, but unlike WT-CFTR, D110C/K892C-CFTR currents slowly increased over the full duration of the experiment (&#e07a;20 min; Fig. 11 A, c). Login to comment
373 Both WTand D110C/K892C-CFTR currents could be completely abolished with removal of ATP and PKA from the intracellular solution (Fig. 11 A, d). Login to comment
374 Representative I-V plots at the outset of recording and at 5 and 20 min in WTand D110C/K892C-CFTR are shown in the middle panel of Fig. 11 A. Login to comment
375 Unlike WT-CFTR, D110C/K892C-CFTR exhibited strong inward rectification in symmetrical 150 mM Cl&#e032; solution. Login to comment
376 The current increase in D110C/K892C-CFTR after ATP and PKA activation was likely caused by DTT-mediated breaking of disulfide bonds between D110C and K892C, allowing more channels to be activated and resulting in a higher current amplitude. Login to comment
377 Meanwhile, the data also suggest that not all D110C/K892C-CFTR channels formed disulfide bonds in the resting state and that some channels could be activated by ATP and PKA in the absence of DTT. Login to comment
378 As a control, in the absence of DTT, D110C/K892C-CFTR macroscopic current reached plateau in &#e07a;5 min (like WT-CFTR) and was maintained or slightly decreased in the next &#e07a;20 min (Fig. S7 A). Login to comment
384 Figure 11.ߓ D110C forms a spontaneous disulfide bond with K892C when channels are in the closed state. Login to comment
385 (A) Representative macropatch currents of WTand D110C/K892C-CFTR were recorded in inside-out mode with symmetrical 150 mM Cl&#e032; solution. Login to comment
388 I-V plots of currents at times a-c for both WTand D110C/K892C-CFTR are shown in the middle panel. Login to comment
392 (B) 1 mM DTT further activated D110C/K892C-CFTR current but not WT-CFTR current in TEVC recording condition. Login to comment
393 Representative traces (left) and summary data (right) for macroscopic currents measured from WTand D110C/K892C-CFTR with addition of 1 mM DTT in the presence of ISO. Login to comment
397 **, P < 0.01 compared with ISO1 in n = 4 for WT-CFTR and n = 5 for D110C/K892C-CFTR experiments. Login to comment
398 (C) ISO plus DTT failed to further activate D110C/K892C-CFTR current in oocytes pretreated with DTT in TEVC recording condition. Login to comment
399 Representative trace (left) and summary data (right) for macroscopic currents measured from D110C/K892C-CFTR with prior addition of 1 mM DTT for 3 min in ND96 solution. Current levels in the summary data are given relative to control conditions before first exposure to ISO and normalized to maximal current in response to ISO1 (Imax). Login to comment
428 Meanwhile, D110 appears to form a salt bridge with K892 of ECL4 and the D110C/K892C spontaneous disulfide bond can only be formed when the channel is in the C0 state (0-ns snapshot in our molecular dynamics simulation; Rahman et al., 2013). Login to comment
430 To further test the existence of a possible open state salt bridge between R117 and E1126, we again asked whether cysteines engineered at positions 117 and 1126 might form a disulfide bond as in R104C/E116C- or D110C/K892C-CFTR. Login to comment
467 In the present work, we demonstrated that D110C/K892C-CFTR forms a spontaneous disulfide bond and appeared to lock the channel into the C0 state. Login to comment
493 In the current study, we identified two spontaneous disulfide bonds in CFTR formed after introduction of cysteines at positions 110 and 892 (a closed state disulfide bond in D110C/K892C-CFTR) or 104 and 116 (an open state disulfide bond in R104C/E116C-CFTR). Login to comment
495 The D110C/K892C disulfide bond was maintained regardless of CFTR gating energy until the reducing agent DTT was added to break it. Login to comment
497 These data suggest that D110C/K892C-CFTR forms a very strong disulfide bond with dissociation energy that must be greater than that of ATP-dependent NBD dimer formation. Login to comment