ABCC7 p.Leu333Cys
ClinVar: |
c.997C>T
,
p.Leu333Phe
D
, Likely pathogenic
|
CF databases: |
c.997C>T
,
p.Leu333Phe
(CFTR1)
?
,
|
Predicted by SNAP2: | A: N (53%), C: N (61%), D: D (91%), E: D (85%), F: D (59%), G: D (85%), H: D (85%), I: N (72%), K: D (85%), M: N (72%), N: D (85%), P: D (85%), Q: D (80%), R: D (85%), S: D (75%), T: D (53%), V: N (78%), W: D (85%), Y: D (80%), |
Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N, |
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[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.
No. Sentence Comment
93 Both Cd2ϩ and MTSEA had significant effects on the conductances of only five (I331C, L333C, R334C, K335C, and T338C) of the 26 Cys-substituted channels examined.
X
ABCC7 p.Leu333Cys 18056267:93:91
status: NEW100 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.
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ABCC7 p.Leu333Cys 18056267:100:231
status: NEW127 For example, whereas L333C in the Glu1371 (WT) channel was inhibited by either Cd2ϩ or MTSEA, neither reagent was particularly effective when this mutation was present in the Gln1371 background 200 150 100 50 0 µS 15001000500 s IBMX Cd 2+ MTSEA DTT -80 -60 -40 -20 0 % Change in conductance I331C L333C R334C K335C T338C Cd 2+ aM Cd 2+ bM Cd 2+ uM A B FIGURE 2.
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ABCC7 p.Leu333Cys 18056267:127:21
status: NEWX
ABCC7 p.Leu333Cys 18056267:127:308
status: NEW131 B, summary of effects of Cd2ϩ on MTSEA-modified I331C, L333C, R334C, K335C, and T338C channels.
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ABCC7 p.Leu333Cys 18056267:131:61
status: NEW135 MTSEA 1371Q 600 400 200 µS 200150100500 s Cd 2+ 1371E -40 0 40 % Change in conductance I331C L333C R334C K335C T338C 1371E 1371Q * * * -80 -60 -40 -20 0 % Change in conductance I331C L333C R334C K335C T338C * * * 1371Q 800 600 400 µS 2001000 s MTSEA 1371E B D E 1 2 30 s1 pAWT; Po=0.18 A 3 1 2 100 s1 pAE1371Q; Po=0.94 C FIGURE 3.
X
ABCC7 p.Leu333Cys 18056267:135:98
status: NEWX
ABCC7 p.Leu333Cys 18056267:135:188
status: NEW142 B, effect of Cd2ϩ on whole cell conductance of L333C-CFTR in 1371E (WT; blue) and in 1371Q (red) backgrounds.
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ABCC7 p.Leu333Cys 18056267:142:53
status: NEW153 The differences between Glu1371 and Gln1371 backgrounds in the effects of Cd2ϩ and MTSEA on I331C, L333C, R334C, K335C, and T338C channels are summarized in Fig. 3 (C and E), respectively.
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ABCC7 p.Leu333Cys 18056267:153:105
status: NEW159 In contrast, I331C, L333C, and K335C reacted faster in the Glu1371 background (Fig. 4, B and C).
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ABCC7 p.Leu333Cys 18056267:159:20
status: NEW160 These results reveal clearly that modification of I331C, L333C, and K335C by both these reagents was much slower in the Gln1371 mutational background than in the WT Glu1371 channels.
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ABCC7 p.Leu333Cys 18056267:160:57
status: NEW163 L333C showed a relatively smaller 10-fold 100 75 50 4803602401200 s 1371E; 100 µM 1371Q; 100 µM 100 90 80 70 240180120600 s 1371E; 1 mM 1371Q; 1 mM 120 100 80 120600 s 1371E; 10 µM 1371Q; 1 mM 100 75 50 25 180120600 s 1371Q; 10 µM 1371E; 10 µM 100 90 80 180120600 s 1371Q; 1 mM 1371E; 1 mM 100 75 50 4803602401200 s 1371E; 100 µM 1371Q; 100 µM 9060300 s 1371E; 10 µM 1371Q; 1 mM 180120600 s 1371E; 10 µM 1371Q; 10 µM 100 75 50 25 180120600 s 1371E; 10 µM 1371Q; 10 µM 140 130 120 110 100 4530150 s 1371Q; 10 µM 1371E; 10 µM FIGURE 4.
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ABCC7 p.Leu333Cys 18056267:163:0
status: NEW178 All of the residues except L333C had varying but significant differences in the magnitude of the functional effect by MTS reagents.
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ABCC7 p.Leu333Cys 18056267:178:27
status: NEW185 However, I331C and L333C channels had a significantly faster modification rate, when minimally active.
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ABCC7 p.Leu333Cys 18056267:185:19
status: NEW186 When stimulated by 0.02 mM IBMX, both I331C and L333C reacted nearly 25 times faster with MTSEA and nearly 10-20 times faster with MTSES.
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ABCC7 p.Leu333Cys 18056267:186:48
status: NEW188 EvidenceforTM6MovementAssociatedwithChannelGating- The state-dependent reactivity of the MTS reagents with I331C, L333C, and K335C channels could indicate a change in the water accessibility of these residues caused by a conformational change in TM6 or by an alteration in the local environment surrounding these residues.
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ABCC7 p.Leu333Cys 18056267:188:114
status: NEW192 For two of the three mutants, I331C and L333C, modification with MTSET profoundly affected channel gating (Fig. 7A).
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ABCC7 p.Leu333Cys 18056267:192:40
status: NEW193 The open probabilities of MTSET-modified I331C and L333C channels were significantly smaller than those of unmodified channels.
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ABCC7 p.Leu333Cys 18056267:193:51
status: NEW196 These results indicate that MTSET reduces the whole cell conductance of I331C- and L333C-expressing oocytes by inhibiting channel gating and not by affecting channel permeation properties.
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ABCC7 p.Leu333Cys 18056267:196:83
status: NEW197 Kinetic analyses of channel gating revealed that the decrease in open probability of MTSET-modified I331C and L333C channels was primarily because of an increase in the mean interburst duration of the A B 1.00.50.0 G0.02/ G1 I331C L333C R334C K335C T338C 200 100 0 µS 8006004002000 s 0.02 1 IBMX (mM) C -100 100 % Change in conductance I331C L333C R334C K335C T338C 0.02 mM IBMX 1 mM IBMX * * * * -80 -60 -40 -20 0 % Change in conductance I331C L333C R334C K335C T338C * * * MTSEA MTSES FIGURE5.EffectsofMTSEA,andMTSESdependonCFTRactivationlevels.
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ABCC7 p.Leu333Cys 18056267:197:110
status: NEWX
ABCC7 p.Leu333Cys 18056267:197:231
status: NEWX
ABCC7 p.Leu333Cys 18056267:197:347
status: NEWX
ABCC7 p.Leu333Cys 18056267:197:450
status: NEW216 Although both studies identified I331C, L333C, R334C, and K335C as accessible to MTS reagents, we find that MTSEA increased the conductance of R334C- and K335C-expressing oocytes, whereas it was reported in the previous study to decrease channel currents.
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ABCC7 p.Leu333Cys 18056267:216:40
status: NEW223 It is possible that this mutation rather than the open 150 125 100 %G/Gi 600 s K335C I-1.0; 10 µM I-0.02; 10 µM 10 1 10 2 10 3 10 4 Modification rate (M -1 s -1 ) I331C L333C R334C K335C T338C 100 50 %G/Gi 3002001000 s I-1.0; 100 µM I-0.02;10 µM MTSEA I331CL333CR334CK335CT338C 100 75 50 25 0 %G/Gi 180120600 s I-0.02; 10 µM I-1.0; 10 µM 200 150 100 %G/Gi 120600 s I-0.02; 10 µM I-1.0; 10 µM 100 75 50 %G/Gi 3602401200 s I-1.0; 100 µM I-0.02; 10 µM 100 80 60 %G/Gi 9060300 s K335C I-1.0; 10 µM I-0.02; 10 µM 100 50 %G/Gi 180120600 s T338C I-1.0; 10 µM I-0.02; 10 µM 10 1 10 2 10 3 10 4 Modification rate (M -1 s -1 ) I331C L333C R334C K335C T338C MTSES 100 75 50 25 %G/Gi 120600 s I-1.0; 10 µM I-0.02; 10 µM 100 75 50 %G/Gi 3602401200 s I-1.0; 100 µM I-0.02; 10 µM 100 75 %G/Gi 180120600 s I-0.02; 100 µM I-1.0; 1 mM A B FIGURE 6.
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ABCC7 p.Leu333Cys 18056267:223:179
status: NEWX
ABCC7 p.Leu333Cys 18056267:223:693
status: NEW240 It must be pointed out that under low IBMX concentrations, a 5-fold decrease in CFTR Po cannot account for the entire differences in reactivity of I331C and L333C.
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ABCC7 p.Leu333Cys 18056267:240:157
status: NEW242 Hence, a small fraction of the increased reactivity of I331C, and L333C at low IBMX concentrations could be due to a relief from this block, although such an increase in reactivity is not observed for R334C and T338C.
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ABCC7 p.Leu333Cys 18056267:242:66
status: NEW258 The openprobabilityandmeanburstandinterbursttimes(meansϮS.E.)forI331C,L333C,K335C,andWTchannels in untreated (-MTSET) and MTSET-treated (ϩMTSET) conditions are shown.
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ABCC7 p.Leu333Cys 18056267:258:76
status: NEW[hide] Novel residues lining the CFTR chloride channel po... J Membr Biol. 2009 Apr;228(3):151-64. Epub 2009 Apr 19. Fatehi M, Linsdell P
Novel residues lining the CFTR chloride channel pore identified by functional modification of introduced cysteines.
J Membr Biol. 2009 Apr;228(3):151-64. Epub 2009 Apr 19., [PMID:19381710]
Abstract [show]
Substituted cysteine accessibility mutagenesis (SCAM) has been used widely to identify pore-lining amino acid side chains in ion channel proteins. However, functional effects on permeation and gating can be difficult to separate, leading to uncertainty concerning the location of reactive cysteine side chains. We have combined SCAM with investigation of the charge-dependent effects of methanethiosulfonate (MTS) reagents on the functional permeation properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. We find that cysteines substituted for seven out of 21 continuous amino acids in the eleventh and twelfth transmembrane (TM) regions can be modified by external application of positively charged [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium [2-sulfonatoethyl] MTS (MTSES). Modification of these cysteines leads to changes in the open channel current-voltage relationship at both the macroscopic and single-channel current levels that reflect specific, charge-dependent effects on the rate of Cl(-) permeation through the channel from the external solution. This approach therefore identifies amino acid side chains that lie within the permeation pathway. Cysteine mutagenesis of pore-lining residues also affects intrapore anion binding and anion selectivity, giving more information regarding the roles of these residues. Our results demonstrate a straightforward method of screening for pore-lining amino acids in ion channels. We suggest that TM11 contributes to the CFTR pore and that the extracellular loop between TMs 11 and 12 lies close to the outer mouth of the pore.
Comments [show]
None has been submitted yet.
No. Sentence Comment
177 Effects of external MTS reagents on the gating of CFTR cysteine mutants (I331C, L333C) have been described (Beck et al. 2008) but would presumably not be noticed using our experimental approach.
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ABCC7 p.Leu333Cys 19381710:177:80
status: NEW[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.
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.
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ABCC7 p.Leu333Cys 19754156:52:315
status: NEW130 Figure 2 contains an example of results obtained with L333C CFTR on a wt background (L333C/wt).
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ABCC7 p.Leu333Cys 19754156:130:54
status: NEWX
ABCC7 p.Leu333Cys 19754156:130:85
status: NEW136 FIGURE 2: Functional impact of covalent labeling of L333C CFTR was charge-independent.
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ABCC7 p.Leu333Cys 19754156:136:52
status: NEW140 Reactions of MTSETþ and MTSES- with L333C/wt CFTR channels were reversed by exposure to DTT, but the rate of reversal was slower in the case of MTSES- -modified channels, as expected due to the unfavorable electrostatic interaction between the negatively charged adduct and the anionic DTT.
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ABCC7 p.Leu333Cys 19754156:140:41
status: NEW271 Beck et al. (9) reported that modification of I331C or L333C CFTR channels results in a profound reduction in open probability, and the authors suggested that these sites may experience significant movement during the gating cycle.
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ABCC7 p.Leu333Cys 19754156:271:55
status: NEW[hide] Alternating access to the transmembrane domain of ... J Biol Chem. 2012 Mar 23;287(13):10156-65. Epub 2012 Feb 1. Wang W, Linsdell P
Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7).
J Biol Chem. 2012 Mar 23;287(13):10156-65. Epub 2012 Feb 1., [PMID:22303012]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.
Comments [show]
None has been submitted yet.
No. Sentence Comment
146 The rate of modification of L333C and K335C, also at the extracellular end of TM6, was also decreased in an E1371Q background, suggesting slower modification of open, compared with closed channels (26).
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ABCC7 p.Leu333Cys 22303012:146:28
status: NEW147 However, these cysteines in the outer pore region (L333C, R334C, and K335C) are not modified by intracellular MTS reagents under any conditions (17, 27), suggesting that unlike T338C they cannot move to a position that is accessible to large cytoplasmic substances.
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ABCC7 p.Leu333Cys 22303012:147:51
status: NEW152 L102C, like T338C, becomes apparently more accessible to internal cysteine reactive reagents in open channels (Fig. 6B), but is inaccessible to extracellular MTSES (Fig. FIGURE 4.
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ABCC7 p.Leu333Cys 22303012:152:28
status: NEW153 Rate of modification of T338C by external MTSES.
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ABCC7 p.Leu333Cys 22303012:153:51
status: NEW[hide] Probing the structural and functional domains of t... J Bioenerg Biomembr. 1997 Oct;29(5):453-63. Akabas MH, Cheung M, Guinamard R
Probing the structural and functional domains of the CFTR chloride channel.
J Bioenerg Biomembr. 1997 Oct;29(5):453-63., [PMID:9511930]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) forms an anion-selective channel involved in epithelial chloride transport. Recent studies have provided new insights into the structural determinants of the channel's functional properties, such as anion selectivity, single-channel conductance, and gating. Using the scanning-cysteine-accessibility method we identified 7 residues in the M1 membrane-spanning segment and 11 residues in and flanking the M6 segment that are exposed on the water-accessible surface of the protein; many of these residues may line the ion-conducting pathway. The pattern of the accessible residues suggests that these segments have a largely alpha-helical secondary structure with one face exposed in the channel lumen. Our results suggest that the residues at the cytoplasmic end of the M6 segment loop back into the channel, narrowing the lumen, and thereby forming both the major resistance to ion movement and the charge-selectivity filter.
Comments [show]
None has been submitted yet.
No. Sentence Comment
115 The ratio of the reaction rates of MTSES- /MTSET+ with L333C, the most extracellular residue tested, is also 0.08, suggesting that there is no charge selectivity for the movement of the MTS reagents from the extracellular solution to L333C.
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ABCC7 p.Leu333Cys 9511930:115:55
status: NEWX
ABCC7 p.Leu333Cys 9511930:115:234
status: NEW116 Thus, in order to normalize for the difference in the intrinsic reaction rates of the two MTS reagents we divide the ratio of the rates at a given residue by the ratio of the reaction rates with L333C.
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ABCC7 p.Leu333Cys 9511930:116:195
status: NEW148 The lack of significant electrical distance to the residues from L333C to S341C (Fig. 3A) implies that the MTS reagents do not pass through the electrical field to reach these residues.
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ABCC7 p.Leu333Cys 9511930:148:65
status: NEW[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.
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).
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ABCC7 p.Leu333Cys 9089437:107:753
status: NEW112 Furthermore, the voltage dependence of the rates of reaction of MTSES- is opposite to the voltage t a b l e i i Second-order Rate Constants at Holding Potential ϭ 0 mV and Ratios of Rate Constants at V ϭ 0 mV (kmtses/kmtset) 1 2* k0,MTSES 3* k0,MTSET 4‡ k0,MTSES 5§ kES/kET Mutant (M-1 S-1) (M-1 S-1) k0,MTSET L333C 2-MEʈ 4300 51000 0.08 1 L333 68 828 0.08 1 R334 53 376 0.14 2 K335 47 455 0.1 1 F337 96 991 0.1 1 S341 72 727 0.1 1 T351 250 219 1.14 14 R352 48 324 0.15 2 Q353 213 102 2.09 26 *Columns 2, 3 are the rate constants for MTSES- and MTSETϩ at V ϭ 0 mV.
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ABCC7 p.Leu333Cys 9089437:112:334
status: NEW115 The same ratio is observed for the rates of reaction with L333C indicating that there is no charge selectivity for the reaction with this residue.
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ABCC7 p.Leu333Cys 9089437:115:58
status: NEW116 §Column 5 is the anion to cation selectivity ratio derived by dividing the data in Column 4 by the ratio obtained for the rates of reaction with L333C.
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ABCC7 p.Leu333Cys 9089437:116:150
status: NEW117 The ratio of the rate constants (kmtses/kmtset) for reaction with an exposed cysteine relative to the ratio of the rate constants for the reaction with L333C gives a measure of the anion to cation selectivity at the level of an exposed cysteine.
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ABCC7 p.Leu333Cys 9089437:117:152
status: NEW134 Note that the electrical distance to the residues from L333C to S341C is close to zero and that the electrical distance to R352C is smaller than the electrical distance to the adjacent residues.
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ABCC7 p.Leu333Cys 9089437:134:55
status: NEW155 lar residue tested, L333C, the ratio of the rates of reaction of MTSES-/MTSETϩ is also 0.08 (Table II, column 4); this suggests that there is no charge selectivity for access of the MTS reagents to this residue from the extracellular solution.
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ABCC7 p.Leu333Cys 9089437:155:20
status: NEW156 To account for this difference in the intrinsic rates of reaction of the two MTS reagents, we divided the ratio of the rates at a given residue by the ratio of the rates of reaction with L333C.
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ABCC7 p.Leu333Cys 9089437:156:187
status: NEW[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.
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.
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ABCC7 p.Leu333Cys 8744306:91:330
status: NEW109 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.
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ABCC7 p.Leu333Cys 8744306:109:191
status: NEW90 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.
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ABCC7 p.Leu333Cys 8744306:90:330
status: NEW108 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.
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ABCC7 p.Leu333Cys 8744306:108:191
status: NEW