ABCC7 p.Lys95Cys
Predicted by SNAP2: | A: D (75%), C: D (75%), D: D (91%), E: D (85%), F: D (85%), G: D (85%), H: D (53%), I: D (80%), L: D (80%), M: D (75%), N: D (80%), P: D (91%), Q: D (75%), R: N (66%), S: D (63%), T: D (80%), V: D (80%), W: D (91%), Y: D (71%), |
Predicted by PROVEAN: | A: N, C: D, D: N, E: N, F: D, G: D, H: N, I: D, L: D, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: D, |
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[hide] Location of a common inhibitor binding site in the... J Biol Chem. 2005 Mar 11;280(10):8945-50. Epub 2005 Jan 5. Linsdell P
Location of a common inhibitor binding site in the cytoplasmic vestibule of the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Biol Chem. 2005 Mar 11;280(10):8945-50. Epub 2005 Jan 5., 2005-03-11 [PMID:15634668]
Abstract [show]
Chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is inhibited by a broad range of organic anions that enter the channel pore from its cytoplasmic end, physically occluding the Cl- permeation pathway. These open channel blocker molecules are presumed to bind within a relatively wide pore inner vestibule that shows little discrimination between different large anions. The present study uses patch clamp recording to identify a pore-lining lysine residue, Lys-95, that acts to attract large blocker molecules into this inner vestibule. Mutations that remove the fixed positive charge associated with this amino acid residue dramatically weaken the blocking effects of five structurally unrelated open channel blockers (glibenclamide, 4,4'-dinitrostilbene-2,2'-disulfonic acid, lonidamine, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and taurolithocholate-3-sulfate) when applied to the cytoplasmic face of the membrane. Mutagenesis of Lys-95 also induced amino acid side chain charge-dependent rectification of the macroscopic current-voltage relationship, consistent with the fixed positive charge on this residue normally acting to attract Cl- ions from the intracellular solution into the pore. These results identify Lys-95 as playing an important role in attracting permeant anions into the channel pore inner vestibule, probably by an electrostatic mechanism. This same electrostatic attraction mechanism also acts to attract larger anionic molecules into the relatively wide inner vestibule, where these substances bind to block Cl- permeation. Thus, structurally diverse open channel blockers of CFTR appear to share a common molecular mechanism of action that involves interaction with a positively charged amino acid side chain located in the inner vestibule of the pore.
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No. Sentence Comment
91 These results, using a number of different amino acid substitutions of Lys-95, strongly suggest that side chain charge at this position is important in controlling the apparent affinity of glibenclamide block; the apparent Kd at -100 mV was not affected in the charge-conservative K95R but was significantly increased in charge-neutralizing mutants (K95A, K95C, K95Q) and most strongly increased in the charge-reversing K95E mutant.
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ABCC7 p.Lys95Cys 15634668:91:356
status: NEW[hide] Regulation of conductance by the number of fixed p... J Gen Physiol. 2010 Mar;135(3):229-45. Epub 2010 Feb 8. Zhou JJ, Li MS, Qi J, Linsdell P
Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore.
J Gen Physiol. 2010 Mar;135(3):229-45. Epub 2010 Feb 8., [PMID:20142516]
Abstract [show]
Rapid chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is dependent on the presence of fixed positive charges in the permeation pathway. Here, we use site-directed mutagenesis and patch clamp recording to show that the functional role played by one such positive charge (K95) in the inner vestibule of the pore can be "transplanted" to a residue in a different transmembrane (TM) region (S1141). Thus, the mutant channel K95S/S1141K showed Cl(-) conductance and open-channel blocker interactions similar to those of wild-type CFTR, thereby "rescuing" the effects of the charge-neutralizing K95S mutation. Furthermore, the function of K95C/S1141C, but not K95C or S1141C, was inhibited by the oxidizing agent copper(II)-o-phenanthroline, and this inhibition was reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between these two introduced cysteine side chains. These results suggest that the amino acid side chains of K95 (in TM1) and S1141 (in TM12) are functionally interchangeable and located closely together in the inner vestibule of the pore. This allowed us to investigate the functional effects of increasing the number of fixed positive charges in this vestibule from one (in wild type) to two (in the S1141K mutant). The S1141K mutant had similar Cl(-) conductance as wild type, but increased susceptibility to channel block by cytoplasmic anions including adenosine triphosphate, pyrophosphate, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and Pt(NO(2))(4)(2-) in inside-out membrane patches. Furthermore, in cell-attached patch recordings, apparent voltage-dependent channel block by cytosolic anions was strengthened by the S1141K mutation. Thus, the Cl(-) channel function of CFTR is maximal with a single fixed positive charge in this part of the inner vestibule of the pore, and increasing the number of such charges to two causes a net decrease in overall Cl(-) transport through a combination of failure to increase Cl(-) conductance and increased susceptibility to channel block by cytosolic substances.
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No. Sentence Comment
17 Furthermore, the function of K95C/S1141C, but not K95C or S1141C, was inhibited by the oxidizing agent copper(II)-o-phenanthroline, and this inhibition was reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between these two introduced cysteine side chains.
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ABCC7 p.Lys95Cys 20142516:17:29
status: NEWX
ABCC7 p.Lys95Cys 20142516:17:50
status: NEW131 (B) Mean fractional current remaining after the addition of CuPhe as a function of voltage in wild type (), K95C (), S1141C (), and K95C/S1141C ().
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ABCC7 p.Lys95Cys 20142516:131:116
status: NEWX
ABCC7 p.Lys95Cys 20142516:131:156
status: NEW132 Data values for K95C/S1141C were significantly different from wild type, K95C, or S1141C (P < 0.05 in each case) at all voltages examined.
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ABCC7 p.Lys95Cys 20142516:132:16
status: NEWX
ABCC7 p.Lys95Cys 20142516:132:73
status: NEW133 (C) Mean fractional current remaining after the addition of CuPhe at +80 mV for different channel variants as indicated, and for K95C/S1141C after washing with normal bath solution (wash) or with bath solution supplemented with 5 mM DTT (wash + DTT).
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ABCC7 p.Lys95Cys 20142516:133:129
status: NEW135 (D) Example leak-subtracted macroscopic I-V relationships for cys-less K95C/S1141C-CFTR recorded under the same conditions as in A.
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ABCC7 p.Lys95Cys 20142516:135:71
status: NEW144 In contrast, each of the mutants, K95C, S341C, and S1141C (all in a cys-less background), was strongly sensitive to both MTSES and MTSET.
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ABCC7 p.Lys95Cys 20142516:144:34
status: NEW146 In contrast, MTSET inhibited currents carried by cys-less S341C and cys-less S1141C, but potentiated currents carried by cys-less K95C.
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ABCC7 p.Lys95Cys 20142516:146:130
status: NEW148 In contrast, in K95C, deposition of positive charge by reaction with MTSET may replace the function of the positive charge at this site that is lost as a consequence of the K95C mutation.
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ABCC7 p.Lys95Cys 20142516:148:16
status: NEWX
ABCC7 p.Lys95Cys 20142516:148:173
status: NEW149 Consistent with this idea, MTSET modification converts the cys-less K95C I-V relationship from outwardly rectified (before modification) to linear or mildly inwardly rectified after modification (Fig. 4 B).
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ABCC7 p.Lys95Cys 20142516:149:68
status: NEW152 The strong reactivity of cys-less K95C, S341C, and S1141C to intracellular MTSES and MTSET is consistent with the cysteine side chains introduced at these positions being exposed within the aqueous inner vestibule of the pore.
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ABCC7 p.Lys95Cys 20142516:152:34
status: NEW154 The K95C/S1141C double mutant generated small macroscopic currents that showed outward rectification under symmetrical Cl concentration conditions (Fig. 3), as observed with all mutations that remove the charge at K95 (Linsdell, 2005), including K95C (Fig. 3).
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ABCC7 p.Lys95Cys 20142516:154:4
status: NEWX
ABCC7 p.Lys95Cys 20142516:154:254
status: NEW155 K95C/S1141C currents in inside-out patches were insensitive to the application of 5 mM of the reducing agent dithiothreitol (DTT; not depicted), suggesting that spontaneous disulfide bond formation between the two cysteine side chains is either negligible or without functional consequence.
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ABCC7 p.Lys95Cys 20142516:155:0
status: NEW156 However, the oxidizing reagent CuPhe, which has been used to induce disulfide bond formation between introduced cysteines in other parts of the CFTR protein (Mense et al., 2006; Loo et al., 2008; Serohijos et al., 2008), led to a strong reduction in current amplitude in K95C/ S1141C (Fig. 3, A-C), suggesting a functional modification of the protein.
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ABCC7 p.Lys95Cys 20142516:156:271
status: NEW158 Interestingly, neither wild-type CFTR currents nor the single mutants K95C or S1141C appeared sensitive to CuPhe under these conditions (Fig. 3, A-C), consistent with this agent acting by causing cross-linking of the two cysteine side chains introduced at these positions.
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ABCC7 p.Lys95Cys 20142516:158:70
status: NEW159 Inhibition of K95C/S1141C by CuPhe was only partially reversed by washing; however, the degree of reversibility was significantly enhanced by the inclusion of 5 mM DTT in the wash solution (Fig. 3 C), consistent with CuPhe inhibition reflecting some oxidative process.
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ABCC7 p.Lys95Cys 20142516:159:14
status: NEW161 A K95C/S341C double mutant did not yield functional currents in inside-out patches either without or after treatment with 5 mM DTT.
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ABCC7 p.Lys95Cys 20142516:161:2
status: NEW162 Although these results suggest that K95C can be cross-linked to S1141C, they are potentially confounded by the presence of endogenous cysteine side chains in the CFTR protein.
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ABCC7 p.Lys95Cys 20142516:162:36
status: NEW164 As shown in Fig. 3 D, cys-less K95C/S1141C also generated small, outwardly rectified currents in inside-out membrane patches that showed the same apparent sensitivity to CuPhe as that described above for these mutations in a wild-type background.
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ABCC7 p.Lys95Cys 20142516:164:31
status: NEW165 On average, the application of CuPhe reduced current amplitude in cys-less K95C/S1141C by 84.7 ± 5.2% at +80 mV (n = 5).
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ABCC7 p.Lys95Cys 20142516:165:75
status: NEW167 K95C/S1141C channel investigated the S1141K mutant at the macroscopic current level using depolarizing voltage ramp protocols like those used in Fig. 1, it became apparent that channel function had been altered in a way we had not anticipated from our initial single-channel experiments (see Fig. 2).
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ABCC7 p.Lys95Cys 20142516:167:0
status: NEW260 Furthermore, we suggest that irreversible inhibition of channel function in the K95C/S1141C double mutant by the oxidizing agent CuPhe (Fig. 3) most likely reflects formation of a disulfide bridge between these two pore-lining cysteine side chains (Fig. 4).
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ABCC7 p.Lys95Cys 20142516:260:80
status: NEW[hide] Functional arrangement of the 12th transmembrane r... Pflugers Arch. 2011 Oct;462(4):559-71. Epub 2011 Jul 28. Qian F, El Hiani Y, Linsdell P
Functional arrangement of the 12th transmembrane region in the CFTR chloride channel pore based on functional investigation of a cysteine-less CFTR variant.
Pflugers Arch. 2011 Oct;462(4):559-71. Epub 2011 Jul 28., [PMID:21796338]
Abstract [show]
The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) alpha-helices, arranged into two pseudo-symmetrical groups of six. While TM6 in the N-terminal TMs is known to line the pore and to make an important contribution to channel properties, much less is known about its C-terminal counterpart, TM12. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of TM12 in a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM12 residues N1138, M1140, S1141, T1142, Q1144, W1145, V1147, N1148, and S1149 when applied to the cytoplasmic side of open channels. Cysteines sensitive to internal MTS reagents were not modified by extracellular [2-(trimethylammonium)ethyl] MTS, consistent with MTS reagent impermeability. Both S1141C and T1142C could be modified by intracellular [2-sulfonatoethyl] MTS prior to channel activation; however, N1138C and M1140C, located deeper into the pore from its cytoplasmic end, were modified only after channel activation. Comparison of these results with previous work on CFTR-TM6 allows us to develop a model of the relative positions, functional contributions, and alignment of these two important TMs lining the CFTR pore. We also propose a mechanism by which these seemingly structurally symmetrical TMs make asymmetric contributions to the functional properties of the channel pore.
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No. Sentence Comment
140 In this respect, the slow rate of modification observed in N1138C (Fig. 3b) is similar to that we reported for P99C and L102C in TM1 [41] and T338C and S341C in TM6 [9], and the much higher modification rate constant for T1142C, S1141C, and (to a lesser extent) M1140C is closer to that reported for K95C in TM1 [41] and I344C, V345C, and M348C in TM6 [9].
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ABCC7 p.Lys95Cys 21796338:140:300
status: NEW151 Thus, a disulfide bridge can be formed between K95C in TM1 and S1141C in TM12, suggesting that the β carbon distance is in the range of ~5-8 Å for these two introduced cysteines [45].
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ABCC7 p.Lys95Cys 21796338:151:47
status: NEW[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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No. Sentence Comment
112 On mutants K95C and K335C interact with methanethiosulfo- the basis of these data, the minimum diameter of the nate (MTS) reagents, and mutations that eliminate the CFTR pore was estimated to be Ç5.3 A˚ (77), similar to positive charge at K335 reduce single-channel conduc- that reported for other Cl0 channels (10, 20, 55).
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ABCC7 p.Lys95Cys 9922375:112:11
status: NEW[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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No. Sentence Comment
475 In three TM1 mutants, G91C, K95C, Q98C, all of which fall on the same face of a predicted TM1 a- ter`` is close to the cytoplasmic end of the pore and that R352 may play a role in determining charge selectivity forhelix, the conductance was irreversibly altered by either MTSES0 or MTSEA0 .
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ABCC7 p.Lys95Cys 9922376:475:28
status: NEW[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.
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No. Sentence Comment
130 Beck et al., (2008) also studied R334C CFTR channels expressed in Xenopus oocytes using a protocol similar to that employed here, but failed to detect increased reactivity toward externally-applied MTSEA+ in the activated state.
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ABCC7 p.Lys95Cys 22923500:130:10
status: NEW158 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.
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ABCC7 p.Lys95Cys 22923500:158:312
status: NEW[hide] Conformational change opening the CFTR chloride ch... Biochim Biophys Acta. 2012 Mar;1818(3):851-60. Epub 2012 Jan 2. Wang W, Linsdell P
Conformational change opening the CFTR chloride channel pore coupled to ATP-dependent gating.
Biochim Biophys Acta. 2012 Mar;1818(3):851-60. Epub 2012 Jan 2., [PMID:22234285]
Abstract [show]
Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are controlled by ATP binding and hydrolysis by its nucleotide binding domains (NBDs). This is presumed to control opening of a single "gate" within the permeation pathway, however, the location of such a gate has not been described. We used patch clamp recording to monitor access of cytosolic cysteine reactive reagents to cysteines introduced into different transmembrane (TM) regions in a cysteine-less form of CFTR. The rate of modification of Q98C (TM1) and I344C (TM6) by both [2-sulfonatoethyl] methanethiosulfonate (MTSES) and permeant Au(CN)(2)(-) ions was reduced when ATP concentration was reduced from 1mM to 10muM, and modification by MTSES was accelerated when 2mM pyrophosphate was applied to prevent channel closure. Modification of K95C (TM1) and V345C (TM6) was not affected by these manoeuvres. We also manipulated gating by introducing the mutations K464A (in NBD1) and E1371Q (in NBD2). The rate of modification of Q98C and I344C by both MTSES and Au(CN)(2)(-) was decreased by K464A and increased by E1371Q, whereas modification of K95C and V345C was not affected. These results suggest that access from the cytoplasm to K95 and V345 is similar in open and closed channels. In contrast, modifying ATP-dependent channel gating alters access to Q98 and I344, located further into the pore. We propose that ATP-dependent gating of CFTR is associated with the opening and closing of a gate within the permeation pathway at the level of these pore-lining amino acids.
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No. Sentence Comment
4 Modification of K95C (TM1) and V345C (TM6) was not affected by these manoeuvres.
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ABCC7 p.Lys95Cys 22234285:4:16
status: NEW6 The rate of modification of Q98C and I344C by both MTSES and Au(CN)2 - was decreased by K464A and increased by E1371Q, whereas modification of K95C and V345C was not affected.
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ABCC7 p.Lys95Cys 22234285:6:143
status: NEW52 Example timecourses of macroscopic currents (measured at -50 mV during brief voltage excursions from a holding potential of 0 mV) carried by K95C, Q98C, I344C and V345C as indicated, in inside-out membrane patches. Current amplitudes were measured every 6 s following attainment of stable current amplitude after channel activation. Channels were activated with PKA (20 nM) and either a high concentration of ATP (1 mM; in (A) and (C)-(E)) or a low concentration of ATP (10 μM; (B)).
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ABCC7 p.Lys95Cys 22234285:52:141
status: NEW55 In each panel, MTSES (20 μM for K95C, I344C and V345C, and 200 μM for Q98C; see Materials and methods) was applied to the cytoplasmic face of the patch at time zero (as indicated by the hatched bar at the bottom of each panel).
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ABCC7 p.Lys95Cys 22234285:55:38
status: NEW60 Additional mutations were introduced into the cys-less background using the QuikChange site-directed mutagenesis -200 -150 -100 -50 0 I (pA) Time (s) K95C A) 1 mM ATP -180 -120 -60 0 Q98C -400 -300 -200 -100 0 -200 -150 -100 -50 0 I344C -300 -200 -100 0 -500 -400 -300 -200 -100 0 V345C -250 -200 -150 -100 -50 0 -300 -200 -100 0 -600 -400 -200 0 -750 -500 -250 0 -600 -400 -200 0 -800 -600 -400 -200 0 I (pA) Time (s) 20 µM MTSES 20 µM MTSES 20 µM MTSES200 µM MTSES C) 1 mM ATP + 2 mM PPi E) E1371Q (1 mM ATP) I (pA) Time (s) D) K464A (1 mM ATP) B) 10 µM ATP -100 -75 -50 -25 0 -200 -150 -100 -50 0 -80 -60 -40 -20 0 -80 -60 -40 -20 0 -120 -90 -60 -30 0 -80 -60 -40 -20 0 -60 -40 -20 0 -300 -200 -100 0 I (pA) Time (s) I (pA) Time (s) 0 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 system (Agilent Technologies, Santa Clara, CA, USA) and verified by DNA sequencing.
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ABCC7 p.Lys95Cys 22234285:60:150
status: NEW91 As described previously, at K95C and Q98C (TM1) and at I344C and V345C (TM6), current amplitude is decreased by treatment 100 1000 10000 1 mM ATP 10 µM ATP 1 mM ATP + 2 mM PPi K95C Q98C I344C V345C * * ModificationRateConstant(M-1 s-1 )ModificationRateConstant(M-1 s-1 ) A B K95C Q98C I344C V345C 100 1000 10000 Cys-less +K464A +E1371Q * * * * * * Fig. 3.
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ABCC7 p.Lys95Cys 22234285:91:28
status: NEWX
ABCC7 p.Lys95Cys 22234285:91:181
status: NEWX
ABCC7 p.Lys95Cys 22234285:91:280
status: NEW100 Inspection of these example timecourses indicates that, while such manipulations have no effect on the rate of modification in K95C or V345C, the rate of modification is altered in both Q98C and I344C (Fig. 2A-C).
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ABCC7 p.Lys95Cys 22234285:100:127
status: NEW101 Quantification of the mean modification rate constant (as described in Materials and methods) demonstrates that decreasing ATP concentration from 1 mM (Fig. 2A) to 10 μM (Fig. 2B) to decrease channel opening rate significantly decreases the rate of modification in Q98C and I344C (~2.0-fold decrease in modification rate constant; Pb0.01), whereas the rate of modification of K95C and V345C was apparently unaffected (P>0.2) (Fig. 3A).
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ABCC7 p.Lys95Cys 22234285:101:382
status: NEW102 Conversely, treatment with PPi (2 mM; Fig. 2C) to inhibit channel closure and increase open probability significantly increases the rate of modification in Q98C and I344C (2.5-2.8-fold increase in modification rate constant; Pb0.01) but has no effect on the rate of modification of K95C and V345C (P>0.4) (Fig. 3A).
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ABCC7 p.Lys95Cys 22234285:102:282
status: NEW103 These results suggest that pharmacological manipulation of NBD function results in changes in the accessibility of Q98C and I344C-but not K95C or V345C-to cytoplasmic MTSES.
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ABCC7 p.Lys95Cys 22234285:103:138
status: NEW111 The K464A mutation significantly decreased the rate of MTSES modification at Q98C and I344C (2.5-2.9-fold decrease in modification rate constant; Pb0.005) but had no effect on the rate of modification at K95C or V345C (P>0.5) (Fig. 3B).
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ABCC7 p.Lys95Cys 22234285:111:204
status: NEW112 Conversely, the E1371Q mutation significantly increased the rate of MTSES modification at Q98C and I344C (3.0-3.1-fold increase in modification rate constant; Pb0.02) but had no effect on the rate of modification at K95C or V345C (P>0.25) (Fig. 3B).
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ABCC7 p.Lys95Cys 22234285:112:216
status: NEW113 These results therefore suggest that altering NBD function non-pharmacologically by mutagenesis alters accessibility of Q98C and I344C to cytoplasmic MTSES, whereas accessibility of K95C and V345C are unaffected by NBD-driven channel gating.
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ABCC7 p.Lys95Cys 22234285:113:182
status: NEW145 In fact, we found that currents carried by K95C, Q98C, and I344C were potently inhibited by much lower concentrations of Au(CN)2 - (200 nM-2 μM; Fig. 6).
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ABCC7 p.Lys95Cys 22234285:145:43
status: NEW146 Both K95C and I344C were rapidly inhibited by 200 nM Au(CN)2 - (Fig. 6), reflecting a high modification rate constant (Fig. 7).
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ABCC7 p.Lys95Cys 22234285:146:5
status: NEW150 We therefore compared the rate of Au(CN)2 - inhibition in K95C, Q98C and I344C at two different ATP concentrations (10 μM and 1 mM), as well as in channels also bearing the NBD mutations K464A or E1371Q (Fig. 6).
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ABCC7 p.Lys95Cys 22234285:150:58
status: NEW151 Quantification of the mean modification rate constant demonstrated that decreasing ATP -300 -200 -100 0 -400 -300 -200 -100 0 -200 -150 -100 -50 0 -60 -40 -20 0 -120 -80 -40 0 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 -100 -75 -50 -25 0 -90 -60 -30 0 -90 -60 -30 0 -120 -80 -40 0 A) 1 mM ATP I (pA) I (pA) I (pA) Time (s) 200 nM Au(CN)2 C) K464A (1 mM ATP) D) E1371Q (1 mM ATP) 2 µM Au(CN)2 200 nM Au(CN)2 I (pA) Time (s) Time (s) -200 -150 -100 -50 0 -90 -60 -30 0 -120 -80 -40 0 K95C Q98C I344C B) 10 µM ATP Time (s) Fig. 6. Timecourse of modification by Au(CN)2 - .
X
ABCC7 p.Lys95Cys 22234285:151:600
status: NEW153 Reporter cysteines (K95C, Q98C, and I344C as indicated) were examined in isolation (A, B) or combined with the NBD mutations K464A (C) or E1371Q (D).
X
ABCC7 p.Lys95Cys 22234285:153:20
status: NEW154 In each panel, Au(CN)2 - (200 nM for K95C and I344C, and 2 μM for Q98C; see Materials and methods) was applied to the cytoplasmic face of the patch at time zero (as indicated by the hatched bar at the bottom of each panel).
X
ABCC7 p.Lys95Cys 22234285:154:37
status: NEW156 concentration to 10 μM significantly decreased the rate of Au(CN)2 - modification of Q98C and I344C (1.7-1.8-fold decrease in modification rate constant; Pb0.005) but had no effect on the rate of modification at K95C (P>0.4) (Fig. 7).
X
ABCC7 p.Lys95Cys 22234285:156:218
status: NEW157 In addition, the K464A mutation significantly decreased the rate of Au(CN)2 - modification of Q98C and I344C (1.4-1.5-fold decrease in modification rate constant with 1 mM ATP; Pb0.005) but had no effect on the rate of modification at K95C (P>0.5) (Fig. 7).
X
ABCC7 p.Lys95Cys 22234285:157:235
status: NEW158 Conversely, the E1371Q mutation significantly increased the rate of Au(CN)2 - modification at Q98C and I344C (2.8-3.5-fold increase in modification rate constant; Pb0.01) but had no effect on the rate of modification at K95C (P>0.2) (Fig. 7).
X
ABCC7 p.Lys95Cys 22234285:158:220
status: NEW165 For two introduced cysteine residues-K95C in TM1 and V345C in TM6-the rate of modification by cytoplasmic reagents was independent of ATP-dependent channel gating (Figs. 3, 6), suggesting that access to these residues is similar both in open channels and in closed channels.
X
ABCC7 p.Lys95Cys 22234285:165:37
status: NEW173 As pointed out above, the lack of apparent state-dependence of modification in K95C and V345C suggests that the rate of modification at these sites is similar in closed channels.
X
ABCC7 p.Lys95Cys 22234285:173:79
status: NEW201 While K95C, Q98C and I344C were rapidly inhibited by low concentrations of cytoplasmic Au(CN)2 - (Fig. 6), V345C showed similar Au(CN)2 - sensitivity as cys-less CFTR (data not shown).
X
ABCC7 p.Lys95Cys 22234285:201:6
status: NEW[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.
No. Sentence Comment
19 Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation.
X
ABCC7 p.Lys95Cys 21746847:19:5
status: NEW22 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.
X
ABCC7 p.Lys95Cys 21746847:22:39
status: NEW71 In contrast, macroscopic currents carried by four mutants, K95C, Q98C, P99C, and L102C, were found to be significantly and rapidly sensitive to the application of both MTSES and MTSET (Figs. 1-3).
X
ABCC7 p.Lys95Cys 21746847:71:59
status: NEW96 Fig. S2 shows the lack of sensitivity to the reducing agent DTT of macroscopic currents carried by the double-cysteine mutant channels K95C/ I344C and Q98C/I344C.
X
ABCC7 p.Lys95Cys 21746847:96:135
status: NEW98 As shown in Fig. 3 A, MTSES modification was rapid in K95C, even when a low concentration of MTSES (20 µM) was used, and considerably slower in L102C (using 200 µM MTSES).
X
ABCC7 p.Lys95Cys 21746847:98:54
status: NEW105 (B) Example leak-subtracted I-V relationships for cys-less CFTR, K95C, Q98C, P99C, L102C, and R104C, recorded from inside-out membrane patches after maximal channel activation with 20 nM PKA, 1 mM ATP, and 2 mM PPi.
X
ABCC7 p.Lys95Cys 21746847:105:65
status: NEW112 As shown in Fig. 4 A, patches excised from MTSET-pretreated cells expressing K95C, Q98C, P99C, or L102C all gave macroscopic currents that were increased in amplitude after the addition of 2 mM constants was that modification was faster for cysteines introduced closer to the intracellular end of TM1, and slower for cysteines located more deeply along the axis of TM1 (Fig. 3 B).
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ABCC7 p.Lys95Cys 21746847:112:77
status: NEW123 (A) Example time courses of macroscopic currents (measured at 50 mV during brief voltage excursions from a holding potential of 0 mV) carried by K95C (left) and L102C (right) as indicated, in inside-out membrane patches.
X
ABCC7 p.Lys95Cys 21746847:123:153
status: NEW125 In each case, MTSES (20 µM for K95C and 200 µM for L102C) was applied to the cytoplasmic face of the patch at time zero (as indicated by the hatched bar at the bottom of each panel).
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ABCC7 p.Lys95Cys 21746847:125:36
status: NEW128 Asterisks indicate a significant difference from MTSES modification of K95C (P < 0.005), and daggers indicate a significant difference from MTSES modification of the same mutant (P < 0.05).
X
ABCC7 p.Lys95Cys 21746847:128:71
status: NEW136 Whereas K95C channels were again rendered insensitive to a test exposure to MTSES, again consistent with them having been covalently modified during pretreatment, currents carried by Q98C, P99C, MTSET to the intracellular solution.
X
ABCC7 p.Lys95Cys 21746847:136:8
status: NEW138 These results suggest that none of K95C, Q98C, P99C, or L102C can be modified covalently by extracellular MTSET.
X
ABCC7 p.Lys95Cys 21746847:138:35
status: NEW141 We used a similar approach to determine if K95C, Q98C, P99C, and L102C could be modified by MTSES pretreatment.
X
ABCC7 p.Lys95Cys 21746847:141:43
status: NEW148 Proximity and alignment of TMs 1 and 6 The results described in Fig. 5, suggesting that K95C is accessible to cytoplasmic MTSES in nonactivated channels but that Q98C is accessible only in activated channels, imply that K95 and Q98 may lie close to the putative barrier within the pore that we recently proposed to regulate access from the cytoplasmic solution (El Hiani and Linsdell, 2010).
X
ABCC7 p.Lys95Cys 21746847:148:88
status: NEW150 These results, which are summarized quantitatively in Fig. 5 C, suggest that although K95C can be modified by MTSES before channel activation, Q98C, P99C, and L102C are modified by MTSES only very slowly, if at all, in channels that have not been activated by PKA and ATP.
X
ABCC7 p.Lys95Cys 21746847:150:86
status: NEW162 (A-C) Example leak-subtracted I-V relationships for K95C/I344C (A), Q98C/I344C (B), and Q98C/M348C (C) after channel activation with 20 nM PKA and 1 mM ATP.
X
ABCC7 p.Lys95Cys 21746847:162:52
status: NEW166 Note that cys-less CFTR, the single mutants K95C, Q98C, or I344C, and the double mutant Q98C/M348C were all insensitive to CuPhe under these conditions.
X
ABCC7 p.Lys95Cys 21746847:166:44
status: NEW167 Also note that CuPhe had a stronger inhibitory effect on currents carried by K95C/I344C when measured at +80 mV compared with 80 mV; this same apparent voltage dependence was previously reported for K95C/S1141C under similar experimental conditions (Zhou et al., 2010).
X
ABCC7 p.Lys95Cys 21746847:167:77
status: NEWX
ABCC7 p.Lys95Cys 21746847:167:207
status: NEW170 (E) Mean effects of CuPhe (black bars), CuPhe followed by washing with normal bath solution (white bars), and CuPhe followed by DTT (gray bars) on macroscopic current amplitude in K95C/I344C (left) and Q98C/I344C (right) at +80 mV.
X
ABCC7 p.Lys95Cys 21746847:170:180
status: NEW172 Mean of data from three to seven patches is shown in D and E. both K95C/I344C and Q98C/I344C by CuPhe was not reversed by washing CuPhe from the bath; however, partial reversal was seen when 5 mM DTT was applied in the continued presence of CuPhe (Fig. 6, A, B, and E), consistent with CuPhe inhibition of these channels reflecting some oxidative process.
X
ABCC7 p.Lys95Cys 21746847:172:70
status: NEW173 The results shown in Fig. 6 suggest that disulfide bond formation can occur between K95C and I344C and between Q98C and I344C after channel activation.
X
ABCC7 p.Lys95Cys 21746847:173:84
status: NEW175 After patch excision, inside-out patches from cells expressing either K95C/I344C or Q98C/ I344C were treated with cytoplasmic CuPhe for 2 min, after which CuPhe was washed from the bath and currents were activated using PKA and ATP, as usual.
X
ABCC7 p.Lys95Cys 21746847:175:70
status: NEW178 Both K95C/I344C and Q98C/I344C channel currents were also potently inhibited by the addition of Cu2+ ions alone (without phenanthroline) to the bath (Fig. 8).
X
ABCC7 p.Lys95Cys 21746847:178:5
status: NEW180 Each of the single mutants K95C, Q98C, and I344C showed reversible "paired" mutants with one cysteine introduced into each of TM1 (at K95 or Q98) and TM6 (at I344 and V345).
X
ABCC7 p.Lys95Cys 21746847:180:27
status: NEW181 Unfortunately, the double mutants K95C/V345C and Q98C/V345C did not yield functional currents when expressed in BHK cells, even after treatment with DTT to break any possible disulfide bonds; a similar lack of functional expression was previously reported for K95C/S341C (Zhou et al., 2010).
X
ABCC7 p.Lys95Cys 21746847:181:34
status: NEWX
ABCC7 p.Lys95Cys 21746847:181:260
status: NEW182 However, K95C/ I344C, Q98C/I344C, and Q98C/M348C did generate macroscopic PKA- and ATP-dependent currents in inside-out patches.
X
ABCC7 p.Lys95Cys 21746847:182:9
status: NEW184 However, the oxidizing agent CuPhe, which has previously been used to induce disulfide bond formation between introduced cysteines in other parts of the CFTR protein (Mense et al., 2006; Loo et al., 2008; Serohijos et al., 2008; Zhou et al., 2010), led to a strong reduction in current amplitude in both K95C/I344C and Q98C/I344C (Fig. 6).
X
ABCC7 p.Lys95Cys 21746847:184:304
status: NEW185 Neither cys-less CFTR nor the single mutants K95C, Q98C, or I344C appeared sensitive to CuPhe under these conditions (Fig. 6 D), consistent with this agent acting by causing disulfide bond formation between the two introduced cysteine side chains in the double mutants K95C/I344C and Q98C/I344C.
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ABCC7 p.Lys95Cys 21746847:185:45
status: NEWX
ABCC7 p.Lys95Cys 21746847:185:269
status: NEW188 (A and B) Example leak-subtracted I-V relationships for K95C/I344C (A) and Q98C/I344C (B) after channel activation with 20 nM PKA and 1 mM ATP in inside-out patches that had been pretreated with CuPhe for 2 min, and then washed to remove CuPhe.
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ABCC7 p.Lys95Cys 21746847:188:56
status: NEW200 Our results concerning the accessibility of cysteines introduced into TM1 are summarized, and compared inhibition by Cu2+ that was of intermediate potency between the cys-less background and the double mutants K95C/I344C and Q98C/I344C.
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ABCC7 p.Lys95Cys 21746847:200:210
status: NEW228 Thus, the side chains of TM1 mutants K95C, Q98C, P99C, and L102C that we identified as accessible to MTS reagents applied from the inside (Fig. 2) were not accessible to MTSET applied to the outside (Fig. 4), whereas R104C, previously shown to be modified by external MTS reagents (Zhou et al., 2008), was not modified by internal MTSES or MTSET (Fig. 2).
X
ABCC7 p.Lys95Cys 21746847:228:37
status: NEW238 Although we have not investigated the state dependence of MTSES modification in TM1 in such great detail, our present results suggest a similar arrangement in which K95C can readily be modified before channel activation (Fig. 5), whereas Q98C, P99C, and L102C are modified rapidly after channel activation (Fig. 3) but very slowly if at all before activation (Fig. 5).
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ABCC7 p.Lys95Cys 21746847:238:165
status: NEW242 Changes in channel function after the addition of the oxidizing agent CuPhe, that were not reversed by removal of this agent, were taken as evidence for the formation of a disulfide bridge between K95C in TM1 and I344C in TM6, and between Q98C in TM1 and I344C in TM6 (Fig. 6).
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ABCC7 p.Lys95Cys 21746847:242:197
status: NEW256 For comparison, the MTSES modification rate constant for P99C and L102C (Fig. 3) was similar to that of T338C and S341C in TM6 (El Hiani and Linsdell, 2010) (all between 100 and 150 M1 s1 ), and the modification rate constant for K95C was comparable to, or slightly greater than, that of I344C, V345C, and M348C (El Hiani and Linsdell, 2010) (all between 2,000 and 4,000 M1 s1 ).
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ABCC7 p.Lys95Cys 21746847:256:246
status: NEW261 If this is the case, the similar discrepancy between MTSES and MTSET modification rate constants at all sites tested implies that the location of anion-cation discrimination that underlies this discrepancy may lie between the cytoplasmic mouth of the pore and the most accessible cysteine residue, namely, that at K95C.
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ABCC7 p.Lys95Cys 21746847:261:314
status: NEW264 This kind of information is necessary to develop and validate three-dimensional structuralmodelsoftheporeregion.Previously,weshowed that a disulfide bond could be formed between K95C (in TM1) and S1141C (in TM12) (Zhou et al., 2010).
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ABCC7 p.Lys95Cys 21746847:264:178
status: NEW[hide] Amino acid residues lining the chloride channel of... J Biol Chem. 1994 May 27;269(21):14865-8. Akabas MH, Kaufmann C, Cook TA, Archdeacon P
Amino acid residues lining the chloride channel of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 1994 May 27;269(21):14865-8., [PMID:7515047]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator forms a chloride channel that is regulated by phosphorylation and intracellular ATP levels. The structure of the channel-forming domains is undetermined. To identify the residues lining this channel we substituted cysteine, one at a time, for 9 consecutive residues (91-99) in the M1 membrane-spanning segment. The cysteine substitution mutants were expressed in Xenopus oocytes. We determined the accessibility of the engineered cysteine to charged, sulfhydryl-specific methanethiosulfonate reagents added extracellularly. We assume that, among residues in membrane-spanning segments, only those lining the channel will be accessible to react with these hydrophilic reagents and that such a reaction would irreversibly alter conduction through the channel. Only the cysteines substituted for Gly-91, Lys-95, and Gln-98 were accessible to the reagents. We conclude that these residues are in the channel lining. The periodicity of these residues is consistent with an alpha-helical secondary structure.
Comments [show]
None has been submitted yet.
No. Sentence Comment
69 Application of the MTS reagents irreversibly alteredthe CFTR-induced currents of three of the cysteine substitution mutants, G91C, K95C, and Q98C.
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ABCC7 p.Lys95Cys 7515047:69:131
status: NEW72 20 and 3C) and Q98C by 32 2 4% (n= 9) and potentiated the current of the mutant K95C by 108 2 22%(n = 4) (Figs.
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ABCC7 p.Lys95Cys 7515047:72:80
status: NEW75 The anionic reagent, MTSES-, had no effect on the K95C and G91C mutants (Fig. 3,A andB).To determine whether thelack of effect was due to inability to react with Cys-95 or lack of effect followingreaction, we sequentially applied MTSES- and MTSEA+;MTSES- did not prevent the potentiationof the current by MTSEA+(data notshown).
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ABCC7 p.Lys95Cys 7515047:75:50
status: NEW82 The effect ofthe MTS reagentson wild typeCFTR and on thethreechannel-liningmutants GSlC, K95C,and QSSC.
X
ABCC7 p.Lys95Cys 7515047:82:89
status: NEW86 A and C are from oocytesinjectedwith wild type CFTR B, Q98C; D,K95C; E, G91C.
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ABCC7 p.Lys95Cys 7515047:86:63
status: NEW97 The application ofMTSEA' to the mutant K95C increased the CFTR-induced current.
X
ABCC7 p.Lys95Cys 7515047:97:39
status: NEW101 1 MINMTS-EA, 8 MIN C K95C 5 D K95C 5 I -50 0 50 100 150-50 0 50 100 150 CHANGEINCURRENT (X) FIG.3.
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ABCC7 p.Lys95Cys 7515047:101:21
status: NEWX
ABCC7 p.Lys95Cys 7515047:101:30
status: NEW110 Based on the accessibility of the cysteine-substitution mutants G91C,K95C and Q98C to the MTS reagents, we infer that the side chains of the corresponding wild type residues, Gly-91,Lys-95, and Gln-98, line the channel ofCFTR.
X
ABCC7 p.Lys95Cys 7515047:110:69
status: NEW[hide] Tuning of CFTR chloride channel function by locati... Biophys J. 2012 Oct 17;103(8):1719-26. doi: 10.1016/j.bpj.2012.09.020. Epub 2012 Oct 16. El Hiani Y, Linsdell P
Tuning of CFTR chloride channel function by location of positive charges within the pore.
Biophys J. 2012 Oct 17;103(8):1719-26. doi: 10.1016/j.bpj.2012.09.020. Epub 2012 Oct 16., [PMID:23083715]
Abstract [show]
High unitary Cl(-) conductance in the cystic fibrosis transmembrane conductance regulator Cl(-) channel requires a functionally unique, positively charged lysine residue (K95) in the inner vestibule of the channel pore. Here we used a mutagenic approach to investigate the ability of other sites in the pore to host this important positive charge. The loss of conductance observed in the K95Q mutation was >50% rescued by substituting a lysine for each of five different pore-lining amino acids, suggesting that the exact location of the fixed positive charge is not crucial to support high conductance. Moving the positive charge also restored open-channel blocker interactions that are lost in K95Q. Introducing a second positive charge in addition to that at K95 did not increase conductance at any site, but did result in a striking increase in the strength of block by divalent Pt(NO(2))(4)(2-) ions. Based on the site dependence of these effects, we propose that although the exact location of the positive charge is not crucial for normal pore properties, transplanting this charge to other sites results in a diminution of its effectiveness that appears to depend on its location along the axis of the pore.
Comments [show]
None has been submitted yet.
No. Sentence Comment
126 This relative location of amino acids is also supported by experimental evidence that disulfide bonds can be formed between cysteine side chains substituted for K95 and S1141 (8), as well as between K95C and I344C, and between Q98C and I344C (13).
X
ABCC7 p.Lys95Cys 23083715:126:199
status: NEW[hide] Cysteine scanning of CFTR's first transmembrane se... Biophys J. 2013 Feb 19;104(4):786-97. doi: 10.1016/j.bpj.2012.12.048. Gao X, Bai Y, Hwang TC
Cysteine scanning of CFTR's first transmembrane segment reveals its plausible roles in gating and permeation.
Biophys J. 2013 Feb 19;104(4):786-97. doi: 10.1016/j.bpj.2012.12.048., [PMID:23442957]
Abstract [show]
Previous cysteine scanning studies of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have identified several transmembrane segments (TMs), including TM1, 3, 6, 9, and 12, as structural components of the pore. Some of these TMs such as TM6 and 12 may also be involved in gating conformational changes. However, recent results on TM1 seem puzzling in that the observed reactive pattern was quite different from those seen with TM6 and 12. In addition, whether TM1 also plays a role in gating motions remains largely unknown. Here, we investigated CFTR's TM1 by applying methanethiosulfonate (MTS) reagents from both cytoplasmic and extracellular sides of the membrane. Our experiments identified four positive positions, E92, K95, Q98, and L102, when the negatively charged MTSES was applied from the cytoplasmic side. Intriguingly, these four residues reside in the extracellular half of TM1 in previously defined CFTR topology; we thus extended our scanning to residues located extracellularly to L102. We found that cysteines introduced into positions 106, 107, and 109 indeed react with extracellularly applied MTS probes, but not to intracellularly applied reagents. Interestingly, whole-cell A107C-CFTR currents were very sensitive to changes of bath pH as if the introduced cysteine assumes an altered pKa-like T338C in TM6. These findings lead us to propose a revised topology for CFTR's TM1 that spans at least from E92 to Y109. Additionally, side-dependent modifications of these positions indicate a narrow region (L102-I106) that prevents MTS reagents from penetrating the pore, a picture similar to what has been reported for TM6. Moreover, modifications of K95C, Q98C, and L102C exhibit strong state dependency with negligible modification when the channel is closed, suggesting a significant rearrangement of TM1 during CFTR's gating cycle. The structural implications of these findings are discussed in light of the crystal structures of ABC transporters and homology models of CFTR.
Comments [show]
None has been submitted yet.
No. Sentence Comment
11 Moreover, modifications of K95C, Q98C, and L102C exhibit strong state dependency with negligible modification when the channel is closed, suggesting a significant rearrangement of TM1 during CFTR`s gating cycle.
X
ABCC7 p.Lys95Cys 23442957:11:27
status: NEW82 This observed decrease of macroscopic currents is due to covalent modification of the engineered cysteines by the reagent as the effect persisted even after a complete removal of MTSES. Similar observations were made for K95C- and Q98C-CFTR.
X
ABCC7 p.Lys95Cys 23442957:82:221
status: NEW108 We next tested the accessibility to external MTSES on three positions identified by experiments with inside-out patches, namely K95C, Q98C, and L102C, in the same manner and all three positions turned out nonreactive (data not shown).
X
ABCC7 p.Lys95Cys 23442957:108:128
status: NEW152 For Q98C and K95C mutant channels, the increases in the mean current amplitude following MTSET modification were ~2- and 6-fold, respectively.
X
ABCC7 p.Lys95Cys 23442957:152:13
status: NEW153 Because the single-channel conductance was drastically decreased in K95C-CFTR, we were not able to assess the gating effect of MTSET modification.
X
ABCC7 p.Lys95Cys 23442957:153:68
status: NEW165 State-dependent modification of E92C-, K95C-, Q98C-, and L102C-CFTR The observation that MTSET modification of Q98C and L102C alters CFTR gating suggests that TM1 indeed participates in gating motions of CFTR.
X
ABCC7 p.Lys95Cys 23442957:165:39
status: NEW172 However, Fig. 6 A shows a representative recording of current response for K95C-CFTR mutants.
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ABCC7 p.Lys95Cys 23442957:172:75
status: NEW196 Third, in the report by Wang et al. (35), K95C, but not Q98C, P99C, or L102C, can react with internal MTSES even before the channel is activated by PKA and ATP, implying a regulated barrier between positions 95 and 98.
X
ABCC7 p.Lys95Cys 23442957:196:42
status: NEW203 (A) A representative recording for MTSES modification in the absence of ATP for K95C/Cysless channels.
X
ABCC7 p.Lys95Cys 23442957:203:80
status: NEW206 (C) Summary of the modification rates for K95C, Q98C, and L102C in the presence of ATP (solid squares).
X
ABCC7 p.Lys95Cys 23442957:206:42
status: NEW[hide] Metal bridges illuminate transmembrane domain move... J Biol Chem. 2014 Oct 10;289(41):28149-59. doi: 10.1074/jbc.M114.593103. Epub 2014 Aug 20. El Hiani Y, Linsdell P
Metal bridges illuminate transmembrane domain movements during gating of the cystic fibrosis transmembrane conductance regulator chloride channel.
J Biol Chem. 2014 Oct 10;289(41):28149-59. doi: 10.1074/jbc.M114.593103. Epub 2014 Aug 20., [PMID:25143385]
Abstract [show]
Opening and closing of the cystic fibrosis transmembrane conductance regulator are controlled by ATP binding and hydrolysis by the cytoplasmic nucleotide-binding domains. Different conformational changes in the channel pore have been described during channel opening and closing; however, the relative importance of these changes to the process of gating the pore is not known. We have used patch clamp recording to identify high affinity Cd(2+) bridges formed between pairs of pore-lining cysteine residues introduced into different transmembrane alpha-helices (TMs). Seven Cd(2+) bridges were identified forming between cysteines in TMs 6 and 12. Interestingly, each of these Cd(2+) bridges apparently formed only in closed channels, and their formation stabilized the closed state. In contrast, a single Cd(2+) bridge identified between cysteines in TMs 1 and 12 stabilized the channel open state. Analysis of the pattern of Cd(2+) bridge formation in different channel states suggests that lateral separation and convergence of different TMs, rather than relative rotation or translation of different TMs, is the key conformational change that causes the channel pore to open and close.
Comments [show]
None has been submitted yet.
No. Sentence Comment
51 To investigate potential Cd2af9; bridges formed between pore-lining cysteine side chains exposed in the inner vestibule of the CFTR pore, we combined individual cysteines that we previously found to be accessible to cytoplasmically applied methanethiosulfonate reagents in three important pore-lining TMs: TM1 (K95C, Q98C) (13), TM6 (I344C, V345C, M348C, A349C) (15), and TM12 (M1140C, S1141C, T1142C, Q1144C, W1145C, V1147C, N1148C) (16), to generate a total of 50 double cysteine mutants (8 TM1:TM6; 14 TM1:TM12; 28 TM6:TM12).
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ABCC7 p.Lys95Cys 25143385:51:314
status: NEW