ABCMdb

ABCC7 p.Arg347Asp

ClinVar:	c.1039C>T , p.Arg347Cys ? , not provided c.1040G>A , p.Arg347His D , Pathogenic c.1040G>T , p.Arg347Leu D , Pathogenic c.1040G>C , p.Arg347Pro D , Pathogenic
CF databases:	c.1040G>C , p.Arg347Pro D , CF-causing ; CFTR1: This mutation destroys a Hha I restriciton site and creates an NcoI site and occurred in a family diagnosed as PS. The mutation have been identified and analyzed using the SSCP technique. c.1040G>A , p.Arg347His D , CF-causing ; CFTR1: The patient is of Italian origin and carries the [delta]F508 mutation on the other chromosome. Initially we thought this was the same mutation as R347 because it destroys the same hhai site; however, R347H does not create the NcoI site. c.1040G>T , p.Arg347Leu (CFTR1) D , A nucleotide change, G->T at position 1172, was detected leading to R347L. The other chromosome carries a [delta]F508. This mutation was found on one chromosome among 150 CF chromosomes screened. c.1039C>T , p.Arg347Cys (CFTR1) ? , This mutation was identified by DGGE and direct sequencing.
Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (71%), I: D (95%), K: D (95%), L: D (80%), M: D (95%), N: D (95%), P: D (75%), Q: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: N, F: D, G: D, 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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[hide] Transmembrane helix 11 of multidrug resistance pro... Biochemistry. 2004 Jul 27;43(29):9413-25. Zhang DW, Nunoya K, Vasa M, Gu HM, Theis A, Cole SP, Deeley RG
Transmembrane helix 11 of multidrug resistance protein 1 (MRP1/ABCC1): identification of polar amino acids important for substrate specificity and binding of ATP at nucleotide binding domain 1.
Biochemistry. 2004 Jul 27;43(29):9413-25., 2004-07-27 [PMID:15260484]

Abstract [show]
Human multidrug resistance protein 1 (MRP1) is an ATP binding cassette (ABC) transporter that confers resistance to many natural product chemotherapeutic agents and can transport structurally diverse conjugated organic anions. MRP1 has three polytopic transmembrane domains (TMDs) and a total of 17 TM helices. Photolabeling and mutagenesis studies of MRP1 indicate that TM11, the last helix in the second TMD, may form part of the protein's substrate binding pocket. We have demonstrated that certain polar residues within a number of TM helices, including Arg(593) in TM11, are determinants of MRP1 substrate specificity or overall activity. We have now extended these analyses to assess the functional consequences of mutating the remaining seven polar residues within and near TM11. Mutations Q580A, T581A, and S585A in the predicted outer leaflet region of the helix had no detectable effect on function, while mutation of three residues close to the membrane/cytoplasm interface altered substrate specificity. Two of these mutations affected only drug resistance. N597A increased and decreased resistance to vincristine and VP-16, respectively, while S605A decreased resistance to vincristine, VP-16 and doxorubicin. The third, S604A, selectively increased 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport. In contrast, elimination of the polar character of the residue at position 590 (Asn in the wild-type protein) uniformly impaired the ability of MRP1 to transport potential physiological substrates and to confer resistance to three different classes of natural product drugs. Kinetic and photolabeling studies revealed that mutation N590A not only decreased the affinity of MRP1 for cysteinyl leukotriene 4 (LTC(4)) but also substantially reduced the binding of ATP to nucleotide binding domain 1 (NBD1). Thus, polar interactions involving residues in TM11 influence not only the substrate specificity of MRP1 but also an early step in the proposed catalytic cycle of the protein.

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319 Since there are many functional similarities between the cooperative interactions of the NBDs of MRP1 and CFTR, it is possible that the CFTR R347D mutation may be affecting the same step in the catalytic cycle as the N590A mutation. Login to comment
316 A nonconservative mutation in CFTR, R347D, that results in a mild form of disease was recently found to affect the ATPase activity of the protein by decreasing its Vmax for ATP 3-5-fold (44). Login to comment
317 R347D is predicted to be in the aqueous pore of the channel in the inner leaflet region of TM6. Login to comment

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

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

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402 R347D altered GSH inhibition and reduced ATPase activity by decreasing nucleotide turnover rather than affinity [194]. Login to comment

[hide] Cystic fibrosis-associated mutations at arginine 3... J Biol Chem. 1999 Feb 26;274(9):5429-35. Cotten JF, Welsh MJ
Cystic fibrosis-associated mutations at arginine 347 alter the pore architecture of CFTR. Evidence for disruption of a salt bridge.
J Biol Chem. 1999 Feb 26;274(9):5429-35., 1999-02-26 [PMID:10026154]

Abstract [show]
Arginine 347 in the sixth transmembrane domain of cystic fibrosis transmembrane conductance regulator (CFTR) is a site of four cystic fibrosis-associated mutations. To better understand the function of Arg-347 and to learn how mutations at this site disrupt channel activity, we mutated Arg-347 to Asp, Cys, Glu, His, Leu, or Lys and examined single-channel function. Every Arg-347 mutation examined, except R347K, had a destabilizing effect on the pore, causing the channel to flutter between two conductance states. Chloride flow through the larger conductance state was similar to that of wild-type CFTR, suggesting that the residue at position 347 does not interact directly with permeating anions. We hypothesized that Arg-347 stabilizes the channel through an electrostatic interaction with an anionic residue in another transmembrane domain. To test this, we mutated anionic residues (Asp-924, Asp-993, and Glu-1104) to Arg in the context of either R347E or R347D mutations. Interestingly, the D924R mutation complemented R347D, yielding a channel that behaved like wild-type CFTR. These data suggest that Arg-347 plays an important structural role in CFTR, at least in part by forming a salt bridge with Asp-924; cystic fibrosis-associated mutations disrupt this interaction.

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No. Sentence Comment
1 To better understand the function of Arg-347 and to learn how mutations at this site disrupt channel activity, we mutated Arg-347 to Asp, Cys, Glu, His, Leu, or Lys and examined single-channel function. Login to comment
5 To test this, we mutated anionic residues (Asp-924, Asp-993, and Glu-1104) to Arg in the context of either R347E or R347D mutations. Login to comment
6 Interestingly, the D924R mutation complemented R347D, yielding a channel that behaved like wild-type CFTR. Login to comment
24 To better understand the role of Arg-347 in CFTR structure and function, we examined the effect of mutating Arg-347 to cysteine, aspartic acid, glutamic acid, lysine, and leucine on CFTR conductance. Login to comment
25 We examined the cytosolic pH (pHc)-dependent behavior of CFTR-R347H and that of the other residue 347 mutants both with (R347C, R347D, R347E, and R347K) and without (R347L) a pHc-titratable residue. Login to comment
86 Visual inspection suggested that the lifetimes of OL and OB states were also influenced by the nature of the residue at position 347: R347E and R347H tended to have longer dwell times in the OL and OB states, whereas R347L, R347C, and R347D tended to display shorter dwell times. Login to comment
89 For R347D, the lifetime in the OB state was so short that a discrete OB was not apparent on the all-points histogram; instead, as pHc decreased, a shoulder developed on the OL state distribution in the all-points histogram (Fig. 1). Login to comment
113 The observable pK (0 mV) for the equilibrium between OL and OB of R347E and R347H were 6.4 and 6.3, respectively. The faster kinetics of R347D, R347C, and R347L made dwell-time analysis for these mutants less reliable. Login to comment
117 Fig. 3B shows that R347C, R347D, and R347L did not reach a peak variance over the range of pHc studied, suggesting that their apparent pK is less than 5.0-5.5. Login to comment
119 Single-channel I-V relationships for R347E (OL and OB states), R347H (OL and OB states), R347K, R347D/D924R, and wild-type CFTR at pHc 6.0. n ϭ 2-4 at each data point. Login to comment
136 B, open-channel current variance of the R347C, R347D, R347L, and R347E mutants versus pHc. Login to comment
148 The Phenotype of R347D Is Suppressed by the D924R Mutation-The data suggest that Arg-347 and Lys-347 may stabilize the structure of the pore; in their absence, the channel "flickers" between two conductance states. Login to comment
153 To identify the Arg-347 interaction partner, we replaced Arg-347 with an anionic residue (R347E or R347D) and introduced an arginine residue in the place of candidate partners in a salt bridge. Login to comment
154 We studied the conductance properties of the following double mutants: R347D/D924R, R347D/D993R, and R347E/E1104R. Login to comment
155 The R347D/D993R and R347E/E1104R mutants each had two conductance states with pHc-dependent behavior (Fig. 5). Login to comment
156 For R347D/D993R the increased entry into the OB state was apparent as a shoulder on the amplitude histogram at pHc 5.5. Login to comment
157 Accordingly, for R347D/D993R and R347E/E1104R the current variance in the open state increased with decreasing pHc (Fig. 5B). Login to comment
158 Qualitatively, the lifetimes of the OL and OB conductance states in the R347D/D993R and R347E/E1104R were similar to that of the R347D and R347E mutants, respectively. The amplitude of the OL state was larger for both of these double mutants as compared with the single mutants (Figs. Login to comment
161 In contrast to the other double mutants, the R347D/D924R mutant did not display the pHc-dependent flicker found in the R347D single mutant (Fig. 5, A and B), and there was no effect of pH on open-channel variance (Fig. 5B). Login to comment
163 These data suggest that the D924R mutation compensates for or rescues the phenotype of the R347D mutation. Login to comment
179 A, single-channel current tracings from excised, inside-out membrane patches containing R347E/E1104R, R347D/D924R, and R347D/D993R. Login to comment
181 B, current variance of R347E/E1104R, R347D/D924R, and R347D/D993R at the indicated pHc was collected as in Fig. 3. Login to comment
199 Second, and more importantly, we found that a second-site complementary mutation at position 924 (D924R) largely eliminated the pHc-dependent flickering phenotype of the R347D mutation and restored current amplitude to near wild-type values. Login to comment
207 The studies of R347D/D924R are consistent with a salt bridge between Arg-347 and Asp-924 and thus an interaction between M6 and M8. Login to comment

[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
218 The mechanism for the decreased I- conductance compared to those of the other halide ions for both WT and R352C is not known, although in the R347D mutant the effect is eliminated (2). Login to comment
225 This I- -induced change appears to result from I-binding to the CFTR protein and is eliminated by the R347D mutation (2). Login to comment

[hide] Structural and ionic determinants of 5-nitro-2-(3-... Br J Pharmacol. 1999 May;127(2):369-76. Walsh KB, Long KJ, Shen X
Structural and ionic determinants of 5-nitro-2-(3-phenylprophyl-amino)-benzoic acid block of the CFTR chloride channel.
Br J Pharmacol. 1999 May;127(2):369-76., [PMID:10385235]

Abstract [show]
1. The goals of this study were to identify the structural components required for arylaminobenzoate block of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and to determine the involvement of two positively charged amino acid residues, found within the channel, in drug binding. 2. Wild-type and mutant CFTR chloride channels were expressed in Xenopus oocytes and CFTR currents measured using the two microelectrode voltage clamp. Block of the wild-type CFTR current by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) occurred in a voltage-dependent manner with preferential inhibition of the inward currents (Kd = 166 microM at -90 mV). 3. Removal of the phenyl ring from the aliphatic chain of NPPB, with the compound 2-butylamino-5-nitrobenzoic acid, caused only a small change in CFTR inhibition (Kd = 243 microM), while addition of an extra phenyl ring at this position (5-nitro-2-(3,3-diphenylpropylamino)-benzoic acid) increased drug potency (Kd = 58 microM). In contrast, removal of the benzoate ring (2-amino-4-phenylbutyric acid) or the 5-nitro group (2-(3-phenylpropylamino)-benzoic acid) of NPPB severely limited drug block of the wild-type channel. 4. NPPB inhibition of CFTR currents in oocytes expressing the mutants K335E and R347E also occurred in a voltage-dependent manner. However, the Kds for NPPB block were increased to 371 and 1573 microM, for the K335E and R347E mutants, respectively. 5. NPPB block of the inward wild-type CFTR current was reduced in the presence of 10 mM of the permeant anion SCN-. 6. These studies present the first step in the development of high affinity probes to the CFTR channel.

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148 Furthermore, the R347D construct lacks multi-ion pore behaviour; a property measured with the wild-type channel in mixtures of Cl7 and SCN7 (Tabcharani et al., 1993; Linsdell et al., 1997). Login to comment
149 Unlike the wild-type channel, the R347D mutant is insensitive to block by high concentrations of internally applied 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) (Linsdell & Hanrahan, 1996a). Login to comment
162 Interestingly, this eect of SCN7 is absent in the R347D mutant (Tabcharani et al., 1993). Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... J Gen Physiol. 1999 Dec;114(6):799-818. Smith SS, Steinle ED, Meyerhoff ME, Dawson DC
Cystic fibrosis transmembrane conductance regulator. Physical basis for lyotropic anion selectivity patterns.
J Gen Physiol. 1999 Dec;114(6):799-818., [PMID:10578016]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel exhibits lyotropic anion selectivity. Anions that are more readily dehydrated than Cl exhibit permeability ratios (P(S)/P(Cl)) greater than unity and also bind more tightly in the channel. We compared the selectivity of CFTR to that of a synthetic anion-selective membrane [poly(vinyl chloride)-tridodecylmethylammonium chloride; PVC-TDMAC] for which the nature of the physical process that governs the anion-selective response is more readily apparent. The permeability and binding selectivity patterns of CFTR differed only by a multiplicative constant from that of the PVC-TDMAC membrane; and a continuum electrostatic model suggested that both patterns could be understood in terms of the differences in the relative stabilization of anions by water and the polarizable interior of the channel or synthetic membrane. The calculated energies of anion-channel interaction, derived from measurements of either permeability or binding, varied as a linear function of inverse ionic radius (1/r), as expected from a Born-type model of ion charging in a medium characterized by an effective dielectric constant of 19. The model predicts that large anions, like SCN, although they experience weaker interactions (relative to Cl) with water and also with the channel, are more permeant than Cl because anion-water energy is a steeper function of 1/r than is the anion-channel energy. These large anions also bind more tightly for the same reason: the reduced energy of hydration allows the net transfer energy (the well depth) to be more negative. This simple selectivity mechanism that governs permeability and binding acts to optimize the function of CFTR as a Cl filter. Anions that are smaller (more difficult to dehydrate) than Cl are energetically retarded from entering the channel, while the larger (more readily dehydrated) anions are retarded in their passage by "sticking" within the channel.

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No. Sentence Comment
290 In the case of CFTR, it is possible to envision two sorts of CFTR pores: those that bind anions, exemplified by the wild-type channel, and those that do not, exemplified by mutant CFTRs like G314E or Q (Mansoura et al., 1998) and R347D (Tabcharani et al., 1993). Login to comment
288 In the case of CFTR, it is possible to envision two sorts of CFTR pores: those that bind anions, exemplified by the wild-type channel, and those that do not, exemplified by mutant CFTRs like G314E or Q (Mansoura et al., 1998) and R347D (Tabcharani et al., 1993). Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... J Biol Chem. 2000 Feb 11;275(6):3729-32. Akabas MH
Cystic fibrosis transmembrane conductance regulator. Structure and function of an epithelial chloride channel.
J Biol Chem. 2000 Feb 11;275(6):3729-32., 2000-02-11 [PMID:10660517]

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No. Sentence Comment
57 These effects were eliminated in the M6 mutant R347D, and Arg-347, therefore, was hypothesized to be at or near an anion binding site (35). Login to comment

[hide] Permeation through the CFTR chloride channel. J Exp Biol. 2000 Jul;203(Pt 13):1947-62. McCarty NA
Permeation through the CFTR chloride channel.
J Exp Biol. 2000 Jul;203(Pt 13):1947-62., [PMID:10851114]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) protein forms a Cl(-) channel found in the plasma membranes of many epithelial cells, including those of the kidney, gut and conducting airways. Mutation of the gene encoding CFTR is the primary defect in cystic fibrosis, a disease that affects approximately 30 000 individuals in the United States alone. Alteration of CFTR function also plays an important role in the pathophysiology of secretory diarrhea and polycystic kidney disease. The basic mechanisms of permeation in this channel are not well understood. It is not known which portions of the protein contribute to forming the pore or which amino acid residues in those domains are involved in the biophysical processes of ion permeation. In this review, I will discuss (i) the present understanding of ion transport processes in the wild-type CFTR channel, (ii) the experimental approaches currently being applied to investigate the pore, and (iii) a proposed structure that takes into account the present data on mechanisms of ion selectivity in the CFTR channel and on blockade of the pore by open-channel blockers.

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No. Sentence Comment
169 Mutation R347D at the putative cytoplasmic end of TM6 reduced the affinity for DIDS. Login to comment
170 However, R347D has recently been shown to grossly perturb the conformation of the pore by disruption of a salt bridge (Cotten and Welsh, 1999). Login to comment

[hide] Two mild cystic fibrosis-associated mutations resu... J Biol Chem. 2001 Mar 23;276(12):9045-9. Epub 2000 Dec 15. Clain J, Fritsch J, Lehmann-Che J, Bali M, Arous N, Goossens M, Edelman A, Fanen P
Two mild cystic fibrosis-associated mutations result in severe cystic fibrosis when combined in cis and reveal a residue important for cystic fibrosis transmembrane conductance regulator processing and function.
J Biol Chem. 2001 Mar 23;276(12):9045-9. Epub 2000 Dec 15., 2001-03-23 [PMID:11118444]

Abstract [show]
The number of complex cystic fibrosis transmembrane conductance regulator (CFTR) genotypes identified as having double-mutant alleles with two mutations inherited in cis has been growing. We investigated the structure-function relationships of a severe cystic fibrosis (CF)-associated double mutant (R347H-D979A) to evaluate the contribution of each mild mutation to the phenotype. CFTR mutants expressed in HeLa cells were analyzed for protein biosynthesis and Cl(-) channel activity. Our data show that R347H is associated with mild defective Cl(-) channel activity and that the D979A defect leads to misprocessing. The mutant R347H-D979A combines both defects for a dramatic decrease in Cl(-) current. To decipher the molecular mechanism of this phenotype, single and double mutants with different charge combinations at residues 347 and 979 were constructed as charged residues were involved in this complex genotype. These studies revealed that residue 979, located in the third cytoplasmic loop, is critical for CFTR processing and Cl(-) channel activity highlighting the role of charged residues. These results have also important implications for CF, as they show that two mutations in cis can act in concert to alter dramatically CFTR function contributing to the wide phenotypic variability of CF disease.

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101 Charge-reversal Mutants-Taking into account the functional defects that result when Arg-347 and Asp-979 are each replaced with an uncharged amino acid such as His (uncharged at pH 7.3) and Ala (R347H and D979A), we constructed additional mutants with different charge combinations at residues 347 and 979, including the R347D-D979R double mutant in which the positive and negative charges were swapped. Login to comment
102 The processing of R347D was similar to those of R347H and the wild-type (Fig. 3, open bars). Login to comment
118 essing of D979R, R347H-D979R, and R347D-D979R was differently impaired (Fig. 3; gray bars). Login to comment
123 The Cl- current of R347D-D979R (2.8 Ϯ 1.7 pA/pF; n ϭ 9) was not significantly different from those of D979R and R347H-D979A (Fig. 2D). Login to comment
124 This result is consistent with the poor amount of R347D-D979R protein at the cell surface. Login to comment
143 First, several lines of experimental evidence indicate that there is probably no direct salt bridge between Arg-347 and Asp-979: (i) removal of either the positive charge at position 347 (R347H and R347D) or the negative charge at position 979 (D979A, D979V, and D979R) has different effects on CFTR processing; (ii) the double-neutral (R347H-D979A) and reversed-charged (R347D-D979R) replacements for Arg-347 and Asp-979 do not lead to the recovery of wild-type processing. Login to comment

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

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

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No. Sentence Comment
4 Mutations of residues lining the channel pore (e.g. R347D) are typically thought to cause disease by altering the interaction of Cl- with the pore. Login to comment
5 However, in the present study we show that the R347D mutation and diphenylamine-2-carboxylate (an open pore inhibitor) also inhibit CFTR ATPase activity, revealing a novel mechanism for cross-talk from the pore to the catalytic domains. Login to comment
7 Finally, we demonstrate that glutathione (GSH) inhibits CFTR ATPase and that this inhibition is altered in the CFTR-R347D variant. Login to comment
102 The Pore Mutant CFTR-R347D Exhibits Altered ATPase Activity-Direct evidence for communication between the chloride channel pore and the catalytic domains of CFTR came from assessments of the ATPase activity of a disease-causing variant of CFTR bearing an amino acid substitution (Arg to Asp) at position 347 within the putative pore region. Login to comment
105 However, the specific activity of PKA-phosphorylated CFTR-R347D was FIG. 1. Login to comment
125 The increase in ATPase activity associated with PKA-treated CFTR-R347D samples was not due to PKA itself, since treatment with this enzyme conferred less than 8% of the total activity measured for the phosphorylated mutant (see legend to Fig. 5A for details). Login to comment
127 Whereas the Vmax of phosphorylated wild type CFTR protein is about 50 nmol/mg/min (18), the Vmax determined for phosphorylated R347D protein is 1.1 nmol/mg/min (Fig. 5B, Table I). Login to comment
129 Defective ATPase activity by CFTR-R347D mutant was not due to FIG. 4. Login to comment
140 Characterization of the effect of CFTR-R347D mutation on the ATPase activity. Login to comment
142 The mean Ϯ S.E. is shown for nine phosphorylated and nonphosphorylated wild type CFTR preparations. For CFTR-R347D preparations, each bar represents the activity of duplicate values. Login to comment
144 PKA phosphorylation of liposomes alone, treated in the same manner as CFTR-R347D preparations, accounted for less than 8% of the ATPase activity observed for the phosphorylated CFTR-R347D protein (0.16 nmol of ATP hydrolyzed/2 h versus 2.03 nmol of ATP hydrolyzed/2 h). Login to comment
146 Panel B, MgATP dependence of the catalytic activity of purified and either phosphorylated or nonphosphorylated CFTR-R347D protein. Login to comment
149 Each sample contained ϳ8 ␮g of purified, reconstituted CFTR-R347D protein, and duplicate or triplicate samples were assessed. Login to comment
150 Panel C, effect of ionic strength on the ATPase activity of phosphorylated wild type CFTR and CFTR-R347D proteins. Login to comment
152 Duplicate samples of wild type protein and triplicate samples of CFTR-R347D were studied. Login to comment
153 global misfolding of the nucleotide binding folds, as the apparent affinity of the phosphorylated mutant for MgATP was comparable, even somewhat higher, than previously reported for phosphorylated wild type protein (Km ϭ 0.1 mM for CFTR-R347D versus Km ϭ 0.3 mM for wild type protein, respectively (18)). Login to comment
154 The pore blocker DPC exerted a similar inhibitory effect on the ATPase activity of phosphorylated CFTR-R347D, as it did on the ATPase activity of wild type protein (67.3 Ϯ 6.1% of control versus 66.8 Ϯ 3.6% of control, respectively). Login to comment
155 These results support previous reports suggesting that DPC interacts with a pore-lining residue other than Arg-347, probably Ser341 (44), and that the R347D mutation does not disrupt DPC binding to this site by inducing global misfolding. Login to comment
156 Moreover, a similar inhibitory trend was observed for increasing salt concentration on the ATPase activity of R347D protein (Fig. 5C), as compared with the wild type protein. Login to comment
167 Finally, we observed a significant difference in the inhibitory effect of GSH (10 mM) on the ATPase activity of wild type and CFTR-R347D proteins, 39 and 63% of untreated controls, respectively (Fig. 6C, p ϭ 0.04). Login to comment
190 Certain organic anions including GSH, GSSG, and gluconate have been previously shown to block chloride ion flux through TABLE I Kinetic parameters of PKA-phosphorylated and non-phosphorylated wild-type and R347D proteins Km Vmax ϩPKA -PKA ϩPKA -PKA mM nmol/mg/min Wild typea 0.3 1.0 54 51 R347D 0.1 1.1 1.1 0.2 a Data for wild type CFTR were reported in our previous studies (18). Login to comment
194 We found further evidence for direct communication between the pore domain and NBDs of CFTR when investigating the catalytic activity of CFTR-R347D, a disease-causing variant with a mutation in TM6 associated with mild disease (45). Login to comment
195 The R347D mutation led to inhibition of the ATPase activity of CFTR, suggesting that the region in which this arginine resides participates in the physical communication between the pore and the NBDs. This residue is thought to reside in the inner vestibule of the CFTR channel and has been implicated in anion binding within the pore (45, 50). Login to comment
198 Both the interaction of DPC with the pore and mutation of the putative pore-lining residue, R347D, induced similar changes in the catalytic activity of phosphorylated CFTR, namely, both of these perturbations caused a 3-5-fold decrease in the Vmax of the enzyme. Login to comment
211 Second, mutation of the pore-lining residue R347D led to a change in the extent of blockade of CFTR ATPase activity by GSH. Login to comment
221 Effect of glutathione on the catalytic activity of wild type and CFTR-R347D proteins. Login to comment
228 Panel C, effect of 10 mM GSH on the catalytic activity of phosphorylated wild type (WT, open) and CFTR-R347D (hatched) proteins relative to control (no GSH). Login to comment
236 Acknowledgments-We thank Dr. Mary Corey (Research Institute, Hospital for Sick Children) for the assistance with the statistical analysis and Dr. Johanna Rommens for providing us with cDNA coding for the mutant R347D (Research Institute, Hospital for Sick Children). Login to comment

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

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

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34 The hypothesis that anion permeability and anion binding are separable facets of the permeation process in the CFTR Cl¦ channel is supported by the fact that several mutations within the pore have been shown to alter anion binding without strongly affecting anion permeability (e.g. K335E, Anderson et al. 1991; R347D, Tabcharani et al. 1993; G314E, Mansoura et al. 1998). Login to comment

[hide] Identification of a region of strong discriminatio... Am J Physiol Lung Cell Mol Physiol. 2001 Oct;281(4):L852-67. McCarty NA, Zhang ZR
Identification of a region of strong discrimination in the pore of CFTR.
Am J Physiol Lung Cell Mol Physiol. 2001 Oct;281(4):L852-67., [PMID:11557589]

Abstract [show]
The variety of methods used to identify the structural determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator Cl(-) channel has made it difficult to assemble the data into a coherent framework that describes the three-dimensional structure of the pore. Here, we compare the relative importance of sites previously studied and identify new sites that contribute strongly to anion selectivity. We studied Cl(-) and substitute anions in oocytes expressing wild-type cystic fibrosis transmembrane conductance regulator or 12-pore-domain mutants to determine relative permeability and relative conductance for 9 monovalent anions and 1 divalent anion. The data indicate that the region of strong discrimination resides between T338 and S341 in transmembrane 6, where mutations affected selectivity between Cl(-) and both large and small anions. Mutations further toward the extracellular end of the pore only strongly affected selectivity between Cl(-) and larger anions. Only mutations at S341 affected selectivity between monovalent and divalent anions. The data are consistent with a narrowing of the pore between the extracellular end and a constriction near the middle of the pore.

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169 For instance, the R347D mutation has been shown to have nonspecific effects due to destruction of a salt bridge (8). Login to comment

[hide] Point mutations in the pore region directly or ind... Pflugers Arch. 2002 Mar;443(5-6):739-47. Epub 2001 Dec 8. Gupta J, Linsdell P
Point mutations in the pore region directly or indirectly affect glibenclamide block of the CFTR chloride channel.
Pflugers Arch. 2002 Mar;443(5-6):739-47. Epub 2001 Dec 8., [PMID:11889571]

Abstract [show]
The sulfonylurea glibenclamide is a relatively potent inhibitor of the CFTR Cl(-) channel. This inhibition is thought to be via an open channel block mechanism. However, nothing is known about the physical nature of the glibenclamide-binding site on CFTR. Here we show that mutations in the pore-forming 6th and 12th transmembrane regions of CFTR affect block by intracellular glibenclamide, confirming previous suggestions that glibenclamide enters the pore in order to block the channel. Two mutations in the 6th transmembrane region, F337A and T338A, significantly weakened glibenclamide block, consistent with a direct interaction between glibenclamide and this region of the pore. Interestingly, two mutations in the 12th transmembrane region (N1138A and T1142A) significantly strengthened block. These two mutations also abolished the dependence of block on the extracellular Cl(-) concentration, which in wild-type CFTR suggests an interaction between Cl(-) and glibenclamide within the channel pore that limits block. We suggest that mutations in the 12th transmembrane region strengthen glibenclamide block not by directly altering interactions between glibenclamide and the pore walls, but indirectly by reducing interactions between Cl(-) ions and glibenclamide within the pore. This work demonstrates that glibenclamide binds within the CFTR channel pore and begins to define its intrapore binding site.

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147 Am J Physiol 271:C628-C634 20. Linsdell P, Hanrahan JW (1997) Interaction of channel blockers with R347D-CFTR [abstract]. Login to comment

[hide] CFTR is a monomer: biochemical and functional evid... J Membr Biol. 2002 Jul 1;188(1):55-71. Chen JH, Chang XB, Aleksandrov AA, Riordan JR
CFTR is a monomer: biochemical and functional evidence.
J Membr Biol. 2002 Jul 1;188(1):55-71., 2002-07-01 [PMID:12172647]

Abstract [show]
Although the CFTR protein alone is sufficient to generate a regulated chloride channel, it is unknown how many of the polypeptides form the channel. Using biochemical and functional assays, we demonstrate that the CFTR polypeptide is a monomer. CFTR sediments as a monomer in a linear, continuous sucrose gradient. Cells co-expressing different epitope-tagged CFTR provide no evidence of co-assembly in immunoprecipitation and nickel affinity binding experiments. Co-expressed wild-type and DF508 CFTR are without influence on each other in their ability to progress through the secretory pathway, suggesting they do not associate in the endoplasmic reticulum. No hybrid conducting single channels are seen in planar lipid bilayers with which membrane vesicles from cells co-expressing similar amounts of two different CFTR conduction species have been fused.

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54 Missense mutations S341A and R347D were generated by site-directed mutagenesis (Stratagene) and cloned into CFTR-M2 by replacing the A¯II-HpaI fragment, and into M2-CFTR by replacing the XbaI-HpaI fragment. Login to comment
79 To obtain approximately equal expression of dierent epitope-tagged CFTR-conduction variants, BHK cells were transiently cotransfected with cDNA in the following ratios: 6:1, S341A-M2:WT-HSV;7:5,R347D-M2:WT-HSV;1:1,S341A-M2:TT338, 339AA-HSV;3:11,R347D-M2:TT338,339AA-HSV;6:1,M2-S341A: HSV-WT; and 1:1,M2-R347D:HSV-WT. Login to comment
180 Mutations S341A and R347D (single-letter amino acid) dramatically lower chloride conductance (Tabcharani et al., 1993; McDonough et al., 1994) while the double mutation TT338, 339AA enhances chloride conduction (Linsdell et al., 1997). Login to comment
193 To the C-terminal ends of CFTR, we attached the M2 epitope to S341A and R347D and the HSV epitope to wild type and TT338, 339AA (S341A-M2, R347D-M2, WT-HSV, and TT338, 339AA-HSV, respectively). Login to comment
194 Single-channel chord conductances for S341A-M2, R347D-M2, WT-HSV, and TT338, 339AA-HSV at À100 mV were (in pS): 2.2, 5.1, 14.3, and 15.6, respectively (Figs. Login to comment
205 (C) Amplitude histograms and single-channel recordings of low-conduction mutants S341A and R347D C-terminally tagged with M2 (S341A-M2 and R347D-M2, respectively), and high-conduction variants WT and TT338, 339AA C-terminally tagged with HSV (WT-HSV and TT338, 339AA-HSV, respectively). Login to comment
207 (D) Single-channel current-voltage relationships of S341A-M2, R347D-M2, WT-HSV, and TT338,339A-HSV. Login to comment
210 (E) Tabulation of single-channel conductances from microsomes containing a low-conducting (S341A.M2 or R347D-M2) and a high-conducting species (WT-HSV or TT338,339AA-HSV). Login to comment
215 To assess CFTR stoichiometry, we co-expressed approximately equal amounts of a low-(S341A-M2 or R347D-M2) and a high-conduction species (WT-HSV or TT338,339AA-HSV) for single-channel analysis (Fig. 4E). Login to comment
223 (C) Amplitude histograms and single-channel recordings of S341A and R347D N-terminally tagged with M2 (M2-S341A and M2-R347D, respectively), and WT N-terminally tagged with HSV (HSV-WT). Login to comment
225 (D) Single-channel current-voltage relationships of M2-S341A, M2-R347D, and HSV-WT. Login to comment
227 Intermediate conducting channels were infrequently observed; membrane vesicles containing WT-HSV plus S341A-M2 or R347D-M2 produced 9.5 pS and 7.5 pS channels, and vesicles containing TT338,339AA-HSV plus S341A-M2 or R347D-M2 yielded 12 pS, 8 pS, and 2 pS channels. Login to comment
228 These channels were most likely subconductances of WT-HSV (9.5 pS and 7.5 pS channels), TT338,339AA-HSV (12 pS and 8 pS), and R347D-M2 (2 pS) since they were seen with microsomes containing each of these species alone. Login to comment
235 The chord conductances of M2-S341A, M2-R347D, and HSV-WT at À100 mV (in pS) were 2.2, 5.1, and 14.6, respectively (Fig. 5C and 5D). Login to comment
236 When co-expressed, microsomes containing HSV-WT and either M2-S341A or M2-R347D produced conductances that were predominantly channels of each constituent in their main conductance states and infrequently their subconductance states (Fig. 5E). Login to comment
237 The possibility that these intermediate conductances resulted from a hybrid assembly of multiple CFTR molecules was £ 3.3% and £ 2.1% for HSV-WT plus M2-S341A and M2-R347D, respectively. Login to comment
244 (E) Tabulation of single-channel conductances from microsomes containing HSV-WT and either M2-S341A or M2-R347D. Login to comment
247 Co-expressed CFTR proteins do not form a hybrid channel (95% CI £ 3.3% for M2-S341A and HSV-WT, and £ 2.1% for M2-R347D and HSV-WT). Login to comment

[hide] CFTR directly mediates nucleotide-regulated glutat... EMBO J. 2003 May 1;22(9):1981-9. Kogan I, Ramjeesingh M, Li C, Kidd JF, Wang Y, Leslie EM, Cole SP, Bear CE
CFTR directly mediates nucleotide-regulated glutathione flux.
EMBO J. 2003 May 1;22(9):1981-9., 2003-05-01 [PMID:12727866]

Abstract [show]
Studies have shown that expression of cystic fibrosis transmembrane conductance regulator (CFTR) is associated with enhanced glutathione (GSH) efflux from airway epithelial cells, implicating a role for CFTR in the control of oxidative stress in the airways. To define the mechanism underlying CFTR-associated GSH flux, we studied wild-type and mutant CFTR proteins expressed in Sf9 membranes, as well as purified and reconstituted CFTR. We show that CFTR-expressing membrane vesicles mediate nucleotide-activated GSH flux, which is disrupted in the R347D pore mutant, and in the Walker A K464A and K1250A mutants. Further, we reveal that purified CFTR protein alone directly mediates nucleotide-dependent GSH flux. Interestingly, although ATP supports GSH flux through CFTR, this activity is enhanced in the presence of the non-hydrolyzable ATP analog AMP-PNP. These findings corroborate previous suggestions that CFTR pore properties can vary with the nature of the nucleotide interaction. In conclusion, our data demonstrate that GSH flux is an intrinsic function of CFTR and prompt future examination of the role of this function in airway biology in health and disease.

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2 We show that CFTR-expressing membrane vesicles mediate nucleotide-activated GSH ¯ux, which is disrupted in the R347D pore mutant, and in the Walker A K464A and K1250A mutants. Login to comment
7 Keywords: CFTR/glutathione/puri®ed protein/R347D pore mutant/Walker A mutants Introduction Cystic ®brosis (CF) is a lethal autosomal recessive disease caused by mutations within the cystic ®brosis transmembrane conductance regulator (CFTR) gene (Boat et al., 1989). Login to comment
63 As seen in Figure 3A (insert), the R347D variant was expressed well in Sf9 membranes. Login to comment
64 We compared [35S]GSH uptake by vesicles containing either phosphorylated wild-type or R347D CFTR proteins in the presence of 1 mM GSH, as this concentration is close to the Km(GSH) and is known not to have any non-speci®c effects on non-pore regions of CFTR. Login to comment
88 In the current study, we found that [35S]GSH uptake was signi®cantly decreased in membrane vesicles expressing the R347D mutant protein, relative to those expressing wild-type CFTR, with rates of GSH ¯ux of 207 and 498 pmol/mg CFTR/h, respectively (Figure 3A; P < 0.0001). Login to comment
91 Kinetic analyses revealed that vesicles with the R347D mutation exhibited an ~75% decrease in the Vmax of GSH uptake compared with vesicles with wild-type CFTR, corresponding to 176 and 612 pmol GSH/mg CFTR/h, respectively (Table I). Login to comment
92 These analyses also suggest that GSH interaction with the pore of the R347D mutant is ~4 times stronger than that with the wild-type pore, with Km(GSH) values of 0.11 and 0.47 mM, respectively. Login to comment
93 Modi®ed af®nity of GSH for the mutant channel is consistent with previous reports, suggesting altered interaction of GSH with the R347D pore (Kogan et al., 2001). Login to comment
100 Kinetic parameters of GSH uptake by Sf9 membrane vesicles containing PKA-phosphorylated wild-type or R347D CFTR proteins, in the presence of MgAMP-PNP Variables Wild type R347D Vmax (pmol/mg CFTR/h) 612 176 Km (mM) 0.47 0.11 Fig. 4. Login to comment
108 Comparison of GSH ¯ux by wild-type CFTR versus CFTR R347D protein. Login to comment
109 (A) Membrane vesicles expressing phosphorylated wild-type or R347D CFTR proteins were incubated with 20 nM [35S]GSH and 1 mM cold GSH, in the presence of MgAMP-PNP. Login to comment
111 Values are expressed as the mean T SEM (n = 5 for wild-type CFTR, n = 2 for R347D). Login to comment
112 Inset: expression of CFTR in membranes from Sf9 cells transfected with wild-type or R347D CFTR constructs. Login to comment
115 (B) Effect of increasing substrate concentration on [35S]GSH uptake by vesicles expressing phosphorylated wild-type CFTR or the R347D variant, in the presence of MgAMP-PNP. Login to comment
117 [35S]GSH uptake by vesicles expressing the R347D protein was also obtained by subtracting GSH uptake values of vesicles with no CFTR from those of vesicles expressing the R347D variant. Login to comment
118 Curve ®tting was performed by non-linear regression analysis, using the Michaelis±Menten equation, to yield the following kinetic parameters for the R347D mutant: Km = 0.11 mM, Vmax = 176 pmol GSH/mg CFTR/h, r2 = 0.97. Login to comment
194 An Sf9 cell pellet (0.5 l) expressing either recombinant CFTR-His proteins (wild type or mutant: R347D, K464A, K1250A) or no CFTR was solubilized in 30 ml of homogenization buffer containing 250 mM sucrose, 50 mM Tris±HCl, 0.25 mM CaCl2 pH 7.5 and protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany). 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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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] 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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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
132 As found for R347A, the mutants R347C and R347D also showed a normal rate of anion transport but altered sensitivity to CFTRinh-172 (Figures 2A and 2B). Login to comment
136 To further investigate the importance of the salt bridge, we generated a double mutant, R347D/D924R, in which the positions of the charged amino acids are inverted. Login to comment
137 Interestingly, in contrast with D924R, the double mutant was able to transport anions but showed a low sensitivity to CFTRinh-172, with an estimated Ki greater than 50 μM (Figures 2A and 2B), similar to that of the single R347D mutant. Login to comment
139 Wild-type protein and the R347D mutant showed a normal pattern of electrophoretic mobility with a prevalent abundance of the band C which represents the fully glycosylated mature form of CFTR. Login to comment
141 In contrast with R347D, the D924R mutation produced a partial defect in maturation, with increased band B intensity compared with band C. Interestingly, this defect seemed to be corrected in the double mutant R347D/D924R (Figure 2C). 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
144 The R347A and R347D mutants showed Figure 2 Mutagenesis of Arg347 and Asp924 residues (A) Rate of I- transport measured in COS-7 cells transfected with the indicated constructs. Login to comment
146 (B) CFTRinh-172 dose-response relationships for wild-type, R347D and R347D/D924R CFTR. Login to comment
155 The calculated Ki for wild-type CFTR, R347A and R347D was 0.85 +- 0.13 μM (n = 8), 17.35 +- 3.90 μM (n = 13) and 53.10 +- 4.74 μM (n = 6) respectively (Figure 3E). 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
163 Dose-responses carried out on wild-type CFTR and R347D cells showed identical sensitivity to GlyH-101 with a Ki of 5.02 +- 0.90 μM (n = 6) and 4.99 +- 0.66 μM (n = 6) respectively (Figures 4A-4C). Login to comment
164 We also tested the sensitivity of wild-type and R347D to a zwitterionic, net neutral analogue of CFTRinh-172, thiazo N-O, in which the carboxyphenyl group is replaced by an oxido-4-pyridinyl group (Figure 5A). Login to comment
165 Although less potent than CFTRinh-172, the thiazo N-O compound caused a dose-dependent decrease of wild-type CFTR currents (Ki = 31.42 +- 7.30 μM, n = 4), but very weak inhibition of the R347D mutant (Figures 5B and 5C). Login to comment
170 Despite the use of a CFTRinh-172 concentration an order of magnitude higher than the half-effective concentration for wild-type CFTR, the Cl- currents Figure 4 CFTR Cl- current inhibition by GlyH-101 (A and B) Representative traces showing transepithelial Cl- currents measured in FRT cells with stable expression of wild-type (WT) and R347D-CFTR. Cells were first stimulated with 20 μM forskolin to activate CFTR and then were tested with increasing concentrations of GlyH-101. Login to comment
190 (B) Representative recordings showing effect of increasing concentrations of thiazo N-O on wild-type and R347D activity. Login to comment
202 Interestingly, when we generated the double mutant R347D/D924R, in which the positions of positive and negative charges are inverted but the salt bridge is maintained [25], we Figure 6 Patch-clamp analysis of CFTR inhibition by CFTRinh-172 (A and C) Superimposed membrane currents recorded from cells expressing wild-type and the R347A mutant at membrane potentials between -100 and +100 mV. Login to comment
208 On the other hand, we found that the double mutant R347D/D924R did not behave as the wild-type CFTR in terms of CFTRinh-172 sensitivity but was more similar to single Arg347 mutants. Login to comment
211 We hypothesized that the negatively charged carboxyl group in CFTRinh-172 interacts with the positive charge of Arg347 , such that the effects of Arg347 mutations could be explained by loss of electrostatic attraction (R347A) or generation of electrostatic repulsion (R347D). Login to comment
212 Accordingly, the activity of the neutral thiazo N-O compound had to be less affected by the R347D mutation. Login to comment
213 However, the potency of this compound on the R347D mutant was reduced, as found for CFTRinh-172, providing evidence against a direct electrostatic interaction between the CFTRinh-172 carboxyl group and Arg347 . Login to comment
219 For example, the R347D mutation has been shown to alter ATPase activity in NBDs [35], a finding that points to strong conformational coupling between TMDs and NBDs. Login to comment
220 However, it is important to point out that in the present study the R347D mutant, although being poorly inhibited by CFTRinh-172, showed an unaltered sensitivity to GlyH-101, an open-channel blocker acting from the extracellular side [8]. Login to comment

[hide] Mutations at arginine 352 alter the pore architect... J Membr Biol. 2008 Mar;222(2):91-106. Epub 2008 Apr 18. Cui G, Zhang ZR, O'Brien AR, Song B, McCarty NA
Mutations at arginine 352 alter the pore architecture of CFTR.
J Membr Biol. 2008 Mar;222(2):91-106. Epub 2008 Apr 18., [PMID:18421494]

Abstract [show]
Arginine 352 (R352) in the sixth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) previously was reported to form an anion/cation selectivity filter and to provide positive charge in the intracellular vestibule. However, mutations at this site have nonspecific effects, such as inducing susceptibility of endogenous cysteines to chemical modification. We hypothesized that R352 stabilizes channel structure and that charge-destroying mutations at this site disrupt pore architecture, with multiple consequences. We tested the effects of mutations at R352 on conductance, anion selectivity and block by the sulfonylurea drug glipizide, using recordings of wild-type and mutant channels. Charge-altering mutations at R352 destabilized the open state and altered both selectivity and block. In contrast, R352K-CFTR was similar to wild-type. Full conductance state amplitude was similar to that of wild-type CFTR in all mutants except R352E, suggesting that R352 does not itself form an anion coordination site. In an attempt to identify an acidic residue that may interact with R352, we found that permeation properties were similarly affected by charge-reversing mutations at D993. Wild-type-like properties were rescued in R352E/D993R-CFTR, suggesting that R352 and D993 in the wild-type channel may interact to stabilize pore architecture. Finally, R352A-CFTR was sensitive to modification by externally applied MTSEA+, while wild-type and R352E/D993R-CFTR were not. These data suggest that R352 plays an important structural role in CFTR, perhaps reflecting its involvement in forming a salt bridge with residue D993.

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19 Tabcharani et al. (1993) showed that anomalous mole-fraction behavior in mixtures of SCN- and Cl- was lost in R347D-CFTR and that this mutation also reduced single-channel conductance. Login to comment
24 The D924R mutation in TM8 complemented the R347D mutation, reverting the channel to WT behavior, allowingtheauthorstoconcludethatR347functionsatleastin part by forming a salt bridge with D924 (Cotten and Welsh 1999). Login to comment
265 Also, near the predicted cytoplasmic end of the CFTR pore, substitutions of the arginine at position 347 by any residue other than lysine destabilized the pore structure, while the double mutation R347D/ D924R recovered open state stability, suggesting that R347 formed a salt bridge with D924 in the wild-type channel (Cotten and Welsh 1999). Login to comment

[hide] Atomic model of human cystic fibrosis transmembran... Cell Mol Life Sci. 2008 Aug;65(16):2594-612. Mornon JP, Lehn P, Callebaut I
Atomic model of human cystic fibrosis transmembrane conductance regulator: membrane-spanning domains and coupling interfaces.
Cell Mol Life Sci. 2008 Aug;65(16):2594-612., [PMID:18597042]

Abstract [show]
We describe herein an atomic model of the outward-facing three-dimensional structure of the membrane-spanning domains (MSDs) and nucleotide-binding domains (NBDs) of human cystic fibrosis transmembrane conductance regulator (CFTR), based on the experimental structure of the bacterial transporter Sav1866. This model, which is in agreement with previous experimental data, highlights the role of some residues located in the transmembrane passages and directly involved in substrate translocation and of some residues within the intracellular loops (ICL1-ICL4) making MSD/NBD contacts. In particular, our model reveals that D173 ICL1 and N965 ICL3 likely interact with the bound nucleotide and that an intricate H-bond network (involving especially the ICL4 R1070 and the main chain of NBD1 F508) may stabilize the interface between MSD2 and the NBD1F508 region. These observations allow new insights into the ATP-binding sites asymmetry and into the molecular consequences of the F508 deletion, which is the most common cystic fibrosis mutation.

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187 Subsequent mutagenesis work involving acidic residues located in different TM helices has shown that the D924R mutation could complement the R347D mutation, suggesting that these two residues may form a salt bridge. Login to comment

[hide] The V510D suppressor mutation stabilizes DeltaF508... Biochemistry. 2010 Aug 3;49(30):6352-7. Loo TW, Bartlett MC, Clarke DM
The V510D suppressor mutation stabilizes DeltaF508-CFTR at the cell surface.
Biochemistry. 2010 Aug 3;49(30):6352-7., 2010-08-03 [PMID:20590134]

Abstract [show]
Deletion of Phe508 (DeltaF508) in the first nucleotide-binding domain (NBD1) of CFTR causes cystic fibrosis. The mutation severely reduces the stability and folding of the protein by disrupting interactions between NBD1 and the second transmembrane domain (TMD2). We found that replacement of Val510 with acidic residues (but not neutral or positive residues) promoted maturation of DeltaF508-CFTR with V510D more efficiently than V510E. Promotion of DeltaF508-CFTR maturation did not require NBD2 as introduction of V510D into a DeltaNBD2/DeltaF508-CFTR mutant restored maturation to levels similar to that of full-length protein. The V510D mutation increased the half-life of mature DeltaF508-CFTR at the cell surface by about 5-fold to resemble the half-life of wild-type CFTR. It was also observed that introduction of the V510R/R1070D mutations into DeltaF508-CFTR also promoted maturation whereas the V510D/R1070A mutations did not. We propose that the V510D mutation in NBD1 promotes maturation and stabilizes DeltaF508-CFTR at the cell surface through formation of a salt bridge with Arg1070 in TMD2.

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160 It was found that mutation of Arg347 to neutral amino acids or Asp destabilized channel function but the D924R mutation complemented R347D to yield a channel that behaved like wild-type CFTR. Login to comment

[hide] ABC transporter-facilitated ATP conductive transpo... Am J Physiol. 1999 Jan;276(1 Pt 1):C1-8. Schwiebert EM
ABC transporter-facilitated ATP conductive transport.
Am J Physiol. 1999 Jan;276(1 Pt 1):C1-8., [PMID:9886914]

Abstract [show]
The concept that the cystic fibrosis (CF) transmembrane conductance regulator, the protein product of the CF gene, can conduct larger multivalent anions such as ATP as well as Cl- is controversial. In this review, I examine briefly past findings that resulted in controversy. It is not the goal of this review to revisit these disparate findings in detail. Rather, I focus intently on more recent studies, current studies in progress, and possible future directions that arose from the controversy and that may reconcile this issue. Important questions and hypotheses are raised as to the physiological roles that ATP-binding cassette (ABC) transporter-facilitated ATP transport and signaling may play in the control of epithelial cell function. Perhaps the identification of key biological paradigms for ABC transporter-mediated extracellular nucleotide signaling may unify and guide the CF research community and other research groups interested in ABC transporters toward understanding why ABC transporters facilitate ATP transport.

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66 Both Cl- and gluconate currents were inhibited by insertion of two well-known Cl- conduction mutations, K335E and R347D, suggesting that CFTR itself is transporting gluconate (26). 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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193 Moreover, the mutant R347D significantly weak- main have had little discernible effect on conduction and permeation, although they have frequently had profoundened the binding of DNDS and DIDS to CFTR, suggesting that R347 contributes to the binding site for disulfonic effects on gating behavior (see below). Login to comment
232 Because the mutant R347D significantly weakened the activity of CFTR Cl0 channels by using excised inside-out membrane patches from cells expressing recombinantbinding of DNDS and DIDS to CFTR, R347 likely contributes to this site (74). 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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337 Block by DNDS was nearly makes them potentially very useful probes of the pore abolished in the R347D CFTR construct, whereas that by interior. The CFTR, and anion channels in general, tend DIDS was reduced but still readily apparent. Login to comment
350 Sucrose when R347 in TM6 was substituted with aspartic acid (R347D). Login to comment
357 R347D construct, and if a histidine was substituted for R347 (R347H), the blocking effect of SCN (XSCN Å 0.075)The magnitude of the voltage dependence was consistent with the ion experiencing from 30 to 60% of the transmem- was greatly enhanced at pH 5.5, suggesting that the presence of the positive charge is important for the high-affin-brane potential as it accessed the binding site. Login to comment
359 The single-channel conductance of R347D was reduced to Ç50% of wild-type CFTR and washanced when [Cl]o was reduced as expected if the two ions compete for a site in the pore. Login to comment
361 Linsdell et al. (95) used rate theory models to simulate the anomalous moleuncharged, intracellular osmolytes, sucrose, sorbitol, and fraction effect seen with SCN and its absence in R347D It is of interest in this regard that there is evidence for tight binding of SCN to another well-characterizedCFTR. Login to comment
559 In a subsequent study, Linsdell and Hanrahan (93) showed that block by DIDS and DNDS was attenuated inbinding on channel structure was underscored by the finding that the dose-dependent activation of TM5 and the R347D CFTR. Login to comment

[hide] Pharmacology of CFTR chloride channel activity. Physiol Rev. 1999 Jan;79(1 Suppl):S109-44. Schultz BD, Singh AK, Devor DC, Bridges RJ
Pharmacology of CFTR chloride channel activity.
Physiol Rev. 1999 Jan;79(1 Suppl):S109-44., [PMID:9922378]

Abstract [show]
Pharmacology of CFTR Chloride Channel Activity. Physiol. Rev. 79, Suppl.: S109-S144, 1999. - The pharmacology of cystic fibrosis transmembrane conductance regulator (CFTR) is at an early stage of development. Here we attempt to review the status of those compounds that modulate the Cl- channel activity of CFTR. Three classes of compounds, the sulfonylureas, the disulfonic stilbenes, and the arylaminobenzoates, have been shown to directly interact with CFTR to cause channel blockade. Kinetic analysis has revealed the sulfonylureas and arylaminobenzoates interact with the open state of CFTR to cause blockade. Suggestive evidence indicates the disulfonic stilbenes act by a similar mechanism but only from the intracellular side of CFTR. Site-directed mutagenesis studies indicate the involvement of specific amino acid residues in the proposed transmembrane segment 6 for disulfonic stilbene blockade and segments 6 and 12 for arylaminobenzoate blockade. Unfortunately, these compounds (sulfonylureas, disulfonic stilbenes, arylaminobenzoate) also act at a number of other cellular sites that can indirectly alter the activity of CFTR or the transepithelial secretion of Cl-. The nonspecificity of these compounds has complicated the interpretation of results from cellular-based experiments. Compounds that increase the activity of CFTR include the alkylxanthines, phosphodiesterase inhibitors, phosphatase inhibitors, isoflavones and flavones, benzimidazolones, and psoralens. Channel activation can arise from the stimulation of the cAMP signal transduction cascade, the inhibition of inactivating enzymes (phosphodiesterases, phosphatases), as well as the direct binding to CFTR. However, in contrast to the compounds that block CFTR, a detailed understanding of how the above compounds increase the activity of CFTR has not yet emerged.

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183 Consistent kidney cells expressing wild-type or R347D CFTR. Login to comment
192 Most importantly, DIDS is a known antagonist of purinergic receptors (53, 54, 56, 104, 105, had previously shown that the R347D mutation reduces the single-channel conductance, eliminates channel116, 249, 257, 366, 430, 443). Login to comment

[hide] Divergent CFTR orthologs respond differently to th... Am J Physiol Cell Physiol. 2012 Jan 1;302(1):C67-76. doi: 10.1152/ajpcell.00225.2011. Epub 2011 Sep 21. Stahl M, Stahl K, Brubacher MB, Forrest JN Jr
Divergent CFTR orthologs respond differently to the channel inhibitors CFTRinh-172, glibenclamide, and GlyH-101.
Am J Physiol Cell Physiol. 2012 Jan 1;302(1):C67-76. doi: 10.1152/ajpcell.00225.2011. Epub 2011 Sep 21., [PMID:21940661]

Abstract [show]
Comparison of diverse orthologs is a powerful tool to study the structure and function of channel proteins. We investigated the response of human, killifish, pig, and shark cystic fibrosis transmembrane conductance regulator (CFTR) to specific inhibitors of the channel: CFTR(inh)-172, glibenclamide, and GlyH-101. In three systems, including organ perfusion of the shark rectal gland, primary cultures of shark rectal gland tubules, and expression studies of each ortholog in cRNA microinjected Xenopus laevis oocytes, we observed fundamental differences in the sensitivity to inhibition by these channel blockers. In organ perfusion studies, shark CFTR was insensitive to inhibition by CFTR(inh)-172. This insensitivity was also seen in short-circuit current experiments with cultured rectal gland tubular epithelial cells (maximum inhibition 4 +/- 1.3%). In oocyte expression studies, shark CFTR was again insensitive to CFTR(inh)-172 (maximum inhibition 10.3 +/- 2.5% at 25 muM), pig CFTR was insensitive to glibenclamide (maximum inhibition 18.4 +/- 4.4% at 250 muM), and all orthologs were sensitive to GlyH-101. The amino acid residues considered responsible by previous site-directed mutagenesis for binding of the three inhibitors are conserved in the four CFTR isoforms studied. These experiments demonstrate a profound difference in the sensitivity of different orthologs of CFTR proteins to inhibition by CFTR blockers that cannot be explained by mutagenesis of single amino acids. We believe that the potency of the inhibitors CFTR(inh)-172, glibenclamide, and GlyH-101 on the CFTR chloride channel protein is likely dictated by the local environment and the three-dimensional structure of additional residues that form the vestibules, the chloride pore, and regulatory regions of the channel.

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231 Indeed, the R347D mutation alters ATPase activity in the NBDs, suggesting that R347 might be involved in conformational coupling between the TMDs and the NBDs (26). Login to comment

[hide] The patch-clamp and planar lipid bilayer technique... J Cyst Fibros. 2004 Aug;3 Suppl 2:101-8. Sheppard DN, Gray MA, Gong X, Sohma Y, Kogan I, Benos DJ, Scott-Ward TS, Chen JH, Li H, Cai Z, Gupta J, Li C, Ramjeesingh M, Berdiev BK, Ismailov II, Bear CE, Hwang TC, Linsdell P, Hug MJ
The patch-clamp and planar lipid bilayer techniques: powerful and versatile tools to investigate the CFTR Cl- channel.
J Cyst Fibros. 2004 Aug;3 Suppl 2:101-8., [PMID:15463939]

Abstract [show]
Using the patch-clamp (PC) and planar lipid bilayer (PLB) techniques the molecular behaviour of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel can be visualised in real-time. The PC technique is a highly powerful and versatile method to investigate CFTR's mechanism of action, interaction with other proteins and physiological role. Using the PLB technique, the structure and function of CFTR can be investigated free from the influence of other proteins. Here we discuss how these techniques are employed to investigate the CFTR Cl- channel with special emphasis on its permeation, conduction and gating properties.

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146 Importantly, as with all mutagenesis studies, there are important caveats: mutations within the CFTR pore might cause indirect and potentially global changes in pore architecture (e.g., R347D, [20]). Login to comment
142 Importantly, as with all mutagenesis studies, there are important caveats: mutations within the CFTR pore might cause indirect and potentially global changes in pore architecture (e.g., R347D, [20]). Login to comment

[hide] Adenosine triphosphate-dependent asymmetry of anio... J Gen Physiol. 1998 Apr;111(4):601-14. Linsdell P, Hanrahan JW
Adenosine triphosphate-dependent asymmetry of anion permeation in the cystic fibrosis transmembrane conductance regulator chloride channel.
J Gen Physiol. 1998 Apr;111(4):601-14., [PMID:9524141]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) forms a tightly regulated channel that mediates the passive diffusion of Cl- ions. Here we show, using macroscopic current recording from excised membrane patches, that CFTR also shows significant, but highly asymmetrical, permeability to a broad range of large organic anions. Thus, all large organic anions tested were permeant when present in the intracellular solution under biionic conditions (PX/PCl = 0.048-0.25), whereas most were not measurably permeant when present in the extracellular solution. This asymmetry was not observed for smaller anions. ATPase inhibitors that "lock" CFTR channels in the open state (pyrophosphate, 5'-adenylylimidodiphosphate) disrupted the asymmetry of large anion permeation by allowing their influx from the extracellular solution, which suggests that ATP hydrolysis is required to maintain asymmetric permeability. The ability of CFTR to allow efflux of large organic anions represents a novel function of CFTR. Loss of this function may contribute to the pleiotropic symptoms seen in cystic fibrosis.

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18 m e t h o d s Experiments were carried out on baby hamster kidney (BHK) or Chinese hamster ovary (CHO) cells stably expressing either wild-type or mutant (K335E or R347D) CFTR (Tabcharani et al., 1991, 1993; Linsdell and Hanrahan, 1996a). Login to comment
131 To determine whether gluconate currents were carried directly via CFTR, we examined gluconate efflux mediated by two low conductance CFTR pore mutants, R347D and K335E (Tabcharani et al., 1993). Login to comment
133 Both R347D (Fig. 7 A) and K335E (Fig. 7 B) had similar permeabilities to gluconate in the intracellular solution under biionic conditions to that of wild-type CFTR (PGluconate/PCl ϭ 0.069 Ϯ 0.010, n ϭ 9, for R347D and 0.064 Ϯ 0.008, n ϭ 7, for K335E), suggesting that relative permeability to large organic anions from the intracellular solution is not disrupted in either of these mutants. Login to comment
148 3.9 fA (n ϭ 3) for R347D and 18.1 Ϯ 3.4 fA (n ϭ 5) for K335E, in both cases significantly smaller than wild type under these conditions (P Ͻ 0.05, two-tailed t test). Login to comment
160 Both R347D (A) and K335E (B) mediate macroscopic gluconate efflux with a similar apparent gluconate permeability to wild type (see Figs. 1 A, 2 B, 3, B and C, 5 D, and 8, A and B). Login to comment
161 (C and D) Relationship between mean gluconate current (I) and current variance (␴2) at -50 mV under symmetrical ionic conditions for R347D (C) and K335E (D), calculated as described in Fig. 6 A. Login to comment
210 However, anion export in BHK cell patches was due to CFTR itself and not the result of modification of an anion transporter endogenous to these cells, since the apparent unitary gluconate current amplitude was significantly reduced in two CFTR mutants with reduced Cl- conductance, R347D and K335E (Fig. 7). Login to comment
249 We thank Shu-Xian Zheng and Jie Liao for technical assistance and Dr. J.M. Rommens (Hospital for Sick Children, Toronto, Ontario, Canada) for providing R347D and K335E cDNA. Login to comment
372 Interaction of channel blockers with R347D-CFTR. Login to comment
151 To determine whether gluconate currents were carried directly via CFTR, we examined gluconate efflux mediated by two low conductance CFTR pore mutants, R347D and K335E (Tabcharani et al., 1993). Login to comment
153 Both R347D (Fig. 7 A) and K335E (Fig. 7 B) had similar permeabilities to gluconate in the intracellular solution under biionic conditions to that of wild-type CFTR (PGluconate/PCl 5 0.069 6 0.010, n 5 9, for R347D and 0.064 6 0.008, n 5 7, for K335E), suggesting that relative permeability to large organic anions from the intracellular solution is not disrupted in either of these mutants. Login to comment
168 3.9 fA (n 5 3) for R347D and 18.1 6 3.4 fA (n 5 5) for K335E, in both cases significantly smaller than wild type under these conditions (P , 0.05, two-tailed t test). Login to comment
180 Both R347D (A) and K335E (B) mediate macroscopic gluconate efflux with a similar apparent gluconate permeability to wild type (see Figs. 1 A, 2 B, 3, B and C, 5 D, and 8, A and B). Login to comment
181 (C and D) Relationship between mean gluconate current (I) and current variance (s2) at 250 mV under symmetrical ionic conditions for R347D (C) and K335E (D), calculated as described in Fig. 6 A. Login to comment
230 However, anion export in BHK cell patches was due to CFTR itself and not the result of modification of an anion transporter endogenous to these cells, since the apparent unitary gluconate current amplitude was significantly reduced in two CFTR mutants with reduced Cl2 conductance, R347D and K335E (Fig. 7). Login to comment
269 We thank Shu-Xian Zheng and Jie Liao for technical assistance and Dr. J.M. Rommens (Hospital for Sick Children, Toronto, Ontario, Canada) for providing R347D and K335E cDNA. Login to comment
391 Interaction of channel blockers with R347D-CFTR. Login to comment

[hide] Cystic fibrosis: channel, catalytic, and folding p... J Bioenerg Biomembr. 1997 Oct;29(5):429-42. Seibert FS, Loo TW, Clarke DM, Riordan JR
Cystic fibrosis: channel, catalytic, and folding properties of the CFTR protein.
J Bioenerg Biomembr. 1997 Oct;29(5):429-42., [PMID:9511928]

Abstract [show]
The identification and characterization of the CFTR gene and protein have provided not only a major impetus to the dissection of the molecular pathophysiology of cystic fibrosis (CF) but also a new perspective on the structure and function of the large superfamily of membrane transport proteins to which it belongs. While the mechanism of the active vectorial translocation of many hydrophobic substrates by several of these transporters remains nearly as perplexing as it has for several decades, considerable insight has been gained into the control of the bidirectional permeation of chloride ions through a single CFTR channel by the phosphorylation of the R-domain and ATP interactions at the two nucleotide binding domains. However, details of these catalytic and allosteric mechanisms remain to be elucidated and await the replacement of two-dimensional conceptualizations with three dimensional structure information. Secondary and tertiary structure determination is required both for the understanding of the mechanism of action of the molecule and to enable a more complete appreciation of the misfolding and misprocessing of mutant CFTR molecules. This is the primary cause of the disease in the majority of the patients and hence understanding the details of the cotranslational interactions with multiple molecular chaperones, the ubiquitin-proteasome pathway and other components of the quality control machinery at the endoplasmic reticulum could provide a basis for the development of new therapeutic interventions.

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50 This anomalous mole fraction effect can be observed for CFTR, but is abolished if residue Arg 347 is mutated to Asp, suggesting that this site is involved in the interaction with permeant anions. Login to comment

[hide] Multi-Ion mechanism for ion permeation and block i... J Gen Physiol. 1997 Oct;110(4):365-77. Linsdell P, Tabcharani JA, Hanrahan JW
Multi-Ion mechanism for ion permeation and block in the cystic fibrosis transmembrane conductance regulator chloride channel.
J Gen Physiol. 1997 Oct;110(4):365-77., [PMID:9379169]

Abstract [show]
The mechanism of Cl ion permeation through single cystic fibrosis transmembrane conductance regulator (CFTR) channels was studied using the channel-blocking ion gluconate. High concentrations of intracellular gluconate ions cause a rapid, voltage-dependent block of CFTR Cl channels by binding to a site approximately 40% of the way through the transmembrane electric field. The affinity of gluconate block was influenced by both intracellular and extracellular Cl concentration. Increasing extracellular Cl concentration reduced intracellular gluconate affinity, suggesting that a repulsive interaction occurs between Cl and gluconate ions within the channel pore, an effect that would require the pore to be capable of holding more than one ion simultaneously. This effect of extracellular Cl is not shared by extracellular gluconate ions, suggesting that gluconate is unable to enter the pore from the outside. Increasing the intracellular Cl concentration also reduced the affinity of intracellular gluconate block, consistent with competition between intracellular Cl and gluconate ions for a common binding site in the pore. Based on this evidence that CFTR is a multi-ion pore, we have analyzed Cl permeation and gluconate block using discrete-state models with multiple occupancy. Both two- and three-site models were able to reproduce all of the experimental data with similar accuracy, including the dependence of blocker affinity on external Cl (but not gluconate) ions and the dependence of channel conductance on Cl concentration. The three-site model was also able to predict block by internal and external thiocyanate (SCN) ions and anomalous mole fraction behavior seen in Cl/SCN mixtures.

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45 three-site model was also able to predict all of the effects of SCN- on CFTR permeation previously described for both wild-type and R347D CFTR (Tabcharani et al., 1993). Login to comment
156 SCN- block is not seen in a pore mutant form of CFTR, R347D (Tabcharani et al., 1993), suggesting that this amino acid may contribute to the SCN- binding site. Login to comment
157 Since R347D also has a Cl- conduc- tance of only ‫%05ف‬ of wild-type CFTR, it was suggested Figure 10. Comparison of experimental data with theoretical values predicted by the three-site model. Login to comment
170 Three-Site, Multi-Occupancy Model for R347D CFTR Block of CFTR by internal SCN-, and the anomalous mole fraction dependence of conductance seen in Cl-/ SCN- mixtures are lost in the low conductance pore mutant R347D CFTR (Tabcharani et al., 1993). Login to comment
171 We wanted to see if the three-site model developed above for SCN- could be modified to describe the permeation properties of R347D CFTR. Login to comment
173 As shown in Fig. 13, specific changes in the height of the second barrier and both adjacent wells near the cytoplasmic end of both the Cl- and SCN- energy profiles were able to reproduce the reduction in Cl- con- ductance (Fig. 13 C), loss of blockade by 10 mM internal SCN- (Fig. 13 C), and the loss of anomalous mole fraction behavior (Fig. 13 D) seen in R347D CFTR. Login to comment
192 A three-site energy profile for R347D CFTR. Login to comment
193 (A and B) Best fit energy profiles for Cl- (A) and SCN- (B) in wild-type (solid lines) and R347D CFTR (dashed lines). Login to comment
194 (C) The three-site model of Fig. 13, A and B predicts the reduced conductance of R347D in symmetrical 150 mM NaCl (᭺) and the lack of block by 10 mM intracellular SCN- (᭹). Login to comment
195 (D) Loss of anomalous mole fraction dependence of conductance in R347D. Login to comment
207 The fact that specific modifications of the model can also reproduce the effects of the pore mutation R347D (Fig. 13) also suggest it may serve as a useful starting point in structure-function studies. Login to comment

[hide] Halide permeation in wild-type and mutant cystic f... J Gen Physiol. 1997 Oct;110(4):341-54. Tabcharani JA, Linsdell P, Hanrahan JW
Halide permeation in wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels.
J Gen Physiol. 1997 Oct;110(4):341-54., [PMID:9379167]

Abstract [show]
Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22 degrees C or 8.7 pS at 37 degrees C. The current-voltage relationship became linear when patches were excised into symmetrical, -tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22 degrees C to 10.9 pS at 37 degrees C. The conductance at 22 degrees C was approximately 1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl activity was hyperbolic and well fitted by a Michaelis-Menten-type function having a of approximately 38 mM and maximum conductance of 10 pS at 22 degrees C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P/P = 0.003-0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P(1.8) > P(1. 3) > P(1.0) > P(0.17), consistent with a "weak field strength" selectivity site. The same sequence was obtained for external halides, although inward F flow was not observed. Iodide currents were protocol dependent and became blocked after 1-2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I/Cl permeability ratio (P/P< 0.4). The switch to low I permeability was enhanced at potentials that favored Cl entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl and I ions may influence I permeation and be responsible for the wide range of P/P ratios that have been reported for the CFTR channel. The low P/P ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR displays a "weak field strength" anion selectivity sequence.

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12 The switch to low I- permeability was enhanced at potentials that favored Cl- entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Login to comment
33 This switch to low I- permeability was accelerated by holding the membrane at potentials that favored Cl- entry into the channel, and was not observed in R347D CFTR, a mutant shown previously to lack multi-ion pore behavior in mixtures of Cl- and thiocyanate. Login to comment
35 m e t h o d s Cells Chinese hamster ovary (CHO) cells expressing wild-type CFTR or the mutant R347D (arginine 347 in the sixth predicted membrane spanning region mutated to aspartate) have been described previously (Tabcharani et al., 1991; Chang et al., 1993; Tabcharani et al., 1993). Login to comment
169 To assess the possible role of this residue in I- permeation and block, we studied the I- selectivity of a mutant in which this residue was converted to aspartate (R347D). Login to comment
173 Most significantly, the permeability ratio PI/PCl was not protocol dependent in the R347D mutant. Login to comment
174 Iodide currents through R347D could be measured indefinitely after Cl- cur- rents had been allowed to flow for several minutes. Login to comment
175 The inability of R347D to distinguish between I- and Cl- ions suggests arg347 may contribute to the selectivity filter. Login to comment
214 In this regard, the mutant R347D had normal anion:cation permeability ratios (Linsdell and Hanrahan, unpublished observations) as did R347E (Anderson et al., 1991), but arg352, which has also been proposed to form part of the anion selectivity filter, remains a candidate. Login to comment
232 Selectivity of the CFTR channel mutant R347D. Login to comment
234 (B) Mean current-voltage relationships obtained when patches containing the R347D mutant are bathed externally with NaCl solution and internally with (᭹) NaCl or (᭺) NaI solution. Login to comment
236 Cl- conductance of the R347D mutant was reduced by almost half in symmetrical Cl solution, as reported previously (Tabcharani et al., 1993). Login to comment
265 The PI/PCl ratio obtained for R347D under biionic conditions is intermediate between Iunbl and Ibl in the wild-type channel, and is consistent with the value of 0.9 reported previously for R347E (Anderson et al., 1991). Login to comment
277 Halide selectivity in CFTR cannot be attributed exclusively to the region around arg347; however, because high PI/PCl ratios have been reported previously for other pore mutants (K95D and K335E; Anderson et al., 1991), and because R347D retains some preference for Br- over Cl- and selects strongly against F- (our unpublished observations). Login to comment
278 PI/PCl was altered by a mutation that abolished the anomalous mole fraction effect (AMFE, R347D); however, these properties are not strictly correlated because K335E also had high PI/PCl in a previous study (Anderson et al., 1991) and yet displays an AMFE in SCN--Cl- mixtures (Tabcharani et al., 1993). Login to comment
15 The switch to low I2 permeability was enhanced at potentials that favored Cl2 entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Login to comment
37 m e t h o d s Cells Chinese hamster ovary (CHO) cells expressing wild-type CFTR or the mutant R347D (arginine 347 in the sixth predicted membrane spanning region mutated to aspartate) have been described previously (Tabcharani et al., 1991; Chang et al., 1993; Tabcharani et al., 1993). Login to comment
179 To assess the possible role of this residue in I2 permeation and block, we studied the I2 selectivity of a mutant in which this residue was converted to aspartate (R347D). Login to comment
184 Most significantly, the permeability ratio PI/PCl was not protocol dependent in the R347D mutant. Login to comment
185 Iodide currents through R347D could be measured indefinitely after Cl2 currents had been allowed to flow for several minutes. Login to comment
186 The inability of R347D to distinguish between I2 and Cl2 ions suggests arg347 may contribute to the selectivity filter. Login to comment
231 In this regard, the mutant R347D had normal anion:cation permeability ratios (Linsdell and Hanrahan, unpublished observations) as did R347E (Anderson et al., 1991), but arg352, which has also been proposed to form part of the anion selectivity filter, remains a candidate. Login to comment
249 Selectivity of the CFTR channel mutant R347D. Login to comment
251 (B) Mean current-voltage relationships obtained when patches containing the R347D mutant are bathed externally with NaCl solution and internally with (d) NaCl or (s) NaI solution. Login to comment
253 Cl2 conductance of the R347D mutant was reduced by almost half in symmetrical Cl solution, as reported previously (Tabcharani et al., 1993). Login to comment
290 The PI/PCl ratio obtained for R347D under biionic conditions is intermediate between Iunbl and Ibl in the wild-type channel, and is consistent with the value of 0.9 reported previously for R347E (Anderson et al., 1991). Login to comment
305 Halide selectivity in CFTR cannot be attributed exclusively to the region around arg347; however, because high PI/PCl ratios have been reported previously for other pore mutants (K95D and K335E; Anderson et al., 1991), and because R347D retains some preference for Br2 over Cl2 and selects strongly against F2 (our unpublished observations). Login to comment
306 PI/PCl was altered by a mutation that abolished the anomalous mole fraction effect (AMFE, R347D); however, these properties are not strictly correlated because K335E also had high PI/PCl in a previous study (Anderson et al., 1991) and yet displays an AMFE in SCN2-Cl2 mixtures (Tabcharani et al., 1993). Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... J Gen Physiol. 1997 Oct;110(4):337-9. Dawson DC, Smith SS
Cystic fibrosis transmembrane conductance regulator. Permeant ions find the pore.
J Gen Physiol. 1997 Oct;110(4):337-9., [PMID:9379166]

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30 Nor was it seen in R347D CFTR, a construct previously shown to lack the anomalous dependence of channel conductance on the mole fraction of SCN characteristic of wild-type CFTR (Tabcharani et al., 1993). Login to comment
31 Interestingly, however, the R347D construct was characterized by an intermediate value of PI/PCl (~1.0) and exhibited a reduced conductance in the presence of Ias expected if this ion binds more tightly than Clin the pore of the mutant protein. Login to comment
44 The two-site model was also used to simulate the block of CFTR by SCN-, but a three-site model was used to generate the anomalous mole fraction effect previously reported, as well as the properties of the mutant, R347D, in which the anomalous mole fraction effect is lost. Login to comment

[hide] Rectification of cystic fibrosis transmembrane con... Biophys J. 1996 Nov;71(5):2458-66. Zhao J, Zerhusen B, Xie J, Drumm ML, Davis PB, Ma J
Rectification of cystic fibrosis transmembrane conductance regulator chloride channel mediated by extracellular divalent cations.
Biophys J. 1996 Nov;71(5):2458-66., [PMID:8913585]

Abstract [show]
We report here distinct rectification of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel reconstituted in lipid bilayer membranes. Under the symmetrical ionic condition of 200 mM KCl (with 1 mM MgCl2 in cis intracellular and 0 MgCl2 in trans extracellular solutions, pH in both solutions buffered at 7.4 with 10 mM HEPES), the inward currents (intracellular-->extracellular chloride movement) through a single CFTR channel were approximately 20% larger than the outward currents. This inward rectification of the CFTR channel was mediated by extracellular divalent cations, as the linear current-voltage relationship of the channel could be restored through the addition of millimolar concentrations of MgCl2 or CaCl2 to the trans solution. The dose responses for [Mg]zero and [Ca]zero had half-dissociation constants of 152 +/- 72 microM and 172 +/- 40 microM, respectively. Changing the pH buffer from HEPES to N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid did not alter rectification of the CFTR channel. The nonlinear conductance property of the CFTR channel seemed to be due to negative surface charges on the CFTR protein, because in pure neutral phospholipid bilayers, clear rectification of the channel was also observed when the extracellular solution did not contain divalent cations. The CFTR protein contains clusters of negatively charged amino acids on several extracellular loops joining the transmembrane segments, which could constitute the putative binding sites for Ca and Mg.

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190 The studies of Tabcharani et al. (1993) showed that TM6 is also involved in the conduction process of the CFTR channel, as point mutations of K335E and R347D altered the multi-ion pore behavior of the CFTR channel. Login to comment
191 The studies of Tabcharani et al. (1993) showed that TM6 is also involved in the conduction process of the CFTR channel, as point mutations of K335E and R347D altered the multi-ion pore behavior of the CFTR channel. Login to comment

[hide] Disulphonic stilbene block of cystic fibrosis tran... J Physiol. 1996 Nov 1;496 ( Pt 3):687-93. Linsdell P, Hanrahan JW
Disulphonic stilbene block of cystic fibrosis transmembrane conductance regulator Cl- channels expressed in a mammalian cell line and its regulation by a critical pore residue.
J Physiol. 1996 Nov 1;496 ( Pt 3):687-93., [PMID:8930836]

Abstract [show]
1. The disulphonic stilbenes 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) were shown to cause a voltage-dependent inhibition of macroscopic cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents expressed in baby hamster kidney cells when applied to the cytoplasmic face of the membrane. These compounds are known to be relatively ineffective at blocking CFTR from the extracellular side of the membrane. 2. Mutation of a positively charged arginine, previously suggested to be located in the channel pore (R347), to a negatively charged aspartate significantly reduced the affinity of block by both DNDS and DIDS, suggesting that this residue contributes to the binding site for disulphonic stilbenes. 3. It is suggested that the CFTR Cl- channel may contain a relatively large inner vestibule in which a number of large anions bind and block Cl- permeation. Arginine 347 may be involved in anion binding within this region.

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14 METHODS Preparation and culture of cells Experiments were carried out on baby hamster kidney (BHK) cells stably expressing either wild-type or R347D CFTR. Login to comment
16 R347D pNUT-CFTR DNA (provided by Dr J. M. Rommens, Hospital for Sick Children, Toronto, Canada) was transfected into subconfluent BHK cells using Lipofectamine reagent (Life Technologies, Burlington, Canada) according to the manufacturer's directions. Login to comment
65 Effect of blockers on R347D-CFTR Previous studies have identified arginine 347 in the sixth membrane-spanning region of CFTR as contributing to an important anion-binding site close to the cytoplasmic end of the Cl- channel pore (Tabcharani et al. 1993). Login to comment
66 Mutation of this positively charged residue to a negatively charged aspartate (the R347D mutant) reduces channel Cl- conductance, eliminates channel block by thiocyanate A B 1.0 0-8 0-6 0-4 0-2 0-0 200 1M DNDS -- i (pA) 200 FM DNDS 1*0 - 0-8 - 0-6 - 0-4 - 0-2 - -50 V (mV) (SCN-) ions, and abolishes anomalous mole fraction behaviour seen in Cl--SCN- mixtures (Tabeharani et al. 1993). Login to comment
67 We examined the effects of 200 FM internal DNDS or DIDS on macroscopic currents carried by R347D-CFTR channels stably expressed in BHK cells (Fig. 3A). Login to comment
69 The effect of the R347D mutation on block by DNDS and DIDS is illustrated further in Fig. 3B, which shows mean i/iO in the presence of 200 /IM DNDS or DIDS as a function of membrane potential for both wild-type and R347D-CFTR. Login to comment
72 Effects of intracellular DNDS and DIDS on macroscopic R347D-CFTR C1- currents A, macroscopic R347D current-voltage relationships before (0) and after (0) addition of 200 FM DNDS (left) or 200 jM DIDS (right). Login to comment
73 B, comparison of the effects of 200 FM DNDS (left) or 200 uM DIDS (right) on wild-type (0) and R347D currents (0). Login to comment
78 Mean parameters of block of wild-type and R347D-CFTR by 200 FM intracellular DNDS or DIDS DNDS DIDS Kd(O) z Kd(O) z (/SM) (/tM) Wild-type 162 + 16 (7) 34 + 3 (7) 77 + 15 (4) 16 + 2 (4) R347D 1382 + 267 (7)** 8 + 5 (7)** 260 + 71 (6)* 15 ± 2 (6) Values for Kd(O) and z' were calculated from each experiment using eqn (2). Login to comment
83 R347D-CFTR had a significantly lower affinity than wild-type for both DNDS and DIDS, with a greater than 8-fold increase in Kd(O) for DNDS and a greater than 3-fold increase in Kd(O) for DIDS observed in this mutant. Login to comment
84 DNDS block of R347D-CFTR was only very weakly voltage dependent, significantly less so than for wild-type, whereas DIDS block showed a similar voltage dependence for both wild-type and R347D. Login to comment
105 The dramatic reduction in affinity for both DNDS and DIDS in the pore mutant R347D-CFTR suggests that the positively charged residue arginine 347 contributes to the binding site for both these molecules in the pore. Login to comment
113 Although arginine 347 appears to contribute to the binding site for both DNDS and DIDS, it is possible that DIDS (but not DNDS) is also able to interact with another site on the channel, modulating its blocking effects on wild-type CFTR and allowing some residual blocking action on R347D-CFTR. Login to comment
191 Acknowledgements WAe would like to thiank Shu-Xian Zheng for constructing the R347D-CFTR BHK cell line, Jie Liao for maintaining the cell cultures and Dr Johanna Rommens for providing R347D CFTR DNA. Login to comment

[hide] cAMP- and Ca2+-independent activation of cystic fi... J Biol Chem. 1996 Jul 5;271(27):16171-9. Becq F, Verrier B, Chang XB, Riordan JR, Hanrahan JW
cAMP- and Ca2+-independent activation of cystic fibrosis transmembrane conductance regulator channels by phenylimidazothiazole drugs.
J Biol Chem. 1996 Jul 5;271(27):16171-9., [PMID:8663098]

Abstract [show]
Patch-clamp, iodide efflux, and biochemical techniques were used to evaluate the ability of phenylimidazothiazoles to open normal and mutated cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels and to investigate the mechanism of activation. As reported previously for bromotetramisole, levamisole activated wild-type CFTR channels stably expressed in Chinese hamster ovary cells in the absence of other secretagogues and without elevating intracellular cAMP or calcium. The protein kinase A (PKA) inhibitor N - (2-(p-bromocinnamylamino)ethyl)-5-isoquinolinesul-fonamid e abolished activation by forskolin but only partially inhibited stimulation by levamisole, suggesting the involvement of other kinases. CFTR channels bearing mutations at multiple phosphorylation sites, in the membrane domains, and in the first nucleotide binding domain (including the disease-causing mutations G551D and DeltaF508) all responded to phenylimidazothiazoles. Moreover, levamisole and bromotetramisole increased the activity of wild-type and mutant channels already exposed to PKA + MgATP, consistent with the inhibition of a constitutive, membrane-associated phosphatase activity. We conclude that phenylimidazothiazole drugs can open normal and mutated CFTR channels by stabilization of phosphoforms of CFTR that are produced by basal activity of PKA and alternative protein kinases. If similar stimulation is observed in humans in vivo, phenylimidazothiazoles may be useful in the development of pharmacological therapies for cystic fibrosis.

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34 27, Issue of July 5, pp. 16171-16179, 1996 (c) 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. MATERIALS AND METHODS Cell Culture-CHO-K1 cells that had been stably transfected with pNUT vector alone (denoted CFTR(-)) or with wild-type CFTR (CFTR(ϩ)) or various mutated versions (G551D, R347D, R117H, and ⌬F508) in pNUT were used (8, 15). Login to comment
153 We also examined a CFTR mutation (R347D) that is at a residue where disease-causing mutations have been identified (R347P and R347H). Login to comment
155 Spontaneous activity of R347D channels was never observed on resting cells (Table I) but was elicited by exposure to 15 ␮M forskolin (80% of patches; Fig. 6A) or l mM levamisole (60% of patches; Fig. 6, B and C). Login to comment
156 The unitary conductance of R347D channels determined after stimulation by forskolin was 2.9 Ϯ 0.2 pS (n ϭ 8) and by levamisole was 2.8 Ϯ 0.3 pS (n ϭ 6; Fig. 6D). Login to comment
163 G551D channel activity in the presence of PKA and ATP was lower than wild-type CFTR (Po ϭ 0.17 Ϯ 0.06, n ϭ 2.6 Ϯ 0.57, n ϭ 3 patches) but was nevertheless increased 2-fold by addition of bromotetramisole in the presence of PKA (Po ϭ 0.36 Ϯ 0.08, n ϭ 6.2 Ϯ 0.56, n ϭ 7 patches). Login to comment
164 Although these values for Po may be overestimated because they are based on the estimated number of channels without locking all the channels open using AMP-PNP, the increase in open probability was consistent and was also observed with the mutants R117H and R347D (data not shown). Login to comment
194 Our results can be summarized as follows: (i) levamisole and bromotetramisole promote the opening of cell-attached CFTR channels in the absence of forskolin, (ii) elevation of intracellular cAMP and Ca2ϩ are not required for this stimulation, (iii) at least four mutant CFTRs (G551D, R117H, R347D, and ⌬F508) can also be activated on-cell by these drugs in the absence of forskolin, (iv) both phenylimidazothiazoles further enhance CFTR channel activity when excised patches were exposed to high levels of PKA, indicating that their target is associated with the membrane, and (v) activation by both drugs requires some kinase activity. Login to comment
196 Functional response of R347D stably expressed in CHO cells. Login to comment
197 Recordings of R347D channels at various potentials in the cell-attached configuration with 15 ␮M forskolin (A) or 1 mM levamisole (C). Login to comment
198 Note the different scales and reduced conductance of R347D mutant. Login to comment
199 B, histogram summarizing fraction of cell-attached patches that contained R347D channel activity in various conditions. Login to comment
200 D, current-voltage relationship of R347D channels in cell-attached patches in the presence of 15 ␮M forskolin (circles) and 1 mM levamisole (squares). Login to comment
206 Frequency Control Levamisole Number of channels Po g pS CFTR 0/15 20/23 12 Ϯ 2.80 0.47 Ϯ 0.05 6.8 Ϯ 0.20 G551D 0/10 31/43 3 Ϯ 0.27 0.35 Ϯ 0.07 5.3 Ϯ 0.30 R117H 0/5 9/13 2 Ϯ 0.32 0.14 Ϯ 0.10 5.7 Ϯ 0.15 R347D 0/4 6/10 4.6 Ϯ 0.40 0.40 Ϯ 0.02 2.8 Ϯ 0.30 ⌬F508 (37 °C) 0/10 0/15 ⌬F508 (23 °C) 0/5 5/8 2.1 Ϯ 0.03 0.13 Ϯ 0.02 6.8 Ϯ 0.14 they are uncompetitive inhibitors of alkaline phosphatase (26, 31, 37). Login to comment
152 We also examined a CFTR mutation (R347D) that is at a residue where disease-causing mutations have been identified (R347P and R347H). Login to comment
154 Spontaneous activity of R347D channels was never observed on resting cells (Table I) but was elicited by exposure to 15 mM forskolin (80% of patches; Fig. 6A) or l mM levamisole (60% of patches; Fig. 6, B and C). Login to comment
193 Our results can be summarized as follows: (i) levamisole and bromotetramisole promote the opening of cell-attached CFTR channels in the absence of forskolin, (ii) elevation of intracellular cAMP and Ca21 are not required for this stimulation, (iii) at least four mutant CFTRs (G551D, R117H, R347D, and DF508) can also be activated on-cell by these drugs in the absence of forskolin, (iv) both phenylimidazothiazoles further enhance CFTR channel activity when excised patches were exposed to high levels of PKA, indicating that their target is associated with the membrane, and (v) activation by both drugs requires some kinase activity. Login to comment
195 Functional response of R347D stably expressed in CHO cells. Login to comment
204 Frequency Control Levamisole Number of channels Po g pS CFTR 0/15 20/23 12 6 2.80 0.47 6 0.05 6.8 6 0.20 G551D 0/10 31/43 3 6 0.27 0.35 6 0.07 5.3 6 0.30 R117H 0/5 9/13 2 6 0.32 0.14 6 0.10 5.7 6 0.15 R347D 0/4 6/10 4.6 6 0.40 0.40 6 0.02 2.8 6 0.30 DF508 (37 &#b0;C) 0/10 0/15 DF508 (23 &#b0;C) 0/5 5/8 2.1 6 0.03 0.13 6 0.02 6.8 6 0.14 Activation of CFTR by Phenylimidazothiazole Drugs they are uncompetitive inhibitors of alkaline phosphatase (26, 31, 37). Login to comment

[hide] Identification of cystic fibrosis transmembrane co... Biophys J. 1996 Jun;70(6):2688-95. Cheung M, Akabas MH
Identification of cystic fibrosis transmembrane conductance regulator channel-lining residues in and flanking the M6 membrane-spanning segment.
Biophys J. 1996 Jun;70(6):2688-95., [PMID:8744306]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride channel that is regulated by phosphorylation and ATP binding. Work by others suggested that some residues in the sixth transmembrane segment (M6) might be exposed in the channel and play a role in ion conduction and selectivity. To identify the residues in M6 that are exposed in the channel and the secondary structure of M6, we used the substituted cysteine accessibility method. We mutated to cysteine, one at a time, 24 consecutive residues in and flanking the M6 segment and expressed these mutants in Xenopus oocytes. We determined the accessibility of the engineered cysteines to charged, lipophobic, sulfhydryl-specific methanethiosulfonate (MTS) reagents applied extracellularly. The cysteines substituted for Ile331, Leu333, Arg334, Lys335, Phe337, Ser341, Ile344, Arg347, Thr351, Arg352, and Gln353 reacted with the MTS reagents, and we infer that they are exposed on the water-accessible surface of the protein. From the pattern of the exposed residues we infer that the secondary structure of the M6 segment includes both alpha-helical and extended regions. The diameter of the channel from the extracellular end to the level of Gln353 must be at least 6 A to allow the MTS reagents to reach these residues.

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190 The multiple ion occupancy effects were eliminated by mutation of Arg347 to Asp or His, and the single-channel conductance was reduced (Tabcharani et al., 1993). Login to comment
188 The multiple ion occupancy effects were eliminated by mutation of Arg347 to Asp or His, and the single-channel conductance was reduced (Tabcharani et al., 1993). Login to comment

[hide] Mutations in the putative pore-forming domain of C... FEBS Lett. 1995 Nov 6;374(3):312-6. Hipper A, Mall M, Greger R, Kunzelmann K
Mutations in the putative pore-forming domain of CFTR do not change anion selectivity of the cAMP activated Cl- conductance.
FEBS Lett. 1995 Nov 6;374(3):312-6., [PMID:7589561]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) apparently forms Cl- channels in apical membranes of secretory epithelial cells. A detailed model describes molecular structure and biophysical properties of CFTR and the impact of various mutations as they occur in cystic fibrosis. In the present report mutations were introduced into the putative 6th alpha-helical transmembrane pore forming domain of CFTR. The mutants were subsequently expressed in Xenopus oocytes by injection of the respective cRNAs. Whole cell (wc) conductances could be reversibly activated by IBMX (1 nmol/l) only in oocytes injected with wild-type (wt) or mutant CFTR but not in oocytes injected with water or antisense CFTR. The activated conductance was partially inhibited by (each 100 mumol/l) DIDS (27%) and glibenclamide (77%), but not by 10 mumol/l NPPB. The following mutations were examined: K335E, R347E, R334E, K335H, R347H, R334H. They did not measurably change the wt-CFTR anion permeability (P) and we conductance (G) sequence of: PCl- > PBr- > P1- and GCl- > GBr- > G1-, respectively. Moreover, anomalous mole fraction behavior for the cAMP activated current could not be detected: neither in wt-CFTR nor in R347E-CFTR. Various mutants for which positively charged amino acids were replaced by histidines (K335H, R347H, R334H) did not show pH sensitivity of the IBMX activated wc conductance. We, therefore, cannot confirm previous results. CFTR might have a different molecular structure than previously suggested or it might act as a regulator of ion conductances.

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21 (vi) Furthermore, anomalous mole fraction behavior of wt-CFTR was abolished in one mutant of CFTR (R347D) [7]. Login to comment
112 Comparable wc measurements were performed in the present study (K335E, R347E compared to R347D in [7]) with SCN- and C1- present in the extracellular bath solution at different concentration ratios. Login to comment

[hide] The CFTR chloride channel of mammalian heart. Annu Rev Physiol. 1995;57:387-416. Gadsby DC, Nagel G, Hwang TC
The CFTR chloride channel of mammalian heart.
Annu Rev Physiol. 1995;57:387-416., [PMID:7539989]

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20 The conclusion that certain residues in M l and M6 line the anion-selective pore is corroborated by three new pieces of evidence: (a) Tabcharani et al (130) found that the charge-switching mutation R347D lowered channel conductance and abolished the anomalous mole-fraction effect seen with mixtures of CI- and SCN- ions, and that channel conductance and anomalous mole-fraction behavior became pH sensitive in the mutant R347H. Login to comment

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

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

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220 This was initially attributed to interaction of this anion with R347 at the cytoplasmic end of TM6 in CFTR, based upon the loss of iodide block in R347D-CFTR. Login to comment
321 Anomalous mole fraction effects (Tabcharani et al., 1993) and protocol-dependent block by iodide (Tabcharani et al., 1997) are lost in R347D-CFTR; this may be due to a severe disruption in secondary structure by the loss of a salt bridge between R347 and D924 (Cotten and Welsh, 1999). Login to comment

[hide] Two salt bridges differentially contribute to the ... J Biol Chem. 2013 Jul 12;288(28):20758-67. doi: 10.1074/jbc.M113.476226. Epub 2013 May 24. Cui G, Freeman CS, Knotts T, Prince CZ, Kuang C, McCarty NA
Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function.
J Biol Chem. 2013 Jul 12;288(28):20758-67. doi: 10.1074/jbc.M113.476226. Epub 2013 May 24., [PMID:23709221]

Abstract [show]
Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.

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82 RESULTS Arg347 Forms a Salt Bridge with Asp924 but Does Not Stabilize the Full Open State-Although Cotten and Welsh first reported that arginine 347 of TM6 forms a salt bridge with aspartic acid 924 of TM8, their results suggested that the double mutation R347D/D924R rescued the channel to a stable open state that exhibits a smaller single channel amplitude, which is reminiscent of the s2 open state of WT-CFTR (14). Login to comment
89 A, representative current samples of WT-, R347A-, R347D-, D924R-, R347K-, and R347D/D924R-CFTR were recorded from excised inside-out patch from Xenopus oocytes with 150 mM Clafa; symmetrical solution in the presence of 1 mM Mg-ATP and 50 nM PKA at VM afd; afa;100 mV (n afd; 4-6 for each mutant). Login to comment
94 R347A-CFTR showed a very long and stable s1 state with very brief openings to s2 or f states, whereas R347D-CFTR only exhibits a long stable s1 state and appears to never get out of s1 (at the resolution of our recording apparatus), as if introduction of negative charge at this position confers electrostatic repulsion with other negative charges in the native channel and thereby greatly interferes with the ability to go beyond the s1 state. Login to comment
96 D924R-CFTR exhibits all three open states in contrast to R347A- and R347D-CFTR, although the stability of the open state is compromised; indeed, the fractional occupancies of both s1 and s2 states are greatly increased in this mutant (Fig. 2B). Login to comment
97 The charge-swapping double mutant R347D/D924R-CFTR exhibited a long stable s2 state with occasional brief openings to s1 and f. Login to comment
101 In addition, breakingthissaltbridgedisruptedthestabilityofthes2andfstates but did not significantly affect s1; therefore, both R347A and R347D showed long stable s1 states, although R347A endeavored to reach the s2 and f states but failed to maintain them, whereas R347D completely lost the ability to open to s2 and f state. Login to comment
109 We therefore hypothesized that Arg347 might also interact with Asp993 to rescue the CFTR channel pore to a stable f state and tested this hypothesis in three double mutants; TABLE 1 Summary of the effects of mutations studied Mutant Main features of open bursts Impact on f state R347A Emphasizes s1 state, brief transitions to s2 and f Can reach f but not stable R347D Emphasizes s1 state, no transitions to s2 and f Cannot reach f D924R Brief transitions to all conductance levels Can reach f but not stable R347K Wild type-like Wild type-like R347D/D924R Emphasizes s2 state, rare and brief transitions to f Can reach f but not stable R352E Opens to all 3 levels; s1 much more stable than in WT, s2 unstable, f unstable Can reach f but not stable D993R Opens to all 3 levels, but none are stable Can reach f but not stable R352E/D993R Wild type-like, with increased transitions to s1 and s2; slightly reduced single-channel conductance Wild type-like R352E/D924R Opens to all 3 levels, but none are stable Can reach f but not stable R347D/D993R Very stable s2; rare and brief transitions to both s1 and f Can reach f but not stable R347A/R352A Opens to all 3 levels; s1 much more stable than in WT, s2 unstable, f unstable Can reach f but not stable R347D/D924R/D993R Opens to all 3 levels; s1 much more stable than in WT, s2 relatively stabilized, f unstable Can reach f but not stable R347D/D924R/R352E/D993R Primarily flickers between s2 and f; s1 much more stable than in WT, slightly reduced single channel conductance Can reach f but not stable FIGURE 3. Login to comment
111 A, representative current samples of R352E/D993R-, R352E/D924R-, and R347D/D993R-CFTR recorded from excised inside-out patches with the same conditions as Fig. 2 (n afd; 3-6 for each mutant). Login to comment
115 Whereas the single channel behavior of R352E/D924R was similar to that of R352E alone, with multiple unstable open states, suggesting that Arg352 and Asp924 do not interact, R347D/D993R was much more like R347D/D924R, with the s2 state dominant (compare Figs. 3 and 2). Login to comment
116 R347D/ D993R-CFTR is able to transition to the f state but sojourns there are even more brief than those seen for the R347D/ D924R. Login to comment
121 In the R347D/ D924R mutant, the positive charge at Arg347 is no longer available to interact with Asp993 . Login to comment
122 Similarly, in the R347D/D993R mutant, the positive charge at Arg347 is no longer available to interact with Asp924 . Login to comment
123 Therefore, we asked whether replacing the negative charge at both Asp924 and Asp993 with a positive charge would allow strong enough interactions with R347D to enable channels to go to the f state. Login to comment
124 This was tested in the triple mutant R347D/D924R/D993R (Fig. 4, A and B). Login to comment
125 Unlike the two double mutants described above (R347D/D924R and R347D/ D993R), the triple mutant exhibited roughly equal occupancy of s1,s2,andfstates;theoccupancyofthes2statewasnotasstableas in either double mutant. Login to comment
141 However, the quadruple mutant R347D/D924R/D993R/R352E did not completely rescue WT behavior (Fig. 4, A and B). Login to comment
146 Representative current samples of R347A/R352A-, R347D/D924R/D993R-, and R347D/D924R/D993R/R352E-CFTR were recorded under the same conditions as in Fig. 3 (n afd; 5-6 for each mutant) (A). Login to comment

[hide] Redox balance in cystic fibrosis. Int J Biochem Cell Biol. 2014 Jul;52:113-23. doi: 10.1016/j.biocel.2014.03.006. Epub 2014 Mar 20. Ziady AG, Hansen J
Redox balance in cystic fibrosis.
Int J Biochem Cell Biol. 2014 Jul;52:113-23. doi: 10.1016/j.biocel.2014.03.006. Epub 2014 Mar 20., [PMID:24657650]

Abstract [show]
The homeostatic balance between oxidants and antioxidants in biological systems is known as redox balance, and is regulated by complex processes. Redox balance regulates many of the known cellular pathways and disease processes. The dysregulation of redox balance can lead to acute or long-term oxidative or reductive stresses that are associated with many of the abnormalities observed in cystic fibrosis (CF). Over the past 5 decades researchers have examined contributors to redox dysregulation, their molecular products, and their impact on ion transport, cell proliferation, inflammation, bacterial killing, and the metabolism of nucleic acids, proteins, and lipids in CF. CF patients exhibit elevated markers of oxidative stress when compared to non-CF healthy controls; however, whether the reported redox imbalance is sufficient to produce pathology has been controversial. In addition, comparisons between CF and non-CF disease controls have been lacking. To better understand the mechanisms which mediate the generation of oxidants and antioxidants in CF and the importance of their balance in effecting oxidative or reductive stress, we will review the determinants of redox balance in the blood, lumen, and cellular compartments. From the perspective of methodological application, we will focus on the approaches most often used to study oxidant and antioxidants in CF, including biochemical, proteomic, metabolomic, and lipidomic studies, with a discussion of the few transcriptomic analyses that predict changes in the expression of regulators of redox. Finally, we will discuss the utility of oxidants and antioxidants as biomarkers of disease and the use of antioxidant therapy in CF.

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1971 In this same report, mutant CFTR (R347D) showed a decreased ability to traffic GSH compared to wild-type. Login to comment