ABCMdb

ABCC7 p.Arg334Ala

ClinVar:	c.1000C>T , p.Arg334Trp D , Pathogenic c.1001G>T , p.Arg334Leu ? , not provided c.1001G>A , p.Arg334Gln ? , not provided
CF databases:	c.1000C>T , p.Arg334Trp D , CF-causing ; CFTR1: This mutation has been found in two Spanish CF chromosomes. One of the patients has the [delta]F508 mutation in the other chromosome and the other patient does not. We have not found this mutation on 30 normal chromosomes with the same haplotype, and in 88 CF chromosomes without the [delta]F508, and in 24 with the [delta]F508. The mutation destroys a MapI site and is easily identified by agarose gel electrophoresis after PCR with intron primers. c.1001G>A , p.Arg334Gln (CFTR1) ? , The above mutation was found by DGGE and direct sequencing in Caucasian patients. c.1001G>T , p.Arg334Leu (CFTR1) D , Missense mutation E334L was detected in a German CBAVD patient who is compound heterozygous for the R334L and I336K mutations.
Predicted by SNAP2:	A: D (91%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (91%), I: D (95%), K: D (85%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (91%), S: D (91%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: N, M: N, N: N, P: N, Q: N, S: N, T: N, V: D, W: D, Y: D,

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Publications
[hide] CFTR: covalent and noncovalent modification sugges... J Gen Physiol. 2001 Oct;118(4):407-31. Smith SS, Liu X, Zhang ZR, Sun F, Kriewall TE, McCarty NA, Dawson DC
CFTR: covalent and noncovalent modification suggests a role for fixed charges in anion conduction.
J Gen Physiol. 2001 Oct;118(4):407-31., [PMID:11585852]

Abstract [show]
The goal of the experiments described here was to explore the possible role of fixed charges in determining the conduction properties of CFTR. We focused on transmembrane segment 6 (TM6) which contains four basic residues (R334, K335, R347, and R352) that would be predicted, on the basis of their positions in the primary structure, to span TM6 from near the extracellular (R334, K335) to near the intracellular (R347, R352) end. Cysteines substituted at positions 334 and 335 were readily accessible to thiol reagents, whereas those at positions 347 and 352 were either not accessible or lacked significant functional consequences when modified. The charge at positions 334 and 335 was an important determinant of CFTR channel function. Charge changes at position 334--brought about by covalent modification of engineered cysteine residues, pH titration of cysteine and histidine residues, and amino acid substitution--produced similar effects on macroscopic conductance and the shape of the I-V plot. The effect of charge changes at position 334 on conduction properties could be described by electrodiffusion or rate-theory models in which the charge on this residue lies in an external vestibule of the pore where it functions to increase the concentration of Cl adjacent to the rate-limiting portion of the conduction path. Covalent modification of R334C CFTR increased single-channel conductance determined in detached patches, but did not alter open probability. The results are consistent with the hypothesis that in wild-type CFTR, R334 occupies a position where its charge can influence the distribution of anions near the mouth of the pore.

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No. Sentence Comment
187 MTSES, MTSET, or MTSEA (100 ␮M or 1 mM) were added to the perfusate of oocytes expressing R334A or R334Q CFTR and produced no discernible effect on conductance (unpublished data). Login to comment

[hide] Molecular determinants of Au(CN)(2)(-) binding and... J Physiol. 2002 Apr 1;540(Pt 1):39-47. Gong X, Burbridge SM, Cowley EA, Linsdell P
Molecular determinants of Au(CN)(2)(-) binding and permeability within the cystic fibrosis transmembrane conductance regulator Cl(-) channel pore.
J Physiol. 2002 Apr 1;540(Pt 1):39-47., 2002-04-01 [PMID:11927667]

Abstract [show]
Lyotropic anions with low free energy of hydration show both high permeability and tight binding in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore. However, the molecular bases of anion selectivity and anion binding within the CFTR pore are not well defined and the relationship between binding and selectivity is unclear. We have studied the effects of point mutations throughout the sixth transmembrane (TM6) region of CFTR on channel block by, and permeability of, the highly lyotropic Au(CN)(2)(-) anion, using patch clamp recording from transiently transfected baby hamster kidney cells. Channel block by 100 microM Au(CN)(2)(-), a measure of intrapore anion binding affinity, was significantly weakened in the CFTR mutants K335A, F337S, T338A and I344A, significantly strengthened in S341A and R352Q and unaltered in K329A. Relative Au(CN)(2)(-) permeability was significantly increased in T338A and S341A, significantly decreased in F337S and unaffected in all other mutants studied. These results are used to define a model of the pore containing multiple anion binding sites but a more localised anion selectivity region. The central part of TM6 (F337-S341) appears to be the main determinant of both anion binding and anion selectivity. However, comparison of the effects of individual mutations on binding and selectivity suggest that these two aspects of the permeation mechanism are not strongly interdependent.

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82 Two other mutants were also transfected into BHK cells; however, even in the presence of PPi, currents carried by R334A and T351A were too small for proper analysis (not shown). Login to comment

[hide] Molecular determinants and role of an anion bindin... J Physiol. 2003 Jun 1;549(Pt 2):387-97. Epub 2003 Apr 4. Gong X, Linsdell P
Molecular determinants and role of an anion binding site in the external mouth of the CFTR chloride channel pore.
J Physiol. 2003 Jun 1;549(Pt 2):387-97. Epub 2003 Apr 4., 2003-06-01 [PMID:12679372]

Abstract [show]
Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is blocked by highly lyotropic permeant anions which bind tightly within the pore. Here we show that several different substitutions of a positively charged amino acid residue, arginine R334, in the putative outer mouth of the CFTR pore, greatly reduce the block caused by lyotropic Au(CN)2- ions applied to the intracellular side of the channel. Fixed positive charge at this site appears to play a role in Au(CN)2- binding, as judged by multiple substitutions of differently charged amino acid side chains and also by the pH dependence of block conferred by the R334H mutant. However, non-charge-dependent effects also appear to contribute to Au(CN)2- binding. Mutation of R334 also disrupts the apparent electrostatic interaction between intracellular Au(CN)2- ions and extracellular permeant anions, an interaction which normally acts to relieve channel block. All six mutations studied at R334 significantly weakened this interaction, suggesting that arginine possesses a unique ability to coordinate ion-ion interactions at this site in the pore. Our results suggest that lyotropic anions bind tightly to a site in the outer mouth of the CFTR pore that involves interaction with a fixed positive charge. Binding to this site is also involved in coordination of multiple permeant anions within the pore, suggesting that anion binding in the outer mouth of the pore is an important aspect in the normal anion permeation mechanism.

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No. Sentence Comment
51 Previously we reported that the mutant R334A could not be expressed in BHK cells (Gong et al. 2002a); in the course of the present study, we confirmed this previous finding, but did find that, in addition to R334C, five other mutants could be studied (Fig. 4). Login to comment

[hide] Extent of the selectivity filter conferred by the ... Mol Membr Biol. 2003 Jan-Mar;20(1):45-52. Gupta J, Lindsell P
Extent of the selectivity filter conferred by the sixth transmembrane region in the CFTR chloride channel pore.
Mol Membr Biol. 2003 Jan-Mar;20(1):45-52., [PMID:12745925]

Abstract [show]
Point mutations within the pore region of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel have previously been shown to alter the selectivity of the channel between different anions, suggesting that part of the pore may form an anion 'selectivity filter'. However, the full extent of this selectivity filter region and the location of anion binding sites in the pore are currently unclear. As a result, comparisons between CFTR and other classes of Cl(-) channel of known structure are difficult. We compare here the effects of point mutations at each of eight consecutive amino acid residues (arginine 334-serine 341) in the crucial sixth transmembrane region (TM6) of CFTR. Anion selectivity was determined using patch-clamp recording from inside-out membrane patches excised from transiently transfected mammalian cell lines. The results suggest that selectivity is predominantly controlled by a single site involving adjacent residues phenylalanine 337 and threonine 338, and that the selectivity conferred by this 'filter' region is modified by anion binding to flanking sites involving the more extracellular arginine 334 and the more intracellular serine 341. Other residues within this part of the pore play only minor roles in controlling anion permeability and conductance. Our results support a model in which specific TM6 residues make important contributions to a single, localized anion selectivity filter in the CFTR pore, and also contribute to multiple anion binding sites both within and on either side of the filter region.

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No. Sentence Comment
124 In most (six of eight) cases, alanine substitution was employed; however, we have previously found that the mutants R334A [15] and T339A [22] fail to express in BHK cells, and for these residues mutants which gave adequate current expression (R334C, T339V) were studied. Login to comment

[hide] CFTR: what's it like inside the pore? J Exp Zool A Comp Exp Biol. 2003 Nov 1;300(1):69-75. Liu X, Smith SS, Dawson DC
CFTR: what's it like inside the pore?
J Exp Zool A Comp Exp Biol. 2003 Nov 1;300(1):69-75., 2003-11-01 [PMID:14598388]

Abstract [show]
The Cystic Fibrosis Conductance Regulator (CFTR) functions as a cAMP-activated, anion-selective channel, but the structural basis for anion permeation is not well understood. Here we summarize recent studies aimed at understanding how anions move through the CFTR channel, and the nature of the environment anions experience inside the pore. From these studies it is apparent that anion permeability selectivity and anion binding selectivity of the pore are consistent with a model based on a "dielectric tunnel." The selectivity pattern for halides and pseudohalides can be predicted if it is assumed that permeant anions partition between bulk water and a polarizable space that is characterized by an effective dielectric constant of about 19. Covalent labeling of engineered cysteines and pH titration of engineered cysteines and histidines lead to the conclusion that the CFTR anion conduction path includes a positively charged outer vestibule. A residue in transmembrane segment 6 (TM6) (R334) appears to reside in the outer vestibule of the CFTR pore where it creates a positive electrostatic potential that enhances anion conduction.

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No. Sentence Comment
118 Changing the charge at position 334 either by modification of R334C/T338H CFTR with polar thiol reactive reagents or by amino acid substitution (R334A/T338C) shifts the titration curve in a direction that was predicted on the basis of a nearby positive charge being able to stabilize a titratable group (Liu et al., 2001). Login to comment

[hide] Direct comparison of the functional roles played b... J Biol Chem. 2004 Dec 31;279(53):55283-9. Epub 2004 Oct 25. Ge N, Muise CN, Gong X, Linsdell P
Direct comparison of the functional roles played by different transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Biol Chem. 2004 Dec 31;279(53):55283-9. Epub 2004 Oct 25., 2004-12-31 [PMID:15504721]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel contains 12 transmembrane (TM) regions that are presumed to form the channel pore. However, little is known about the relative functional contribution of different TM regions to the pore. We have used patch clamp recording to investigate the functional consequences of point mutations throughout the six transmembrane regions in the N-terminal part of the CFTR protein (TM1-TM6). A range of specific functional assays compared the single channel conductance, anion binding, and anion selectivity properties of different channel variants. Overall, our results suggest that TM1 and -6 play dominant roles in forming the channel pore and determining its functional properties, with TM5 perhaps playing a lesser role. In contrast, TM2, -3, and -4 appear to play only minor supporting roles. These results define transmembrane regions 1 and 6 as major contributors to the CFTR channel pore and have strong implications for emerging structural models of CFTR and related ATP-binding cassette proteins.

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No. Sentence Comment
45 Previously we showed that the TM6 mutant R334A did not express but characterized several other Arg-334 mutants (10, 28); in the present study we have used the charge conservative R334K mutant. Login to comment

[hide] Evidence for direct CFTR inhibition by CFTR(inh)-1... Biochem J. 2008 Jul 1;413(1):135-42. Caci E, Caputo A, Hinzpeter A, Arous N, Fanen P, Sonawane N, Verkman AS, Ravazzolo R, Zegarra-Moran O, Galietta LJ
Evidence for direct CFTR inhibition by CFTR(inh)-172 based on Arg347 mutagenesis.
Biochem J. 2008 Jul 1;413(1):135-42., 2008-07-01 [PMID:18366345]

Abstract [show]
CFTR (cystic fibrosis transmembrane conductance regulator) is an epithelial Cl- channel inhibited with high affinity and selectivity by the thiazolidinone compound CFTR(inh)-172. In the present study, we provide evidence that CFTR(inh)-172 acts directly on the CFTR. We introduced mutations in amino acid residues of the sixth transmembrane helix of the CFTR protein, a domain that has an important role in the formation of the channel pore. Basic and hydrophilic amino acids at positions 334-352 were replaced with alanine residues and the sensitivity to CFTR(inh)-172 was assessed using functional assays. We found that an arginine-to-alanine change at position 347 reduced the inhibitory potency of CFTR(inh)-172 by 20-30-fold. Mutagenesis of Arg347 to other amino acids also decreased the inhibitory potency, with aspartate producing near total loss of CFTR(inh)-172 activity. The results of the present study provide evidence that CFTR(inh)-172 interacts directly with CFTR, and that Arg347 is important for the interaction.

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No. Sentence Comment
111 R334A and S341A showed reduced anion transport, although this was significantly greater than cells transfected with the fluorescent protein alone (Figure 1B). Login to comment
127 CFTR form CFTRinh-172 Ki (μM) Hill coefficient I- influx (mM/s) n Wild-type 1.32 + - 0.25 1.03 + - 0.07 0.1336 + - 0.0107 10 S341A 0.57 + - 0.17 1.21 + - 0.37 0.0297 + - 0.0064 4 T338A 3.20 + - 0.86 1.13 + - 0.20 0.1260 + - 0.0225 4 R347A 44.98 + - 4.71** 0.91 + - 0.04 0.1288 + - 0.0154 7 R334A 2.39 + - 0.74 0.93 + - 017 0.0313 + - 0.062 4 A349S 1.23 + - 0.41 1.11 + - 0.25 0.1500 + - 0.011 4 R347D >50 Not determined 0.1160 + - 0.0136 7 R347D/D924R >50 Not determined 0.1008 + - 0.0504 4 R347C >50 Not determined 0.1437 + - 0.0123 4 Mock 0.003 + - 0.001 10 introduced a mutation at position 349 (an alanine residue replaced by a serine residue). Login to comment
143 FRT cells were stably transfected with wild-type, R334A, R347A and R347D CFTR, and transepithelial Cl- currents were measured. Login to comment
157 For comparison, the sensitivity of the R334A mutant was not significantly altered (Ki = 0.50 +- 0.18 μM, n = 6; Figures 3C and 3E), in agreement with the fluorescence assay results. Login to comment
158 Interestingly, the R347D mutant, although insensitive to CFTRinh-172, was fully inhibited by the open-channel blocker GlyH-101 Figure 3 CFTR Cl- current inhibition by CFTRinh-172 (A-D) Representative traces showing recordings of transepithelial Cl- currents measured in FRT cells with stable expression of wild-type (WT), R347A, R334A and R347D-CFTR. Cells were first stimulated with 20 μM forskolin to activate CFTR and then tested with increasing concentrations of CFTRinh-172. Login to comment

[hide] Differential contribution of TM6 and TM12 to the p... Pflugers Arch. 2012 Mar;463(3):405-18. Epub 2011 Dec 13. Cui G, Song B, Turki HW, McCarty NA
Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers.
Pflugers Arch. 2012 Mar;463(3):405-18. Epub 2011 Dec 13., [PMID:22160394]

Abstract [show]
Previous studies suggested that four transmembrane domains 5, 6, 11, 12 make the greatest contribution to forming the pore of the CFTR chloride channel. We used excised, inside-out patches from oocytes expressing CFTR with alanine-scanning mutagenesis in amino acids in TM6 and TM12 to probe CFTR pore structure with four blockers: glibenclamide (Glyb), glipizide (Glip), tolbutamide (Tolb), and Meglitinide. Glyb and Glip blocked wildtype (WT)-CFTR in a voltage-, time-, and concentration-dependent manner. At V (M) = -120 mV with symmetrical 150 mM Cl(-) solution, fractional block of WT-CFTR by 50 muM Glyb and 200 muM Glip was 0.64 +/- 0.03 (n = 7) and 0.48 +/- 0.02 (n = 7), respectively. The major effects on block by Glyb and Glip were found with mutations at F337, S341, I344, M348, and V350 of TM6. Under similar conditions, fractional block of WT-CFTR by 300 muM Tolb was 0.40 +/- 0.04. Unlike Glyb, Glip, and Meglitinide, block by Tolb lacked time-dependence (n = 7). We then tested the effects of alanine mutations in TM12 on block by Glyb and Glip; the major effects were found at N1138, T1142, V1147, N1148, S1149, S1150, I1151, and D1152. From these experiments, we infer that amino acids F337, S341, I344, M348, and V350 of TM6 face the pore when the channel is in the open state, while the amino acids of TM12 make less important contributions to pore function. These data also suggest that the region between F337 and S341 forms the narrow part of the CFTR pore.

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No. Sentence Comment
57 For simplification, names of mutant channels are often abbreviated (e.g., R334A is R334A-CFTR). Login to comment
119 The major effects of increasing or decreasing sensitivity to Glyb were seen with mutations R334A, K335A, F337A, S341A, I344A, R347A, M348A, V350A, and R352A (Fig. 3 left). Login to comment
125 Mutations R334A and K335A lie in the outer vestibule of the pore of CFTR; surprisingly, the two mutations db ca 2 nA 100 ms 100 ms 2 nA Concentration (μM) 0 200 400 600 800 1000 0.2 0.4 0.6 0.8 1.0 Fractionalblock 100 ms 4 nA Fig. 2 Concentration-dependent block of WT-CFTR by Glyb, Glip and Tolb. Login to comment
133 R334A weakened block while K335A strengthened block by both blockers. Login to comment
135 Also, we reported that R334A and R334C exhibited multiple single-channel conductance levels, including subconductance 1 (s1), subconductance 2 (s2), and full conductance states (f). Login to comment
151 The surprising finding that mutations at six adjacent positions Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** * * * 0.8 0.6 0.4 0.2 0 Fractional block by Glyb50 μM Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** ** ** ** ** ** ** * * * * * * ** ** Fractional block by Tolb300 μM 0.8 0.6 0.4 0.2 0 Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT * ** ** ** ** ** ** ** ** Fractional block by Glip200 μM 0.8 0.6 0.4 0.2 0 Fig. 3 Alanine-scanning in TM6 to identify the amino acids that interact with the three blockers. Login to comment
158 Among the 20 single amino acid mutants of TM12 that we tested in this paper, none of them exhibited significant change in their single-channel conductance compared to WT-CFTR, while we know that mutations R334A, F337A, S341A, R347A, and R352A in TM6 all exhibited significant change in their single-channel conductance [11, 12, 29, and the present manuscript]; these data strongly suggest that TM6 and TM12 do not equally contribute to the pore of CFTR. Login to comment
162 Although mutations R334A and K335A exhibited opposite effects on steady-state block by Glyb and Glip, neither mutation altered initial block (Fig. 5). Login to comment
163 Effects on time-dependent block by mutations R334A and K335A Fractional block by Glip200 μM V1153A D1152A I1151A S1150A S1149A N1148A V1147A A1146S W1145A Q1144A L1143A T1142A S1141A M1140A I1139A N1138A M1137A A1136S L1135A T1134A WT 0 0.2 0.4 0.6 0.8 * ** ** ** ** ** ** * V1153A D1152A I1151A S1150A S1149A N1148A V1147A A1146S W1145A Q1144A L1143A T1142A S1141A M1140A I1139A N1138A M1137A A1136S L1135A T1134A WT 0 0.2 0.4 0.6 0.8 1.0 * * * * * ** ** ** ** Fractional block by Glyb50 μM Fig. 4 Alanine-scanning in TM12 to identify amino acids that interact with Glyb and Glip. Login to comment
166 Double asterisks indicate significantly different compared to WT-CFTR (p<0.01) Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.3 0.2 0.1 0 * * ** ** 0.4 Initial block by 50 μM Glyb Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.4 0.3 0.2 0.1 0 ** ** * Initial block by 200 μM Glip Fig. 5 Initial block of WT-CFTR and selected TM6 mutants by 50 μM Glyb (left) and 200 μM Glip (right) in symmetrical 150 mM Cl- solution. Data are shown only for those mutants which exhibited significant changes in steady-state fractional block according to Fig. 3 (bars show mean±SEM, n=5-10). Login to comment
168 Double asterisks indicate significantly different compared to WT-CFTR (p<0.01) were similar for Glyb and Glip, although the effect of R334A on Glyb was larger than for Glip and the effect of K335A was larger for Glip than Glyb (Fig. 6). Login to comment
193 Probable orientation of drugs in the pore Glyb and Glip are identical molecules along most of their lengths, differing only in the substituents on the ring at the Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT 0.8 0.6 0.2 0 ** ** ** ** Time-dependent block by 50 μμM Glyb Q353A R352A T351A V350A A349S M348A R347A L346A V345A I344A C343A F342A S341A I340A T339A T338A F337A I336A K335A R334A WT ** ** * ** * Time-dependent block by 200 μM Glip 0.4 0.8 0.6 0.2 00.4 Fig. 6 Time-dependent block of WT-CFTR and selected TM6 mutants by 50 μM Glyb (left) and 200 μM Glip (right) in symmetrical 150 mM Cl- solution. Data are shown only for those mutants which exhibited significant changes in fractional block according to Fig. 3 (bars show mean±SEM, n=5-10). Login to comment
221 The effects of mutations R334A and K335A are indirect, likely related to the movement of chloride within the pore, or the stabilization of the outer vestibule. Login to comment

[hide] CFTR: a cysteine at position 338 in TM6 senses a p... Biophys J. 2004 Dec;87(6):3826-41. Epub 2004 Sep 10. Liu X, Zhang ZR, Fuller MD, Billingsley J, McCarty NA, Dawson DC
CFTR: a cysteine at position 338 in TM6 senses a positive electrostatic potential in the pore.
Biophys J. 2004 Dec;87(6):3826-41. Epub 2004 Sep 10., [PMID:15361410]

Abstract [show]
We investigated the accessibility to protons and thiol-directed reagents of a cysteine substituted at position 338 in transmembrane segment 6 (TM6) of CFTR to test the hypothesis that T338 resides in the pore. Xenopus oocytes expressing T338C CFTR exhibited pH-dependent changes in gCl and I-V shape that were specific to the substituted cysteine. The apparent pKa of T338C CFTR was more acidic than that expected for a cysteine or similar simple thiols in aqueous solution. The pKa was shifted toward alkaline values when a nearby positive charge (R334) was substituted with neutral or negatively charged residues, consistent with the predicted influence of the positive charge of R334, and perhaps other residues, on the titration of a cysteine at 338. The relative rates of chemical modification of T338C CFTR by MTSET+ and MTSES- were also altered by the charge at 334. These observations support a model for CFTR that places T338 within the anion conduction path. The apparent pKa of a cysteine substituted at 338 and the relative rates of reaction of charged thiol-directed reagents provide a crude measure of a positive electrostatic potential that may be due to R334 and other residues near this position in the pore.

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184 To investigate the effect of charge at position 334 on the titration behavior of T338C CFTR, we examined the conductance of oocytes expressing double mutants, T338C/R334A, T338C/R334E, and T338C/R334D CFTR. Login to comment
185 Shown in Fig. 8 A are representative titration curves for the conductance for T338C CFTR and two of these double mutants (n ¼ 5 each). Login to comment
186 Neutralizing the charge at 334 (R334A) resulted in a pKa that was more than one pH unit more basic (8.78 6 0.03, n ¼ 4) than that determined for T338C CFTR. Login to comment
187 The substitution of acidic residues, however, did not result in a large additional shift of the apparent pKa to more alkaline values (8.84 6 0.05 for T338C/R334D CFTR, n ¼ 4 and 8.96 6 0.08 for T338C/R334E CFTR, n ¼ 5). Login to comment
221 FIGURE 8 The pH-induced changes in the conductances of oocytes expressing T338C/R334A, T338C/R334E, T338H, or T338H/R334C CFTR. Login to comment
222 (A) Sample titration curves of conductances of oocytes expressing T338C CFTR (solid circles), T338C/R334A (open squares), or T338C/ R334E CFTRs (shaded triangles). Login to comment
340 A comparison of the apparent pKa of T338C CFTR with that of the double mutant, T338C/R334A, suggests that the amino acid substitution at position 334 shifted the pKa from ;7.4 to 8.8 or ;1.4 units. Login to comment
342 A comparison of T338C/R334A (pKa ¼ 8.8) with T338C/R334E (pKa ¼ 8.9), would suggest a further change in Cq o of ;ÿ6 mV. Login to comment
347 Prediction of the electrostatic effects of R334 If we ignore the possible effects of structural changes in the CFTR protein produced by amino acid substitution, then the change in Cq o calculated from the difference in the pKa of T338C/R334 and T338C/R334A can be taken to be a crude measure of CR334 o ; the component of Cq o due to the native arginine, and we can compare the value derived experimentally with that predicted on the basis of first principles. Login to comment
223 (A) Sample titration curves of conductances of oocytes expressing T338C CFTR (solid circles), T338C/R334A (open squares), or T338C/ R334E CFTRs (shaded triangles). Login to comment
344 A comparison of T338C/R334A (pKa &#bc; 8.8) with T338C/R334E (pKa &#bc; 8.9), would suggest a further change in Cq o of ;6 mV. Login to comment
348 The absolute values of Cq o must be interpreted with caution since we do not know to what extent structural differences between these two mutants might have resulted from the amino acid substitution (see below), but both of the measurements used to estimate the electrostatic potential of the vestibule indicated an asymmetry between the impact of basic and acidic residues at 334. Prediction of the electrostatic effects of R334 If we ignore the possible effects of structural changes in the CFTR protein produced by amino acid substitution, then the change in Cq o calculated from the difference in the pKa of T338C/R334 and T338C/R334A can be taken to be a crude measure of CR334 o ; the component of Cq o due to the native arginine, and we can compare the value derived experimentally with that predicted on the basis of first principles. Login to comment

[hide] Maximization of the rate of chloride conduction in... Arch Biochem Biophys. 2004 Jun 1;426(1):78-82. Gong X, Linsdell P
Maximization of the rate of chloride conduction in the CFTR channel pore by ion-ion interactions.
Arch Biochem Biophys. 2004 Jun 1;426(1):78-82., [PMID:15130785]

Abstract [show]
Multi-ion pore behaviour has been identified in many Cl(-) channel types but its biophysical significance is uncertain. Here, we show that mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel that disrupt anion-anion interactions within the pore are associated with drastically reduced single channel conductance. These results are consistent with models suggesting that rapid Cl(-) permeation in CFTR results from repulsive ion-ion interactions between Cl(-) ions bound concurrently inside the pore. Naturally occurring mutations that disrupt these interactions can result in cystic fibrosis.

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No. Sentence Comment
38 Two other mutants, R334A and R334T, did not express any detectable Cl current in BHK cells. Login to comment

[hide] Three charged amino acids in extracellular loop 1 ... J Gen Physiol. 2014 Aug;144(2):159-79. doi: 10.1085/jgp.201311122. Epub 2014 Jul 14. Cui G, Rahman KS, Infield DT, Kuang C, Prince CZ, McCarty NA
Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR.
J Gen Physiol. 2014 Aug;144(2):159-79. doi: 10.1085/jgp.201311122. Epub 2014 Jul 14., [PMID:25024266]

Abstract [show]
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) bears six extracellular loops (ECL1-6); ECL1 is the site of several mutations associated with CF. Mutation R117H has been reported to reduce current amplitude, whereas D110H, E116K, and R117C/L/P may impair channel stability. We hypothesized that these amino acids might not be directly involved in ion conduction and permeation but may contribute to stabilizing the outer vestibule architecture in CFTR. We used cRNA injected oocytes combined with electrophysiological techniques to test this hypothesis. Mutants bearing cysteine at these sites were not functionally modified by extracellular MTS reagents and were blocked by GlyH-101 similarly to WT-CFTR. These results suggest that these three residues do not contribute directly to permeation in CFTR. In contrast, mutants D110R-, E116R-, and R117A-CFTR exhibited instability of the open state and significantly shortened burst duration compared with WT-CFTR and failed to be locked into the open state by AMP-PNP (adenosine 5'-(beta,gamma-imido) triphosphate); charge-retaining mutants showed mainly the full open state with comparably longer open burst duration. These interactions suggest that these ECL1 residues might be involved in maintaining the outer pore architecture of CFTR. A CFTR homology model suggested that E116 interacts with R104 in both the closed and open states, D110 interacts with K892 in the fully closed state, and R117 interacts with E1126 in the open state. These interactions were confirmed experimentally. The results suggest that D110, E116, and R117 may contribute to stabilizing the architecture of the outer pore of CFTR by interactions with other charged residues.

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154 ECL1 mutations shift the reversal potential in macroscopic currents To further verify that these ECL1 amino acids do not strongly or directly affect ion conduction and permeation, we compared the reversal potentials (Vrev) of D110R-, K114D-, E116R-, and R117A-CFTR with WT-CFTR and with the R334A mutant, which has been shown to have a profound effect on Vrev compared with WT-CFTR, consistent with the role of R334 in providing charge in the outer mouth of the open channel. Login to comment
156 E116R- and R117A-CFTR exhibited significantly right-shifted reversal potentials compared with WT-CFTR, but the effects were not as large as for R334A-CFTR. Login to comment
159 As previously reported, R334A-CFTR exhibited outward rectification but with a significantly reduced rectification ratio: 1.33 &#b1; 0.14 (n = 9, P < 0.01). Login to comment
224 Tab l e 1 Reversal potentials of WT-CFTR and mutants in ND96 bath solution CFTR n Vrev mV WT 14 &#e032;27.75 &#b1; 0.78 R334A 6 &#e032;12.15 &#b1; 1.64a R117A 6 &#e032;22.51 &#b1; 0.85a E116R 5 &#e032;21.45 &#b1; 1.14a K114D 5 &#e032;24.68 &#b1; 3.22 D110R 5 &#e032;27.64 &#b1; 3.29 R104E 5 &#e032;21.15 &#b1; 1.08a R899C 4 &#e032;25.30 &#b1; 3.94 D891C 6 &#e032;25.81 &#b1; 2.44 K892E 5 &#e032;23.70 &#b1; 3.62 E1124R 5 &#e032;18.32 &#b1; 0.43a E1126R 5 &#e032;20.67 &#b1; 3.16b R117E/E1126R 6 &#e032;23.06 &#b1; 1.37b R104E/E116R 6 &#e032;27.17 &#b1; 1.08 Values are mean &#b1; SEM. Login to comment

[hide] Murine and human CFTR exhibit different sensitivit... Am J Physiol Lung Cell Mol Physiol. 2015 Oct 1;309(7):L687-99. doi: 10.1152/ajplung.00181.2015. Epub 2015 Jul 24. Cui G, McCarty NA
Murine and human CFTR exhibit different sensitivities to CFTR potentiators.
Am J Physiol Lung Cell Mol Physiol. 2015 Oct 1;309(7):L687-99. doi: 10.1152/ajplung.00181.2015. Epub 2015 Jul 24., [PMID:26209275]

Abstract [show]
Development of therapeutic molecules with clinical efficacy as modulators of defective CFTR includes efforts to identify potentiators that can overcome or repair the gating defect in mutant CFTR channels. This has taken a great leap forward with the identification of the potentiator VX-770, now available to patients as "Kalydeco." Other small molecules with different chemical structure also are capable of potentiating the activity of either wild-type or mutant CFTR, suggesting that there are features of the protein that may be targeted to achieve stimulation of channel activity by structurally diverse compounds. However, neither the mechanisms by which these compounds potentiate mutant CFTR nor the site(s) where these compounds bind have been identified. This knowledge gap partly reflects the lack of appropriate experimental models to provide clues toward the identification of binding sites. Here, we have compared the channel behavior and response to novel and known potentiators of human CFTR (hCFTR) and murine (mCFTR) expressed in Xenopus oocytes. Both hCFTR and mCFTR were blocked by GlyH-101 from the extracellular side, but mCFTR activity was increased with GlyH-101 applied directly to the cytoplasmic side. Similarly, glibenclamide only exhibited a blocking effect on hCFTR but both blocked and potentiated mCFTR in excised membrane patches and in intact oocytes. The clinically used CFTR potentiator VX-770 transiently increased hCFTR by approximately 13% but potentiated mCFTR significantly more strongly. Our results suggest that mCFTR pharmacological sensitivities differ from hCFTR, which will provide a useful tool for identifying the binding sites and mechanism for these potentiators.

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131 WT 0.0 0.1 0.2 0.3 Fractional inhibition by 2.5 &#b5;M GlyH-101 # R334C R334A T338A R352A # # # 0.4 0.5 0.4 &#b5;A 50 s ND96 ISO ISO+ GlyH ND96 ISO R334C- hCFTR A B C D ND96 ISO ISO+ GlyH ND96 ISO T338A-hCFTR 1 &#b5;A 50 s 1.0 &#b5;A 50 s ND96 ISO ISO+ GlyH ND96 ISO WT-hCFTR Fig. 5. Login to comment
155 We have previously reported with the TEVC technique 1.0 &#b5;A 50 s ND96 ISO ISO+ GlyH ND96 ISO WT-mCFTR Fractional inhibition by GlyH 0.0 0.1 0.3 0.4 0.2 0.5 20 40 60 80 100 Concentration (&#b5;M) Kd = 32.39 &#b5;M Fractional activation by GlyH 0.0 0.1 0.3 0.4 0.2 20 40 60 80 100 Concentration (&#b5;M) Kd = 103.56 &#b5;M 0.5 A B C D E Fractional activation by 25 &#b5;M GlyH-101 0.0 0.3 0.1 0.2 F Fractional inhibition by 25 &#b5;M GlyH-101 0.0 0.6 0.2 0.4 # * # 0.5 &#b5;A 50 s R334A-mCFTR ND96 ISO ISO+ GlyH ND96 ISO Fig. 6. Login to comment
162 D: representative current trace of R334A-mCFTR recorded at VM afd; afa;60 mV. Login to comment
163 Extracellular 25 òe;M GlyH-101 failed to block but only potentiated R334A-mCFTR. Summary data for fractional inhibition (E) and fractional potentiation (F) of WT-, V100L-, R147H-, I201V-, R334A-, and T338A-mCFTR with 25 òe;M GlyH-101. Login to comment
180 GlyH-101 at 2.5 òe;M blocked WT-hCFTR 28% at VM afd; afa;60 mV in the continuing presence of 10 òe;M ISO (used to activate CFTR via the beta2-adrenergic receptor; see MATERIALS AND METHODS), but the blocking effect was completely lost with both the R334A and R334C mutations (Fig. 5). Login to comment
200 A representative current trace of R334A-mCFTR is shown in Fig. 6D. GlyH-101 completely lost its blocking effect and only exhibited the potentiation function at R334A-mCFTR. Summary data for inhibition are shown in Fig. 6E. GlyH-101 displayed significantly strengthened block of V100L-mCFTR and distinctly weakened block of I201V- mCFTR. Login to comment
311 In fact, we can separate the inhibitory and stimulatory effects of GlyH-101 on mCFTR because the R334A mutation completely abolished the inhibitory effect of GlyH-101 on mCFTR without affecting potentiation. Login to comment