ABCMdb

ABCC7 p.Cys491Ser

ClinVar:	c.1471T>C , p.Cys491Arg ? , not provided
CF databases:	c.1471T>C , p.Cys491Arg (CFTR1) ? , This misense has been found in a CF patient of North African origin with [delta]F508 on the other CF chromosome. This mutation was found once out of 1460 CF chromosomes screened. c.1472G>C , p.Cys491Ser (CFTR1) ? ,
Predicted by SNAP2:	A: N (82%), D: D (85%), E: D (71%), F: D (71%), G: D (59%), H: D (85%), I: N (57%), K: D (71%), L: D (59%), M: D (59%), N: D (75%), P: D (75%), Q: D (63%), R: D (85%), S: N (66%), T: N (61%), V: N (87%), W: D (91%), Y: D (85%),
Predicted by PROVEAN:	A: 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, S: N, T: N, V: N, W: D, Y: N,

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[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

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307 [138] A455E, P574H cAMP-stimulated apical membrane Cl-currents but current magnitudes were reduced compared to wild-type Electrophysiology of epithelial cells [139] C491S, C1344S, C1355S C491S channels opened almost exclusively to a 3-pS subconductance. Login to comment

[hide] The DeltaF508 mutation disrupts packing of the tra... J Biol Chem. 2004 Sep 17;279(38):39620-7. Epub 2004 Jul 21. Chen EY, Bartlett MC, Loo TW, Clarke DM
The DeltaF508 mutation disrupts packing of the transmembrane segments of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2004 Sep 17;279(38):39620-7. Epub 2004 Jul 21., 2004-09-17 [PMID:15272010]

Abstract [show]
The most common mutation in cystic fibrosis (deletion of Phe-508 in the first nucleotide binding domain (DeltaF508)) in the cystic fibrosis transmembrane conductance regulator (CFTR) causes retention of the mutant protein in the endoplasmic reticulum. We previously showed that the DeltaF508 mutation causes the CFTR protein to be retained in the endoplasmic reticulum in an inactive and structurally altered state. Proper packing of the transmembrane (TM) segments is critical for function because the TM segments form the chloride channel. Here we tested whether the DeltaF508 mutation altered packing of the TM segments by disulfide cross-linking analysis between TM6 and TM12 in wild-type and DeltaF508 CFTRs. These TM segments were selected because TM6 appears to line the chloride channel, and cross-linking between these TM segments has been observed in the CFTR sister protein, the multidrug resistance P-glycoprotein. We first mapped potential contact points in wild-type CFTR by cysteine mutagenesis and thiol cross-linking analysis. Disulfide cross-linking was detected in CFTR mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12), and W356C(TM6)/W1145C(TM12) in a wild-type background. The disulfide cross-linking occurs intramolecularly and was reducible by dithiothreitol. Introduction of the DeltaF508 mutation into these cysteine mutants, however, abolished cross-linking. The results suggest that the DeltaF508 mutation alters interactions between the TM domains. Therefore, a potential target to correct folding defects in the DeltaF508 mutant of CFTR is to identify compounds that promote correct folding of the TM domains.

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57 The construction of Cys-less CFTR (C76S/C126S/C225S/C276S/C343S/C491S/C524S/C590S/C592S/C657S/C832S/C866S/C1344S/C1355S/C1395S/C1400S/C1410S/C1458S) was performed using the following cDNA fragments. Login to comment
58 Point mutations C76/126S were generated in sequence in the PstI (bp 1) 3 XbaI (bp 573) fragment; point mutations C225S/C276S/C343S were generated in sequence in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; point mutations C491S/C524S/C590S/C592S/C657S were generated in sequence in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment; point mutations C832S/C866S were generated in sequence in the ApaI (bp 2333) 3 EcoRI (bp 3643) fragment; point mutations C1344S/C1355S/ C1395S/C1400S/C1410S/C1458S were generated in sequence in the EcoRI (bp 3643) 3 XhoI (bp 4560) fragment, the five insert fragments were then ligated and inserted into the PstI and XhoI sites of plasmid vector pMT21. Login to comment

[hide] Extensive sequencing of the CFTR gene: lessons lea... Hum Genet. 2005 Dec;118(3-4):331-8. Epub 2005 Sep 28. McGinniss MJ, Chen C, Redman JB, Buller A, Quan F, Peng M, Giusti R, Hantash FM, Huang D, Sun W, Strom CM
Extensive sequencing of the CFTR gene: lessons learned from the first 157 patient samples.
Hum Genet. 2005 Dec;118(3-4):331-8. Epub 2005 Sep 28., [PMID:16189704]

Abstract [show]
Cystic fibrosis (CF) is one of the most common monogenic diseases affecting Caucasians and has an incidence of approximately 1:3,300 births. Currently recommended screening panels for mutations in the responsible gene (CF transmembrane regulator gene, CFTR) do not detect all disease-associated mutations. Our laboratory offers extensive sequencing of the CFTR (ABCC7) gene (including the promoter, all exons and splice junction sites, and regions of selected introns) as a clinical test to detect mutations which are not found with conventional screening. The objective of this report is to summarize the findings of extensive CFTR sequencing from our first 157 consecutive patient samples. In most patients with classic CF symptoms (18/24, 75%), extensive CFTR sequencing confirmed the diagnosis by finding two disease-associated mutations. In contrast, only 5 of 75 (7%) patients with atypical CF had been identified with two CFTR mutations. A diagnosis of CF was confirmed in 10 of 17 (58%) newborns with either positive sweat chloride readings or positive immunoreactive trypsinogen (IRT) screen results. We ascertained ten novel sequence variants that are potentially disease-associated: two deletions (c.1641AG>T, c.2949_2853delTACTC), seven missense mutations (p.S158T, p.G451V, p.K481E, p.C491S, p.H949L, p.T1036N, p.F1099L), and one complex allele ([p.356_A357del; p.358I]). We ascertained three other apparently novel complex alleles. Finally, several patients were found to carry partial CFTR gene deletions. In summary, extensive CFTR gene sequencing can detect rare mutations which are not found with other screening and diagnostic tests, and can thus establish a definitive diagnosis in symptomatic patients with previously negative results. This enables carrier detection and prenatal diagnosis in additional family members.

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7 We ascertained ten novel sequence variants that are potentially disease-associated: two deletions (c.1641AG>T, c.2949_2853delTACTC), seven missense mutations (p.S158T, p.G451V, p.K481E, p.C491S, p.H949L, p.T1036N, p.F1099L), and one complex allele ([p.356_A357del; p.358I]). Login to comment
92 The p.C491S missense mutation is expected to be a benign variant since it was ascertained during single exon sequencing in an unaffected sibling of a CF patient. Login to comment
95 The PolyPhen prediction tool also suggests that p.C491S is a benign variant (Table 7). Login to comment

[hide] Contribution of the CFTR gene, the pancreatic secr... Clin Genet. 2007 May;71(5):451-7. Tzetis M, Kaliakatsos M, Fotoulaki M, Papatheodorou A, Doudounakis S, Tsezou A, Makrythanasis P, Kanavakis E, Nousia-Arvanitakis S
Contribution of the CFTR gene, the pancreatic secretory trypsin inhibitor gene (SPINK1) and the cationic trypsinogen gene (PRSS1) to the etiology of recurrent pancreatitis.
Clin Genet. 2007 May;71(5):451-7., [PMID:17489851]

Abstract [show]
Acute recurrent/chronic pancreatitis (CP) is a complex multigenic disease. This is a case-control study consisting of 25 Greek patients with CP and a control population of 236 healthy Greek subjects. The whole coding area and neighboring intronic regions of the three genes were screened. Seventeen of 25 patients (68%) had mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene: nine compound heterozygotes with either mild or severe mutations and eight heterozygotes. Four patients (16%) carried CFTR-modulating haplotypes V470-TG11-T5 and V470-TG12-T7. All were negative for PRSS1 gene mutations, while variants c.486C/T and c.738C/T were found in nine patients each, three homozygotes for the minor alleles. Two carried SPINK1 gene mutation p.N34S, one being transheterozygote with CFTR mutation p.F1052V. The promoter variant -253T>C was found in four individuals (one homozygous for the minor allele), all four being transheterozygotes with mutations in the CFTR gene as well. Finally two carried c.272C/T in the 3' untranslated region, one being a p.N34S carrier as well. In total, 80% (20/25) of patients had a molecular defect in one or both of the CFTR and SPINK1 genes, suggesting that mutations/variants in the CFTR plus or minus mutations in the SPINK1, but not the PRSS1 gene, may confer a high risk for recurrent pancreatitis.

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No. Sentence Comment
93 a Additional mutations found in the controls: p.R1162L (1.66%), p.D565G (0.47%), p.A120T (0.47%) and 0.24% each for p.R297Q, p.L997F, p.E826K, p.I807M, p.S495Y and p.C491S. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Biochemistry. 2009 Oct 27;48(42):10078-88. Alexander C, Ivetac A, Liu X, Norimatsu Y, Serrano JR, Landstrom A, Sansom M, Dawson DC
Cystic fibrosis transmembrane conductance regulator: using differential reactivity toward channel-permeant and channel-impermeant thiol-reactive probes to test a molecular model for the pore.
Biochemistry. 2009 Oct 27;48(42):10078-88., 2009-10-27 [PMID:19754156]

Abstract [show]
The sixth transmembrane segment (TM6) of the CFTR chloride channel has been intensively investigated. The effects of amino acid substitutions and chemical modification of engineered cysteines (cysteine scanning) on channel properties strongly suggest that TM6 is a key component of the anion-conducting pore, but previous cysteine-scanning studies of TM6 have produced conflicting results. Our aim was to resolve these conflicts by combining a screening strategy based on multiple, thiol-directed probes with molecular modeling of the pore. CFTR constructs were screened for reactivity toward both channel-permeant and channel-impermeant thiol-directed reagents, and patterns of reactivity in TM6 were mapped onto two new, molecular models of the CFTR pore: one based on homology modeling using Sav1866 as the template and a second derived from the first by molecular dynamics simulation. Comparison of the pattern of cysteine reactivity with model predictions suggests that nonreactive sites are those where the TM6 side chains are occluded by other TMs. Reactive sites, in contrast, are generally situated such that the respective amino acid side chains either project into the predicted pore or lie within a predicted extracellular loop. Sites where engineered cysteines react with both channel-permeant and channel-impermeant probes occupy the outermost extent of TM6 or the predicted TM5-6 loop. Sites where cysteine reactivity is limited to channel-permeant probes occupy more cytoplasmic locations. The results provide an initial validation of two, new molecular models for CFTR and suggest that molecular dynamics simulation will be a useful tool for unraveling the structural basis of anion conduction by CFTR.

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No. Sentence Comment
42 The Cys-less CFTR construct (C76S, C126S, C225S, C276S, C343S, C491S, C524S, C590L, C592L, C657S, C832S, C866S, C1344S, C1355S, C1395S, C1400S, C1410S, C1458S) was a gift from Drs. Martin Mense and David Gadsby and was used in their pGEMHE vector previously described (13). Login to comment

[hide] Cystic fibrosis genetic counseling difficulties du... J Cyst Fibros. 2012 Jul;11(4):344-8. doi: 10.1016/j.jcf.2012.01.004. Epub 2012 Feb 11. Poulou M, Fylaktou I, Fotoulaki M, Kanavakis E, Tzetis M
Cystic fibrosis genetic counseling difficulties due to the identification of novel mutations in the CFTR gene.
J Cyst Fibros. 2012 Jul;11(4):344-8. doi: 10.1016/j.jcf.2012.01.004. Epub 2012 Feb 11., [PMID:22326559]

Abstract [show]
BACKGROUND: The Cystic Fibrosis database includes amongst the 1893 gene mutations and polymorphisms a lot of missense mutations, the disease status of which still remains unproven. In populations with high rates of CFTR mutation heterogeneity, molecular diagnosis is difficult often causing counseling difficulties especially in cases of rare and/or novel mutations. METHODS: Approaches to counseling in cases of novel variants. RESULTS: Thirty-seven novel variants (4 synonymous, 24 missense, 2 frameshift and 10 intronic substitutions) were identified and evaluated with the help of in silico tools. CONCLUSIONS: In a diagnostic environment the answers have to be given within a specific timeframe, the in silico tools in combination with the phenotype offer some help but their diagnostic value is limited and cannot be used in isolation for the determination of the severity of the mutation.

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62 (3) Disease causing Atypical CF 15 14 (13) c.2450GNT p.Gly817Val Benign T 0.31 Path. (4) Polymorphism CBAVD 16 11 (10) c.1472GNC p.Cys491Ser Benign T 0.59 Neut. Login to comment
73 Disruption of ESE motifs/creation of ESS motifs Acc increased (0.44/0.50) Acc gained 0.51 p.V1318G c.3953TNG No change New donor site (48.15/74.98) (+55.73%) if used causes reduction of exon size (-11nt) Donor increased (0.31/0.99) p.K1165T c.3494ANC No change Minor changes No change p.S977C c.2930CNG No change Minor changes No change p.I521F c.1561ANT No change Minor changes No change p.V1212F c.3634GNT No change Disruption of ESE motifs Donor gained 0.36 p.F319V c.955TNG No change Minor changes Acc increased (0.42/0.61) p.G817V c.2450GNT No change Minor changes No change p.C491S c.1472GNC No change Disruption of ESE motifs Acceptor sites created p.I336L c.1006ANC No change Disruption of ESE motifs No change p.F305V c.913TNG Increased score for SRp55 Disruption of ESS motifs Donor gained (0.99) p.S511C c.1532CNG Decreased score for SC35 Disruption of ESS motifs No change p.S1311N c.3932GNA Changes on ss scores Disruption of ESS motifs Donor increased (0.50/0.63) p.L926F c.2778GNT No change Creation of ESS motifs Changes on ss scores p.A1225V c.3674CNT No change Disruption of ESE motif/creation of ESS motif Donor increased (0.72/0.99) p.F533L c.1597TNC No change Creation of ESE motifs/disruption of ESS motifs No change p.Q1209H c.3627ANC No change Minor changes No change c.2490+3ANG No change Minor changes No change c.2909-36TNC Decrease of donor ss Disruption of ESS motifs Acc increased (0.33/0.48)/donor increased (0.74/0.94) c.2909-10TNC Change for SRp55 best hit (3.97/5.16) Disruption of ESS motifs No change c.4137-21GNT No change Disruption of ESE and creation of ESS motifs Acc increased (0.86/0.96) c.1116+4ANT No change WT donor site disrupted Marginal changes on donor ss c.2988+30TNC No change Disruption of ESE motif/creation of ESS motif No change c.1680-27GNA No change Disruption of ESS motifs No change c.2620-24CNG No change Creation of ESE motifs Donor gained 0.96 c.2620-18delT No change New donor ss/changes on ESE and ESS motifs Acc increased (0.41/0.55) c.2658-8CNG Decreased score for acc site Minor changes Marginal changes on ss p.L1227L c.3681ANG No change Changes on ESE and ESS motifs No change p.R1158R c.3472CNA No change Creation of ESE motifs/creation of ESS motifs Marginal changes on ss p.D1275D c.3825TNC No change No change Donor increased (0.56/0.87) p.L346L c.1036CNT No change No change No change Abbreviations: ESE: exonic splicing enhancer, HSF: human splicing finder, ESS: exonic splicing silencer, WT: wild type, Mut: mutant, ss: splicing site. Login to comment
75 Disruption of ESE motifs/creation of ESS motifs Acc increased (0.44/0.50) Acc gained 0.51 p.V1318G c.3953TNG No change New donor site (48.15/74.98) (+55.73%) if used causes reduction of exon size (-11nt) Donor increased (0.31/0.99) p.K1165T c.3494ANC No change Minor changes No change p.S977C c.2930CNG No change Minor changes No change p.I521F c.1561ANT No change Minor changes No change p.V1212F c.3634GNT No change Disruption of ESE motifs Donor gained 0.36 p.F319V c.955TNG No change Minor changes Acc increased (0.42/0.61) p.G817V c.2450GNT No change Minor changes No change p.C491S c.1472GNC No change Disruption of ESE motifs Acceptor sites created p.I336L c.1006ANC No change Disruption of ESE motifs No change p.F305V c.913TNG Increased score for SRp55 Disruption of ESS motifs Donor gained (0.99) p.S511C c.1532CNG Decreased score for SC35 Disruption of ESS motifs No change p.S1311N c.3932GNA Changes on ss scores Disruption of ESS motifs Donor increased (0.50/0.63) p.L926F c.2778GNT No change Creation of ESS motifs Changes on ss scores p.A1225V c.3674CNT No change Disruption of ESE motif/creation of ESS motif Donor increased (0.72/0.99) p.F533L c.1597TNC No change Creation of ESE motifs/disruption of ESS motifs No change p.Q1209H c.3627ANC No change Minor changes No change c.2490+3ANG No change Minor changes No change c.2909-36TNC Decrease of donor ss Disruption of ESS motifs Acc increased (0.33/0.48)/donor increased (0.74/0.94) c.2909-10TNC Change for SRp55 best hit (3.97/5.16) Disruption of ESS motifs No change c.4137-21GNT No change Disruption of ESE and creation of ESS motifs Acc increased (0.86/0.96) c.1116+4ANT No change WT donor site disrupted Marginal changes on donor ss c.2988+30TNC No change Disruption of ESE motif/creation of ESS motif No change c.1680-27GNA No change Disruption of ESS motifs No change c.2620-24CNG No change Creation of ESE motifs Donor gained 0.96 c.2620-18delT No change New donor ss/changes on ESE and ESS motifs Acc increased (0.41/0.55) c.2658-8CNG Decreased score for acc site Minor changes Marginal changes on ss p.L1227L c.3681ANG No change Changes on ESE and ESS motifs No change p.R1158R c.3472CNA No change Creation of ESE motifs/creation of ESS motifs Marginal changes on ss p.D1275D c.3825TNC No change No change Donor increased (0.56/0.87) p.L346L c.1036CNT No change No change No change Abbreviations: ESE: exonic splicing enhancer, HSF: human splicing finder, ESS: exonic splicing silencer, WT: wild type, Mut: mutant, ss: splicing site. Login to comment

[hide] Cysteine residues in the nucleotide binding domain... Biophys J. 2002 Mar;82(3):1278-92. Harrington MA, Kopito RR
Cysteine residues in the nucleotide binding domains regulate the conductance state of CFTR channels.
Biophys J. 2002 Mar;82(3):1278-92., [PMID:11867445]

Abstract [show]
Gating of cystic fibrosis transmembrane conductance regulator (CFTR) channels requires intermolecular or interdomain interactions, but the exact nature and physiological significance of those interactions remains uncertain. Subconductance states of the channel may result from alterations in interactions among domains, and studying mutant channels enriched for a single conductance type may elucidate those interactions. Analysis of CFTR channels in inside-out patches revealed that mutation of cysteine residues in NBD1 and NBD2 affects the frequency of channel opening to the full-size versus a 3-pS subconductance. Mutating cysteines in NBD1 resulted in channels that open almost exclusively to the 3-pS subconductance, while mutations of cysteines in NBD2 decreased the frequency of subconductance openings. Wild-type channels open to both size conductances and make fast transitions between them within a single open burst. Full-size and subconductance openings of both mutant and wild-type channels are similarly activated by ATP and phosphorylation. However, the different size conductances open very differently in the presence of a nonhydrolyzable ATP analog, with subconductance openings significantly shortened by ATPgammaS, while full-size channels are locked open. In wild-type channels, reducing conditions increase the frequency and decrease the open time of subconductance channels, while oxidizing conditions decrease the frequency of subconductance openings. In contrast, in the cysteine mutants studied, altering redox potential has little effect on gating of the subconductance.

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No. Sentence Comment
72 C491S, C524S, C1344/1355S, and C491/524S mutants were inserted into a pcDNA3.1 expression vector and transiently transfected into HEK 293 cells using calcium phosphate. Login to comment
91 In inside-out patch clamp recordings, C491S mutant channels show openings to two different conductance levels, with the majority of channel openings to a subconductance of ϳ3 pS (Fig. 2, Table 1). Login to comment
93 The subconductance openings of the C491S mutant are much shorter than the wild-type subconductance, as shown by comparing the dwell time histograms in Fig. 1 C with that in Fig. 2 C. Login to comment
95 Despite this difference, the C491S subconductance openings appeared to be very similar to the wild-type subconductance in current amplitudes and in requirement for phosphorylation. Login to comment
96 As shown in Fig. 3, C491S mutant channels, like the wild-type subconductance, require phosphorylation by PKA for high-probability openings. Login to comment
98 While the C491S single mutation had occasional full-size openings along with subconductance openings, mutating both cysteine residues in NBD1 resulted in a channel that almost never opened to the full-size conductance (Table 1; Fig. 4, A and B). Login to comment
104 As shown by the dwell time histograms in Fig. 4 C, the long component of the open time distributions for the C491/524S and C-QUAD-S mutants is much shorter than the wild-type subconductance, and closer to that of the C491S mutant. Login to comment
105 Mutation of C524 has little effect on channel gating While CFTR channels carrying the C491S mutation either alone or in combination with C524S or C1344/1355S open almost exclusively to a 3 pS subconductance, channels carry- ing only the C524S mutation exhibit conductance similar to wild-type channels. Login to comment
106 Like the wild-type channel, nearly every patch of C524S channels gates to the full-size openings, although, like wild-type channels, subconductance openings do appear (Table 1). Login to comment
117 In the presence of a mixture of ATP and ATP␥S, full-size channels are "locked open": opening in long bursts that can last for minutes (Anderson et al., 1991b; Baukrowitz et al., FIGURE 2 C491S-CFTR channels open most frequently to a 3-pS subconductance. Login to comment
118 (A) One-minute sample traces of inside-out patches from HEK 293 cells expressing the C491S mutant of CFTR showing the two modes of gating of the channel. Login to comment
122 (C) Open dwell time histogram of 6000 opening and closing events of C491S channels in nine separate patches. Login to comment
124 (D) Current-voltage relationship of subconductance channels from C491S CFTR. Login to comment
131 Two other mutants (C491S, C491/524S) were tested for the effect of ATP␥S on subconductance openings (Fig. 5 C). Login to comment
132 Both the C491S and C491/524S mutants have open dwell times that are shorter than wild-type subconductance, and these dwell times were not significantly altered by the presence of ATP␥S (p Ͼ 0.05; Kolmogorov-Smirnov). Login to comment
134 This is in contrast FIGURE 3 Increased phosphorylation by PKA increases the frequency of subconductance openings in C491S patches. Login to comment
135 (A) One-minute sample traces of an inside-out patch from an HEK 293 cell expressing C491S mutant CFTR with and without the addition of PKA. Login to comment
138 The patch was held at 75 mV. (B) Graph of open probability versus time for the patch shown in A. TABLE 1 Mutations of cysteine residues in NBD1 increase the proportion of patches with subconductance openings, while mutations of cysteines in NBD2 decrease the proportion of patches with subconductance openings Mutation Patches with Subconductance Patches with Full Size Total Patches Percent with Subconductance Percent with Full Size Wild-type 34 49 49 69 100 C491S 16 5 18 89 28 C491/524S 10 1 13 77 8 C524S 2 5 6 33 83 C1344/1355S 8 30 30 27 100 C-QUAD-S 19 0 30 63 0 The number of inside-out patches containing frequent full-size and/or subconductance channel openings were counted. Login to comment
143 Moreover, the presence of ATP␥S shortens the open dwell time of wild-type channels to approximately that of channels containing the C491S mutant, suggesting that the altered behavior of the C491S channel may be related to changes in ATP hydrolysis at NBF1. Login to comment
181 In some cases the channel transitions to the subconductance without appearing to close, while sometimes it opens directly to the subconductance state and then transitions to the full-size channel. This type of rapid transition between full-size and subconductance was not observed in recordings of wild-type channels in reducing conditions, nor in the C491S, C491/524S, or C- QUAD-S mutants in which the subconductance state makes up the majority of channel openings observed. Login to comment
183 Effect of mutation of C1344 and C1355 on subconductance frequency and redox sensitivity Mutation of cysteine residues in the second nucleotide binding domain by themselves had much less of an effect on channel gating than the C491S mutation. Login to comment
206 The 3-pS subconductance of channels containing the C491S mutant alone or with other cysteine mutations was similar to the subconductance observed in recordings from patches containing wild-type CFTR channels, although with a shortened open dwell time. Login to comment
208 The effect of the C491S mutation on control of channel conductance (enriching for subconductance at the expense of full-size openings) was unexpected; however, the specificity of this mutation for that effect is supported by several pieces of data. Login to comment
221 With channels containing the C491S mutation opening almost exclusively to a subconductance with gating properties very different from the full-size channel, it is difficult to directly relate the effect of redox potential on gating kinetics of the mutants compared to the wild-type channel. Login to comment
244 Our data suggest that subconductance opening may be related to a decreased hydrolysis by the first nucleotide binding domain because mutation of C491S in NBD1 results in an increase in subconductance frequency at the expense of the full-size openings. Recently published work with truncation mutants of CFTR has suggested that the CFTR channel might be "double-barreled," with one pore producing the full-size conductance and a second, independently gated pore producing a 3-4-pS subconductance (Yue et al., 2000). Login to comment
256 The frequency of subconductance openings in CFTR channels with the C491S mutation may mean that this region of the molecule is important for the type of intermolecular interactions observed by other groups to be important for high-frequency opening of the full-size channel. Login to comment
73 C491S, C524S, C1344/1355S, and C491/524S mutants were inserted into a pcDNA3.1 expression vector and transiently transfected into HEK 293 cells using calcium phosphate. Login to comment
92 In inside-out patch clamp recordings, C491S mutant channels show openings to two different conductance levels, with the majority of channel openings to a subconductance of b03;3 pS (Fig. 2, Table 1). Login to comment
94 The subconductance openings of the C491S mutant are much shorter than the wild-type subconductance, as shown by comparing the dwell time histograms in Fig. 1 C with that in Fig. 2 C. Login to comment
97 As shown in Fig. 3, C491S mutant channels, like the wild-type subconductance, require phosphorylation by PKA for high-probability openings. Login to comment
99 While the C491S single mutation had occasional full-size openings along with subconductance openings, mutating both cysteine residues in NBD1 resulted in a channel that almost never opened to the full-size conductance (Table 1; Fig. 4, A and B). Login to comment
119 (A) One-minute sample traces of inside-out patches from HEK 293 cells expressing the C491S mutant of CFTR showing the two modes of gating of the channel. Login to comment
123 (C) Open dwell time histogram of 6000 opening and closing events of C491S channels in nine separate patches. Login to comment
125 (D) Current-voltage relationship of subconductance channels from C491S CFTR. Login to comment
133 Both the C491S and C491/524S mutants have open dwell times that are shorter than wild-type subconductance, and these dwell times were not significantly altered by the presence of ATPॹS (p b0e; 0.05; Kolmogorov-Smirnov). Login to comment
136 (A) One-minute sample traces of an inside-out patch from an HEK 293 cell expressing C491S mutant CFTR with and without the addition of PKA. Login to comment
140 TABLE 1 Mutations of cysteine residues in NBD1 increase the proportion of patches with subconductance openings, while mutations of cysteines in NBD2 decrease the proportion of patches with subconductance openings Mutation Patches with Subconductance Patches with Full Size Total Patches Percent with Subconductance Percent with Full Size Wild-type 34 49 49 69 100 C491S 16 5 18 89 28 C491/524S 10 1 13 77 8 C524S 2 5 6 33 83 C1344/1355S 8 30 30 27 100 C-QUAD-S 19 0 30 63 0 The number of inside-out patches containing frequent full-size and/or subconductance channel openings were counted. Login to comment
145 Moreover, the presence of ATPॹS shortens the open dwell time of wild-type channels to approximately that of channels containing the C491S mutant, suggesting that the altered behavior of the C491S channel may be related to changes in ATP hydrolysis at NBF1. Login to comment
185 Effect of mutation of C1344 and C1355 on subconductance frequency and redox sensitivity Mutation of cysteine residues in the second nucleotide binding domain by themselves had much less of an effect on channel gating than the C491S mutation. Login to comment
210 The effect of the C491S mutation on control of channel conductance (enriching for subconductance at the expense of full-size openings) was unexpected; however, the specificity of this mutation for that effect is supported by several pieces of data. Login to comment
223 With channels containing the C491S mutation opening almost exclusively to a subconductance with gating properties very different from the full-size channel, it is difficult to directly relate the effect of redox potential on gating kinetics of the mutants compared to the wild-type channel. Login to comment
247 Our data suggest that subconductance opening may be related to a decreased hydrolysis by the first nucleotide binding domain because mutation of C491S in NBD1 results in an increase in subconductance frequency at the expense of the full-size openings. Recently published work with truncation mutants of CFTR has suggested that the CFTR channel might be "double-barreled," with one pore producing the full-size conductance and a second, independently gated pore producing a 3-4-pS subconductance (Yue et al., 2000). Login to comment
259 The frequency of subconductance openings in CFTR channels with the C491S mutation may mean that this region of the molecule is important for the type of intermolecular interactions observed by other groups to be important for high-frequency opening of the full-size channel. It is conceivable that the cysteine residues in NBD1, particularly C491, could be important in the intermolecular associations that stabilize a dimer of the channel and allow it to produce full-size openings. Login to comment

[hide] CFTR: Ligand exchange between a permeant anion ([A... Biophys J. 2006 Sep 1;91(5):1737-48. Epub 2006 Jun 9. Serrano JR, Liu X, Borg ER, Alexander CS, Shaw CF 3rd, Dawson DC
CFTR: Ligand exchange between a permeant anion ([Au(CN)2]-) and an engineered cysteine (T338C) blocks the pore.
Biophys J. 2006 Sep 1;91(5):1737-48. Epub 2006 Jun 9., [PMID:16766608]

Abstract [show]
Previous attempts to identify residues that line the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have utilized cysteine-substituted channels in conjunction with impermeant, thiol-reactive reagents like MTSET+ and MTSES-. We report here that the permeant, pseudohalide anion [Au(CN)2]- can also react with a cysteine engineered into the pore of the CFTR channel. Exposure of Xenopus oocytes expressing the T338C CFTR channel to as little as 100 nM [Au(CN)2]- produced a profound reduction in conductance that was not reversed by washing but was reversed by exposing the oocytes to a competing thiol like DTT (dithiothreitol) and 2-ME (2-mercaptoethanol). In detached, inside out patches single-channel currents were abolished by [Au(CN)2]- and activity was not restored by washing [Au(CN)2]- from the bath. Both single-channel and macroscopic currents were restored, however, by exposing [Au(CN)2]- -blocked channels to excess [CN]-. The results are consistent with the hypothesis that [Au(CN)2]- can participate in a ligand exchange reaction with the cysteine thiolate at 338 such that the mixed-ligand complex, with a charge of -1, blocks the anion conduction pathway.

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23 MATERIALS AND METHODS Mutagenesis and in vitro transcription The Cys-less CFTR construct (C76S, C126S, C225S, C276S, C343S, C491S, C524S, C590L, C592L, C657S, C832S, C866S, C1344S, C1355S, C1395S, C1400S, C1410S, C1458S) was a gift from Drs. Martin Mense and Submitted December 28, 2005, and accepted for publication May 19, 2006. Login to comment

[hide] Deletion of Phenylalanine 508 in the First Nucleot... J Biol Chem. 2015 Sep 18;290(38):22862-78. doi: 10.1074/jbc.M115.641134. Epub 2015 Jul 6. Chong PA, Farber PJ, Vernon RM, Hudson RP, Mittermaier AK, Forman-Kay JD
Deletion of Phenylalanine 508 in the First Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Increases Conformational Exchange and Inhibits Dimerization.
J Biol Chem. 2015 Sep 18;290(38):22862-78. doi: 10.1074/jbc.M115.641134. Epub 2015 Jul 6., [PMID:26149808]

Abstract [show]
Deletion of Phe-508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) results in destabilization of the domain, intramolecular interactions involving the domain, and the entire channel. The destabilization caused by F508del manifests itself in defective channel processing and channel gating defects. Here, we present NMR studies of the effect of F508del and the I539T stabilizing mutation on NBD1 dynamics, with a view to understanding these changes in stability. Qualitatively, F508del NMR spectra exhibit significantly more peak broadening than WT spectra due to the enhanced intermediate time scale (millisecond to microsecond) motions in the mutant. Unexpectedly, studies of fast (nanosecond to picosecond) motions revealed that F508del NBD1 tumbles more rapidly in solution than WT NBD1. Whereas F508del tumbles at a rate nearly consistent with the monomeric state, the WT protein tumbles significantly more slowly. Paramagnetic relaxation enhancement experiments confirm that NBD1 homodimerizes in solution in the expected head-to-tail orientation. NMR spectra of WT NBD1 reveal significant concentration-dependent chemical shift perturbations consistent with NBD1 dimerization. Chemical shift analysis suggests that the more rapid tumbling of F508del is the result of an impaired ability to dimerize. Based on previously published crystal structures and NMR spectra of various NBD1 mutants, we propose that deletion of Phe-508 affects Q-loop conformational sampling in a manner that inhibits dimerization. These results provide a potential mechanism for inhibition of channel opening by F508del and support the dimer interface as a target for cystic fibrosis therapeutics.

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288 Of note, we were not able to express or purify NBD1 using the previously published mutations C491S, C524S, C590V, and C592V (12), probably because these mutations destabilize NBD1. Login to comment