ABCMdb

ABCC7 p.Pro99Leu

Admin's notes:	Class II-III (maturation defect, gating defect) Veit et al.
ClinVar:	c.296C>T , p.Pro99Leu ? , not provided
CF databases:	c.296C>T , p.Pro99Leu (CFTR1) ? , This mutation was found together with [delta]F508 in a patient with extreme mild symptoms. It was found by SSCP and sequencing.
Predicted by SNAP2:	A: D (85%), C: D (85%), D: D (80%), E: D (95%), F: D (91%), G: D (91%), H: D (91%), I: D (91%), K: D (95%), L: D (91%), M: D (91%), N: D (91%), Q: D (91%), R: D (95%), S: D (85%), T: D (85%), V: D (91%), W: D (95%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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II-III (maturation defect, gating defect) <a href="https://www.ncbi.nlm.nih.gov/pubmed/26823392">Veit et al.</a>
admin on 2016-08-19 15:16:22

Publications
[hide] Arg352 is a major determinant of charge selectivit... Biochemistry. 1999 Apr 27;38(17):5528-37. Guinamard R, Akabas MH
Arg352 is a major determinant of charge selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel.
Biochemistry. 1999 Apr 27;38(17):5528-37., 1999-04-27 [PMID:10220340]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator forms an anion-selective channel. We previously showed that charge selectivity, the ability to discriminate between anions and cations, occurs near the cytoplasmic end of the channel. The molecular determinants of charge selectivity, however, are unknown. We investigated the role of Arg352, a residue flanking the predicted cytoplasmic end of the M6 segment, in the mechanism of charge selectivity. We determined the Cl- to Na+ permeability ratio (PCl/PNa) from the reversal potential measured in a 10-fold NaCl gradient. For the wild type, PCl/PNa was 36 (range of 28-51). For the R352H mutant, PCl/PNa was dependent on cytoplasmic pH. At pH 5.4, the PCl/PNa was 33 (range of 27-41), similar to that of the wild type, but at pH 7.2, where the histidine should be largely uncharged, PCl/PNa was 3 (range of 2.9-3.1). For the R352C and R352Q mutants, PCl/PNa was 7 (range of 6-8) and 4 (range of 3.5-4.4), respectively. Furthermore, Na+ which does not carry a significant fraction of the current through the wild type is measurably conducted through R352Q. Thus, the charge of the side chain at position 352 is a strong determinant of charge selectivity. In the wild type, the positive charge on Arg352 contributes to an electrostatic potential in the channel that forms a barrier to cation permeation. Mutation of Arg352 did not alter the halide selectivity sequence. Selectivity among halides must involve other residues.

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No. Sentence Comment
283 The mutation P99L also changed the halide permeability ratios (37); however, this is presumably due to effects of the mutation on protein conformation because this residue is not on the water-accessible surface of the protein (18, 19). Login to comment

[hide] DHPLC screening of cystic fibrosis gene mutations. Hum Mutat. 2002 Apr;19(4):374-83. Ravnik-Glavac M, Atkinson A, Glavac D, Dean M
DHPLC screening of cystic fibrosis gene mutations.
Hum Mutat. 2002 Apr;19(4):374-83., [PMID:11933191]

Abstract [show]
Denaturing high performance liquid chromatography (DHPLC) using ion-pairing reverse phase chromatography (IPRPC) columns is a technique for the screening of gene mutations. In order to evaluate the potential utility of this assay method in a clinical laboratory setting, we subjected the PCR products of 73 CF patients known to bear CFTR mutations to this analytic technique. We used thermal denaturation profile parameters specified by the MELT program tool, made available by Stanford University. Using this strategy, we determined an initial analytic sensitivity of 90.4% for any of 73 known CFTR mutations. Most of the mutations not detected by DHPLC under these conditions are alpha-substitutions. This information may eventually help to improve the MELT algorithm. Increasing column denaturation temperatures for one or two degrees above those recommended by the MELT program allowed 100% detection of CFTR mutations tested. By comparing DHPLC methodology used in this study with the recently reported study based on Wavemaker 3.4.4 software (Transgenomic, Omaha, NE) [Le Marechal et al., 2001) and with previous SSCP analysis of CFTR mutations [Ravnik-Glavac et al., 1994] we emphasized differences and similarities in order to refine the DHPLC system and discuss the relationship to the alternative approaches. We conclude that the DHPLC method, under optimized conditions, is highly accurate, rapid, and efficient in detecting mutations in the CFTR gene and may find high utility in screening individuals for CFTR mutations. Hum Mutat 19:374-383, 2002. Published 2002 Wiley-Liss, Inc.

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No. Sentence Comment
42 The following mutations have been studied: exon 3: W57G, R74W, R75Q, G85E, 394delTT, 405+ 1G>A; exon 4: E92X, P99L, 441delA, 444delA, 457TAT>G, D110H, R117C, R117H, A120T, 541delC, 544delCA, Q151X, 621+1G>T, 662- 2A>C; exon 7: 1078delT, F331L, R334W, I336K, R347C, R347P, A349V, R352Q, 1221delCT; exon 10: S492F, Q493X, 1609delCA, deltaI507, deltaF508; exon 11: G542X, S549N, G551D, R553X, A559T, R560K, R560T; exon 13: K716X, Q685X, G628R, L719X; exon 17b: H1054D, G1061R, 3320ins5, R1066H, R1066L, R1070Q, 3359delCT, L1077P, H1085R, Y1092X; exon 19: R1162X, 3659delC, 3662delA, 3667del4, 3737delA, I1234V, S1235R, 3849G>A; exon 20: 3860ins31,S1255X,3898insC,3905insT,D1270N, W1282X, Q1291R; and exon 21: N1303H, N1303K, W1316X. Login to comment

[hide] Cystic fibrosis diagnosed in an elderly man. Respiration. 2004 Jan-Feb;71(1):98-100. Gilljam M, Bjorck E
Cystic fibrosis diagnosed in an elderly man.
Respiration. 2004 Jan-Feb;71(1):98-100., [PMID:14872121]

Abstract [show]
Although most patients with cystic fibrosis (CF) are diagnosed in early childhood, the diagnosis may be delayed for patients with mild symptoms or single-organ disease. We describe a man with known infertility and a history of productive cough diagnosed with CF at 61 years of age. The patient carries two mutations and one variant in the CF transmembrane conductance regulator gene: 394delTT, P99L and R75Q. During a 15-year follow-up time he has developed significant pulmonary disease.

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No. Sentence Comment
21 Genetic testing later confirmed two CFTR gene mutations, 394delTT and P99L, and the R75Q variant. Login to comment
33 P99L is a missense mutation in exon 4 that was originally described in a patient with the 'F508 mutation on the other allele and presenting with very mild CF [7]. Login to comment

[hide] Misprocessing of the CFTR protein leads to mild cy... Hum Mutat. 2005 Apr;25(4):360-71. Clain J, Lehmann-Che J, Dugueperoux I, Arous N, Girodon E, Legendre M, Goossens M, Edelman A, de Braekeleer M, Teulon J, Fanen P
Misprocessing of the CFTR protein leads to mild cystic fibrosis phenotype.
Hum Mutat. 2005 Apr;25(4):360-71., [PMID:15776432]

Abstract [show]
Cystic fibrosis (CF) is mainly caused by mutations that interfere with the biosynthetic folding of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The aim of this study was to determine the mechanism of dysfunction of a disease-causing mutation associated with variable phenotypes. In order to attain these objectives, we studied the effect of the p.L206W mutation on CFTR protein production and function, and we examined the genotype-phenotype correlation of [p.L206W]+[p.F508del] patients. We showed that p.L206W is a processing (class II) mutation since the CFTR biosynthetic pathway was severely impaired, whereas single-channel measurements indicated ion conductance similar to the wild-type protein. These data raise the larger question of the phenotypic variability of class II mutants, including p.F508del. Since multiple potential partners could modify the processing of the CFTR protein during its course to the cell surface, environmental and other genetic factors might contribute to this variability.

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No. Sentence Comment
274 p.P99L, p.R117H, p.R334W, and p.R347D/H/P form ClÀ channels with altered permeation properties but are processed normally and are therefore indexed as class IV mutants [Sheppard et al., 1993, 1996; Tabcharani et al., 1993]. Login to comment

[hide] Application of high-resolution single-channel reco... Methods Mol Biol. 2011;741:419-41. Cai Z, Sohma Y, Bompadre SG, Sheppard DN, Hwang TC
Application of high-resolution single-channel recording to functional studies of cystic fibrosis mutants.
Methods Mol Biol. 2011;741:419-41., [PMID:21594800]

Abstract [show]
The patch-clamp technique is a powerful and versatile method to investigate the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, its malfunction in disease and modulation by small molecules. Here, we discuss how the molecular behaviour of CFTR is investigated using high-resolution single-channel recording and kinetic analyses of channel gating. We review methods used to quantify how cystic fibrosis (CF) mutants perturb the biophysical properties and regulation of CFTR. By explaining the relationship between macroscopic and single-channel currents, we demonstrate how single-channel data provide molecular explanations for changes in CFTR-mediated transepithelial ion transport elicited by CF mutants.

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No. Sentence Comment
297 For example, the CF mutant, P99L, in the first transmembrane segment attenuated the single-channel conductance (wild-type, 7.72 ± 0.22 pS (means ± SEM; n = 4); P99L, 4.97 ± 0.24 pS (n = 5, p < 0.0001)) and altered the anion selectivity sequence (wild-type, Br- ≥ Cl- > I-; P99L, Br- ≥ Cl- = I-) (62). Login to comment

[hide] Structure and function of the CFTR chloride channe... Physiol Rev. 1999 Jan;79(1 Suppl):S23-45. Sheppard DN, Welsh MJ
Structure and function of the CFTR chloride channel.
Physiol Rev. 1999 Jan;79(1 Suppl):S23-45., [PMID:9922375]

Abstract [show]
Structure and Function of the CFTR Chloride Channel. Physiol. Rev. 79, Suppl.: S23-S45, 1999. - The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ABC transporter family that forms a novel Cl- channel. It is located predominantly in the apical membrane of epithelia where it mediates transepithelial salt and liquid movement. Dysfunction of CFTR causes the genetic disease cystic fibrosis. The CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. Here we review the structure and function of this unique channel, with a focus on how the various domains contribute to channel function. The MSDs form the channel pore, phosphorylation of the R domain determines channel activity, and ATP hydrolysis by the NBDs controls channel gating. Current knowledge of CFTR structure and function may help us understand better its mechanism of action, its role in electrolyte transport, its dysfunction in cystic fibrosis, and its relationship to other ABC transporters.

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No. Sentence Comment
124 When the basic arginine at this position was re- gle-channel Cl0 conductance in the rank order: wild type ' P99G ú P99L ' P99A (119). Login to comment

[hide] CFTR: mechanism of anion conduction. Physiol Rev. 1999 Jan;79(1 Suppl):S47-75. Dawson DC, Smith SS, Mansoura MK
CFTR: mechanism of anion conduction.
Physiol Rev. 1999 Jan;79(1 Suppl):S47-75., [PMID:9922376]

Abstract [show]
CFTR: Mechanism of Anion Conduction. Physiol. Rev. 79, Suppl.: S47-S75, 1999. - The purpose of this review is to collect together the results of recent investigations of anion conductance by the cystic fibrosis transmembrane conductance regulator along with some of the basic background that is a prerequisite for developing some physical picture of the conduction process. The review begins with an introduction to the concepts of permeability and conductance and the Nernst-Planck and rate theory models that are used to interpret these parameters. Some of the physical forces that impinge on anion conductance are considered in the context of permeability selectivity and anion binding to proteins. Probes of the conduction process are considered, particularly permeant anions that bind tightly within the pore and block anion flow. Finally, structure-function studies are reviewed in the context of some predictions for the origin of pore properties.

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No. Sentence Comment
585 A patient mutation, P99L, was associated with reduced single-chan-tion reduced the single-channel conductance by Ç30%. Login to comment

[hide] Cystic fibrosis phenotype associated with pancreat... J Biol Chem. 1997 Nov 28;272(48):30563-6. Fanen P, Labarthe R, Garnier F, Benharouga M, Goossens M, Edelman A
Cystic fibrosis phenotype associated with pancreatic insufficiency does not always reflect the cAMP-dependent chloride conductive pathway defect. Analysis of C225R-CFTR and R1066C-CFTR.
J Biol Chem. 1997 Nov 28;272(48):30563-6., [PMID:9374552]

Abstract [show]
We have previously screened the cystic fibrosis transmembrane conductance regulator (CFTR) gene and identified new disease-causing mutations. C225R and R1066C are both associated with pancreatic insufficiency, but the former mutation is associated with mild and unusual lung disease, whereas the latter is associated with severe lung disease. In the present study, we expressed these mutants heterologously in HeLa cells, and we analyzed protein synthesis by immunoprecipitation and chloride channel function by using a halide-sensitive fluorescent dye, 6-methoxy-N-ethylquinolinium. Immunoprecipitation and functional studies showed that cells transfected with C225R-CFTR exhibit cAMP-dependent chloride fluxes; C225R-CFTR protein is poorly expressed but fully glycosylated and can be compared with R117H-CFTR. R1066C-CFTR protein is not correctly processed and, unlike DeltaF508-CFTR, this defect cannot be corrected by reduced temperature or overexpression in butyrate-treated cells; defective processing may occur at a different step in the biosynthetic pathway. These results point to two different mechanisms underlying the same pancreatic status and suggest that it is unwise to use pancreatic sufficiency and insufficiency to define mild and severe cystic fibrosis (CF) disease, respectively. Finally, the experimental model described here may be helpful to predict the pulmonary status of CF patients bearing mutations located in putative membrane-spanning domains of the CFTR protein.

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No. Sentence Comment
101 Six CF-associated mutations (P99L, R117H, P205S, R334W, R347P, and R347H) located in putative membrane-spanning domains that have already been analyzed for their functional properties (2-5) were all associated with a mild phenotype (pancreatic sufficiency, PS). Login to comment

[hide] Probing the structural and functional domains of t... J Bioenerg Biomembr. 1997 Oct;29(5):453-63. Akabas MH, Cheung M, Guinamard R
Probing the structural and functional domains of the CFTR chloride channel.
J Bioenerg Biomembr. 1997 Oct;29(5):453-63., [PMID:9511930]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) forms an anion-selective channel involved in epithelial chloride transport. Recent studies have provided new insights into the structural determinants of the channel's functional properties, such as anion selectivity, single-channel conductance, and gating. Using the scanning-cysteine-accessibility method we identified 7 residues in the M1 membrane-spanning segment and 11 residues in and flanking the M6 segment that are exposed on the water-accessible surface of the protein; many of these residues may line the ion-conducting pathway. The pattern of the accessible residues suggests that these segments have a largely alpha-helical secondary structure with one face exposed in the channel lumen. Our results suggest that the residues at the cytoplasmic end of the M6 segment loop back into the channel, narrowing the lumen, and thereby forming both the major resistance to ion movement and the charge-selectivity filter.

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No. Sentence Comment
143 A similar mechanism is likely involved in the alteration of the halide permeability sequence by the P99L mutation (Sheppard et al., 1996) because Pro99 is also not a channel-lining residue (Akabas et al., 1994b). Login to comment

[hide] Function of Xenopus cystic fibrosis transmembrane ... J Biol Chem. 1996 Oct 11;271(41):25184-91. Price MP, Ishihara H, Sheppard DN, Welsh MJ
Function of Xenopus cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels and use of human-Xenopus chimeras to investigate the pore properties of CFTR.
J Biol Chem. 1996 Oct 11;271(41):25184-91., [PMID:8810276]

Abstract [show]
To explore the relationship between structure and function in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, we studied Xenopus CFTR. We found that the anion permeability sequence of cAMP-activated Cl- currents in the apical membrane of Xenopus A6 epithelia differed from that of cAMP-activated Cl- currents in human epithelia expressing CFTR. To understand the molecular basis for this difference and to learn whether CFTR from another species would have properties similar to human CFTR, we assembled a full-length Xenopus CFTR cDNA from A6 cells. Expression of Xenopus CFTR in HeLa cells generated cAMP-activated whole-cell currents and cAMP-dependent protein kinase-activated single channels that resembled those of human CFTR with the exception that the anion permeability sequence was different (Br- = I- > Cl- in Xenopus CFTR and Br- = Cl- > I- in human). In addition, the single-channel conductance of Xenopus CFTR was increased. To investigate protein regions that account for these differences, we constructed chimeric proteins by replacing either the first or second membrane-spanning domain of human CFTR with the equivalent region of Xenopus CFTR (hX1-6 and hX7-12, respectively) and examined their function in HeLa cells. We found that the anion permeability sequence (Br- = I- > Cl-) and single-channel conductance of hX1-6 resembled that of Xenopus CFTR expressed in HeLa cells, whereas hX7-12 had properties like those of human CFTR. However, the gating of hX1-6 showed a flickery behavior. The altered gating of hX1-6 was attributed to residues in the first extracellular loop of Xenopus CFTR because mutation of residues in that region to the corresponding residues of human CFTR produced gating behavior similar to that of human CFTR. These data suggest that sequence differences in the first membrane-spanning domains are responsible for the differences in the permeation properties of human and Xenopus CFTR and that the first extracellular loop influences channel gating.

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No. Sentence Comment
260 In MSD1, two mutations (P99L and K335E) that alter both anion permeability and conductance have been identified (15, 30); these are conserved in human and Xenopus CFTR. Login to comment
279 In MSD1, two mutations (P99L and K335E) that alter both anion permeability and conductance have been identified (15, 30); these are conserved in human and Xenopus CFTR. Login to comment

[hide] Contribution of proline residues in the membrane-s... J Biol Chem. 1996 Jun 21;271(25):14995-5001. Sheppard DN, Travis SM, Ishihara H, Welsh MJ
Contribution of proline residues in the membrane-spanning domains of cystic fibrosis transmembrane conductance regulator to chloride channel function.
J Biol Chem. 1996 Jun 21;271(25):14995-5001., [PMID:8663008]

Abstract [show]
Proline residues located in membrane-spanning domains of transport proteins are thought to play an important structural role. In the cystic fibrosis transmembrane conductance regulator (CFTR), the predicted transmembrane segments contain four prolines: Pro99, Pro205, Pro324, and Pro1021. These residues are conserved across species, and mutations of two (P99L and P205S) are associated with cystic fibrosis. To evaluate the contribution of these prolines to CFTR Cl- channel function, we mutated each residue individually to either alanine or glycine or mutated all four simultaneously to alanine (P-Quad-A). We also constructed the two cystic fibrosis-associated mutations. cAMP agonists stimulated whole cell Cl- currents in HeLa cells expressing the individual constructs that resembled those produced by wild-type CFTR. However, the amount of current was decreased in the rank order: wild-type CFTR = Pro324 > Pro1021 > Pro99 >/= Pro205 mutants. The anion selectivity sequence of the mutants (Br- >/= Cl- > I-) resembled wild-type except for P99L (Br- >/= Cl- = I-). Although the Pro99, Pro324, and Pro1021 mutants produced mature protein, the amount of mature protein was much reduced with the Pro205 mutants, and the P-Quad-A made none. Because the Pro99 constructs produced mature protein but had altered whole cell currents, we investigated their single-channel properties. Mutant channels were regulated like wild-type CFTR; however, single-channel conductance was decreased in the rank order: wild-type CFTR >/= P99G > P99L >/= P99A. These results suggest that proline residues in the transmembrane segments are important for CFTR function, Pro205 is critical for correct protein processing, and Pro99 may contribute either directly or indirectly to the Cl- channel pore.

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No. Sentence Comment
2 These residues are conserved across species, and mutations of two (P99L and P205S) are associated with cystic fibrosis. Login to comment
6 The anion selectivity sequence of the mutants (Br- Cl- > I- ) resembled wild-type except for P99L (Br- Cl- ‫؍‬ I- ). Login to comment
9 Mutant channels were regulated like wild-type CFTR; however, single-channel conductance was decreased in the rank order: wild-type CFTR P99G > P99L P99A. Login to comment
23 These residues are conserved across species and mutations of two (P99L and P205S) are associated with cystic fibrosis (CF) (12, 13). Login to comment
24 Because P99L and P205S are associated with a milder clinical phenotype characterized by retention of some pancreatic function (termed pancreatic sufficiency) (2), these mutants may retain some residual Cl- channel function. Based on the above information, the aims of this study were to determine the contribution of proline residues located within the MSDs of CFTR to Cl- channel function and to understand how P99L and P205S cause a loss of Cl- channel function. Login to comment
76 Cyclic AMP agonists activated whole cell currents in cells expressing each of the individual proline to alanine or glycine mutations and in cells expressing the CF-associated mutations, P99L and P205S. Login to comment
77 As an example, Fig. 2 shows data from studies of P99A, P99G, and P99L; qualitatively similar results were obtained with the Pro205 , Pro324 , and Pro1021 mutants (data not shown). Login to comment
85 However, the CF-associated mutation P99L had an altered anion selectivity sequence, Br- Ն Cl- ϭ I- (Fig. 3 and Table I). Login to comment
119 Reversal potentials (Erev) of baseline subtracted cAMP-activated whole cell currents were P99A, -30 Ϯ 1 mV (n ϭ 7); P99G, -34 Ϯ 2 mV (n ϭ 5); and P99L, -24 Ϯ 3 mV (n ϭ 6). Login to comment
122 Mutant n Px/PCl Gx/GCl Br- Cl- IBr- ClI- CFTR 5 1.18 Ϯ 0.08 1.00 0.73 Ϯ 0.05 1.27 Ϯ 0.16 1.00 0.61 Ϯ 0.08 P99A 7 0.98 Ϯ 0.03 1.00 0.70 Ϯ 0.06 1.04 Ϯ 0.05 1.00 0.72 Ϯ 0.05 P99G 5 1.06 Ϯ 0.02 1.00 0.75 Ϯ 0.08 1.04 Ϯ 0.07 1.00 0.66 Ϯ 0.05 P99L 5 1.21 Ϯ 0.07 1.00 1.06 Ϯ 0.07 1.33 Ϯ 0.11 1.00 0.95 Ϯ 0.08 P205A 4 1.09 Ϯ 0.07 1.00 0.64 Ϯ 0.09 0.95 Ϯ 0.04 1.00 0.46 Ϯ 0.11 P205G 5 1.09 Ϯ 0.05 1.00 0.45 Ϯ 0.05 1.05 Ϯ 0.03 1.00 0.44 Ϯ 0.06 P205S 2 1.01 Ϯ 0.01 1.00 0.55 Ϯ 0.28 1.09 Ϯ 0.09 1.00 0.59 Ϯ 0.08 P324A 7 1.08 Ϯ 0.04 1.00 0.72 Ϯ 0.06 1.15 Ϯ 0.07 1.00 0.60 Ϯ 0.08 P324G 6 1.12 Ϯ 0.07 1.00 0.69 Ϯ 0.04 1.22 Ϯ 0.14 1.00 0.57 Ϯ 0.04 P1021A 3 1.15 Ϯ 0.17 1.00 0.73 Ϯ 0.11 1.17 Ϯ 0.10 1.00 0.47 Ϯ 0.19 P1021G 7 1.17 Ϯ 0.06 1.00 0.78 Ϯ 0.02 1.21 Ϯ 0.08 1.00 0.59 Ϯ 0.06 though for P99G the reduction was small, for P99A and P99L the effect was marked. Login to comment
126 The conductance for P99G was 7.31 Ϯ 0.24 pS (n ϭ 5), not significantly different from wild type (p ϭ 0.26). Login to comment
127 In contrast, the conductances of P99A and P99L were significantly decreased at 4.66 Ϯ 0.25 pS (n ϭ 5, p Ͻ 0.0001) and 4.97 Ϯ 0.24 pS (n ϭ 5, p Ͻ 0.0001), respectively. Login to comment
129 Because the single-channel current amplitudes of P99A and P99L were much reduced and because in most cases the patches of membrane contained large numbers of channels, we could not accurately measure single-channel kinetics. Login to comment
145 The number of cells responding to cAMP agonists with Cl- current activation relative to the total number of cells tested for each construct was: CFTR (8/16; 50%), P99A (11/12; 92%), P99G (9/19; 47%), P99L (10/19; 53%), P205A (7/12; 58%), P205G (5/9; 56%), P205S (7/20; 35%), P324A (9/18; 50%), P324G (9/22; 41%), P1021A (8/18; 44%), and P1021G (7/16; 44%). Login to comment
176 Similar results were observed with P99G and P99L; n Ͼ 5 for each mutant. Login to comment
177 B, representative single-channel recordings are from excised inside-out membrane patches from HeLa cells transiently expressing wild-type CFTR, P99A, P99G, and P99L. Login to comment
180 C, single-channel I-V relationships of CFTR (circles), P99A (squares), P99G (triangles), and P99L (inverted triangles. Login to comment
194 When Pro99 was mutated to leucine, the channel lost its ability to discriminate between Cl- and I- . Login to comment
199 Substitution of alanine, glycine, and leucine at Pro99 decreased single-channel conductance in the rank order: wild-type CFTR Ն P99G Ͼ P99L Ն P99A. Login to comment
206 Implications for Cystic Fibrosis-P99L and P205S are CF mutations located in MSD1 that are associated with a milder (pancreatic sufficiency) clinical phenotype (12, 13). Login to comment
207 Our studies of the processing and function of P99L and P205S explain why these mutants generate less Cl- current than wild-type CFTR. Login to comment
208 Loss of Cl- channel function caused by P205S was predominantly a result of defective protein processing; whereas that caused by P99L was a consequence of both defective protein processing and altered Cl- channel function. Login to comment
215 The present results complement and extend our previous study of mild CF mutants located in MSD1 (R117H, R334W, and R347P). Login to comment
218 Interestingly, P99L forms a Cl- channel with altered pore properties and is also misprocessed. Login to comment
221 We thank Dr. M. Schwartz (Department of Clinical Genetics, University Hospital, Copenhagen, Denmark) for generously providing details of the clinical phenotype of the patient with the P99L mutation prior to publication. Login to comment
78 As an example, Fig. 2 shows data from studies of P99A, P99G, and P99L; qualitatively similar results were obtained with the Pro205 , Pro324 , and Pro1021 mutants (data not shown). Login to comment
87 However, the CF-associated mutation P99L had an altered anion selectivity sequence, Br2 $ Cl2 5 I2 (Fig. 3 and Table I). Login to comment
123 Reversal potentials (Erev) of baseline subtracted cAMP-activated whole cell currents were P99A, 230 6 1 mV (n 5 7); P99G, 234 6 2 mV (n 5 5); and P99L, 224 6 3 mV (n 5 6). Login to comment
131 In contrast, the conductances of P99A and P99L were significantly decreased at 4.66 6 0.25 pS (n 5 5, p , 0.0001) and 4.97 6 0.24 pS (n 5 5, p , 0.0001), respectively. Login to comment
133 Because the single-channel current amplitudes of P99A and P99L were much reduced and because in most cases the patches of membrane contained large numbers of channels, we could not accurately measure single-channel kinetics. Login to comment
149 The number of cells responding to cAMP agonists with Cl2 current activation relative to the total number of cells tested for each construct was: CFTR (8/16; 50%), P99A (11/12; 92%), P99G (9/19; 47%), P99L (10/19; 53%), P205A (7/12; 58%), P205G (5/9; 56%), P205S (7/20; 35%), P324A (9/18; 50%), P324G (9/22; 41%), P1021A (8/18; 44%), and P1021G (7/16; 44%). Login to comment
181 Similar results were observed with P99G and P99L; n . Login to comment
183 B, representative single-channel recordings are from excised inside-out membrane patches from HeLa cells transiently expressing wild-type CFTR, P99A, P99G, and P99L. Login to comment
186 C, single-channel I-V relationships of CFTR (circles), P99A (squares), P99G (triangles), and P99L (inverted triangles. Login to comment
200 When Pro99 was mutated to leucine, the channel lost its ability to discriminate between Cl2 and I2 . Login to comment
214 Implications for Cystic Fibrosis-P99L and P205S are CF mutations located in MSD1 that are associated with a milder (pancreatic sufficiency) clinical phenotype (12, 13). Login to comment
216 Loss of Cl2 channel function caused by P205S was predominantly a result of defective protein processing; whereas that caused by P99L was a consequence of both defective protein processing and altered Cl2 channel function. Login to comment
226 Interestingly, P99L forms a Cl2 channel with altered pore properties and is also misprocessed. Login to comment
230 We thank Dr. M. Schwartz (Department of Clinical Genetics, University Hospital, Copenhagen, Denmark) for generously providing details of the clinical phenotype of the patient with the P99L mutation prior to publication. Login to comment

[hide] Understanding how cystic fibrosis mutations disrup... Int J Biochem Cell Biol. 2014 Jul;52:47-57. doi: 10.1016/j.biocel.2014.04.001. Epub 2014 Apr 13. Wang Y, Wrennall JA, Cai Z, Li H, Sheppard DN
Understanding how cystic fibrosis mutations disrupt CFTR function: from single molecules to animal models.
Int J Biochem Cell Biol. 2014 Jul;52:47-57. doi: 10.1016/j.biocel.2014.04.001. Epub 2014 Apr 13., [PMID:24727426]

Abstract [show]
Defective epithelial ion transport is the hallmark of the life-limiting genetic disease cystic fibrosis (CF). This abnormality is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the ATP-binding cassette transporter that functions as a ligand-gated anion channel. Since the identification of the CFTR gene, almost 2000 disease-causing mutations associated with a spectrum of clinical phenotypes have been reported, but the majority remain poorly characterised. Studies of a small number of mutations including the most common, F508del-CFTR, have identified six general mechanisms of CFTR dysfunction. Here, we review selectively progress to understand how CF mutations disrupt CFTR processing, stability and function. We explore CFTR structure and function to explain the molecular mechanisms of CFTR dysfunction and highlight new knowledge of disease pathophysiology emerging from large animal models of CF. Understanding CFTR dysfunction is crucial to the development of transformational therapies for CF patients.

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2011 For example, the CF mutation P99L, which affects a residue that faces away from the pore (Akabas et al., 1994; Gao et al., 2013, but see Wang et al., 2011), had an altered anion selectivity sequence (wild-type: Br- ࣙ Cl- > I-; P99L: Br- ࣙ Cl- = I-) and reduced single-channel conductance (Sheppard et al., 1996). Login to comment