ABCMdb

ABCC7 p.Phe337Cys

Predicted by SNAP2:	A: D (71%), C: D (66%), D: D (91%), E: D (91%), G: D (75%), H: D (75%), I: D (71%), K: D (91%), L: D (53%), M: D (66%), N: D (85%), P: D (91%), Q: D (85%), R: D (85%), S: D (85%), T: D (85%), V: D (75%), W: D (85%), Y: D (71%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: N,

[switch to compact view]
Comments [show]

None has been submitted yet.

Publications
[hide] Conformational changes in a pore-lining helix coup... J Biol Chem. 2008 Feb 22;283(8):4957-66. Epub 2007 Dec 3. Beck EJ, Yang Y, Yaemsiri S, Raghuram V
Conformational changes in a pore-lining helix coupled to cystic fibrosis transmembrane conductance regulator channel gating.
J Biol Chem. 2008 Feb 22;283(8):4957-66. Epub 2007 Dec 3., 2008-02-22 [PMID:18056267]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ATP-binding cassette transporters in that it functions as an ion channel. In CFTR, ATP binding opens the channel, and its subsequent hydrolysis causes channel closure. We studied the conformational changes in the pore-lining sixth transmembrane segment upon ATP binding by measuring state-dependent changes in accessibility of substituted cysteines to methanethiosulfonate reagents. Modification rates of three residues (resides 331, 333, and 335) near the extracellular side were 10-1000-fold slower in the open state than in the closed state. Introduction of a charged residue by chemical modification at two of these positions (resides 331 and 333) affected CFTR single-channel gating. In contrast, modifications of pore-lining residues 334 and 338 were not state-dependent. Our results suggest that ATP binding induces a modest conformational change in the sixth transmembrane segment, and this conformational change is coupled to the gating mechanism that regulates ion conduction. These results may establish a structural basis of gating involving the dynamic rearrangement of transmembrane domains necessary for vectorial transport of substrates in ATP-binding cassette transporters.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
100 The oocytes 750 500 250 0 µS 180012006000 s IBMX MTSEA Cd 2+ DTT 200 100 0 µS 180012006000 s IBMX DTT Cd 2+ MTSEA A B C -100 -80 -60 -40 -20 0 20 40 % Change in conductance Y325C A326C L327C I328C K329C G330C I331C I332C L333C R334C K335C I336C F337C T338C T339C I340C S341C F342C WT I344C V345C R347C M348C A349C V350C T351C Q353C * * * * * Cd 2+ 1mM MTSEA 1mM D FIGURE 1. Login to comment
218 Finally, the MTSEA reactivity was restricted to only five of twenty-six residues in and flanking TM6 in our study, whereas in the earlier study, residues F337C, S341C, I344C, R347C, T351C, R352C, and Q353C were also shown to be accessible to MTS reagents. Login to comment

[hide] State-dependent access of anions to the cystic fib... J Biol Chem. 2008 Mar 7;283(10):6102-9. Epub 2007 Dec 31. Fatehi M, Linsdell P
State-dependent access of anions to the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Biol Chem. 2008 Mar 7;283(10):6102-9. Epub 2007 Dec 31., 2008-03-07 [PMID:18167343]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is gated by intracellular factors; however, conformational changes in the channel pore associated with channel activation have not been identified. We have used patch clamp recording to investigate the state-dependent accessibility of substituted cysteine residues in the CFTR channel pore to a range of cysteine-reactive reagents applied to the extracellular side of the membrane. Using functional modification of the channel current-voltage relationship as a marker of modification, we find that several positively charged reagents are able to penetrate deeply into the pore from the outside irrespective of whether or not the channels have been activated. In contrast, access of three anionic cysteine-reactive reagents, the methanesulfonate sodium (2-sulfonatoethyl)methanesulfonate, the organic mercurial p-chloromercuriphenylsulfonic acid, and the permeant anion Au(CN)(2)(-), to several different sites in the pore is strictly limited prior to channel activation. This suggests that in nonactivated channels some ion selectivity mechanism exists to exclude anions yet permit cations into the channel pore from the extracellular solution. We suggest that activation of CFTR channels involves a conformational change in the pore that removes a strong selectivity against anion entry from the extracellular solution. We propose further that this conformational change occurs in advance of channel opening, suggesting that multiple distinct closed pore conformations exist.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
75 Another mutant, F337C, became significantly more inwardly rectified in the presence of MTSES but was apparently not affected by inclusion of MTSET in the pipette solution (Fig. 3). Login to comment
90 Conformational Change in the Pore on Activation of CFTR 6104 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 10•MARCH , shape when included in the pipette solution or when preincubated with cells (or, in the case of F337C, the same lack of effect) (Fig. 4A). Login to comment
114 F, wild type (both panels); E, R334C (left); Ⅺ, K335C (left); ‚, F337C (right); ƒ, T338C (right); छ, S341C (right) (mean of data from 3-9 patches). Login to comment
115 All MTS treatments led to significant changes in rectification ratio (p Ͻ 0.05) except MTSET-wild type, MTSES-wild type, and MTSET-F337C. Login to comment
142 In contrast, F337C was only very weakly inhibited by Au(CN)2 - either with or without cAMP prestimulation. Login to comment
143 Alteration of I-V shape in F337C by MTSES (Figs. Login to comment
147 Whatever the explanation, this lack of effect appears to be specific for F337C because the four other reactive cysteine mutants studied could be modified functionally by all reagents tested. Login to comment
189 E, the mean change in CFTR macroscopic conductance for R334C, K335C, F337C, and S341C following addition of KCN without (white bars) or with (black bars) cAMP pretreatment is shown (mean of data from 4-5 patches). Login to comment

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

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

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
52 We proposed that these spontaneous changes, that are not seen in either wt or Cys-less CFTR, reflect the coordination of trace Table 1: Percent Change in Oocyte Conductance in the Presence of Compounda MTSETþ MTSES- [Ag(CN)2]- [Au(CN)2]- G330C O O O O I331C -51.6 ( 6.3 -28.9 ( 2.1 -63.1 ( 8.8 O I332C O O O O L333C -58.5 ( 4.8 -47.5 ( 7.6 -83.1 ( 2.2 O R334C þ76.9 ( 11.3 -84.4 ( 1.5 -67.4 ( 7.4 -41.4 ( 3.1 K335C þ10.7 ( 2.4 -37.3 ( 1.5 -29.1 ( 6.4 -54.6 ( 4.7 I336C -54.4 ( 7.9 -75.0 ( 0.6 -81.2 ( 10.5 O F337C O O -89.6 ( 1.9 -90.1 ( 1.3 T338C -37.1 ( 3.3 -85.4 ( 2.5 -75.0 ( 5.2 -88.3 ( 1.6 T339C O O -24.5 ( 7.2 O I340C O O -93.8 ( 1.0 O S341C O O -49.3 ( 4.8 O F342C O O -84.7 ( 1.8 O C343 O O O O I344C O O -66.9 ( 9.3 -77.9 ( 2.1 V345C O O -49.1 ( 9.3 O L346C O O O O R347C O O O O M348C O O -47.9 ( 8.8 -50.1 ( 3.3 A349C O O -19.0 ( 2.0 O V350C O O O O T351C O O O O R352C O O -77.5 ( 1.3 O Q353C O O -72.6 ( 4.5 -76.7 ( 2.8 a Values are means ( SE of three or more oocytes. Login to comment
147 Lack of Reactivity of F337C CFTR toward MTSETþ and MTSES- . Login to comment
149 Beck et al. (9) reported no reactivity of F337C on a wt CFTR background (F337/wt CFTR) toward MTSEAþ or MTSES- , but Cheung and Akabas (5, 6) and Fatehi and Linsdell (10) reported reactivity toward both MTSETþ and MTSES- . Login to comment
150 Accordingly, we examined the reactivity toward MTS reagents of F337C CFTR (wt and Cys-less backgrounds) carefully to ensure that any change in conductance observed in the presence of an MTS reagent met our criteria for a thiol-disulfide exchange reaction. Login to comment
151 In some experiments weobserved that exposureofoocytes expressing F337C/wt CFTR to MTSETþ or MTSES- produced decreases in conductance.Someofthesechangeswerereversed by simplywashing off the compound while others persisted to a variable extent after washing. Login to comment
152 We determined, however, that these variable effects of exposure to MTSET- or MTSES- were not due to thiol-disulfide exchange reactions.3 Reactivity of F337C CFTR toward Channel-Permeant Probes. Login to comment
153 The reactivity of F337C/wt CFTR toward the channel-permeant probes, although similar to that seen previously with T338C/wt CFTR (12), differed significantly in detail. Login to comment
154 Exposure of oocytes expressing F337C/wt CFTR to 1 mM [Au(CN)2]- produced a profound inhibition that was not reversed by superfusing the oocytes with a [Au(CN)2]- -free solution (Figure 3). Login to comment
155 After inhibition of F337C conductance by [Au- (CN)2]- , exposure of oocytes to a competing thiol, 2-ME, did not reverse the inhibition of conductance as previously seen with T338C/wtCFTR(12), butthe inhibition wasrelievedbyexposing the oocyte to a solution containing 1 mM KCN as expected from the high-affinity liganding of Au(1) by the cyanide anion (12). Login to comment
156 Similar results were obtained with oocytes expressing F337C/ Cys-less CFTR, confirming that the cysteine at 337 is the site of the reaction with [Au(CN)2]- (Supporting Information Figure S3). Login to comment
157 F337C/Cys-less CFTR was also reactive toward the second, channel-permeant probe, [Ag(CN)2]- . Login to comment
165 That this could reflect the size and/or polarity of the latter reagents is suggested by the observation that S341C, like F337C CFTR, clearly reacts with a smaller MTS compound, MMTS (not shown). Login to comment
167 The inhibition was readily and FIGURE 3: Selective reactivity of F337C CFTR. Login to comment
281 Note the lack of consistent results reported for F337C, S341C, I344C, R347C, T351C, R352C, and Q353C (shaded). Login to comment

[hide] Dual roles of the sixth transmembrane segment of t... J Gen Physiol. 2010 Sep;136(3):293-309. Bai Y, Li M, Hwang TC
Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation.
J Gen Physiol. 2010 Sep;136(3):293-309., [PMID:20805575]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is the only member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily that functions as a chloride channel. Previous work has suggested that the external side of the sixth transmembrane segment (TM6) plays an important role in governing chloride permeation, but the function of the internal side remains relatively obscure. Here, on a cysless background, we performed cysteine-scanning mutagenesis and modification to screen the entire TM6 with intracellularly applied thiol-specific methanethiosulfonate reagents. Single-channel amplitude was reduced in seven cysteine-substituted mutants, suggesting a role of these residues in maintaining the pore structure for normal ion permeation. The reactivity pattern of differently charged reagents suggests that the cytoplasmic part of TM6 assumes a secondary structure of an alpha helix, and that reactive sites (341, 344, 345, 348, 352, and 353) reside in two neighboring faces of the helix. Although, as expected, modification by negatively charged reagents inhibits anion permeation, interestingly, modification by positively charged reagents of cysteine thiolates on one face (344, 348, and 352) of the helix affects gating. For I344C and M348C, the open time was prolonged and the closed time was shortened after modification, suggesting that depositions of positive charges at these positions stabilize the open state but destabilize the closed state. For R352C, which exhibited reduced single-channel amplitude, modifications by two positively charged reagents with different chemical properties completely restored the single-channel amplitude but had distinct effects on both the open time and the closed time. These results corroborate the idea that a helix rotation of TM6, which has been proposed to be part of the molecular motions during transport cycles in other ABC transporters, is associated with gating of the CFTR pore.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
82 7 out of the 25 mutant channels exhibited a reduced single-channel current amplitude, including, from extracellular to intracellular, R334C, K335C, F337C, T338C, S341C, R347C, and R352C (Fig. 2). Login to comment
95 Single-channel amplitude: cysless/WT,0.46±0.005pA(n=5); cysless/K355C, 0.28 ± 0.011 pA (n = 4); cysless/F337C, 0.19 ± 0.008 pA (n = 3). Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Biochemistry. 2012 Mar 20;51(11):2199-212. Epub 2012 Mar 7. Norimatsu Y, Ivetac A, Alexander C, Kirkham J, O'Donnell N, Dawson DC, Sansom MS
Cystic fibrosis transmembrane conductance regulator: a molecular model defines the architecture of the anion conduction path and locates a "bottleneck" in the pore.
Biochemistry. 2012 Mar 20;51(11):2199-212. Epub 2012 Mar 7., [PMID:22352759]

Abstract [show]
We developed molecular models for the cystic fibrosis transmembrane conductance regulator chloride channel based on the prokaryotic ABC transporter, Sav1866. Here we analyze predicted pore geometry and side-chain orientations for TM3, TM6, TM9, and TM12, with particular attention being paid to the location of the rate-limiting barrier for anion conduction. Side-chain orientations assayed by cysteine scanning were found to be from 77 to 90% in accord with model predictions. The predicted geometry of the anion conduction path was defined by a space-filling model of the pore and confirmed by visualizing the distribution of water molecules from a molecular dynamics simulation. The pore shape is that of an asymmetric hourglass, comprising a shallow outward-facing vestibule that tapers rapidly toward a narrow "bottleneck" linking the outer vestibule to a large inner cavity extending toward the cytoplasmic extent of the lipid bilayer. The junction between the outer vestibule and the bottleneck features an outward-facing rim marked by T338 in TM6 and I1131 in TM12, consistent with the observation that cysteines at both of these locations reacted with both channel-permeant and channel-impermeant, thiol-directed reagents. Conversely, cysteines substituted for S341 in TM6 or T1134 in TM12, predicted by the model to lie below the rim of the bottleneck, were found to react exclusively with channel-permeant reagents applied from the extracellular side. The predicted dimensions of the bottleneck are consistent with the demonstrated permeation of Cl(-), pseudohalide anions, water, and urea.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
218 The dagger denotes that F337C was previously reported by our group to be unreactive toward MTSET+ and MTSES- . Login to comment
338 Recently, however, we used temperature increases from 22 to 37 °C to effect substantial changes in the conformation of the outer vestibule of the CFTR pore that were evident from markedly increased rates of reaction of MTSES- with cysteines substituted at positions 334 and 336-338 at 37 °C.62 In the case of F337C CFTR, a cysteine that was unreactive toward externally applied MTSES- at 22 °C was highly reactive at 37 °C. Despite these substantial changes in conformation, however, the position of the size selectivity barrier was unchanged. Login to comment

[hide] Locating a Plausible Binding Site for an Open Chan... Mol Pharmacol. 2012 Aug 24. Norimatsu Y, Ivetac A, Alexander C, O'Donnell N, Frye L, Sansom MS, Dawson DC
Locating a Plausible Binding Site for an Open Channel Blocker, GlyH-101, in the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator.
Mol Pharmacol. 2012 Aug 24., [PMID:22923500]

Abstract [show]
High-throughput screening has led to the identification of small-molecule blockers of the CFTR chloride channel, but the structural basis of blocker binding remains to be defined. We recently developed molecular models of the CFTR channel based on homology to the bacterial transporter, Sav1866, that could permit blocker binding to be analyzed in silico. The models accurately predicted the existence of a narrow region in the pore that is a likely candidate for the binding site of an open-channel pore blocker like GlyH-101, thought to act by entering the channel from the extracellular side. As a more stringent test of predictions of the CFTR pore model, we applied induced-fit, virtual ligand docking techniques to identify potential binding sites for GlyH-101 within the CFTR pore. The highest scoring, docked position was near two pore-lining residues, F337 and T338, and the rate of reaction of anionic thiol-directed reagents with cysteines substituted at either of these positions was slowed in the presence of the blocker, consistent with the predicted repulsive effect of the net negative charge on GlyH-101. When a bulky phenylalanine that forms part of the predicted binding pocket (F342) was replaced with alanine, the apparent affinity of the blocker increased by approximately 200 fold. A Molecular Mechanics-Generalized Born/Surface Area (MM-GB/SA) analysis of GlyH-101 binding predicted that substitution of F342 with alanine would substantially increase blocker affinity, primarily due to decreased intramolecular strain within the blocker-protein complex. This study suggests that GlyH-101 blocks the CFTR channel by binding within the pore bottleneck.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
108 In this example, externally-applied [Au(CN)2]- was reacted with F337C CFTR. Login to comment
115 Figure 3C and 3D contain the time courses for the reactions of [Au(CN)2]- with F337C and T338C CFTR pre-and post-activation. Login to comment
141 Figure 5C and 5D summarize the inhibition of F337C CFTR and T338C CFTR by [Au(CN)2]- in the presence and in the absence of GlyH-101. Login to comment
144 In Figure 5E and 5F the measured second order rate constants for covalent modification of F337C and T338C CFTR are plotted versus GlyH-101 concentration. Login to comment
151 We also studied the reaction of I1131C CFTR with MTSES- . Login to comment
159 The presence of negative charges near I1131 is consistent with the observed slow reaction of I1131C CFTR with MTSES- (20 M-1 sec-1 ; Figure 7) which is more than 100-fold less than that seen for a Cys at 338 (3.3 x 103 M-1 sec-1 ; Figure 6). Login to comment
160 As reported previously (Norimatsu et al., 2012) the macroscopic conductance of I1131C CFTR was increased by depositing the negatively charged sulfonic acid group via reaction with MTSES- . Login to comment
209 Second, alkylation of T338C CFTR with IAM, which results in covalent addition of an acetamide moiety predicted by the model to create a steric clash with GlyH-101, significantly reduced the apparent binding affinity of GlyH-101. Login to comment
210 In contrast, alkylation of F337C CFTR with IAM is not predicted by the molecular model to cause a steric clash and does not markedly alter GlyH-101 block. Login to comment
220 The state-dependent reactivity of T338C CFTR observed in the current study is consistent with the finding of Beck et al., (2008) that MTSES- reacts slightly faster with a high open probability mutant T338C/E1371Q CFTR than with T338C/wt CFTR.2 Mornon et al., (2009) created a homology model of CFTR based on the inward-facing conformation of a prokaryotic transporter, MsbA (Ward et al., 2007) (PDB code: 3B5X). Login to comment
224 A conformational change of this sort would be consistent with the state-dependent reactivity of F337C and T338C CFTR observed in the current study. Login to comment
225 The MsbA-based model of Mornon et al., (2009) also predicts that the side chain of R334 protrudes into the external aqueous environment, and when R334 is mutated to a cysteine in the MsbA-based model of Mornon et al., (2009) using Maestro (version 9.1, Schrödinger LLC), the reactive thiolate is clearly accessible from the extracellular solution (Figure 9C), consistent with the closed state reactivity of R334C observed in the current study and by Zhang et al., (2005). Login to comment
226 On the other hand, the mechanism that renders R334C CFTR unreactive in the conducting state of CFTR is not clear. Login to comment
158 Figure 3, C and D, contains the time courses for the reactions of [Au(CN)2]afa; with the F337C and T338C CFTRs before TABLE 1 EC50 at 0 mV (mean afe; S.E.M.) for GlyH-101 for wt and mutant CFTRs, with and without modification with iodoacetamide CFTR EC50 at 0 mV òe;M wt 1.1 afe; 0.11 (n afd; 4) K95C 1.4 afe; 0.35 (n afd; 4) F337C 1.8 afe; 0.06 (n afd; 3) F337C af9; iodoacetamide 2.4 afe; 0.29 (n afd; 3) T338C 3.7 afe; 0.27 (n afd; 3) T338C af9; iodoacetamide 24 afe; 2.6 (n afd; 3) Fig. 3. Login to comment
162 The subsequent application of [Au(CN)2]afa; almost completely abolished F337C CFTR conductance. Login to comment
163 C and D, time courses of the decreases in normalized conductance as a result of F337C (C) and T338C (D) modifications with [Au(CN)2]afa; . Login to comment
165 For the F337C CFTR, the abscissa represents cumulative [Au(CN)2]afa; exposure time. Login to comment
167 The reaction rate for the F337C CFTR before activation of the channels was b03;20 times slower than the rate after activation. Login to comment
171 The second-order reaction rate constants for the F337C CFTR before and after activation were 1.5 and 26 Mafa;1 safa;1 , respectively. Login to comment
206 Figure 5, C and D, summarizes the inhibition of the F337C and T338C CFTRs by [Au(CN)2]afa; in the presence and absence of GlyH-101. Login to comment
215 C and D, F337C (C) and T338C (D) CFTR channels were protected by 10 òe;M GlyH-101 from reactions with [Au(CN)2]afa; . Login to comment
217 The F337C CFTR was reacted with 600 òe;M [Au(CN)2]afa; and the T338C CFTR was reacted with 5 òe;M [Au(CN)2]afa; in the presence and absence of 10 òe;M GlyH-101. Login to comment
318 In contrast, alkylation of the F337C CFTR with iodoacetamide was not predicted by the molecular model to cause a steric clash and did not alter GlyH-101 blockade markedly. Login to comment
350 A conformational change of this sort would be consistent with the state-dependent reactivity of the F337C and T338C CFTRs observed in the current study. Login to comment

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

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

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
183 F337C-and F337E-CFTR exhibited significantly altered reversal potential, relative permeability, and relative conductance compared to WT-CFTR (Supplementary Tables 1, 2, 3), as did F337A-, S-, Y-, and L-CFTR [24]. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Biochemistry. 2011 Nov 29;50(47):10311-7. Epub 2011 Nov 4. Liu X, Dawson DC
Cystic fibrosis transmembrane conductance regulator: temperature-dependent cysteine reactivity suggests different stable conformers of the conduction pathway.
Biochemistry. 2011 Nov 29;50(47):10311-7. Epub 2011 Nov 4., [PMID:22014307]

Abstract [show]
Cysteine scanning has been widely used to identify pore-lining residues in mammalian ion channels, including the cystic fibrosis transmembrane conductance regulator (CFTR). These studies, however, have been typically conducted at room temperature rather than human body temperature. Reports of substantial effects of temperature on gating and anion conduction in CFTR channels as well as an unexpected pattern of cysteine reactivity in the sixth transmembrane segment (TM6) prompted us to investigate the effect of temperature on the reactivity of cysteines engineered into TM6 of CFTR. We compared reaction rates at temperatures ranging from 22 to 37 degrees C for cysteines placed on either side of an apparent size-selective accessibility barrier previously defined by comparing reactivity toward channel-permeant and channel-impermeant, thiol-directed reagents. The results indicate that the reactivity of cysteines at three positions extracellular to the position of the accessibility barrier, 334, 336, and 337, is highly temperature-dependent. At 37 degrees C, cysteines at these positions were highly reactive toward MTSES(-), whereas at 22 degrees C, the reaction rates were 2-6-fold slower to undetectable. An activation energy of 157 kJ/mol for the reaction at position 337 is consistent with the hypothesis that, at physiological temperature, the extracellular portion of the CFTR pore can adopt conformations that differ significantly from those that can be accessed at room temperature. However, the position of the accessibility barrier defined empirically by applying channel-permeant and channel-impermeant reagents to the extracellular aspect of the pore is not altered. The results illuminate previous scanning results and indicate that the assay temperature is a critical variable in studies designed to use chemical modification to test structural models for the CFTR anion conduction pathway.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
12 Cheung and Akabas1,2 reported reactivity of F337C CFTR toward MTSEA+ , MTSET+ , and MTSES- . Login to comment
13 Fatehi et al.5 reported reactivity of F337C CFTR toward MTSES- but not MTSET+ , while Beck et al.6 reported no reactivity toward MTSEA+ at this locus. Login to comment
48 ■ RESULTS The representative experiments compiled in Figure 1 illustrate the dramatic effect of increased temperature on the rate of reaction of MTSES- with F337C CFTR. Login to comment
50 Increased temperature dramatically increased the reactivity of F337C CFTR toward MTSES- . Login to comment
51 Oocytes expressing F337C CFTR were activated using a stimulatory cocktail (10 μM Isop and 1 mM IBMX, hatched bar and cross hairs) and then exposed to 1 mM 2-ME to reverse any spurious reactions of the substituted cysteine.11 (A) Exposure to 1 mM MTSES- (dark gray bar and circles) produced no reaction, but 1 mM [Au(CN)2]- (black bar and circles) produced profound inhibition that was not reversed by 1 mM 2-ME but was reversed by 1 mM KCN (white bar and triangles). Login to comment
58 In many such experiments conducted at room temperature, no change in conductance was detectable upon exposure of an oocyte expressing F337C CFTR to 1 mM MTSES- , even for periods exceeding 10 min. Login to comment
60 In this instance, exposure of the oocyte expressing F337C CFTR to MTSES- at room temperature evoked what at first appeared to be a very slow rate of reaction, but the decline spontaneously reversed upon removal of the reagent from the superfusate. Login to comment
62 The inhibition following the reaction of [Au(CN)2]- was not reversed by exposure to a competing thiol, 2-ME, but was readily reversed by exposure to the high-affinity metal ligand, CN- , as expected from previous studies.10,12 Figure 1B depicts an experiment in which an oocyte expressing F337C CFTR was exposed to 1 mM MTSES- at 22 °C, and the superfusate temperature was then increased to 32 °C in the presence of the mixed disulfide. Login to comment
67 An oocyte expressing F337C CFTR was heated to 37 °C for 10 min and then cooled to 22 °C. Login to comment
71 This result indicates that, although reaction of MTSES- with F337C CFTR was readily detectable at 37 °C, exposure to the elevated temperature did not result in any irreversible change in the channel. Login to comment
86 In the case of R334 and I336, exposure to MTSES- (3 μM and 1 mM, respectively) produced reactions at 22 °C that were slow but, unlike that of F337C CFTR, readily discernible at room temperature. Login to comment
93 As expected, the apparent activation energies varied widely, ranging from being that expected for disulfide exchange15,16 for T338C CFTR to values in the range of those generally associated with protein conformational change.17-20 Figure 6 summarizes the inhibition by MTSES- of CFTR conductance at 22 °C (27 °C for F337C CFTR) and 37 °C in oocytes expressing CFTR constructs bearing substituted cysteines at positions extracellular to (334 and 336-338) and cytoplasmic to (339-342 and 344) the apparent accessibility cutoff defined by Alexander et al.10 It is apparent that, despite the dramatic increases in the reaction rates of cysteines extracellular to the cutoff, the position of the cutoff was unchanged at 37 °C. Login to comment
97 Temperature Dependence of MTSES- Modification kMTSES (M-1 s-1 ) mutant 22 °C 30 °C 32 °C 37 °C Ea (kJ/mol) R334C 2648 ± 259 (n = 3) 9411 ± 1210 (n = 5) 18407 ± 3240 (n = 3) 98 I336C 1.2a 2.3 ± 0.1 (n = 3) 6.9 ± 0.4 (n = 4) 88 F337C 2.6 ± 0.7 (27 °C)b (n = 3) 5.1 ± 1.2 (n = 3) 19.4 ± 4.4 (n = 4) 157 T338C 4067 ± 573 (n = 5) 7192 ± 370 (n = 4) 7972 ± 1019 (n = 6) 35 a Value from ref 10. b The reaction rate was undetectable at 22 °C, so the value determined at 27 °C was used. Login to comment
106 Arrhenius plots for (A) R334C, (B) I336C, (C) F337C, and (D) T338C CFTR. Login to comment
124 This finding does not, in and of itself, resolve the discrepancy between our data and those of Cheung and Akabas1,2 and Fatehi et al.,5 who reported reactivity of F337C CFTR toward externally applied MTSES- at room temperature, but the observation of a slow, but discernable reaction rate at 27 °C (Table 1) raises the possibility of some, as yet unidentified, condition of their experiments that allows the CFTR channel to access conformations at temperatures within the 22 °C-27 °C range in which the thiol-disulfide exchange reaction can occur at position 337. Login to comment
129 The percent block of CFTR conductance by MTSES- was defined as the change in conductance induced by MTSES- , ΔgES, divided by the conductance at time zero of exposure, gt=0, at 22 °C (27 °C for F337C, white bars) and 37 °C (dark gray bars). Login to comment

[hide] Alignment of transmembrane regions in the cystic f... J Gen Physiol. 2011 Aug;138(2):165-78. Epub 2011 Jul 11. Wang W, El Hiani Y, Linsdell P
Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Gen Physiol. 2011 Aug;138(2):165-78. Epub 2011 Jul 11., [PMID:21746847]

Abstract [show]
Different transmembrane (TM) alpha helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl(-) channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84-T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
214 In open CFTR channels, internally applied MTS reagents can penetrate far enough into the pore as to modify L102C in TM1 and F337C in TM6. Login to comment
227 Second, whereas cysteines substituted for TM6 residues in the putative narrow pore region-F337C, T338C, and S341C-could be modified by both intracellular and extracellular MTS reagents (El Hiani and Linsdell, 2010), no residues that could be modified from both sides of the membrane were identified in TM1. Login to comment

[hide] Locating the anion-selectivity filter of the cysti... J Gen Physiol. 1997 Mar;109(3):289-99. Cheung M, Akabas MH
Locating the anion-selectivity filter of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel.
J Gen Physiol. 1997 Mar;109(3):289-99., [PMID:9089437]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator forms an anion-selective channel; the site and mechanism of charge selectivity is unknown. We previously reported that cysteines substituted, one at a time, for Ile331, Leu333, Arg334, Lys335, Phe337, Ser341, Ile344, Arg347, Thr351, Arg352, and Gln353, in and flanking the sixth membrane-spanning segment (M6), reacted with charged, sulfhydryl-specific, methanethiosulfonate (MTS) reagents. We inferred that these residues are on the water-accessible surface of the protein and may line the ion channel. We have now measured the voltage-dependence of the reaction rates of the MTS reagents with the accessible, engineering cysteines. By comparing the reaction rates of negatively and positively charged MTS reagents with these cysteines, we measured the extent of anion selectivity from the extracellular end of the channel to eight of the accessible residues. We show that the major site determining anion vs. cation selectivity is near the cytoplasmic end of the channel; it favors anions by approximately 25-fold and may involve the residues Arg347 and Arg 352. From the voltage dependence of the reaction rates, we calculated the electrical distance to the accessible residues. For the residues from Leu333 to Ser341 the electrical distance is not significantly different than zero; it is significantly different than zero for the residues Thr351 to Gln353. The maximum electrical distance measured was 0.6 suggesting that the channel extends more cytoplasmically and may include residues flanking the cytoplasmic end of the M6 segment. Furthermore, the electrical distance calculations indicate that R352C is closer to the extracellular end of the channel than either of the adjacent residues. We speculate that the cytoplasmic end of the M6 segment may loop back into the channel narrowing the lumen and thereby forming both the major resistance to current flow and the anion-selectivity filter.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
107 We did not measure the reaction rate constants for the most extracellular residue, I331C, because we thought that it was unlikely that the reaction rates would be voltage dependent given the absence of voltage dependence at the adjacent, more cytoplasmic residues. We also did not measure the reaction rate constants for the mutants I344C and R347C because, although MTSEAϩ reacted with these residues, MTSES- and MTSETϩ did not react with these k ψ( )( )ln k Ψ 0=( )( ) zFδ RT/( )-ln ψ= t a b l e i Second-order Rate Constants for the Reaction of the MTS Reagents with the Water-exposed Cysteine Mutants k ES (M-1s-1) k EA (M-1s-1) k ET (M-1s-1) mutant -25 mV -50 mV -75 mV -25 mV -50 mV -75 mV -25 mV -50 mV -75 mV L333C 71 Ϯ 3(3) 71 Ϯ 20(2) 71 Ϯ 23(3) 320 Ϯ 89(2) 320 Ϯ 128(2) 333 Ϯ 139(3) 952 Ϯ 136(2) 1,000 Ϯ 350(2) 1,053 Ϯ 443(2) R334C 48 Ϯ 14(2) 48 Ϯ 6(3) 44 Ϯ 8(4) 145 Ϯ 32(2) 163 Ϯ 7(2) 182 Ϯ 21(3) 444 Ϯ 49(2) 454 Ϯ 124(2) 588 Ϯ 95(3) K335C 36 Ϯ 20(3) 23 Ϯ 11(3) 27 Ϯ 16(3) 222 Ϯ 80(3) 121 Ϯ 51(4) 107 Ϯ 30(3) 217 Ϯ 111(3) 235 Ϯ 28(3) 217 Ϯ 95(4) F337C 91 Ϯ 17(2) 80 Ϯ 22(3) 71 Ϯ 20(4) 222 Ϯ 74(2) 222 Ϯ 86(3) 285 Ϯ 81(3) 740 Ϯ 246(3) 740 Ϯ 82(2) 714 Ϯ 51(2) S341C 56 Ϯ 18(3) 56 Ϯ 40(2) 43 Ϯ 12(3) 93 Ϯ 6(3) 110 Ϯ 22(3) 138 Ϯ 34(3) 690 Ϯ 356(3) 556 Ϯ 246(3) 800 Ϯ 224(4) T351C 100 Ϯ 25(5) 57 Ϯ 6(3) 26 Ϯ 9(6) 146 Ϯ 30(4) 195 Ϯ 42(4) 296 Ϯ 18(3) 308 Ϯ 47(10) 392 Ϯ 78(6) 769 Ϯ 89(5) R352C 42 Ϯ 4(3) 26 Ϯ 4(5) 21 Ϯ 6(4) 105 Ϯ 76(3) 137 Ϯ 46(3) 205 Ϯ 58(2) 417 Ϯ 138(4) 800 Ϯ 128(2) 952 Ϯ 408(2) Q353C 125 Ϯ 23(4) 51 Ϯ 12(4) 42 Ϯ 8(4) 83 Ϯ 24(4) 116 Ϯ 42(4) 160 Ϯ 92(3) 189 Ϯ 48(6) 220 Ϯ 48(3) 625 Ϯ 273(4) residues and therefore we could not determine the charge selectivity at these positions.2 The reaction rate constants that we have measured are between 10-and 500-fold slower than the rates of reaction with sulfhydryls in free solution (Table II) (Stauffer and Karlin, 1994). 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.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
91 Effects of MTS reagents on wild-type cysteines RESULTS in CFTR To identify the residues in and flanking the M6 membrane-spanning segment that are on the water-exposed surface of As reported previously (Akabas et al., 1994b), extracellular applications of the MTS reagents to Xenopus oocytes ex- L2j K329C L. _J *G330C 1331C 1332C L333C R334C K335C 1336C F337C T338C T339C 1340C S341C T342C C343,WT 1344C V345C L346C R347C M348C A349C V350C T351C R352C Q353C 0 2000 4000 6000 8000 0 25 50 PEAK CURRENTS (nA) TIME TO REACH PLATEAU (min) FIGURE 2 Peak CFTR-induced currents and time to reach the plateau current after stimulation with cAMP-activating reagents for 24 cysteine-substitution mutants and wild-type CFTR. Login to comment
109 Accessibility of substituted cysteines to MTSES- A 1-min application of 10 mM MTSES- significantly inhibited the CFIR-induced currents of 9 of the 24 cysteine-substituted mutants (Fig. 4 A), L333C, R334C, K335C, F337C, S341C, R347C, T351C, R352C, and Q353C. Login to comment
90 Effects of MTS reagents on wild-type cysteines RESULTS in CFTR To identify the residues in and flanking the M6 membrane-spanning segment that are on the water-exposed surface of As reported previously (Akabas et al., 1994b), extracellular applications of the MTS reagents to Xenopus oocytes ex- L2j K329C L. _J *G330C 1331C 1332C L333C R334C K335C 1336C F337C T338C T339C 1340C S341C T342C C343,WT 1344C V345C L346C R347C M348C A349C V350C T351C R352C Q353C 0 2000 4000 6000 8000 0 25 50 PEAK CURRENTS (nA) TIME TO REACH PLATEAU (min) FIGURE 2 Peak CFTR-induced currents and time to reach the plateau current after stimulation with cAMP-activating reagents for 24 cysteine-substitution mutants and wild-type CFTR. Login to comment
108 Accessibility of substituted cysteines to MTSES- A 1-min application of 10 mM MTSES- significantly inhibited the CFIR-induced currents of 9 of the 24 cysteine-substituted mutants (Fig. 4 A), L333C, R334C, K335C, F337C, S341C, R347C, T351C, R352C, and Q353C. Login to comment

[hide] Long-range coupling between the extracellular gate... FASEB J. 2015 Nov 25. pii: fj.15-278382. Wei S, Roessler BC, Icyuz M, Chauvet S, Tao B, Hartman JL 4th, Kirk KL
Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.
FASEB J. 2015 Nov 25. pii: fj.15-278382., [PMID:26606940]

Abstract [show]
The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that: 1) all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy and 2) highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced.-Wei, S., Roessler, B. C., Icyuz, M., Chauvet, S., Tao, B., Hartman, J. L., IV, Kirk, K. L. Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
70 Primer sequences for cloning and site-directed mutagenesis Ycf1p Forward cloning primer: CAACACAGGCATGTATATTA- AGAGC Reverse cloning primer: TTAAACTTATGGCGTCAGAG- TTGCC F565A: CATTGACTACTGACTTAGTTGCCCCTGCTTTG- ACTCTGTTC F565S: CATTGACTACTGACTTAGTTTCCCCTGCTTTGA- CTCTGTTC F565L: CATTGACTACTGACTTAGTTTTACCTGCTTTG- ACTCTGTTC G756D: AAGACAAACGAGCTTTTTGATCTCCAGATAAG- GAGATCCC D777N: ACAGCTGGCAAAGGATCATTAAGTAAATAAG- TGTCAGCTC Y1281G: GATCAAGCTCCGGCCTACCACGAGTGGAATA- ATTATTAAAC Yor1p Forward cloning primer: CTAATTGTACATCCGGTTTT- AACC Reverse cloning primer: TTGAGTCATTGCCCTTAA- AATGG F468S: AGGCAACCTGGTAATATTTCTGCCTCTTTATC- TTTATTTC F468A: AGGCAACCTGGTAATATTGCTGCCTCTTTATC- TTTATTTC F468L: AGGCAACCTGGTAATATTCTTGCCTCTTTATC- TTTATTTC G713D: GTGGTATTACTTTATCTGGTGATCAAAAGGCA- CGTATCAATTT Y1222G: ATAGGTAAACCAGGTCTACCGGCAAAATCAA- CATTTTCAA CFTR Forward cloning primer: GAAGAAGCAATGGAAAAA- ATGATTG Reverse cloning primer: TCGGTGAATGTTCTGACCT- TGG F337S: TCATCCTCCGGAAAATATCCACCACCATCTCA- TTCTGC F337A: TCATCCTCCGGAAAATAGCCACCACCATCTCA- TTCTGC F337L: TCATCCTCCGGAAAATATTAACCACCATCTCA- TTCTGC F337C: TCATCCTCCGGAAAATATGCACCACCATCTC- ATTCTGC Immunoblot analysis of CFTR protein expression Expression of the CFTR F337 mutants was verified by immunoblotting as described elsewhere (15). Login to comment
152 Mean percent ATP-free currents 6 SEMs were as follows: WT (0.5 6 0.2%; n = 5); F337L (0.6 6 0.3%; n = 5); F337C (2.5 6 1.4%; n = 5), F337A (9.6 6 1.4%; n = 5), and F337S (15.8 6 4.5%; n = 10). Login to comment
69 Primer sequences for cloning and site-directed mutagenesis Ycf1p Forward cloning primer: CAACACAGGCATGTATATTA- AGAGC Reverse cloning primer: TTAAACTTATGGCGTCAGAG- TTGCC F565A: CATTGACTACTGACTTAGTTGCCCCTGCTTTG- ACTCTGTTC F565S: CATTGACTACTGACTTAGTTTCCCCTGCTTTGA- CTCTGTTC F565L: CATTGACTACTGACTTAGTTTTACCTGCTTTG- ACTCTGTTC G756D: AAGACAAACGAGCTTTTTGATCTCCAGATAAG- GAGATCCC D777N: ACAGCTGGCAAAGGATCATTAAGTAAATAAG- TGTCAGCTC Y1281G: GATCAAGCTCCGGCCTACCACGAGTGGAATA- ATTATTAAAC Yor1p Forward cloning primer: CTAATTGTACATCCGGTTTT- AACC Reverse cloning primer: TTGAGTCATTGCCCTTAA- AATGG F468S: AGGCAACCTGGTAATATTTCTGCCTCTTTATC- TTTATTTC F468A: AGGCAACCTGGTAATATTGCTGCCTCTTTATC- TTTATTTC F468L: AGGCAACCTGGTAATATTCTTGCCTCTTTATC- TTTATTTC G713D: GTGGTATTACTTTATCTGGTGATCAAAAGGCA- CGTATCAATTT Y1222G: ATAGGTAAACCAGGTCTACCGGCAAAATCAA- CATTTTCAA CFTR Forward cloning primer: GAAGAAGCAATGGAAAAA- ATGATTG Reverse cloning primer: TCGGTGAATGTTCTGACCT- TGG F337S: TCATCCTCCGGAAAATATCCACCACCATCTCA- TTCTGC F337A: TCATCCTCCGGAAAATAGCCACCACCATCTCA- TTCTGC F337L: TCATCCTCCGGAAAATATTAACCACCATCTCA- TTCTGC F337C: TCATCCTCCGGAAAATATGCACCACCATCTC- ATTCTGC Immunoblot analysis of CFTR protein expression Expression of the CFTR F337 mutants was verified by immunoblotting as described elsewhere (15). Login to comment
151 Mean percent ATP-free currents 6 SEMs were as follows: WT (0.5 6 0.2%; n = 5); F337L (0.6 6 0.3%; n = 5); F337C (2.5 6 1.4%; n = 5), F337A (9.6 6 1.4%; n = 5), and F337S (15.8 6 4.5%; n = 10). Login to comment

[hide] Localizing a gate in CFTR. Proc Natl Acad Sci U S A. 2015 Feb 24;112(8):2461-6. doi: 10.1073/pnas.1420676112. Epub 2015 Feb 9. Gao X, Hwang TC
Localizing a gate in CFTR.
Proc Natl Acad Sci U S A. 2015 Feb 24;112(8):2461-6. doi: 10.1073/pnas.1420676112. Epub 2015 Feb 9., [PMID:25675504]

Abstract [show]
Experimental and computational studies have painted a picture of the chloride permeation pathway in cystic fibrosis transmembrane conductance regulator (CFTR) as a short narrow tunnel flanked by wider inner and outer vestibules. Although these studies also identified a number of transmembrane segments (TMs) as pore-lining, the exact location of CFTR's gate(s) remains unknown. Here, using a channel-permeant probe, [Au(CN)2](-), we provide evidence that CFTR bears a gate that coincides with the predicted narrow section of the pore defined as residues 338-341 in TM6. Specifically, cysteines introduced cytoplasmic to the narrow region (i.e., positions 344 in TM6 and 1148 in TM12) can be modified by intracellular [Au(CN)2](-) in both open and closed states, corroborating the conclusion that the internal vestibule does not harbor a gate. However, cysteines engineered to positions external to the presumed narrow region (e.g., 334, 335, and 337 in TM6) are all nonreactive toward cytoplasmic [Au(CN)2](-) in the absence of ATP, whereas they can be better accessed by extracellular [Au(CN)2](-) when the open probability is markedly reduced by introducing a second mutation, G1349D. As [Au(CN)2](-) and chloride ions share the same permeation pathway, these results imply a gate is situated between amino acid residues 337 and 344 along TM6, encompassing the very segment that may also serve as the selectivity filter for CFTR. The unique position of a gate in the middle of the ion translocation pathway diverges from those seen in ATP-binding cassette (ABC) transporters and thus distinguishes CFTR from other members of the ABC transporter family.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
78 State-Dependent Reactivity of T338C, F337C, and R334C Implicates the Location of a Gate for CFTR. Login to comment
88 Similar observations were made for F337C-CFTR and R334C-CFTR (Fig. S4 A-D). Login to comment
96 Our previous studies demonstrated that a disease-associated mutation G1349D could decrease the Po of CFTR by ~10-fold (34) without affecting trafficking of the channel (34, 35); we thus engineered this mutation into R334C, K335C, F337C, and T338C backgrounds. Login to comment
192 [Au(CN)2]- , forskolin, with G1349D, /M/s Outside R334C 189 &#b1; 39 - 403 &#b1; 20 537 &#b1; 56 K335C - - 56 &#b1; 9 1,809 &#b1; 201 F337C 437 &#b1; 49 - 20 &#b1; 3 32 &#b1; 6 T338C 752 &#b1; 59 - 1,135 &#b1; 166 118 &#b1; 18 Inside I344C 32 &#b1; 5 37 &#b1; 4 - - N1148C 437 &#b1; 66 2,089 &#b1; 130 - - Residues located extracellularly (extra.) Login to comment