ABCC7 p.Pro99Cys
| ClinVar: |
c.296C>T
,
p.Pro99Leu
?
, not provided
|
| CF databases: |
c.296C>T
,
p.Pro99Leu
(CFTR1)
?
, This mutation was found together with [delta]F508 in a patient with extreme mild symptoms. It was found by SSCP and sequencing.
|
| Predicted by SNAP2: | A: D (85%), C: D (85%), D: D (80%), E: D (95%), F: D (91%), G: D (91%), H: D (91%), I: D (91%), K: D (95%), L: D (91%), M: D (91%), N: D (91%), Q: D (91%), R: D (95%), S: D (85%), T: D (85%), V: D (91%), W: D (95%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Application of high-resolution single-channel reco... Methods Mol Biol. 2011;741:419-41. Cai Z, Sohma Y, Bompadre SG, Sheppard DN, Hwang TC
Application of high-resolution single-channel recording to functional studies of cystic fibrosis mutants.
Methods Mol Biol. 2011;741:419-41., [PMID:21594800]
Abstract [show]
The patch-clamp technique is a powerful and versatile method to investigate the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, its malfunction in disease and modulation by small molecules. Here, we discuss how the molecular behaviour of CFTR is investigated using high-resolution single-channel recording and kinetic analyses of channel gating. We review methods used to quantify how cystic fibrosis (CF) mutants perturb the biophysical properties and regulation of CFTR. By explaining the relationship between macroscopic and single-channel currents, we demonstrate how single-channel data provide molecular explanations for changes in CFTR-mediated transepithelial ion transport elicited by CF mutants.
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No. Sentence Comment
298 However, Akabas et al. (63) concluded that P99 does not line the CFTR pore because the site-directed mutant P99C did not react with methanethiosulfonate reagents.
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ABCC7 p.Pro99Cys 21594800:298:108
status: NEW[hide] Functional arrangement of the 12th transmembrane r... Pflugers Arch. 2011 Oct;462(4):559-71. Epub 2011 Jul 28. Qian F, El Hiani Y, Linsdell P
Functional arrangement of the 12th transmembrane region in the CFTR chloride channel pore based on functional investigation of a cysteine-less CFTR variant.
Pflugers Arch. 2011 Oct;462(4):559-71. Epub 2011 Jul 28., [PMID:21796338]
Abstract [show]
The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) alpha-helices, arranged into two pseudo-symmetrical groups of six. While TM6 in the N-terminal TMs is known to line the pore and to make an important contribution to channel properties, much less is known about its C-terminal counterpart, TM12. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of TM12 in a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM12 residues N1138, M1140, S1141, T1142, Q1144, W1145, V1147, N1148, and S1149 when applied to the cytoplasmic side of open channels. Cysteines sensitive to internal MTS reagents were not modified by extracellular [2-(trimethylammonium)ethyl] MTS, consistent with MTS reagent impermeability. Both S1141C and T1142C could be modified by intracellular [2-sulfonatoethyl] MTS prior to channel activation; however, N1138C and M1140C, located deeper into the pore from its cytoplasmic end, were modified only after channel activation. Comparison of these results with previous work on CFTR-TM6 allows us to develop a model of the relative positions, functional contributions, and alignment of these two important TMs lining the CFTR pore. We also propose a mechanism by which these seemingly structurally symmetrical TMs make asymmetric contributions to the functional properties of the channel pore.
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No. Sentence Comment
140 In this respect, the slow rate of modification observed in N1138C (Fig. 3b) is similar to that we reported for P99C and L102C in TM1 [41] and T338C and S341C in TM6 [9], and the much higher modification rate constant for T1142C, S1141C, and (to a lesser extent) M1140C is closer to that reported for K95C in TM1 [41] and I344C, V345C, and M348C in TM6 [9].
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ABCC7 p.Pro99Cys 21796338:140:111
status: NEW[hide] Structure and function of the CFTR chloride channe... Physiol Rev. 1999 Jan;79(1 Suppl):S23-45. Sheppard DN, Welsh MJ
Structure and function of the CFTR chloride channel.
Physiol Rev. 1999 Jan;79(1 Suppl):S23-45., [PMID:9922375]
Abstract [show]
Structure and Function of the CFTR Chloride Channel. Physiol. Rev. 79, Suppl.: S23-S45, 1999. - The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ABC transporter family that forms a novel Cl- channel. It is located predominantly in the apical membrane of epithelia where it mediates transepithelial salt and liquid movement. Dysfunction of CFTR causes the genetic disease cystic fibrosis. The CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. Here we review the structure and function of this unique channel, with a focus on how the various domains contribute to channel function. The MSDs form the channel pore, phosphorylation of the R domain determines channel activity, and ATP hydrolysis by the NBDs controls channel gating. Current knowledge of CFTR structure and function may help us understand better its mechanism of action, its role in electrolyte transport, its dysfunction in cystic fibrosis, and its relationship to other ABC transporters.
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No. Sentence Comment
128 However, because P99C did(128) could control the pore properties of the CF-associated mutant R347H simply by manipulating pH.
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ABCC7 p.Pro99Cys 9922375:128:17
status: NEW[hide] Alignment of transmembrane regions in the cystic f... J Gen Physiol. 2011 Aug;138(2):165-78. Epub 2011 Jul 11. Wang W, El Hiani Y, Linsdell P
Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Gen Physiol. 2011 Aug;138(2):165-78. Epub 2011 Jul 11., [PMID:21746847]
Abstract [show]
Different transmembrane (TM) alpha helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl(-) channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84-T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore.
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No. Sentence Comment
71 In contrast, macroscopic currents carried by four mutants, K95C, Q98C, P99C, and L102C, were found to be significantly and rapidly sensitive to the application of both MTSES and MTSET (Figs. 1-3).
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ABCC7 p.Pro99Cys 21746847:71:71
status: NEW105 (B) Example leak-subtracted I-V relationships for cys-less CFTR, K95C, Q98C, P99C, L102C, and R104C, recorded from inside-out membrane patches after maximal channel activation with 20 nM PKA, 1 mM ATP, and 2 mM PPi.
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ABCC7 p.Pro99Cys 21746847:105:77
status: NEW112 As shown in Fig. 4 A, patches excised from MTSET-pretreated cells expressing K95C, Q98C, P99C, or L102C all gave macroscopic currents that were increased in amplitude after the addition of 2 mM constants was that modification was faster for cysteines introduced closer to the intracellular end of TM1, and slower for cysteines located more deeply along the axis of TM1 (Fig. 3 B).
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ABCC7 p.Pro99Cys 21746847:112:89
status: NEW136 Whereas K95C channels were again rendered insensitive to a test exposure to MTSES, again consistent with them having been covalently modified during pretreatment, currents carried by Q98C, P99C, MTSET to the intracellular solution.
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ABCC7 p.Pro99Cys 21746847:136:189
status: NEW138 These results suggest that none of K95C, Q98C, P99C, or L102C can be modified covalently by extracellular MTSET.
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ABCC7 p.Pro99Cys 21746847:138:47
status: NEW141 We used a similar approach to determine if K95C, Q98C, P99C, and L102C could be modified by MTSES pretreatment.
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ABCC7 p.Pro99Cys 21746847:141:55
status: NEW150 These results, which are summarized quantitatively in Fig. 5 C, suggest that although K95C can be modified by MTSES before channel activation, Q98C, P99C, and L102C are modified by MTSES only very slowly, if at all, in channels that have not been activated by PKA and ATP.
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ABCC7 p.Pro99Cys 21746847:150:149
status: NEW228 Thus, the side chains of TM1 mutants K95C, Q98C, P99C, and L102C that we identified as accessible to MTS reagents applied from the inside (Fig. 2) were not accessible to MTSET applied to the outside (Fig. 4), whereas R104C, previously shown to be modified by external MTS reagents (Zhou et al., 2008), was not modified by internal MTSES or MTSET (Fig. 2).
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ABCC7 p.Pro99Cys 21746847:228:49
status: NEW238 Although we have not investigated the state dependence of MTSES modification in TM1 in such great detail, our present results suggest a similar arrangement in which K95C can readily be modified before channel activation (Fig. 5), whereas Q98C, P99C, and L102C are modified rapidly after channel activation (Fig. 3) but very slowly if at all before activation (Fig. 5).
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ABCC7 p.Pro99Cys 21746847:238:244
status: NEW256 For comparison, the MTSES modification rate constant for P99C and L102C (Fig. 3) was similar to that of T338C and S341C in TM6 (El Hiani and Linsdell, 2010) (all between 100 and 150 M1 s1 ), and the modification rate constant for K95C was comparable to, or slightly greater than, that of I344C, V345C, and M348C (El Hiani and Linsdell, 2010) (all between 2,000 and 4,000 M1 s1 ).
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ABCC7 p.Pro99Cys 21746847:256:57
status: NEW[hide] Contribution of proline residues in the membrane-s... J Biol Chem. 1996 Jun 21;271(25):14995-5001. Sheppard DN, Travis SM, Ishihara H, Welsh MJ
Contribution of proline residues in the membrane-spanning domains of cystic fibrosis transmembrane conductance regulator to chloride channel function.
J Biol Chem. 1996 Jun 21;271(25):14995-5001., [PMID:8663008]
Abstract [show]
Proline residues located in membrane-spanning domains of transport proteins are thought to play an important structural role. In the cystic fibrosis transmembrane conductance regulator (CFTR), the predicted transmembrane segments contain four prolines: Pro99, Pro205, Pro324, and Pro1021. These residues are conserved across species, and mutations of two (P99L and P205S) are associated with cystic fibrosis. To evaluate the contribution of these prolines to CFTR Cl- channel function, we mutated each residue individually to either alanine or glycine or mutated all four simultaneously to alanine (P-Quad-A). We also constructed the two cystic fibrosis-associated mutations. cAMP agonists stimulated whole cell Cl- currents in HeLa cells expressing the individual constructs that resembled those produced by wild-type CFTR. However, the amount of current was decreased in the rank order: wild-type CFTR = Pro324 > Pro1021 > Pro99 >/= Pro205 mutants. The anion selectivity sequence of the mutants (Br- >/= Cl- > I-) resembled wild-type except for P99L (Br- >/= Cl- = I-). Although the Pro99, Pro324, and Pro1021 mutants produced mature protein, the amount of mature protein was much reduced with the Pro205 mutants, and the P-Quad-A made none. Because the Pro99 constructs produced mature protein but had altered whole cell currents, we investigated their single-channel properties. Mutant channels were regulated like wild-type CFTR; however, single-channel conductance was decreased in the rank order: wild-type CFTR >/= P99G > P99L >/= P99A. These results suggest that proline residues in the transmembrane segments are important for CFTR function, Pro205 is critical for correct protein processing, and Pro99 may contribute either directly or indirectly to the Cl- channel pore.
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No. Sentence Comment
204 Because P99C did not react with sulfhydryl-specific reagents, Akabas and collaborators concluded that Pro99 does not line the channel pore (7).
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ABCC7 p.Pro99Cys 8663008:204:8
status: NEW212 This suggests that the defective processing of the P205S mutant observed in HeLa cells likely accounts for the loss of Cl- channel function in patients bearing this mutation.
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ABCC7 p.Pro99Cys 8663008:212:8
status: NEW[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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No. Sentence Comment
196 Third, in the report by Wang et al. (35), K95C, but not Q98C, P99C, or L102C, can react with internal MTSES even before the channel is activated by PKA and ATP, implying a regulated barrier between positions 95 and 98.
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ABCC7 p.Pro99Cys 23442957:196:62
status: NEW263 (A) Representative single-channel traces for WT/Cysless, A96C/ Cysless, I106C/Cysless, and P99C mutant channels.
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ABCC7 p.Pro99Cys 23442957:263:91
status: NEW