ABCC7 p.Arg104Cys
| Predicted by SNAP2: | A: D (59%), C: D (71%), D: D (85%), E: D (63%), F: D (80%), G: D (59%), H: N (66%), I: D (75%), K: N (87%), L: D (53%), M: N (53%), N: N (61%), P: D (85%), Q: N (66%), S: N (57%), T: N (57%), V: D (75%), W: D (85%), Y: D (53%), |
| Predicted by PROVEAN: | A: N, C: D, D: D, E: N, F: N, G: D, H: N, I: N, K: N, L: N, M: N, N: N, P: D, Q: 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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
4 State-independent modification of R104C and R117C suggests that these residues are located at the outermost part of the pore.
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ABCC7 p.Arg104Cys 18449561:4:34
status: NEW89 The expected side chain charge at R104C and R117C following modification by these reagents mirrored the effects of mutation to Fig. 3 Charge and chloride dependence of current rectification in mutant forms of CFTR.
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ABCC7 p.Arg104Cys 18449561:89:34
status: NEW100 However, using the same MTS pretreatment protocols as in our previous study [2] (see "Materials and methods"), we found that both R104C and R117C could be modified by both MTSET and MTSES prior to channel activation, effectively mimicking the effects of inclusion of these substances in the pipette solution on rectification of the I-V relationship (Fig. 6).
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ABCC7 p.Arg104Cys 18449561:100:130
status: NEW101 This suggests that both positively and negatively charged MTS reagents can modify both R104C and R117C independently of the state of channel activation, a situation that contrasts with R334C, K335C, and other TM6 mutants.
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ABCC7 p.Arg104Cys 18449561:101:87
status: NEW103 As shown in Fig. 7, external application of pCMBS also increased the inward rectification seen in R104C and R117C, consistent with deposition of a negative charge on the cysteine present at these positions.
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ABCC7 p.Arg104Cys 18449561:103:98
status: NEW128 Our present study, together with previous work on R334 [6,8,22,26], has surveyed the effects of removing all permanent positive charges (contributed by arginine and lysine side chains) in the outer TMs and ECLs on the permeation properties of Fig. 6 Modification of R104C and R117C by MTSES is independent of channel activation.
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ABCC7 p.Arg104Cys 18449561:128:266
status: NEW131 b Mean rectification ratios for R104C (left) and R117C (right) under control conditions (open bars) and following modification by MTSET or MTSES using a pretreatment protocol (black bars) or by inclusion in the pipette (gray bars; see Fig. 5).
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ABCC7 p.Arg104Cys 18449561:131:32
status: NEW134 a Example relative current-voltage (IREL-V) relationships for R104C (left) and R117C (right) under control conditions and with MTSET or MTSES present in the pipette solution.
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ABCC7 p.Arg104Cys 18449561:134:62
status: NEW150 Mean of data from four to six patches in both b and c Fig. 7 Modification of R104C and R117C by pCMBS.
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ABCC7 p.Arg104Cys 18449561:150:77
status: NEW152 b Mean rectification ratios for R104C (left) and R117C (right) under control conditions (open bars) and following modification by pCMBS using a pretreatment protocol (black bars) or by inclusion in the pipette (gray bars).
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ABCC7 p.Arg104Cys 18449561:152:32
status: NEW[hide] Adrenoleukodystrophy: subcellular localization and... J Neurochem. 2007 Jun;101(6):1632-43. Takahashi N, Morita M, Maeda T, Harayama Y, Shimozawa N, Suzuki Y, Furuya H, Sato R, Kashiwayama Y, Imanaka T
Adrenoleukodystrophy: subcellular localization and degradation of adrenoleukodystrophy protein (ALDP/ABCD1) with naturally occurring missense mutations.
J Neurochem. 2007 Jun;101(6):1632-43., [PMID:17542813]
Abstract [show]
Mutation in the X-chromosomal adrenoleukodystrophy gene (ALD; ABCD1) leads to X-linked adrenoleukodystrophy (X-ALD), a severe neurodegenerative disorder. The encoded adrenoleukodystrophy protein (ALDP/ABCD1) is a half-size peroxisomal ATP-binding cassette protein of 745 amino acids in humans. In this study, we chose nine arbitrary mutant human ALDP forms (R104C, G116R, Y174C, S342P, Q544R, S606P, S606L, R617H, and H667D) with naturally occurring missense mutations and examined the intracellular behavior. When expressed in X-ALD fibroblasts lacking ALDP, the expression level of mutant His-ALDPs (S606L, R617H, and H667D) was lower than that of wild type and other mutant ALDPs. Furthermore, mutant ALDP-green fluorescence proteins (S606L and H667D) stably expressed in CHO cells were not detected due to rapid degradation. Interestingly, the wild type ALDP co-expressed in these cells also disappeared. In the case of X-ALD fibroblasts from an ALD patient (R617H), the mutant ALDP was not detected in the cells, but appeared upon incubation with a proteasome inhibitor. When CHO cells expressing mutant ALDP-green fluorescence protein (H667D) were cultured in the presence of a proteasome inhibitor, both the mutant and wild type ALDP reappeared. In addition, mutant His-ALDP (Y174C), which has a mutation between transmembrane domain 2 and 3, did not exhibit peroxisomal localization by immunofluorescense study. These results suggest that mutant ALDPs, which have a mutation in the COOH-terminal half of ALDP, including S606L, R617H, and H667D, were degraded by proteasomes after dimerization. Further, the region between transmembrane domain 2 and 3 is important for the targeting of ALDP to the peroxisome.
Comments [show]
None has been submitted yet.
No. Sentence Comment
216 The mutation of R104C and G116R is located in loop1 between TMD1 and 2, Y174 is in loop2 between TMD2 and 3, S342P and Q544R are located in TMD6 and the helical region between Walker A and B, respectively.
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ABCC7 p.Arg104Cys 17542813:216:16
status: NEW234 d R104C was degraded to several fragments when stably expressed in CHO cells (Fig. 4).
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ABCC7 p.Arg104Cys 17542813:234:2
status: NEW248 In the case of mutant ALDP (R104C), several degradation products were detected in the peroxisomal fraction on sucrose-gradient centrifugation (Fig. 4), suggesting that it might be degraded on peroxisomal membranes.
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ABCC7 p.Arg104Cys 17542813:248:28
status: NEW276 Furthermore, we found fragmentation of mutant ALDP (R104C) on peroxisomes which was not inhibited by proteasomes inhibitors, suggesting that additional protease(s) is also involved in the quality control of mutant ALDP.
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ABCC7 p.Arg104Cys 17542813:276:52
status: NEW[hide] [Adrenoleukodystrophy: structure and function of A... Yakugaku Zasshi. 2007 Jan;127(1):163-72. Takahashi N, Morita M, Imanaka T
[Adrenoleukodystrophy: structure and function of ALDP, and intracellular behavior of mutant ALDP with naturally occurring missense mutations].
Yakugaku Zasshi. 2007 Jan;127(1):163-72., [PMID:17202797]
Abstract [show]
Adrenoleukodystrophy (ALD) is an inherited disorder characterized by progressive demyelination of the central nervous system and adrenal dysfunction. The biochemical characterization is based on the accumulation of pathgnomonic amounts of saturated very long-chain fatty acid (VLCFA; C>22) in all tissues, including the brain white matter, adrenal glands, and skin fibroblasts, of the patients. The accumulation of VLCFA in ALD is linked to a mutation in the ALD (ABCD1) gene, an ABC subfamily D member. The ALD gene product, so-called ALDP (ABCD1), is thought to be involved in the transport of VLCFA or VLCFA-CoA into the peroxisomes. ALDP is a half-sized peroxisomal ABC protein and it has 745 amino acids in humans. ALDP is thought to be synthesized on free polysomes, posttranslationally transported to peroxisomes, and inserted into the membranes. During this process, ALDP interacts with Pex19p, a chaperone-like protein for intracellular trafficking of peroxisomal membrane protein (PMP), the complex targets Pex3p on the peroxisomal membranes, and ALDP is inserted into the membranes. After integration into the membranes, ALDP is thought to form mainly homodimers. Here, we chose nine arbitrary mutations of human ALDP with naturally occurring missense mutations and examined the intracellular behavior of their ALDPs. We found that mutant ALDP (S606L, R617H, and H667D) was degraded together with wild-type ALDP by proteasomes. These results suggest that the complex of mutant and wild-type ALDP is recognized as misfolded proteins and degraded by the protein quality control system associated with proteasomes. Further, we found fragmentation of mutant ALDP (R104C) on peroxisomes and it was not inhibited by proteasomes inhibitors, suggesting that an additional protease(s) is also involved in the quality control of mutant ALDP. In addition, mutation of ALDP (Y174C) suggests that a loop between transmembrane domains 2 and 3 is important for the targeting of ALDP to peroxisomes.
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No. Sentence Comment
49 変異型 ALDP の分解過程の解析 新生タンパク質が正しいフォールディングを受け ることは,そのタンパク質の正常な機能発現のため に必須である．遺伝子変異などが存在すると,タン パク質がミスフォールディングされる．このミスフ ォールドタンパクが細胞外へ分泌されたり,細胞内 に蓄積したりすると生体にとって極めて有害になる ため,このようなタンパクはプロテアソーム,リソ ソーム等によって迅速に分解される．ちなみに,嚢 胞性線維症の原因タンパク質 CFTR は細胞膜イオ ンチャネルとして機能する ABC タンパク質である が,変異 CFTR は小胞体膜からプロテアソームに リクルートされ分解されることが報告されてい る．32,33) しかしながら,変異型 ALDP を始めとし て,ペルオキシソーム膜タンパク質についての解析 はほとんど行われていない． 変異型 ALDP の一過性発現と安定過剰発現実験 より,ALDP(S606L, R617H, H667D, R104C)は, プロテアーゼにより分解されていると推定された． そこで,ALDP-GFP(H667D)を発現している CHO 細胞に各種プロテアーゼ阻害剤を処理し,解 析を行った．その結果,プロテアソーム阻害剤であ る lactacystin を処理した細胞では ALDP-GFP 及び ALDP の バ ン ド が 出 現 し た ( Fig. 4 )． 一 方 , leupeptin, AEBSF, E64d には効果がなかった．ま た他のプロテアソーム阻害剤である MG132 も有効 であった．さらにプロテアソーム阻害剤により分解 を逃れた変異型 ALDP-GFP(H667D)の細胞内局 在を蛍光抗体法で観察すると,ペルオキシソームに 局在していることが確認された．一方,変異型 ALDP(R104C)のフラグメント化は上記プロテアー ゼ処理では阻害されなかった． さらに ALD 患者由来細胞の内因性変異 ALDP の分解とプロテアソーム分解系の関与について確認 するため,変異型 ALDP(R617H)を持つ患者由 来線維芽細胞を用いてタンパク分解の阻害実験を行 った．その結果,lactacystin と MG132 処理により, ALDP のバンドが出現した．以上の結果より,ペ ルオキシソーム膜上にはミスフォールドしたタンパ ク質を認識する仕組みが存在し,プロテアソーム及 び他のプロテアーゼを介して排除していることが示 唆された． 一方,山田らは ALD 患者線維芽細胞を［35 S］メチ オニンでパルスチェイスすることにより,変異型 ALDP(G512S, R660W)の分解が E-64 と leupepu- tin により抑制されることを報告している．34) 彼ら の実験ではプロテアソーム阻害剤については実験し ていないので,プロテアソームの関与は不明である が,変異型 ALDP の分解には,複数のプロテアー ゼが関与している可能性がある． 7.
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ABCC7 p.Arg104Cys 17202797:49:2791
status: NEWX
ABCC7 p.Arg104Cys 17202797:49:4585
status: NEW[hide] Alternating access to the transmembrane domain of ... J Biol Chem. 2012 Mar 23;287(13):10156-65. Epub 2012 Feb 1. Wang W, Linsdell P
Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7).
J Biol Chem. 2012 Mar 23;287(13):10156-65. Epub 2012 Feb 1., [PMID:22303012]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.
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No. Sentence Comment
151 For example, L102C in TM1 is modified by internal, but not external MTS reagents (18), a result confirmed by the present results (Figs. 1 and 3), whereas R104C, only 2 residues closer to the external end of TM1, is modified by external, but not internal MTS reagents (28).
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ABCC7 p.Arg104Cys 22303012:151:154
status: NEW157 Modification rate constant for T338C/E1371Q was quantified from experiments using a higher concentration of MTSES (200 M).
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ABCC7 p.Arg104Cys 22303012:157:154
status: NEW163 One apparent problem with this explanation is that a residue only slightly closer to the outer end of TM1 (R104C) is accessible to extracellular, but not intracellular MTS reagents (see above); the model shown in Fig. 7A should put this residue close to Thr-338.
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ABCC7 p.Arg104Cys 22303012:163:107
status: NEW170 Rate of modification of T338C and L102C by external Au(CN)2 - .
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ABCC7 p.Arg104Cys 22303012:170:107
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
70 This list of MTS reagent-insensitive mutants includes R104C (Figs. 1 B and 2), which we have previously shown to be sensitive to modification by externally applied MTSES and MTSET (Zhou et al., 2008).
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ABCC7 p.Arg104Cys 21746847:70:54
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.Arg104Cys 21746847:105:94
status: NEW199 However, R104C was not sensitive to intracellularly applied MTS reagents (Figs. 1 and 2), consistent with the idea that such reagents are not able to permeate the CFTR pore (Alexander et al., 2009; El Hiani and Linsdell, 2010).
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ABCC7 p.Arg104Cys 21746847:199:9
status: NEW217 Interestingly, amino acid side chains only one to two residues closer to the outer ends of these TMs, R104C in TM1 and I336C in TM6, can be modified by external, but not internal, MTS reagents (Fig. 9).
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ABCC7 p.Arg104Cys 21746847:217:102
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.Arg104Cys 21746847:228:217
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
97 Opening rates for WTand R104C/ E116C-CFTR were measured as previously described except that R117A-CFTR were significantly lower than WT-CFTR (Fig. 2 D).
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ABCC7 p.Arg104Cys 25024266:97:24
status: NEW254 alone, R104C/E116C-CFTR exhibited very long stable openings with brief closed states and s1 and s2 subconductance states (Fig. 9 B, control).
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ABCC7 p.Arg104Cys 25024266:254:7
status: NEW260 If the long openings of R104C/E116C-CFTR were caused by the formation of a spontaneous disulfide bond, then the reducing agent DTT should break the disulfide bond and modify the channel behavior.
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ABCC7 p.Arg104Cys 25024266:260:24
status: NEW269 We reasoned that if a salt bridge between R104 and E116 is important for stabilizing the open state, the bifunctional linker MTS-2-MTS may lock the cysteine-substituted double mutant R104C/E116C-CFTR into the full open state by covalently binding to both engineered cysteines.
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ABCC7 p.Arg104Cys 25024266:269:183
status: NEW283 To test this idea further, we preincubated oocytes expressing R104C/E116C-CFTR with 5 mM MTSET+ for Figure 9.ߓ E116 forms a salt bridge with R104 in both the closed and open states.
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ABCC7 p.Arg104Cys 25024266:283:62
status: NEW286 (B) Two cysteines engineered at positions 104 and 116 (R104C/E116C) form a spontaneous disulfide bond when CFTR is in the open state.
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ABCC7 p.Arg104Cys 25024266:286:55
status: NEW287 Representative single-channel trace of R104C/E116C-CFTR recorded with the same conditions as A.
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ABCC7 p.Arg104Cys 25024266:287:39
status: NEW290 +DTT in pipette: R104C/E116C-CFTR recorded with 1 mM DTT in the extracellular pipette solution (left, middle trace).
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ABCC7 p.Arg104Cys 25024266:290:17
status: NEW291 In the bottom trace, oocytes expressing R104C/ E116C-CFTR were incubated in solution containing 5 mM MTSET+ over 10 min before single-channel current recording (+MTSET; left, bottom trace).
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ABCC7 p.Arg104Cys 25024266:291:40
status: NEW293 Mean fraction of open burst duration is plotted at right for R104C/E116C-CFTR under three different experimental conditions, for each of the open conductance states: s1, dark red; s2, orange; and f, light green.
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ABCC7 p.Arg104Cys 25024266:293:61
status: NEW294 (C) Cross-linking R104C to E116C using MTS-2-MTS locks CFTR channels into the closed state.
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ABCC7 p.Arg104Cys 25024266:294:18
status: NEW295 Representative trace (left) and summary data (right) for macroscopic currents measured from R104C/E116C-CFTR with addition of 1 mM MTS-2-MTS in the absence of ISO at VM = &#e032;60 mV.
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ABCC7 p.Arg104Cys 25024266:295:92
status: NEW317 The required proximity for a salt bridge is confirmed by our finding that the thiol groups of two engineered cysteines at these positions are in close enough proximity in the open state to form a spontaneous disulfide bond (&#e07a;2-3 &#c5;); (b) R104C and/or E116C do not contribute directly to ion conduction and permeation through CFTR because both could be modified by MTSET without affecting channel conductance.
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ABCC7 p.Arg104Cys 25024266:317:247
status: NEW319 The R104C/E116C spontaneous open state disulfide bond exhibited the following characteristics: (a) R104C/ E116C-CFTR still required ATP and PKA for activation.
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ABCC7 p.Arg104Cys 25024266:319:4
status: NEWX
ABCC7 p.Arg104Cys 25024266:319:99
status: NEW320 (b) R104C/E116C-CFTR exhibited an intraburst closed state even in the absence of DTT that is long enough to represent true channel closures, suggesting that the spontaneous disulfide bond is not strong enough to lock the channel into the open state but rather the channel is still affected by NBD-mediated gating, although to a much lower degree than the WT.
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ABCC7 p.Arg104Cys 25024266:320:4
status: NEW323 Homology modeling and simulation predict that R104 and E116 might remain very close to each other and form a salt bridge when the channel is in the closed state as well; we therefore asked whether the bifunctional cross-linker MTS-2-MTS would lock R104C/E116C-CFTR closed when applied in the closed state.
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ABCC7 p.Arg104Cys 25024266:323:248
status: NEW324 Representative data are shown in Fig. 9 C. R104C/E116C-CFTR could be reversibly activated and reactivated by ISO (ISO1 and ISO2) without significant decrement.
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ABCC7 p.Arg104Cys 25024266:324:43
status: NEW328 The closed state MTS-2-MTS cross-link bond also showed a clear difference from the R104C/E116C open state spontaneous with a single exponential function with &#e074; = 5.33 min, which suggests that it takes &#e07a;7-8 min for WT-CFTR current to reach its plateau.
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ABCC7 p.Arg104Cys 25024266:328:83
status: NEW422 We found that E116 of ECL1 forms a salt bridge with R104 of TM1 in both the closed and open states, and the two amino acids are very close to each other when the channel is in the open state because R104C only forms a spontaneous disulfide bond with E116C in this state.
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ABCC7 p.Arg104Cys 25024266:422:199
status: NEW430 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.
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ABCC7 p.Arg104Cys 25024266:430:195
status: NEW493 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).
X
ABCC7 p.Arg104Cys 25024266:493:240
status: NEW496 In contrast, the R104C/E116C disulfide bond was occasionally broken during normal NBD-mediated gating in the presence of ATP, although the closed state was rather brief.
X
ABCC7 p.Arg104Cys 25024266:496:17
status: NEW498 In contrast, R104C/E116C forms a relatively weak disulfide bond, most likely because of its dihedral angle being dramatically off from 90&#b0;, and thus the dissociation energy is likely below that of ATP hydrolysis and subsequent NBD dedimerization.
X
ABCC7 p.Arg104Cys 25024266:498:13
status: NEW