ABCMdb

ABCC7 p.Ser1118Phe

ClinVar:	c.3353C>T , p.Ser1118Phe ? , not provided c.3353C>G , p.Ser1118Cys ? , not provided
CF databases:	c.3353C>G , p.Ser1118Cys (CFTR1) D , The mutation was detected by multiplex heteroduplex analysis on the MDE gel matrix. It was found in one Canadian CBAVD patient (second mutation: [delta]F508). c.3353C>T , p.Ser1118Phe (CFTR1) ? , CF patient.
Predicted by SNAP2:	A: N (87%), C: N (87%), D: D (63%), E: D (63%), F: D (53%), G: N (82%), H: D (53%), I: D (63%), K: D (66%), L: N (57%), M: D (63%), N: N (61%), P: N (53%), Q: D (53%), R: D (66%), T: N (82%), V: N (61%), W: D (75%), Y: D (63%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, T: N, V: N, W: D, Y: N,

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Publications
[hide] Perturbation of the pore of the cystic fibrosis tr... J Biol Chem. 2001 Apr 13;276(15):11575-81. Epub 2000 Dec 21. Kogan I, Ramjeesingh M, Huan LJ, Wang Y, Bear CE
Perturbation of the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits its atpase activity.
J Biol Chem. 2001 Apr 13;276(15):11575-81. Epub 2000 Dec 21., 2001-04-13 [PMID:11124965]

Abstract [show]
Mutations in the cystic fibrosis gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) lead to altered chloride (Cl(-)) flux in affected epithelial tissues. CFTR is a Cl(-) channel that is regulated by phosphorylation, nucleotide binding, and hydrolysis. However, the molecular basis for the functional regulation of wild type and mutant CFTR remains poorly understood. CFTR possesses two nucleotide binding domains, a phosphorylation-dependent regulatory domain, and two transmembrane domains that comprise the pore through which Cl(-) permeates. Mutations of residues lining the channel pore (e.g. R347D) are typically thought to cause disease by altering the interaction of Cl(-) with the pore. However, in the present study we show that the R347D mutation and diphenylamine-2-carboxylate (an open pore inhibitor) also inhibit CFTR ATPase activity, revealing a novel mechanism for cross-talk from the pore to the catalytic domains. In both cases, the reduction in ATPase correlates with a decrease in nucleotide turnover rather than affinity. Finally, we demonstrate that glutathione (GSH) inhibits CFTR ATPase and that this inhibition is altered in the CFTR-R347D variant. These findings suggest that cross-talk between the pore and nucleotide binding domains of CFTR may be important in the in vivo regulation of CFTR in health and disease.

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No. Sentence Comment
176 With regards to CFTR, there is indirect evidence supporting interaction between permeation and gating, in that certain mutations in the transmembrane segments of CFTR, namely S1118F in TM11 (33) and the disease-causing mutant R117H (50), exhibit altered channel open times. Login to comment

[hide] Relationship between anion binding and anion perme... J Physiol. 2001 Feb 15;531(Pt 1):51-66. Linsdell P
Relationship between anion binding and anion permeability revealed by mutagenesis within the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Physiol. 2001 Feb 15;531(Pt 1):51-66., 2001-02-15 [PMID:11179391]

Abstract [show]
1. Anion binding within the pores of wild-type and mutant cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, expressed in two different mammalian cell lines, was assayed using patch clamp recording. Specifically, experiments measured both the conductance of different anions and the ability of other permeant anions to block Cl- permeation through the pore. 2. Under symmetrical ionic conditions, wild-type CFTR channels showed the conductance sequence Cl- > NO3- > Br- > or = formate > F- > SCN- congruent to ClO4-. 3. High SCN- conductance was not observed, nor was there an anomalous mole fraction effect of SCN- on conductance under the conditions used. Iodide currents could not be measured under symmetrical ionic conditions, but under bi-ionic conditions I- conductance appeared low. 4. Chloride currents through CFTR channels were blocked by low concentrations (10 mM) of SCN-, I- and ClO4-, implying relatively tight binding of these anions within the pore. 5. Two mutations in CFTR which alter the anion permeability sequence, F337S and T338A, also altered the anion conductance sequence. Furthermore, block by SCN-, I- and ClO4- were weakened in both mutants. Both these effects are consistent with altered anion binding within the pore. 6. The effects of mutations on anion permeability and relative anion conductance suggested that, for most anions, increased permeability was associated with increased conductance. This indicates that the CFTR channel pore does not achieve its anion selectivity by selective anion binding within the mutated region. Instead, it is suggested that entry of anions into the region around F337 and T338 facilitates their passage through the pore. In wild-type CFTR channels, anion entry into this crucial pore region is probably dominated by anion hydration energies.

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No. Sentence Comment
254 Interestingly, S1118 in TM11 has been suggested to occupy a position similar to that of T338 in TM6, and the mutations S1118A and S1118F cause small alterations in anion permeability (Zhang et al. 2000). Login to comment

[hide] Asymmetric structure of the cystic fibrosis transm... Biochemistry. 2001 Jun 5;40(22):6620-7. Gupta J, Evagelidis A, Hanrahan JW, Linsdell P
Asymmetric structure of the cystic fibrosis transmembrane conductance regulator chloride channel pore suggested by mutagenesis of the twelfth transmembrane region.
Biochemistry. 2001 Jun 5;40(22):6620-7., 2001-06-05 [PMID:11380256]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel contains 12 membrane-spanning regions which are presumed to form the transmembrane pore. Although a number of findings have suggested that the sixth transmembrane region plays a key role in forming the pore and determining its functional properties, the role of other transmembrane regions is currently not well established. Here we assess the functional importance of the twelfth transmembrane region, which occupies a homologous position in the carboxy terminal half of the CFTR molecule to that of the sixth transmembrane region in the amino terminal half. Five residues in potentially important regions of the twelfth transmembrane region were mutated individually to alanines, and the function of the mutant channels was examined using patch clamp recording following expression in mammalian cell lines. Three of the five mutations significantly weakened block of unitary Cl(-) currents by SCN(-), implying a partial disruption of anion binding within the pore. Two of these mutations also caused a large reduction in the steady-state channel mean open probability, suggesting a role for the twelfth transmembrane region in channel gating. However, in direct contrast to analogous mutations in the sixth transmembrane region, all mutants studied here had negligible effects on the anion selectivity and unitary Cl(-) conductance of the channel. The relatively minor effects of these five mutations on channel permeation properties suggests that, despite their symmetrical positions within the CFTR protein, the sixth and twelfth transmembrane regions make highly asymmetric contributions to the functional properties of the pore.

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Sentences [show]
No. Sentence Comment
187 A point mutation in TM11 (S1118F) was recently shown to cause a dramatic reduction in mean channel open burst duration, implying a decrease in the stability of the channel open state (42). Login to comment

[hide] Time-dependent interactions of glibenclamide with ... J Membr Biol. 2004 Oct 1;201(3):139-55. Zhang ZR, Cui G, Zeltwanger S, McCarty NA
Time-dependent interactions of glibenclamide with CFTR: kinetically complex block of macroscopic currents.
J Membr Biol. 2004 Oct 1;201(3):139-55., 2004-10-01 [PMID:15711774]

Abstract [show]
Blockade of the CFTR chloride channel by glibenclamide was studied in Xenopus oocytes using two-electrode voltage-clamp recordings, macropatch recordings, and summations of single-channel currents, in order to test a kinetic model recently developed by us from single-channel experiments. Both the forward and reverse macroscopic reactions, at negative and positive membrane potential V(M), respectively, were slow in comparison to those reactions for other CFTR pore blockers such as DPC and NPPB, resulting in prominent relaxations on the order of tens of milliseconds. The rate of the reverse reaction was voltage-dependent, and dependent on the Cl(-) driving force, while that of the forward reaction was not. In inside-out macropatches, block and relief from block occurred in two distinct phases that differed in apparent affinity. The results are consistent with the presence of multiple glibenclamide binding sites in CFTR, with varying affinity and voltage dependence; they support the kinetic model and suggest experimental approaches for identification of those sites by mutagenesis.

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No. Sentence Comment
300 Unblocked macroscopic currents in WT-CFTR and nearly all mutants studied to date are time-independent (exceptions are S1118F-CFTR and V317E-CFTR, which confer voltage-jump relaxations on currents in the absence of added blockers; Zhang, McDonough & McCarty, 2000b; Zhang et al. 2002). Login to comment

[hide] Novel residues lining the CFTR chloride channel po... J Membr Biol. 2009 Apr;228(3):151-64. Epub 2009 Apr 19. Fatehi M, Linsdell P
Novel residues lining the CFTR chloride channel pore identified by functional modification of introduced cysteines.
J Membr Biol. 2009 Apr;228(3):151-64. Epub 2009 Apr 19., [PMID:19381710]

Abstract [show]
Substituted cysteine accessibility mutagenesis (SCAM) has been used widely to identify pore-lining amino acid side chains in ion channel proteins. However, functional effects on permeation and gating can be difficult to separate, leading to uncertainty concerning the location of reactive cysteine side chains. We have combined SCAM with investigation of the charge-dependent effects of methanethiosulfonate (MTS) reagents on the functional permeation properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. We find that cysteines substituted for seven out of 21 continuous amino acids in the eleventh and twelfth transmembrane (TM) regions can be modified by external application of positively charged [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium [2-sulfonatoethyl] MTS (MTSES). Modification of these cysteines leads to changes in the open channel current-voltage relationship at both the macroscopic and single-channel current levels that reflect specific, charge-dependent effects on the rate of Cl(-) permeation through the channel from the external solution. This approach therefore identifies amino acid side chains that lie within the permeation pathway. Cysteine mutagenesis of pore-lining residues also affects intrapore anion binding and anion selectivity, giving more information regarding the roles of these residues. Our results demonstrate a straightforward method of screening for pore-lining amino acids in ion channels. We suggest that TM11 contributes to the CFTR pore and that the extracellular loop between TMs 11 and 12 lies close to the outer mouth of the pore.

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Sentences [show]
No. Sentence Comment
201 Previously, SCN- permeabil- ity was shown to be significantly decreased in S1118F but unaltered in S1118A (Zhang et al. 2000). Login to comment

[hide] Clinical and molecular characterization of S1118F-... Pediatr Pulmonol. 2009 Oct;44(10):1003-9. Penmatsa H, Frederick CA, Nekkalapu S, Conoley VG, Zhang W, Li C, Kappes J, Stokes DC, Naren AP
Clinical and molecular characterization of S1118F-CFTR.
Pediatr Pulmonol. 2009 Oct;44(10):1003-9., [PMID:19774621]

Abstract [show]
BACKGROUND: Cystic fibrosis is a lethal autosomal recessive disorder usually associated with lung disease, pancreatic insufficiency and high sweat chloride levels. CLINICAL CASE: A patient admitted to Le Bonheur Children's Medical Center (LBCMC, Memphis, TN) showed symptoms of meconium ileus which required exploratory laparotomy, bowel resection and ileostomy. Genotyping showed DeltaF508/I1027T on one chromosome and S1118F on the other. Sweat testing on three different occasions gave negative and intermediate results (22.7, 24.6 mmol/L; 55.1, 58.6 mmol/L and 55.1, 58 mmol/L) and pancreatic elastase testing showed normal levels. OBJECTIVE: To characterize S1118F-CFTR mutation at a molecular level to help understand the associated CF-phenotype. METHODS: Molecular characterization of S1118F-CFTR mutant was studied in HEK-293 cells at 37 degrees C. Various biochemical methods such as Western blotting, real-time PCR, Pulse chase labeling and iodide efflux assay were employed. RESULTS: S1118F-CFTR makes less than 10-15% of mature CFTR (band C) compared to WT-CFTR. The mRNA levels of S1118F-CFTR and WT-CFTR are comparable. S1118F-CFTR is functional but shows about 10-15% of WT-CFTR activity. S1118F-CFTR shows impaired maturation and CF-correctors can increase the amount of mature and functional CFTR by three- to fourfold. CONCLUSION: S1118F-CFTR shows impaired maturation and an individual with S1118F-CFTR paired with DeltaF508-CFTR exhibits atypical CF symptoms with intermediate sweat chloride level and meconium ileus despite documented pancreatic sufficiency.

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No. Sentence Comment
8 Results: S1118F-CFTR makes less than 10-15% of mature CFTR (band C) compared to WT-CFTR. Login to comment
9 The mRNA levels of S1118F-CFTR and WT-CFTR are comparable. Login to comment
10 S1118F-CFTR is functional but shows about 10-15% of WT-CFTR activity. Login to comment
11 S1118F-CFTR shows impaired maturation and CF-correctors can increase the amount of mature and functional CFTR by three- to fourfold. Login to comment
12 Conclusion: S1118F-CFTR shows impaired maturation and an individual with S1118F-CFTR paired with DF508-CFTRexhibits atypical CF symptomswith intermediate sweat chloride level and meconium ileus despite documented pancreatic sufficiency. Login to comment
39 This class of mutations includes C-terminal truncated CFTR which form unstable mature protein with five- to sixfold faster degradation rate than WT-CFTR.2-7 We present herein a clinical case of a CF patient with intermediate sweat chloride levels, pancreatic sufficiency, meconium ileus and mutations in the CFTR gene (DF508 and S1118F). Login to comment
40 The objective of this study is to characterize S1118F-CFTR mutation at protein level to help understand the associated CF-phenotype. Login to comment
42 MATERIALS AND METHODS Genotyping Genotyping was performed at Ambry Genetics (Aliso Viejo, CA) which showed DF508/I1027Ton one chromosome and S1118F on the other. Login to comment
45 Site-directed mutagenesis was performed by a single point mutation in eukaryotic expression vector (pcDNA3) containing WT-CFTR cDNA using Quikchange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and appropriate primers to make S1118F-CFTR mutant. Login to comment
50 Pulse Chase Labeling HEK-293 cells transiently transfected with WTor S1118F-CFTR cDNA were incubated in MEM medium (Invitrogen) without methionine and cysteine for 60 min at 378C. Login to comment
56 Probe designed by Universal ProbeLibrary (Roche, Indianapolis, IN) was used and TATA-box binding protein (TBP) was used as the house keeping gene.10 Iodide Efflux HEK-293 cells were transfected with 2 mg of pcDNA3 WT-CFTR, pcDNA3 S1118F-CFTR or empty vector (pcDNA3) cDNA. Login to comment
65 RESULTS An Individual Harboring S1118F-CFTR Exhibits Atypical CF Symptoms A Caucasian infant was born at 376=7 weeks by cesarean section in Mississippi (USA). Login to comment
72 Genotyping was sent to Ambry Genetics which showed DF508/I1027T on one chromosome and S1118F on the other. Login to comment
81 S1118F-CFTR Makes Less Than 10-15% of Mature CFTR (band C) Compared to WT-CFTR Previous studies in Xenopus oocytes have shown that S1118F-CFTR is a mutant with altered permeation and gating characteristics compared to WT-CFTR.14 It is therefore likely that this mutant belongs to Class IV CFTR mutations. Login to comment
83 Using Quickchange method, we generated S1118F mutation on pcDNA3-CFTR background. Login to comment
87 The data shows that S1118F-CFTR appears as a 180 kDa immunoreactive band (band C; the mature CFTR) but is less than 10-15% of that for WT-CFTR (Fig. 1A). Login to comment
89 Iodide efflux measurements were performed using HEK-293 cells transiently transfected with WTor S1118F-CFTR cDNA. Login to comment
90 As shown in Figure 1B and C, S1118F-CFTR is functional but has much lower activity (10-15%) compared to WT-CFTR. Login to comment
92 In an attempt to define which class this mutation might belong to, we compared S1118F-CFTR with other well-defined classes of CFTR-mutations such as DF508-CFTR (Class II), G551D-CFTR (Class III) and R117H-CFTR (Class IV) and the data is shown in Figure 1D. It is likely that S1118F-CFTR is a trafficking mutant with impaired maturation. Login to comment
93 S1118F-CFTR Shows Impaired Maturation To test if the reduced expression level of S1118F-CFTR is due to reduced maturation efficiency, we performed pulse chase experiments by treating HEK293 cells expressing S1118F-CFTR or WT-CFTR with a brief pulse for 30 min. Login to comment
96 However, the maturation is not efficient for S1118F-CFTR and only a small portion of band B was converted to band C (impaired maturation). Login to comment
97 These biochemical studies are consistent with a mild CF-phenotype observed for individuals harboring S1118F-CFTR as it is a trafficking mutant with impaired maturation efficiency. Login to comment
98 To investigate if S1118F-CFTR mRNA has reduced stability, real-time PCR was performed to compare mRNA Pediatric Pulmonology expression levels in HEK-293 cells transfected with equal amounts of WTor S1118F-CFTR cDNA. Login to comment
99 The results showed that the mRNA expression level for S1118F-CFTR is not reduced compared to WT-CFTR (Fig. 3). Login to comment
100 It is therefore reasonable to propose that S1118F-CFTR does not belong to the class V mutations. Login to comment
101 CF-Correctors Can Increase the Amount and Function of S1118F-CFTR Several Folds To test if S1118F-CFTR can be rescued by using CF-correctors, HEK-293 cells expressing WTor S1118F-CFTR was incubated with CF-corrector C1 [6-(1H- benzomidazol-2-ylsulfanylmethyl)-2-(6-methoxy-4-methyl- quinazolin-2-ylamino)-pyrimidin-4-ol] or C3 [(4-cyclohexyloxy-2-{1-[4-(4-methoxy-benzensulfonyl)-piperazin- 1-yl]-ethyl}-quinazoline], lysed, immunoprecipitated and probed for CFTR. Login to comment
102 The result showed that treatment of HEK-293 cells expressing S1118F-CFTR with these CF-correctors did increase the mature form of CFTR. Login to comment
105 Iodide efflux experiments were performed by using HEK-293 cells expressing S1118F-CFTR. Login to comment
106 As shown in Figure 4B,C, in the presence of CF-Corrector C3, the function of S1118F-CFTR increases two- to fourfold. Login to comment
107 This result is consistent with the Western Blotting result suggesting that S1118F-CFTR mutant can be functionally rescued by using CF-correctors. Login to comment
108 DISCUSSION S1118F-CFTR is a serine-to-phenylalanine mutation in the 11th transmembrane-spanning domain (TMD) of Pediatric Pulmonology Fig. 1. Login to comment
109 Characterization of S1118F-CFTR. Login to comment
110 A: HEK-293 cells were transiently transfected with pcDNA3 containing WT-, S1118F- or DF508-CFTR cDNA, lysed after 48 hr, immunoprecipitated and western blotted for CFTR (a 24-1 mab). Login to comment
111 The experiments were performed 4 times. B: Iodide efflux assay in HEK-293 cells transiently transfected with WTor S1118F-CFTR cDNA. Login to comment
112 PKA activating agonist cocktail (containing 10 mM forskolin, 100 mM IBMX and 200 mM cpt-cAMP) was added to activate CFTR. Line graph is Iodide efflux (nmol/min) as mean of 3 (WT and Control) or 5 (S1118F-CFTR) experiments with standard error mean (SEM). Login to comment
114 D: HEK-293cells weretransiently transfected with pCDNA3 containing WT-, S1118F-, R117H-, G551D- or DF508-CFTR cDNA, lysed 48 hr later, immunoprecipitated (a 24-1 mab) and western blotted for CFTR (a 24-1 mab). Login to comment
119 expressed S1118F-CFTR mutant in stage V Xenopus oocytes and studied the channel properties electrophysiologically. Login to comment
122 S1118F-CFTR shows impaired maturation. Login to comment
123 A: Pulse-chase experiments to monitor the formation of mature protein over a period of 2 hr in HEK-293 cells transfected with WTor S1118F-CFTR cDNA. Login to comment
124 The experiments were performed 4 times. B: Quantitation of mature(band C) andimmature (bandB) protein in WTand S1118F-CFTR from pulse-chase experiment showing impaired maturation of S1118F-CFTR. Login to comment
125 The band B at time ''0`` was considered as 100% for WTand S1118F-CFTR, respectively. Login to comment
127 Real-time PCR for comparing mRNA expression levels in HEK-293 cells transfected with equal amounts of WTand S1118F-CFTR cDNA. Login to comment
129 The change in relative expression levels between WT-CFTR and S1118F-CFTR was found to be insignificant. Login to comment
132 CF-correctors can increase the amount and function of S1118F-CFTR. Login to comment
134 The experiments were repeated for 4 times. B: Iodide efflux assay in HEK-293 cells transiently transfected with S1118F-CFTR with or without CF-corrector C3 treatment (5 mM). Login to comment
142 To help understand this mild CF-phenotype, we used various biochemical methods to characterize S1118F-CFTR mutant at protein level. Login to comment
146 Secondly, to investigate if S1118F-CFTR is functional (as a ClÀ channel), we performed iodide efflux experiments. Login to comment
147 The result showed S1118F-CFTR is functional but has much lower activity compared to WT-CFTR (Fig. 1B,C). Login to comment
148 This functional result is consistent with the protein expression data and with Zhang`s result which showed S1118F-CFTR is a functional ClÀ channel yet with altered permeation and gating characteristics compared to WT-CFTR.14 Thirdly, we compared S1118F-CFTR with other well-defined CF-mutations using Western blotting. Login to comment
150 Based on the observations as described above and Zhang`s result,14 it is therefore reasonable to assume that S1118F-CFTR might be a trafficking mutant with impaired maturation. Login to comment
151 Pulse chase experiments were employed to test the maturation efficiency of S1118F-CFTR. Login to comment
153 Next, to investigate if the impaired maturation is due to the reduced mRNA stability, we performed real-time PCR to compare mRNA expression levels in HEK293 cells transfected with equal amounts of WTor S1118F-CFTR cDNA. Login to comment
154 The results showed that the mRNA expression level for S1118F-CFTR is not reduced compared to WT-CFTR suggesting that S1118F-CFTR does not belong to class V mutations (Fig. 3). Login to comment
158 The use of pharmacological chaperones (CF-correctors) to promote the maturation of such mutants is one of the promising approaches.19-22 To test if the maturation efficiency for S1118F-CFTR can be improved by using CF-correctors, we treated HEK-293 cells expressing S1118F-CFTR with CF-correctors C1 and C3. Login to comment
160 In the presence of CF-corrector C3, the mature band C increased by three- to fourfold over untreated cells and the function of S1118F-CFTR increased two- to fourfold as well in iodide efflux experiments. Login to comment
161 Consistent with a mild CF-phenotype observed for a patient, our studies suggest that S1118F-CFTR is likely to be trafficking mutant with impaired maturation. Login to comment
163 To our knowledge, this is the first study in which S1118F-CFTR mutant was fully characterized at the protein level. Login to comment

[hide] Detecting Common CFTR Mutations by Reverse Dot Blo... Iran J Pediatr. 2011 Mar;21(1):51-7. Dooki MR, Akhavan-Niaki H, Juibary AG
Detecting Common CFTR Mutations by Reverse Dot Blot Hybridization Method in Cystic Fibrosis First Report from Northern Iran.
Iran J Pediatr. 2011 Mar;21(1):51-7., [PMID:23056764]

Abstract [show]
OBJECTIVE: Cystic fibrosis and its distribution vary widely in different countries and/or ethnic groups. Common cystic fibrosis transmembrane conductance regulator (CFTR) mutations were reported from Iran, but the northern population was not or underrepresented in those studies. The aim of this study was to determine the frequency of common CFTR mutations in children from northern Iran. METHODS: Thirty unrelated Iranian cystic fibrosis patients aged less than 11 years and living in Mazandaran province (in Iran) were screened for 5 common CFTR gene mutations. deltaF508, N1303K, G542X, R347H and W1282X using Reverse Dot Blot method. FINDINGS: Only one mutation, DeltaF508, was found in 7 patients accounting for 21.7% (13/60) of alleles. CONCLUSION: These findings can be used for planning future screening and appropriate genetic counseling programs in Iranian CF families.

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No. Sentence Comment
234 Penmatsa H, Frederick CA, Nekkalapu S, et al. Clinical and molecular characterization of S1118F-CFTR. Login to comment
237 Penmatsa H, Frederick CA, Nekkalapu S, et al. Clinical and molecular characterization of S1118F-CFTR. Login to comment

[hide] Interaction between permeation and gating in a put... Biophys J. 2000 Jul;79(1):298-313. Zhang ZR, McDonough SI, McCarty NA
Interaction between permeation and gating in a putative pore domain mutant in the cystic fibrosis transmembrane conductance regulator.
Biophys J. 2000 Jul;79(1):298-313., [PMID:10866956]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel with distinctive kinetics. At the whole-cell level, CFTR currents in response to voltage steps are time independent for wild type and for the many mutants reported so far. Single channels open for periods lasting up to tens of seconds; the openings are interrupted by brief closures at hyperpolarized, but not depolarized, potentials. Here we report a serine-to-phenylalanine mutation (S1118F) in the 11th transmembrane domain that confers voltage-dependent, single-exponential current relaxations and moderate inward rectification of the macroscopic currents upon expression in Xenopus oocytes. At steady state, the S1118F-CFTR single-channel conductance rectifies, corresponding to the whole-cell rectification. In addition, the open-channel burst duration is decreased 10-fold compared with wild-type channels. S1118F-CFTR currents are blocked in a voltage-dependent manner by diphenylamine-2-carboxylate (DPC); the affinity of S1118F-CFTR for DPC is similar to that of the wild-type channel, but blockade exhibits moderately reduced voltage dependence. Selectivity of the channel to a range of anions is also affected by this mutation. Furthermore, the permeation properties change during the relaxations, which suggests that there is an interaction between gating and permeation in this mutant. The existence of a mutation that confers voltage dependence upon CFTR currents and that changes kinetics and permeation properties of the channel suggests a functional role for the 11th transmembrane domain in the pore in the wild-type channel.

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Sentences [show]
No. Sentence Comment
2 Here we report a serine-to-phenylalanine mutation (S1118F) in the 11th transmembrane domain that confers voltage-dependent, single-exponential current relaxations and moderate inward rectification of the macroscopic currents upon expression in Xenopus oocytes. Login to comment
3 At steady state, the S1118F-CFTR single-channel conductance rectifies, corresponding to the whole-cell rectification. Login to comment
5 S1118F-CFTR currents are blocked in a voltage-dependent manner by diphenylamine-2-carboxylate (DPC); the affinity of S1118F-CFTR for DPC is similar to that of the wild-type channel, but blockade exhibits moderately reduced voltage dependence. Login to comment
22 Here we report a serine-to-phenylalanine mutation (S1118F) in TM11 that affects both permeation and gating. Login to comment
23 Most strikingly, the macroscopic S1118F-CFTR currents display voltage-dependent current relaxations; these relaxations are modulated by permeating anions. Login to comment
33 Preparation of oocytes and cRNA CFTR was subcloned into the pALTER vector (Promega), and the S1118F, S1118A, and S1118F/F1111S mutations were made using the Promega Altered Sites protocol (McDonough et al., 1994). Login to comment
80 Unexpectedly, the most obvious consequence of the mutation of S1118F-CFTR is the appearance of relaxations in current density upon stepping to a new membrane potential. Login to comment
85 However, S1118F-CFTR displays voltage-dependent current relaxations in response to protocols applying either depolarizing or hyperpolarizing prepotentials. Login to comment
87 These voltage-jump relaxations lead to a larger conductance for jumps to more negative potentials and to a smaller conductance for jumps in the FIGURE 1 Mutation S1118F-CFTR results in current relaxations. Login to comment
93 (B and C) Families of currents measured in oocytes expressing either wild-type or S1118F-CFTR channels, respectively. Login to comment
104 Note that S1118F-CFTR also displays tail currents (Figs. 1, 2, 6, and 7), which have not been described for WT or any mutant of CFTR studied thus far. Login to comment
116 In WT-CFTR, TM11 contains four phenylalanine residues (Fig. 1 A); mutation of S1118F adds a fifth. Login to comment
117 If this domain is ॷ-helical, as predicted from hydropathy analysis (Riordan et al., 1989), mutation S1118F would then place three phenylalanine residues in close mutual proximity (at positions 1107, 1111, and 1118) and on approximately the same face of the helix. Login to comment
118 To determine whether this imposed pattern was the source of relaxations in S1118F-CFTR, due to introduction of another bulky residue, the double mutation S1118F/F1111S was constructed. Login to comment
119 These channels displayed relaxations indistinguishable from those of the single FIGURE 2 Relaxations induced in S1118F-CFTR currents were fit to single-exponential decay functions. Login to comment
126 mutant S1118F-CFTR (not shown). Login to comment
132 Gating behavior of single CFTR channels from oocytes expressing WT or S1118F-CFTR was studied in excised patch mode. Login to comment
139 Under these conditions, burst duration was 1392 afe; 77 ms for WT-CFTR (n afd; 441 bursts) and 102 afe; 8 ms for S1118F- FIGURE 3 The current-voltage relations for S1118F-CFTR channel shift during the voltage-jump relaxations. Login to comment
152 Note that S1118F-CFTR channels exhibit much briefer openings than does WT. Login to comment
153 Currents were not observed in phosphorylated WT or S1118F-CFTR channels bathed in solutions lacking ATP (not shown). Login to comment
155 Hence the mean burst duration in S1118F-CFTR is reduced to only 7% that of WT channels. Login to comment
166 We tested whether S1118F-CFTR exhibited alterations in the interaction with DPC, which would be consistent with a pore-lining position for this serine. Login to comment
167 Fig. 6 shows background-subtracted S1118F-CFTR currents for a representative cell before (Fig. 6 A) and several minutes after (Fig. 6 B) bath application of 200 òe;M DPC. Login to comment
171 At afa;100 mV, DPC blocks S1118F-CFTR and WT channels with roughly the same efficacy: the apparent KD (at afa;100 mV) was, respectively, 266 afe; 13 òe;M and 276 afe; 14 òe;M (mean afe; SE, n afd; 6 and 15; data for WT are from McDonough et al., 1994). Login to comment
174 The voltage dependence of the S1118F-CFTR block is significantly less steep than that of the wild type; the apparent binding distance is òa; afd; 0.27 afe; 0.01 (mean afe; SE, n afd; 6) for the mutant versus 0.41 afe; 0.03 for WT channels (McDonough et al., 1994). Login to comment
175 S1118F-CFTR exhibits altered permeation characteristics S1118, according to our alignment, occupies a position in TM11 that is homologous to that of T338 in TM6. Login to comment
179 Because oocytes expressing S1118F-CFTR show voltage-jump relaxations, it was not appropriate to use the more common approach of applying voltage-ramp protocols to generate selectivity data. Login to comment
180 Instead, currents in oocytes expressing WT-, S1118F-, or S1118A-CFTR channels were elicited by stepping for 75 ms from the holding potential of afa;30 mV to a series of test potentials between afa;140 and af9;80 mV in af9;20 mV increments. Login to comment
182 If there are two open states that vary in the voltage dependence of microscopic kinetics, currents measured early in the relaxation and at later times likely represent different mixtures of FIGURE 5 S1118F-CFTR channels show rectification of single-channel conductance. Login to comment
189 Hence currents at each potential were averaged over two time periods: either the first FIGURE 6 Mutation S1118F has mild effects on blockade by DPC. Login to comment
204 (B) Currents in S1118F-CFTR-expressing oocytes, showing pronounced relaxations at depolarizing test potentials. Login to comment
207 Note the prominent tail currents in S1118F-CFTR but not in WT. Login to comment
235 Data are shown for both instantaneous currents and steady-state currents for WT and S1118F-CFTR (Fig. 8, A and B) or S1118A-CFTR (Fig. 8, C and D). Login to comment
236 In Clafa; -containing solutions, before anion substitutions were made, there was a significant difference (p afd; 0.047) between instantaneous and steady-state reversal potentials in S1118F-CFTR (Table 2) compared to the WT (Table 1), but not in S1118A-CFTR. Login to comment
237 This suggests that S1118F-CFTR channels may be less Clafa; -selective than are WT channels. Login to comment
238 Comparing the selectivity pattern in S1118F-CFTR with that of WT, without respect to time dependence, showed significant changes for both relative permeabilities and relative conductances (compare Tables 1 and 2). Login to comment
239 The ability of the large anions (acetate, gluconate, glutamate, and isethionate) to gain access to the pore is altered by mutations S1118A and S1118F, as indicated by mild to significant changes in relative permeabilities for these anions compared to that in WT channels. Login to comment
246 Accordingly, GX/GCl in S1118F-CFTR showed dramatic changes for seven of the nine test anions (Fig. 8 B). Login to comment
247 Relative conductances for the large anions were altered substantially in the S1118F-CFTR. Login to comment
249 S1118F-CFTR also exhibited less block by iodide and thiocyanate, consistent with disruption of the high-affinity binding of these anions in the WT pore. Login to comment
250 Relative conductances were generally less affected in S1118A-CFTR than in S1118F-CFTR. Login to comment
252 FIGURE 8 Selectivity data for WT CFTR as compared to S1118F-CFTR (A and B) and S1118A-CFTR (C and D). Login to comment
260 &#a7; p b0d; 0.01 for S1118F-CFTR or p afd; 0.02 for S1118A-CFTR. Login to comment
263 Nor does relative permeability for S1118F-CFTR (Fig. 8 A) or S1118A-CFTR (Fig. 8 C) show any time dependence. Login to comment
264 However, relative conductances for the largest anions studied (gluconate and glutamate) decreased in S1118F-CFTR during the 75-ms voltage jump (Fig. 8 B;&#a7; indicates p b0d; 0.01). Login to comment
265 Because these effects in S1118F-CFTR were only found for the largest anions tested, we propose that this result signifies a time-dependent change in the diameter of the narrowest part of the pore. We would expect this difference to be magnified in experiments in which Clafa; was replaced entirely with glutamate or gluconate (Khakh and Lester, 1999). Login to comment
269 Fig. 9 shows the current-voltage relations in the presence of chloride and glutamate for one representative experiment in S1118F-CFTR. Login to comment
274 Based upon the following observations, we reasoned that there may be a structural connection between the changes in permeation properties of S1118F-CFTR and the process underlying the voltage jump relaxations: 1) The change in permeation properties after a voltage jump (Figs. 1 and 9) is consistent with the steady-state kinetics of single channels (Fig. 5). Login to comment
278 To address this issue, we asked whether there is any interaction between permeation and gating in S1118F-CFTR channels studied under whole-cell conditions. Login to comment
280 Current-voltage relations in chloride and glutamate for instantaneous currents (--) and steady-state currents (- - -) in S1118F-CFTR were adjusted on the voltage axis so that their reversal potentials were set to zero. Login to comment
284 TABLE 2 Selectivity in S1118F-CFTR and S1118A-CFTR Ion S1118F S1118A Erev (mV) PX/PCl GX/GCl Erev (mV) PX/PCl GX/GCl SCN afa;40.97 afe; 1.20* 1.84 afe; 0.08* 0.57 afe; 0.02* afa;56.44 afe; 0.93 2.59 afe; 0.09 0.20 afe; 0.01 NO3 afa;32.83 afe; 1.69 1.30 afe; 0.06 0.95 afe; 0.02 afa;37.35 afe; 0.87 1.21 afe; 0.04 0.88 afe; 0.01 Br afa;28.28 afe; 1.32 1.07 afe; 0.03 0.88 afe; 0.03* afa;34.53 afe; 0.85 1.12 afe; 0.02 0.77 afe; 0.01 Cl afa;26.14 afe; 1.59* 1.0 1.0 afa;30.63 afe; 0.64 1.0 1.0 I afa;8.46 afe; 1.14 0.52 afe; 0.03* 0.38 afe; 0.03* afa;10.35 afe; 1.26 0.39 afe; 0.01 0.27 afe; 0.05 Acetate 39.64 afe; 1.77* 0.05 afe; 0.01* 0.12 afe; 0.01* 23.24 afe; 1.83* 0.09 afe; 0.01* 0.50 afe; 0.01* Glutamate 23.93 afe; 3.61* 0.16 afe; 0.01* 0.23 afe; 0.01* 19.59 afe; 1.07* 0.09 afe; 0.01* 0.49 afe; 0.01* 0.32 afe; 0.01I * Isethionate 20.24 afe; 3.62* 0.14 afe; 0.03 0.25 afe; 0.02* 23.71 afe; 0.99* 0.09 afe; 0.01* 0.48 afe; 0.01 ClO4 afa;6.42 afe; 1.57* 0.42 afe; 0.02* 0.18 afe; 0.01 27.29 afe; 1.36* 0.06 afe; 0.01* 0.12 afe; 0.01* 0.14 afe; 0.01I * Gluconate 28.26 afe; 3.29* 0.12 afe; 0.02 0.20 afe; 0.01* 20.03 afe; 1.04* 0.10 afe; 0.01* 0.50 afe; 0.01* 0.27 afe; 0.01I * Ions are listed in the same order as in Table 1. Login to comment
288 WT or S1118F-CFTR channels were studied using the same voltage-clamp protocol as the one used for the data shown in Fig. 7. Login to comment
295 S1118F-CFTR currents showed a relaxation as expected from previous experiments (Figs. 1, 2, and 7). Login to comment
298 Because SCNafa; blocks mutant channels as it does WT channels (Table 2), we would expect the time constant for S1118F-CFTR channels in the presence of SCNafa; to be reduced as it is for WT channels. Login to comment
299 In contrast, the relaxations in S1118F-CFTR were slowed by this less permeant anion such that the process underlying the relaxations was not complete even by the end of the 75-ms voltage jump. Login to comment
304 In summary, the lengthening of the gating process at positive potentials in S1118F-CFTR follows the relative conductance sequence. Login to comment
306 This implies a distinct connection between permeation and gating in S1118F-CFTR. Login to comment
314 (C) Time constants for the relaxations in WT and S1118F-CFTR channels were calculated by fitting the decay to an exponential function. Login to comment
315 Shown are the time constants (঄) for S1118F-CFTR currents recorded in bath solution containing Clafa; , Brafa; , or SCNafa; . Login to comment
316 surements in S1118F-CFTR show modest current relaxations, on the order of 30%, during voltage jumps between afa;140 and af9;80 mV. Login to comment
343 With respect to block by DPC, S1118F-CFTR had an effect much like that of T338A-CFTR, wherein affinity at afa;100 mV was not changed significantly but the voltage dependence was reduced. Login to comment
344 The absolute difference in the apparent KD (at afa;100 mV) for DPC between wild type and S1118F-CFTR, taken by itself, is too small to conclude that residue S1118 lines the channel pore. Login to comment
346 However, both S1118A-CFTR and S1118F-CFTR altered the selectivity behavior of the pore, suggesting that this position may contribute to the pore walls. Login to comment
347 Relative conductances for many of the substitute anions were altered in S1118F-CFTR. Login to comment
353 Furthermore, S1118F-CFTR also exhibits a reduced single-channel conductance. Login to comment
365 S1118F-CFTR single channels differ doubly from wild-type channels. Login to comment
366 First, S1118F-CFTR openings are much briefer than wild-type openings (burst duration is reduced by 93%). Login to comment
367 The choppy bursts of S1118F-CFTR closely resemble the bursts of mutant R117H, a mutation that causes mild cystic fibrosis (Sheppard et al., 1993). Login to comment
368 Second, the single-channel conductance of S1118F-CFTR rectifies inwardly, in agreement with the steady-state rectification of macroscopic currents. Login to comment
369 The rectification of single-channel steady-state conductance for S1118F-CFTR arises primarily from decreased conductance at positive potentials, because the single-channel conductances for the WT and mutant are nearly equal at afa;100 mV but differ at af9;100 mV. Login to comment
371 That S1118F-CFTR has more defective conduction properties for positive current at depolarizing potentials (i.e., for inward flux of Clafa; ) suggests that the mutated residue affects the channel at a point nearer the extracellular than the cytoplasmic end of the channel. Login to comment
375 The shortened openings in S1118F-CFTR may reflect a destabilization of the global protein structure rather than a specific effect on the pore. Login to comment
383 The single-channel recordings confirm that S1118F-CFTR openings are interrupted to a much greater extent than wild-type openings. Login to comment
389 Thus there is no reason to suspect that the relaxations are produced by voltage-dependent blocking events, and we tentatively favor the explanation that the S1118F-CFTR has altered gating kinetics. Login to comment
391 Although an additional state may have been added to the kinetic scheme for S1118F-CFTR, it is also possible that an existing state has been modified. Login to comment
392 For instance, it will be important to determine if the prominent voltage-dependent fast flicker in WT observed at hyperpolarizing potentials is modified by the S1118F mutation. Login to comment
393 Possible mechanisms S1118F-CFTR exhibits alterations in permeation properties, as might be expected given the location of this mutation, but also exhibits alterations in single-channel gating. Login to comment
397 It seems more likely that a unique process occurs for S1118F-CFTR. Login to comment
401 At the other end of the scale, the S1118F mutation might disrupt the stability of the protein global structure, leading to the observed effects on conduction. Login to comment
405 The relative conductance of the S1118F-CFTR channel to large anions decreases during the course of the relaxations, which may indicate that either the large anions are more capable of blocking the channels in the steady-state conformation, or that Clafa; permeation becomes blocked by an intrinsic portion of the protein in the steady-state conformation. Login to comment
418 The S1118F mutation may provide this reporter group in the form of the Phe side chain and/or slow the relaxations to a rate that is resolvable in macroscopic recordings. Login to comment

[hide] Distribution of CFTR mutations in the Czech popula... J Cyst Fibros. 2013 Sep;12(5):532-7. doi: 10.1016/j.jcf.2012.12.002. Epub 2012 Dec 29. Krenkova P, Piskackova T, Holubova A, Balascakova M, Krulisova V, Camajova J, Turnovec M, Libik M, Norambuena P, Stambergova A, Dvorakova L, Skalicka V, Bartosova J, Kucerova T, Fila L, Zemkova D, Vavrova V, Koudova M, Macek M, Krebsova A, Macek M Jr
Distribution of CFTR mutations in the Czech population: positive impact of integrated clinical and laboratory expertise, detection of novel/de novo alleles and relevance for related/derived populations.
J Cyst Fibros. 2013 Sep;12(5):532-7. doi: 10.1016/j.jcf.2012.12.002. Epub 2012 Dec 29., [PMID:23276700]

Abstract [show]
BACKGROUND: This two decade long study presents a comprehensive overview of the CFTR mutation distribution in a representative cohort of 600 Czech CF patients derived from all regions of the Czech Republic. METHODS: We examined the most common CF-causing mutations using the Elucigene CF-EU2v1 assay, followed by MLPA, mutation scanning and/or sequencing of the entire CFTR coding region and splice site junctions. RESULTS: We identified 99.5% of all mutations (1194/1200 CFTR alleles) in the Czech CF population. Altogether 91 different CFTR mutations, of which 20 were novel, were detected. One case of de novo mutation and a novel polymorphism was revealed. CONCLUSION: The commercial assay achieved 90.7%, the MLPA added 1.0% and sequencing increased the detection rate by 7.8%. These comprehensive data provide a basis for the improvement of CF DNA diagnostics and/or newborn screening in our country. In addition, they are relevant to related Central European populations with lower mutation detection rates, as well as to the sizeable North American "Bohemian diaspora".

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62 There are over 10 million inhabitants in the country, which according to population genetic analyses, is a representative of the CE ethnic composition [3], with significant overlaps with Table 1 (continued) Mutations/HGVS nomenclature/ Mutations/traditional nomenclature, legacy name/ Legacy exon/intron No. of alleles % 65. c.2290CNT R764X# Ex13 1 0.08 66. c.2490+1GNA 2622+1GNA# In13 1 0.08 67. c.2538GNA W846X*# Ex14a 1 0.08 68. c.2551CNT R851X# Ex14a 1 0.08 69. c.2589_2599delAATTTGGTGCT 2721del11 Ex14a 1 0.08 70. c.2705delG 2837delG Ex15 1 0.08 71. c.2789delG 2921delG Ex15 1 0.08 72. c.2803_2813delCTACCACTGGT 2935del11 Ex15 1 0.08 73. c.2856GNC M952I Ex15 1 0.08 74. c.2991GNC L997F# Ex17a 1 0.08 75. c.3106delA 3238delA Ex17a 1 0.08 76. c.3136GNT E1046X Ex17a 1 0.08 77. c.3139GNC G1047R Ex17a 1 0.08 78. c.3196CNT R1066C*# Ex17b 1 0.08 79. c.3196CNG R1066G Ex17b 1 0.08 80. c.3302TNG M1101R Ex17b 1 0.08 81. c.3310GNA E1104K Ex17b 1 0.08 82. c.3353CNT S1118F Ex17b 1 0.08 83. c.3472CNT R1158X*# Ex19 1 0.08 84. c.3587CNG S1196X# Ex19 1 0.08 85. c.3708delT 3840delT Ex19 1 0.08 86. c.3937CNT Q1313X# Ex21 1 0.08 87. c.3971TNC L1324P Ex22 1 0.08 88. c.4003CNT L1335F Ex22 1 0.08 89. c.4004TNC L1335P Ex22 1 0.08 90. c.4097TNA I1366N Ex22 1 0.08 91. c.4426CNT Q1476X Ex24 1 0.08 92. Login to comment

[hide] Relative contribution of different transmembrane s... Pflugers Arch. 2014 Mar;466(3):477-90. doi: 10.1007/s00424-013-1317-x. Epub 2013 Aug 20. Wang W, El Hiani Y, Rubaiy HN, Linsdell P
Relative contribution of different transmembrane segments to the CFTR chloride channel pore.
Pflugers Arch. 2014 Mar;466(3):477-90. doi: 10.1007/s00424-013-1317-x. Epub 2013 Aug 20., [PMID:23955087]

Abstract [show]
The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) alpha-helices, arranged in 2 symmetrical groups of 6. However, those TMs that line the channel pore are not completely defined. We used patch clamp recording to compare the accessibility of cysteine-reactive reagents to cysteines introduced into different TMs. Several residues in TM11 were accessible to extracellular and/or intracellular cysteine reactive reagents; however, no reactive cysteines were identified in TMs 5 or 11. Two accessible residues in TM11 (T1115C and S1118C) were found to be more readily modified from the extracellular solution in closed channels, but more readily modified from the intracellular solution in open channels, as previously reported for T338C in TM6. However, the effects of mutagenesis at S1118 (TM11) on a range of pore functional properties were relatively minor compared to the large effects of mutagenesis at T338 (TM6). Our results suggest that the CFTR pore is lined by TM11 but not by TM5 or TM7. Comparison with previous works therefore suggests that the pore is lined by TMs 1, 6, 11, and 12, suggesting that the structure of the open channel pore is asymmetric in terms of the contributions of different TMs. Although TMs 6 and 11 appear to undergo similar conformational changes during channel opening and closing, the influence of these two TMs on the functional properties of the narrowest region of the pore is clearly unequal.

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209 Previously, it was shown that substitution of S1118 with a much larger phenylalanine residue (S1118F) caused a larger (~35 %) decrease in single channel conductance [46]. Login to comment