ABCMdb

ABCC7 p.His949Tyr

ClinVar:	c.2845C>T , p.His949Tyr D , Pathogenic c.2846A>T , p.His949Leu ? , not provided c.2846A>G , p.His949Arg ? , not provided
CF databases:	c.2846A>G , p.His949Arg (CFTR1) D , The above mutation was found by DGGE and direct sequencing in Caucasian patients. c.2845C>T , p.His949Tyr (CFTR1) ? , The nucleotide change C->T at position 2977 (codon 949 in exon 15, H949Y), has been found in a 60 years old woman with no manifestation of gastrointestinal disease but having a bronchial dilatation. c.2846A>C , p.His949Pro (CFTR1) ? , c.2846A>T , p.His949Leu (CFTR1) ? , The nucleotide change was found by DGGE followed by sequencing of both the strands. This mutation was identified in 4 male patients from Puglia, region of Southern Italy. This mutation was identified in cis with another mutation: H939R. This is a novel complex CFTR allele: H939R + H949L.
Predicted by SNAP2:	A: D (85%), C: D (85%), D: D (95%), E: D (95%), F: D (91%), G: D (91%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (91%), R: D (95%), S: D (91%), T: D (91%), V: D (91%), W: D (95%), Y: D (53%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Two novel mutations in a cystic fibrosis patient o... Hum Genet. 1999 Jun;104(6):511-5. Wagner JA, Vassilakis A, Yee K, Li M, Hurlock G, Krouse ME, Moss RB, Wine JJ
Two novel mutations in a cystic fibrosis patient of Chinese origin.
Hum Genet. 1999 Jun;104(6):511-5., [PMID:10453741]

Abstract [show]
Cystic fibrosis is rare in non-Caucasian populations, and in such populations little is known about the spectrum of mutations and polymorphisms in the CFTR gene. We studied a 23-year-old patient of Chinese ethnicity with sweat chloride values of 104 mM/l, pancreatic sufficiency, an FEV1 60% of normal, sputum cultures positive for Staphylococcus aureus and Burkholderia cepacia, and a history of allergic bronchopulmonary aspergillosis. Genetic screening for 31 common CFTR mutations was negative, leading us to search for unknown mutations using single-strand conformation polymorphism and heteroduplex analysis (SSCP/HA). Two novel mutations were detected. In exon 4, a deletion of 8 bp (451458, deltaGCTTCCTA) causes a frameshift and immediately creates a stop codon. In exon 16, mutation 3041G-->A causes the missense change G970D. Functional analysis using an isotopic flux assay indicated that the G970D mutation retains partial function; western blotting indicated that the protein is glycosylated. The patient is heterozygous for the common polymorphisms (2694T/G) in exon 14a and (GATT)6/7 in intron 6a, indicating that these variants arose in ancestors common to Caucasians and Chinese.

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No. Sentence Comment
97 G970 lies within the third cytoplasmic loop of CFTR (residues 933- 990, between TMD 6 and 7), as do the CF-causing mutations, S945L (Claustres et al. 1993) and H949Y (Ghanem et al. 1994). Login to comment
98 Seibert et al. (1996) examined all three point mutations in the third cytoplasmic loop and determined that S945L and H949Y are trafficking mutations, while G970R is trafficked normally, but shows significantly reduced function when tested with an iodide efflux assay. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Biochemistry. 2000 Apr 4;39(13):3797-803. Chen EY, Bartlett MC, Clarke DM
Cystic fibrosis transmembrane conductance regulator has an altered structure when its maturation is inhibited.
Biochemistry. 2000 Apr 4;39(13):3797-803., 2000-04-04 [PMID:10736180]

Abstract [show]
Inefficient maturation and trafficking to the cell surface of the cystic fibrosis transmembrane conductance regulator (CFTR) is the primary cause of cystic fibrosis. CFTR protein that fails to mature accumulates as an immature core-glycosylated protein and is rapidly degraded. To determine how the structures of mature and immature CFTR are different, we compared the properties of CFTR that had been expressed in the presence or absence of the proteasome inhibitor, MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal). Transient expression of wild-type CFTR in the presence of submicromolar concentrations of MG-132 blocks maturation of the protein. We found that expression of CFTR in the presence of MG-132 trapped the protein in a trypsin-sensitive conformation. In addition, the structure of the carboxyl-terminus of immature and mature CFTR differed as histidine-tagged mature CFTR was preferentially recovered by metal-chelate chromatography. No chloride channel activity was detected when membranes containing immature CFTR were fused with planar lipid bilayers. These results show that expression of CFTR in the presence of MG-132 traps the protein in an altered conformation that may be inactive.

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No. Sentence Comment
200 H949Y is a partially processing-defective mutant that expressed less than 10% of the mature band, relative to wild type (3). Login to comment
224 Membranes were prepared from HEK cells transiently transfected with various CFTR cDNAs: WT, ∆F508, R297Q, H949Y, and G149R. Login to comment

[hide] Aberrant CFTR-dependent HCO3- transport in mutatio... Nature. 2001 Mar 1;410(6824):94-7. Choi JY, Muallem D, Kiselyov K, Lee MG, Thomas PJ, Muallem S
Aberrant CFTR-dependent HCO3- transport in mutations associated with cystic fibrosis.
Nature. 2001 Mar 1;410(6824):94-7., 2001-03-01 [PMID:11242048]

Abstract [show]
Cystic fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Initially, Cl- conductance in the sweat duct was discovered to be impaired in CF, a finding that has been extended to all CFTR-expressing cells. Subsequent cloning of the gene showed that CFTR functions as a cyclic-AMP-regulated Cl- channel; and some CF-causing mutations inhibit CFTR Cl- channel activity. The identification of additional CF-causing mutants with normal Cl- channel activity indicates, however, that other CFTR-dependent processes contribute to the disease. Indeed, CFTR regulates other transporters, including Cl(-)-coupled HCO3- transport. Alkaline fluids are secreted by normal tissues, whereas acidic fluids are secreted by mutant CFTR-expressing tissues, indicating the importance of this activity. HCO3- and pH affect mucin viscosity and bacterial binding. We have examined Cl(-)-coupled HCO3- transport by CFTR mutants that retain substantial or normal Cl- channel activity. Here we show that mutants reported to be associated with CF with pancreatic insufficiency do not support HCO3- transport, and those associated with pancreatic sufficiency show reduced HCO3- transport. Our findings demonstrate the importance of HCO3- transport in the function of secretory epithelia and in CF.

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No. Sentence Comment
186 letters to nature 96 NATURE |VOL 410 |1 MARCH 2001 |www.nature.com HCO3 -/Cl- transportratio 0 0.25 0.50 0.75 1.00 WT I148T G178R R297Q G551D H620Q G970R A1067T G1244E S1255P G1349D E193K G551S A800G H949Y R1070Q Pancreatic insufficient Pancreatic sufficientD648V N CI148T G178R E193K R297Q R117H A1067T R1070Q G1244E S1255P G1349D NBD2 RD H949Y G970R CL4CL3CL2CL1 NBD1 G551D G551S H620Q D648V A800G Figure 3 The HCO3:Cl-transport ratio of CFTR mutants associated with CF. Login to comment

[hide] Genetics of idiopathic disseminated bronchiectasis... Semin Respir Crit Care Med. 2003 Apr;24(2):179-84. Luisetti M, Pignatti PF
Genetics of idiopathic disseminated bronchiectasis.
Semin Respir Crit Care Med. 2003 Apr;24(2):179-84., [PMID:16088537]

Abstract [show]
Bronchiectasis is an abnormal dilation of bronchi, consequent to the destruction of their walls. It is included in the category of obstructive pulmonary diseases, along with chronic obstructive pulmonary disease (COPD), asthma, and cystic fibrosis. In approximately 50% of cases, bronchiectasis is associated with underlying conditions; in the remainder, known causes are not ascertainable (idiopathic bronchiectasis). A search for genetic determinants of this phenotype, with the cystic fibrosis gene as a candidate, has been performed by three independent groups. The results of this search agreed on the association of bronchiectasis with cystic fibrosis gene mutations and polymorphisms. The cystic fibrosis gene is also associated with bronchiectasis due to rheumatoid arthritis and allergic bronchopulmonary aspergillosis. A few other genes have been investigated in idiopathic bronchiectasis, with negative results. Idiopathic bronchiectasis is, therefore, to be considered as an obstructive multifactorial disorder belonging to the category of cystic fibrosis monosymptomatic diseases (or CFTR-opathies), whose pathogenesis is influenced by environmental factors and other undetermined genes.

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42 Greek M/F 11/12 5/16 na Mean age (yrs) 53 Ϯ 15 53 Ϯ 14 na CFTR gene 1 G576A-R668C/L997F 1 ⌬F508/D192N 1 ⌬F508,I1027T mutation 1 ⌬F508/L997F 1 ⌬I507/3849 + 10kb C → T 1 D565G, R668C 1 ⌬F508/- 1 ⌬F508/3849 + 10kb C → T 1 T896I/- 1 R1066C/- 1 H949Y/T1220I 1 I148T/- 1 3667ins4/- 1 ⌬F508/- 1 ⌬F508/S977F 1 R75Q/- 1 2183AA→G 1 M1137V/- 1 L997F/- IVS8-5T 5 5/7 1 5/9 1 5/5 CFTR, cystic fibrosis transmembrane conductance regulator; na, not available. Login to comment

[hide] Rescue of DeltaF508 and other misprocessed CFTR mu... Mol Pharm. 2005 Sep-Oct;2(5):407-13. Loo TW, Bartlett MC, Clarke DM
Rescue of DeltaF508 and other misprocessed CFTR mutants by a novel quinazoline compound.
Mol Pharm. 2005 Sep-Oct;2(5):407-13., [PMID:16196493]

Abstract [show]
Cystic fibrosis (CF) is most commonly caused by deletion of Phe508 in the cystic fibrosis transmembrane conductance regulator protein (DeltaF508 CFTR). The misfolded DeltaF508 CFTR protein is retained in the endoplasmic reticulum (misprocessed mutant) and is rapidly degraded. Studies on misprocessed mutants of P-glycoprotein (P-gp), a sister protein of CFTR, however, have shown that specific substrates and modulators can act as specific chemical/pharmacological chaperones to rescue the protein. A major goal in CF research is the identification of compounds that can be used at low concentrations to rescue misprocessed CFTR mutants. Here, we show that a novel quinazoline derivative, 4-cyclohexyloxy-2-{1-[4-(4-methoxy-benzenesulfonyl)piperazin-1-yl]ethyl}qu inazoline (CF(cor)-325), rescued DeltaF508 CFTR. Incubation of BHK cells stably expressing human DeltaF508 CFTR with 1-10 microM CF(cor)-325 resulted in maturation and delivery of a functional molecule to the cell surface as determined by the iodide efflux assay. The misprocessed CFTR mutants R258G, S945L, and H949Y were also rescued by CF(cor)-325 in either BHK or HEK 293 cells. CF(cor)-325 appeared to be specific for DeltaF508 CFTR because another quinazoline derivative, prazosin, did not rescue the misprocessed CFTR mutants. CF(cor)-325 could also rescue misprocessed mutants of P-gp. The compound was a P-gp inhibitor as it inhibited vinblastine-stimulated ATPase activity. P-gp-mediated vinblastine resistance was also reduced about 10-fold with 300 nM CF(cor)-325. These results show that CF(cor)-325 is a particularly important lead compound for treatment of CF because low concentrations can be used to rescue many misprocessed CFTR mutants.

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No. Sentence Comment
5 Incubation of BHK cells stably expressing human ∆F508 CFTR with 1-10 µM CFcor-325 resulted in maturation and delivery of a functional molecule to the cell surface as determined by the iodide efflux assay. The misprocessed CFTR mutants R258G, S945L, and H949Y were also rescued by CFcor-325 in either BHK or HEK 293 cells. Login to comment
26 Wild-type, ∆F508, H139R, G149R, R258G, S945L, and H949Y CFTR cDNAs were inserted into the pcDNA3 (Invitrogen, Oakville, ON) vector as described previously.13,14 Wild-type and mutant G268V P-gp cDNAs were inserted into the pMT21 vector (Genetics Institute) as described previously.15 Baby hamster kidney (BHK) cells stably expressing CFTR or P-gp were generated by cotransfection with cDNA and pWL-neo (Stratagene, Cedar Creek, TX). Login to comment
132 (C) BHK cells expressing misprocessed CFTR mutants H139R, G149R, R258G, S945L, H949Y, or wild-type CFTR were incubated for 48 h with (+) or without (-) 3 µM CFcor-325. Whole cell extracts were subjected to immunoblot analysis with a rabbit polyclonal antibody against CFTR. Login to comment
144 BHK cells expressing mutants H139R, G149R, and R258G in the first transmembrane domain (TMD1)30 or mutants S945L and H949Y in TMD213 were treated with or without 3 µM CFcor-325 for 48 h. Whole cell SDS extracts were then subjected to immunoblot analysis. Login to comment
145 The presence of CFcor-325 significantly enhanced maturation of mutants R258G, S945L, and H949Y (Figure 2C). Login to comment
178 It was possible to promote maturation of some mutants that had mutations in different domains of CFTR including NBD1 (∆F508), TMD1 (R258G in the second intracellular loop), and TMD2 (S945L and H949Y in the third intracellular loop). Login to comment
24 Wild-type, ∆F508, H139R, G149R, R258G, S945L, and H949Y CFTR cDNAs were inserted into the pcDNA3 (Invitrogen, Oakville, ON) vector as described previously.13,14 Wild-type and mutant G268V P-gp cDNAs were inserted into the pMT21 vector (Genetics Institute) as described previously.15 Baby hamster kidney (BHK) cells stably expressing CFTR or P-gp were generated by cotransfection with cDNA and pWL-neo (Stratagene, Cedar Creek, TX). Login to comment
130 (C) BHK cells expressing misprocessed CFTR mutants H139R, G149R, R258G, S945L, H949Y, or wild-type CFTR were incubated for 48 h with (+) or without (-) 3 µM CFcor-325. Whole cell extracts were subjected to immunoblot analysis with a rabbit polyclonal antibody against CFTR. Login to comment
142 BHK cells expressing mutants H139R, G149R, and R258G in the first transmembrane domain (TMD1)30 or mutants S945L and H949Y in TMD213 were treated with or without 3 µM CFcor-325 for 48 h. Whole cell SDS extracts were then subjected to immunoblot analysis. Login to comment
143 The presence of CFcor-325 significantly enhanced maturation of mutants R258G, S945L, and H949Y (Figure 2C). Login to comment
176 It was possible to promote maturation of some mutants that had mutations in different domains of CFTR including NBD1 (∆F508), TMD1 (R258G in the second intracellular loop), and TMD2 (S945L and H949Y in the third intracellular loop). Login to comment

[hide] New horizons in the treatment of cystic fibrosis. Br J Pharmacol. 2011 May;163(1):173-83. doi: 10.1111/j.1476-5381.2010.01137.x. Cuthbert AW
New horizons in the treatment of cystic fibrosis.
Br J Pharmacol. 2011 May;163(1):173-83. doi: 10.1111/j.1476-5381.2010.01137.x., [PMID:21108631]

Abstract [show]
Cystic fibrosis (CF) is a lethal, recessive, genetic disease affecting approximately 1 in 2500 live births among Caucasians. The CF gene codes for a cAMP/PKA-dependent, ATP-requiring, membrane chloride ion channel, generally found in the apical membranes of many secreting epithelia and known as CFTR (cystic fibrosis transmembrane conductance regulator). There are currently over 1700 known mutations affecting CFTR, many of which give rise to a disease phenotype. Around 75% of CF alleles contain the DeltaF508 mutation in which a triplet codon has been lost, leading to a missing phenylalanine at position 508 in the protein. This altered protein fails to be trafficked to the correct location in the cell and is generally destroyed by the proteasome. The small amount that does reach the correct location functions poorly. Clearly the cohort of patients with at least one DeltaF508 allele are a major target for therapeutic intervention. It is now over two decades since the CF gene was discovered and during this time the properties of CFTR have been intensely investigated. At long last there appears to be progress with the pharmaco-therapeutic approach. Ongoing clinical trials have produced fascinating results in which clinical benefit appears to have been achieved. To arrive at this point ingenious ways have been devised to screen very large chemical libraries for one of two properties: (i) agents promoting trafficking of mutant CFTR to, and insertion into the membrane, and known as correctors or (ii) agents which activate appropriately located mutant CFTR, known as potentiators. The best compounds emerging from these programmes are then used as chemical scaffolds to synthesize other compounds with appropriate pharmaceutical properties, hopefully with their pharmacological activity maintained or even enhanced. In summary, this approach attempts to make the mutant CFTR function in place of the real CFTR. A major function of CFTR in healthy airways is to maintain an adequate airway surface liquid (ASL) layer. In CF the position is further confounded since epithelial sodium channels (ENaC) are no longer regulated and transport salt and water out of the airways to exacerbate the lack of ASL. Thus an additional possibility for treatment of CF is to use agents that inhibit ENaC either alone or as adjuncts to CFTR correctors and/or potentiators. Yet a further way in which a pharmacological approach to CF can be considered is to recruit alternative chloride channels, such as calcium-activated chloride channel (CaCC), to act as surrogates for CFTR. A number of P2Y(2) receptor agonists have been investigated that operate by increasing Ca(2+)(i) which in turn activates CaCC. Some of these compounds are currently in clinical trials. The knowledge base surrounding the structure and function of CFTR that has accumulated in the last 20 years is impressive. Translational research feeding from this is now yielding compounds that provide real prospects for a pharmacotherapy for this disease.

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No. Sentence Comment
251 In one of these, VRT-325 rescued CFTR mutants R258G, S945L and H949Y as well as DF508 CFTR (Loo et al., 2005). Login to comment

[hide] Pharmacological therapy for cystic fibrosis: from ... J Cyst Fibros. 2011 Jun;10 Suppl 2:S129-45. Becq F, Mall MA, Sheppard DN, Conese M, Zegarra-Moran O
Pharmacological therapy for cystic fibrosis: from bench to bedside.
J Cyst Fibros. 2011 Jun;10 Suppl 2:S129-45., [PMID:21658632]

Abstract [show]
With knowledge of the molecular behaviour of the cystic fibrosis transmembrane conductance regulator (CFTR), its physiological role and dysfunction in cystic fibrosis (CF), therapeutic strategies are now being developed that target the root cause of CF rather than disease symptoms. Here, we review progress towards the development of rational new therapies for CF. We highlight the discovery of small molecules that rescue the cell surface expression and defective channel gating of CF mutants, termed CFTR correctors and CFTR potentiators, respectively. We draw attention to alternative approaches to restore epithelial ion transport to CF epithelia, including inhibitors of the epithelial Na(+) channel (ENaC) and activators of the Ca(2+)-activated Cl(-) channel TMEM16A. The expertise required to translate small molecules identified in the laboratory to drugs for CF patients depends on our ability to coordinate drug development at an international level and our ability to provide pertinent biological information using suitable disease models.

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No. Sentence Comment
100 [29] BHK cells F508del, R258G, S945L, H949Y cAMP-stimulated iodide efflux, biochemistry VRT-325 (1-10 μM) rescued F508del-CFTR and other mutants after 48 h incubation of BHK cells at 37°C. VRT-325 also rescues misprocessed P-gp mutants and acts as P-gp inhibitor. Login to comment

[hide] Regulation of Activation and Processing of the Cys... J Biol Chem. 2012 Oct 11. Wang G, Duan DD
Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3.
J Biol Chem. 2012 Oct 11., [PMID:23060444]

Abstract [show]
NEG2, a short C-terminal segment (817-838) of the unique regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, has been reported to regulate CFTR gating in response to cAMP-dependent R domain phosphorylation. The underlying mechanism, however, is unclear. Here, K946 of cytoplasmic loop 3 (CL3) is proposed as counter-ion of D835, D836 or E838 of NEG2 to prevent channel activation by PKA. R764 or R766 of the S768 phosphorylation site of the R domain is proposed to promote channel activation possibly by weakening the putative CL3-NEG2 electrostatic attraction. First, not only D835A, D836A and E838A but also K946A reduced the PKA dependent CFTR activation. Second, both K946D and D835R/D836R/E838R mutants were activated by ATP and curcumin to a different extent. Third, R764A and R766A mutants enhanced the PKA-dependent activation. On the other hand, it is very exciting that D835R/D836R/E838R and K946D/H950D and H950R exhibited normal channel processing and activity while D835R/D836R/E838R/K946D/H950D was misprocessed and silent in response to forskolin. Further, D836R and E838R played a critical role in the asymmetric electrostatic regulation of CFTR processing and S768 phosphorylation may not be involved. Thus, a complex interfacial interaction among CL3, NEG2 and the S768 phosphorylation site may be responsible for the asymmetric electrostatic regulation of CFTR activation and processing.

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152 On the other hand, S945L and H949Y, found in patients with cystic fibrosis, are close to K946 and H950 of CL3 and stop maturation of the protein (31). Login to comment
191 However, S945L and H949Y, found in patients with CF, are close to Lys-946 and His-950 of CL3 and stop maturation of the protein (31). Login to comment

[hide] Conformation, independent of charge, in the R doma... Biophys J. 2000 Mar;78(3):1293-305. Xie J, Zhao J, Davis PB, Ma J
Conformation, independent of charge, in the R domain affects cystic fibrosis transmembrane conductance regulator channel openings.
Biophys J. 2000 Mar;78(3):1293-305., [PMID:10692317]

Abstract [show]
The R domain of cystic fibrosis transmembrane conductance regulator (CFTR), when phosphorylated, undergoes conformational change, and the chloride channel opens. We investigated the contribution of R domain conformation, apart from the changes induced by phosphorylation, to channel opening, by testing the effect of the peptidyl-prolyl isomerase, cyclophilin A, on the CFTR channel. When it was applied after the channel had been opened by PKA phosphorylation, cyclophilin A increased the open probability of wild-type CFTR (from P(o) = 0.197 +/- 0.010 to P(o) = 0.436 +/- 0. 029) by increasing the number of channel openings, not open time. Three highly conserved proline residues in the R domain, at positions 740, 750, and 759, were considered as candidate targets for cyclophilin A. Mutations of these prolines to alanines (P3A mutant) resulted in a channel unresponsive to cyclophilin A but with pore properties similar to the wild type, under strict control of PKA and ATP, but with significantly increased open probability (P(o) = 0.577 +/- 0.090) compared to wild-type CFTR, again due to an increase in the number of channel openings and not open time. Mutation of each of the proline residues separately and in pairs demonstrated that all three proline mutations are required for maximal P(o). When P3A was expressed in 293 HEK cells and tested by SPQ assay, chloride efflux was significantly increased compared to cells transfected with wild-type CFTR. Thus, treatments favoring the trans-peptidyl conformation about conserved proline residues in the R domain of CFTR affect openings of CFTR, above and beyond the effect of PKA phosphorylation.

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272 Although other CFTR mutants have chloride transport in excess of WT CFTR, they are either not processed efficiently (e.g., P574H or H949Y) (Sheppard et al., 1996a, b; Seibert et al., 1996) or open without PKA stimulation (e.g., CFTR-D836X), and thus are not subject to physiologic regulation (Sheppard et al., 1994). Login to comment
274 Although other CFTR mutants have chloride transport in excess of WT CFTR, they are either not processed efficiently (e.g., P574H or H949Y) (Sheppard et al., 1996a, b; Seibert et al., 1996) or open without PKA stimulation (e.g., CFTR-D836X), and thus are not subject to physiologic regulation (Sheppard et al., 1994). Login to comment

[hide] Disease-associated mutations in cytoplasmic loops ... Biochemistry. 1997 Sep 30;36(39):11966-74. Seibert FS, Jia Y, Mathews CJ, Hanrahan JW, Riordan JR, Loo TW, Clarke DM
Disease-associated mutations in cytoplasmic loops 1 and 2 of cystic fibrosis transmembrane conductance regulator impede processing or opening of the channel.
Biochemistry. 1997 Sep 30;36(39):11966-74., [PMID:9305991]

Abstract [show]
Since little is known about the contribution to function of the N-terminal cytoplasmic loops (CL1, residues 139-194; CL2, residues 242-307) of cystic fibrosis transmembrane conductance regulator (CFTR), all nine point mutations identified in CLs 1 and 2 from patients with cystic fibrosis were reconstructed in the expression vector pcDNA3-CFTR and expressed transiently in COS-1 and HEK-293 cells and stably in Chinese hamster ovary (CHO) cells. Four amino acid substitutions retarded production of mature, fully glycosylated CFTR, suggesting that misprocessing of the channel causes the disease symptoms in the affected patients. Protein maturation could not be promoted by cell culture conditions of reduced temperature (26 degrees C). When properly processed mutants were evaluated for functional defects by the iodide efflux method, the G178R- and E193K-CFTR-expressing cell lines showed impaired anion translocation activities. Patch-clamp studies of single channels revealed that E193K variants had a significantly decreased open probability, which resulted from an increase in the mean closed time of the channels. This contrasted with a previous study of disease-associated point mutations in CL3 that mainly affected the mean open time. None of the maturation-competent CL 1 and 2 mutants had altered conductance. Thus, the N-terminal CLs appear not to contribute to the anion translocation pathway of CFTR; rather, mutations in CL1 can impede transition to the open state. Interestingly, the ability of the non-hydrolyzable ATP analogue adenylyl imidodiphosphate (AMP-PNP) to lock the channel into open bursts was abolished by the I148T and G178R amino acid substitutions.

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113 The treatment did not promote processing to a degree detectable by Western blotting (Figure 3); this finding was not unexpected because of all mutations in the CLs examined thus far, only the H949Y-CFTR variant could be rescued to some degree (18, 19, 21; unpublished observations). Login to comment

[hide] Cytoplasmic loop three of cystic fibrosis transmem... J Biol Chem. 1996 Nov 1;271(44):27493-9. Seibert FS, Linsdell P, Loo TW, Hanrahan JW, Riordan JR, Clarke DM
Cytoplasmic loop three of cystic fibrosis transmembrane conductance regulator contributes to regulation of chloride channel activity.
J Biol Chem. 1996 Nov 1;271(44):27493-9., [PMID:8910333]

Abstract [show]
To examine the contribution of the large cytoplasmic loops of the cystic fibrosis transmembrane conductance regulator (CFTR) to channel activity, the three point-mutations (S945L, H949Y, G970R) were characterized that have been detected in the third cytoplasmic loop (CL3, residues 933-990) in patients with cystic fibrosis. Chinese hamster ovary cell lines stably expressing wild-type CFTR or mutant G970R-CFTR yielded polypeptides with apparent masses of 170 kDa as the major products, whereas the major products of mutants S945L-CFTR and H949Y-CFTR had apparent masses of 150 kDa. The 150-kDa forms of CFTR were sensitive to endoglycosidase H digestion, indicating that these mutations interfered with maturation of the protein. Increased levels of mature CFTR (170 kDa) could be obtained for mutant H949Y when cells were grown at a lower temperature (26 degrees C) or incubated in the presence of 10% glycerol. For all mutants, the open probability (P0) of the CFTR channels was significantly altered. S945L-CFTR and G970R-CFTR showed a severe reduction in the P0, whereas the H949Y mutation doubled the P0 relative to wild-type. The changes in P0 predominantly resulted from an alteration of the mean burst durations which suggests that CL3 is involved in obtaining and/or maintaining stability of the open state. In addition, mutants S945L and G970R had current-voltage relationships that were not completely linear over the range +/-80 mV, but showed slight outward rectification. The fact that CL3 mutations can have subtle effects on channel conductance indicates that this region may be physically close to the inner mouth of the pore.

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0 Cytoplasmic Loop Three of Cystic Fibrosis Transmembrane Conductance Regulator Contributes to Regulation of Chloride Channel Activity* (Received for publication, May 3, 1996, and in revised form, August 22, 1996) Fabian S. Seibert‡, Paul Linsdell§¶, Tip W. Loo, John W. Hanrahan§ʈ, John R. Riordan**‡‡, and David M. Clarke§§ From the Medical Research Council Group in Membrane Biology, Departments of Medicine and Biochemistry, University of Toronto, Toronto, Ontario, Canada, M5S 1A8, the §Department of Physiology, McGill University, Montreal, Quebec, Canada, H3G 1Y6, and the **Mayo Graduate School of Medicine and Department of Biochemistry and Molecular Biology, S. C. Johnson Medical Research Center, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259 To examine the contribution of the large cytoplasmic loops of the cystic fibrosis transmembrane conductance regulator (CFTR) to channel activity, the three point-mutations (S945L, H949Y, G970R) were characterized that have been detected in the third cytoplasmic loop (CL3, residues 933-990) in patients with cystic fibrosis. Login to comment
1 Chinese hamster ovary cell lines stably expressing wild-type CFTR or mutant G970R-CFTR yielded polypeptides with apparent masses of 170 kDa as the major products, whereas the major products of mutants S945L-CFTR and H949Y-CFTR had apparent masses of 150 kDa. Login to comment
3 Increased levels of mature CFTR (170 kDa) could be obtained for mutant H949Y when cells were grown at a lower temperature (26 °C) or incubated in the presence of 10% glycerol. Login to comment
5 S945L-CFTR and G970R-CFTR showed a severe reduction in the P0, whereas the H949Y mutation doubled the P0 relative to wild-type. Login to comment
35 The aim of the present study was to examine the functional significance of a previously uninvestigated domain, CL3 (predicted residues: 933-990, connecting TMs 8 and 9 of CFTR; Fig. 1), by characterizing the three different point-mutations that have been identified in CL3 from patients with CF (S945L (Claustres et al., 1993), H949Y (Ghanem et al., 1994), and G970R (Cuppens et al., 1993)). Login to comment
54 Western blotting with the CFTR-specific monoclonal antibody M3A7 (Kartner et al., 1992) demonstrated that wild-type and G970R-mutant CFTRs yielded fully mature protein (170 kDa, band C) as the major product, whereas mutants S945L and H949Y yielded little of the mature form (Fig. 2). Login to comment
58 These findings indicate that the S945L and H949Y mutations cause a defect in the biosynthetic processing pathways of CFTR maturation. Login to comment
63 In the case of H949Y, however, the amount of protein in band C was further increased under both conditions, with a more striking effect observed with 10% glycerol. Login to comment
69 H949Y-CFTR-containing cells exhibited iodide effluxes which were similar to wild-type, although there was much less mature protein than in wild-type expressing cells as judged by Western blotting. Login to comment
70 This suggested that the H949Y mutation may actually produce a hyperactive form of CFTR. Login to comment
116 For each mutant, however, the level of channel activity was clearly different from wild-type CFTR (Fig. 7); both S945L and G970R channels showed a lower level of activity than wild-type, while for H949Y-CFTR activity appeared to be higher than wild-type. Login to comment
117 This was confirmed by channel mean open probability (P0) measurements; S945L-CFTR and G970R-CFTR had significantly lower mean P0 values than wild-type channels, and the mean P0 of H949Y channels was significantly greater than observed for wild-type (Fig. 8A). Login to comment
124 In contrast, H949Y channels had a linear I-V relationship (Fig. 9B) with a conductance similar to wild-type (7.9 Ϯ 0.1 pS; n ϭ 7). Login to comment
128 The most striking effect due to the amino acid substitutions was a drastically altered P0 of the mutant CFTRs relative to wild-type CFTR, with S945L and G970R decreasing the P0 of the channel and H949Y doubling its P0. Login to comment
131 Within the framework of this model, the altered duration of the open state observed in the present study indicates that mutations in CL3 can affect events at NBF2 or affect communication from NBF2 to the pore. Mutations within CL3 can have opposite effects of either prolonging (H949Y) or dramatically decreasing (S945L, G970R) the duration of the open state. Login to comment
146 Notably, one of the CL3 mutations, H949Y, results in a channel which is more active than the one produced by nature. Login to comment
149 As seen for H949Y, this hyperactivity is mainly due to an increase in the mean open time of the channel, whereas conductance of the channel is not affected by the mutation. Login to comment
150 The occurrence of substantial activity for these mutants correlates well with the observation that patients affected by the P574H and H949Y mutations suffer from a less severe form of CF and are pancreatic sufficient (Kerem et al., 1990; Ghanem et al., 1994). Login to comment
154 Specifically, the wild-type-like iodide efflux level together with the low expression level of H949Y-CFTR suggest a very high activity for that mutant, which cannot be fully accounted for by the doubling of the P0 observed in single-channel patch-clamping. However, iodide efflux may overestimate H949Y-CFTR activity if both wild-type CFTR expressing and H949Y-CFTR expressing cells reach an efflux plateau which masks a difference in the activity of their respective CFTR channels. Login to comment
161 Examples of wild-type, S945L, H949Y, and G970R CFTR channel currents recorded from inside-out patches at a membrane potential of -30 mV. Login to comment
180 The only exception thus far is H949Y which already has some expression at the cell surface under normal growth conditions. Login to comment
34 The aim of the present study was to examine the functional significance of a previously uninvestigated domain, CL3 (predicted residues: 933-990, connecting TMs 8 and 9 of CFTR; Fig. 1), by characterizing the three different point-mutations that have been identified in CL3 from patients with CF (S945L (Claustres et al., 1993), H949Y (Ghanem et al., 1994), and G970R (Cuppens et al., 1993)). Login to comment

[hide] Crucial role for phylogenetically conserved cytopl... J Biol Chem. 2013 Aug 2;288(31):22207-18. doi: 10.1074/jbc.M113.476218. Epub 2013 Jun 13. Cheepala SB, Bao J, Nachagari D, Sun D, Wang Y, Zhong TP, Naren AP, Zheng J, Schuetz JD
Crucial role for phylogenetically conserved cytoplasmic loop 3 in ABCC4 protein expression.
J Biol Chem. 2013 Aug 2;288(31):22207-18. doi: 10.1074/jbc.M113.476218. Epub 2013 Jun 13., [PMID:23766510]

Abstract [show]
The ABC transporter ABCC4 is recognized as an ATP-dependent exporter of endogenous substances as well as an increasing variety of anionic chemotherapeutics. A loss-of-function variant of zebrafish Abcc4 was identified with a single amino acid substitution in the cytoplasmic loop T804M. Because this substituted amino acid is highly conserved among ABCC4 orthologs and is located in cytoplasmic loop 3 (CL3), we investigated the impact of this mutation on human and zebrafish Abcc4 expression. We demonstrate that zebrafish Abcc4 T804M or human ABCC4 T796M exhibit substantially reduced expression, coupled with impaired plasma membrane localization. To understand the molecular basis for the localization defect, we developed a homology model of zebrafish Abcc4. The homology model suggested that the bulky methionine substitution disrupted side-chain contacts. Molecular dynamic simulations of a fragment of human or zebrafish CL3 containing a methionine substitution indicated altered helicity coupled with reduced thermal stability. Trifluoroethanol challenge coupled with circular dichroism revealed that the methionine substitution disrupted the ability of this fragment of CL3 to readily form an alpha-helix. Furthermore, expression and plasma membrane localization of these mutant ABCC4/Abcc4 proteins are mostly rescued by growing cells at subphysiological temperatures. Because the cystic fibrosis transmembrane conductance regulator (ABCC7) is closely related to ABCC4, we extended this by engineering certain pathogenic CFTR-CL3 mutations, and we showed they destabilized human and zebrafish ABCC4. Altogether, our studies provide the first evidence for a conserved domain in CL3 of ABCC4 that is crucial in ensuring its proper plasma membrane localization.

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199 Notably, disease-related point mutations in CFTR CL3 at positions S945L and H949Y (Fig. 5A) (44) affected maturation of CFTR (26). Login to comment
238 Furthermore, we extended these studies to show that other mutations in CL3 (analogous to those in CFTR (S945L and H949Y)) that also disrupt its helical properties reduce protein expression. Login to comment
198 Notably, disease-related point mutations in CFTR CL3 at positions S945L and H949Y (Fig. 5A) (44) affected maturation of CFTR (26). Login to comment
237 Furthermore, we extended these studies to show that other mutations in CL3 (analogous to those in CFTR (S945L and H949Y)) that also disrupt its helical properties reduce protein expression. Login to comment

[hide] Cystic fibrosis: the 'bicarbonate before chloride'... Curr Biol. 2001 Jun 26;11(12):R463-6. Wine JJ
Cystic fibrosis: the 'bicarbonate before chloride' hypothesis.
Curr Biol. 2001 Jun 26;11(12):R463-6., [PMID:11448786]

Abstract [show]
The specific effects of some mutations that cause cystic fibrosis suggest that reduced HCO(3)(-) transport is the key to understanding cystic fibrosis pathology. But there is a puzzling discrepancy between measures of CFTR-mediated chloride conductance in expression systems and the sweat chloride values of patients.

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52 Ion transport (% WT) 42 41 69 75 >100 >100 98 + 103 100 + + 120 Pancreatic sufficient Pancreatic insufficient Bicarbonate Chloride - intermediate Chloride - high Unknown WT D648V R117H R1070Q H949Y G551S H620Q I148T A1067T G178R G970R S1255P G1244E G551D G1349D 0 0.5 1 1.5 2 2.5 Current Biology ࢞F508 Dispatch R absence of the vas deferens [16]. Login to comment
77 Published values were found for H949Y [22] and G551S [17] and I148T [13]. Login to comment

[hide] Enhancing the Potency of F508del Correction: A Mul... J Pharmacol Clin Toxicol. 2013 Aug 28;1(1):1007. Kirby EF, Heard AS, Wang XR
Enhancing the Potency of F508del Correction: A Multi-Layer Combinational Approach to Drug Discovery for Cystic Fibrosis.
J Pharmacol Clin Toxicol. 2013 Aug 28;1(1):1007., [PMID:24855632]

Abstract [show]
With better understanding of the cellular and molecular pathophysiology underlying cystic fibrosis (CF), novel drugs are being developed that specifically target the molecular defects of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel on the plasma membrane that causes CF. Starting with cell-based high-throughput screening, small molecules have been identified that are able to fix specific molecular defects of various disease-causing CFTR mutants. With the successful development of ivacaftor, a "potentiator" that enhances CFTR chloride channel activity, new types of small-molecule compounds that "correct" the misfolding and misprocessing of the most common CF-causing mutation, F508del, are actively being sought for. Recent studies focused on the potential mechanisms of action of some of the investigational CFTR "correctors" shed new light on how the F508del mutant can be targeted in an attempt to ameliorate the clinical symptoms associated with CF. A multi-layer combinational approach has been proposed to achieve the high-potency correction necessary for significant clinical outcome. The mechanistic insights obtained from such studies will shape the future therapeutics development for the vast majority of CF patients.

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64 VRT-325 was found to rescue not only F508del CFTR but also other CFTR processing mutants such as R258G, S945L, and H949Y, and processing mutants of P-glycoprotein (P-gp), a drug pump that also belongs to the ABC transporter family [29]. Login to comment