ABCMdb

ABCC7 p.Gln98Cys

ClinVar:	c.293A>G , p.Gln98Arg ? , not provided c.292C>T , p.Gln98* D , Pathogenic c.293A>C , p.Gln98Pro ? , not provided
CF databases:	c.293A>C , p.Gln98Pro (CFTR1) D , This mutation was found by DHPLC and confirmed by sequencing. The adult male patient, from Southern Sweden, carries deltaF508 on the other chromosome. The patient has high sweat chloride (116 mmol/L), bronchiectasis and CBAVD. c.292C>T , p.Gln98* D , CF-causing c.293A>G , p.Gln98Arg (CFTR1) D , This mutation was found in one CF patient from Southern France, who carries [delta]F508 on the other gene. It creates a HaeIII restriction site (N : 290 +78 +70 bp), (m: 153 + 137 + 78 + 70 bp) when using the primers 4i5/4i3 from Zielinski. Also reported by Yoshimura & Azuma on 4/01/1000: This mutation was detected in one of the CFTR alleles of a 15-year old Japanese male patient with cystic fibrosis. He is pancreatic insufficient, has CBAVD, and his sweat chloride was high (74 mmol/L). Another mutation was not found despite the thorough evaluation for his entire 27 exons of the CFTR gene. Interestingly, he was heterozygous at the cDNA 125 in 5'UTR (i.e., 125G/125C), and this is the only difference from his healthy sister who is also heterozygous for Q98R mutation, but 125G/125G, suggesting that 125C may be disease-causing.
Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), R: N (78%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: N, F: D, G: D, H: D, I: D, K: N, L: D, M: D, N: N, P: D, R: N, S: N, T: D, V: D, W: D, Y: D,

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[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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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 . Login to comment

[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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3 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 1 mM to 10 μM, and modification by MTSES was accelerated when 2 mM pyrophosphate was applied to prevent channel closure. Login to comment
6 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. Login to comment
44 Both wild type CFTR and a variant in which all 18 endogenous cysteine residues have been substituted by other inside-out inside-out + ATP and PKA + ATP and PKA B) Q98C + MTSES (6 s) + MTSES (6 s) + MTSES (30 s) + MTSES (30 s) + MTSES (120 s) + MTSES (120 s) C) Q98C/E1371Q 200 pA 500 ms 250 pA 500 ms A) Voltage Protocol -50 -25 0 25 50 V (mV) 500 ms Fig. 1. Login to comment
47 (B) Raw currents carried by Q98C following patch excision from the cell (inside-out), following channel activation with ATP (1 mM) and PKA, and at different times after application of MTSES (200 μM) to the cytoplasmic face of the membrane. Login to comment
48 (C) Raw currents carried by Q98C/E1371Q under these same conditions. Login to comment
49 Note that background (leak) currents are small in Q98C prior to addition of ATP and PKA, whereas in Q98C/E1371Q, as with all constructs bearing the E1371Q mutation, large constitutive currents are observed and are not further increased in amplitude by PKA and ATP (see also Refs. Login to comment
52 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)). Login to comment
55 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). Login to comment
60 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. Login to comment
73 In most cases, 20 μM MTSES and 200 nM Au(CN)2 - were used, however, in channels bearing the Q98C mutation these concentrations were increased to 200 μM MTSES and 200 nM Au(CN)2 - because of the lower rate of modification of a cysteine at this position ([15], and Figs. 3, 6 and 7). Login to comment
75 The similar low apparent modification rate for Q98C by Au(CN)2 - (Figs. 6 and 7) suggests that accessibility of this substance is limited by similar factors as that of MTSES. Login to comment
90 Examples of macroscopic currents recorded from Q98C-CFTR under these conditions are shown in Fig. 1B, and examples of timecourses of MTSES-induced current amplitude change are shown in Fig. 2. Login to comment
91 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. Login to comment
100 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). Login to comment
101 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). Login to comment
102 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). Login to comment
103 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. Login to comment
111 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). Login to comment
112 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). Login to comment
113 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. Login to comment
145 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). Login to comment
147 For Q98C, the modification rate constant was lower, and experiments were carried out using a higher concentration of Au(CN)2 - (2 μM). Login to comment
148 Interestingly, the modification rate constant for MTSES is also lower for Q98C (Fig. 3), probably reflecting its location more deeply into the pore [15]. Login to comment
150 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). Login to comment
151 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 - . Login to comment
153 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). Login to comment
154 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). Login to comment
156 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). Login to comment
157 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). Login to comment
158 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). Login to comment
166 In contrast, nearby residues (Q98C in TM1, I344C in TM6) showed strongly state-dependent accessibility, both to the large MTSES (Fig. 3) and the small, permeant Au(CN)2 - ion (Fig. 7). Login to comment
168 Given the well known effects of these manipulations on channel gating and overall open probability, it seems reasonable to suggest that the rate of modification at Q98C and I344C is positively associated with open probability, suggesting that open channels are modified more easily than are closed channels. Login to comment
174 However, our measurements of time constants of changes in macroscopic current amplitude in channels that are opening and closing do not allow us to estimate the rate of modification at Q98C and I344C in closed channels. Login to comment
175 Previously we suggested that the rate of modification of Q98C [15] and I344C [14] is negligible in inactive channels prior to PKA-dependent phosphorylation. Login to comment
176 It is possible that phosphorylation leads to partial opening of the putative gate and a partial increase in access, and ATP-dependent gating then results in a further increase in access through this region. This could allow slow modification of Q98C and I344C in phosphorylated, but closed, channels. Login to comment
181 Perhaps surprisingly, then, we find the effects of low ATP and the K464A mutation on modification rate constants for Q98C and I344C to be quantitatively similar and in the range of 1.5 to 3-fold (Figs. 3, 7). Login to comment
201 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). Login to comment

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

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

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22 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. Login to comment
71 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). Login to comment
96 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. Login to comment
102 (A) Example time courses of macroscopic currents (measured at +50 mV) carried by cys-less CFTR and Q98C inside-out membrane patches. Login to comment
104 Note that whereas these MTS reagents have no effect on cys-less CFTR current amplitude, they cause rapid inhibition (MTSES) or augmentation (MTSET) of current carried by Q98C. Login to comment
105 (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. Login to comment
112 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). Login to comment
136 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. Login to comment
138 These results suggest that none of K95C, Q98C, P99C, or L102C can be modified covalently by extracellular MTSET. Login to comment
141 We used a similar approach to determine if K95C, Q98C, P99C, and L102C could be modified by MTSES pretreatment. Login to comment
148 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). Login to comment
150 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. Login to comment
162 (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. Login to comment
166 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. Login to comment
168 In contrast, the inhibitory effects of CuPhe on Q98C/I344C were similar when measured at 80 mV or +80 mV. Login to comment
170 (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. Login to comment
172 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. Login to comment
173 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. Login to comment
175 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. Login to comment
178 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). Login to comment
180 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). Login to comment
181 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). Login to comment
182 However, K95C/ I344C, Q98C/I344C, and Q98C/M348C did generate macroscopic PKA- and ATP-dependent currents in inside-out patches. Login to comment
184 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). Login to comment
185 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. Login to comment
186 Furthermore, the lack of effect of CuPhe on Q98C/M348C indicated that not all double-cysteine mutants were CuPhe sensitive, which we take to indicate that only nearby cysteine side chains can be cross-linked by this reagent. Login to comment
188 (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. Login to comment
200 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. Login to comment
206 (A) Example leak-subtracted I-V relationships for cys-less (left), I344C (center), and Q98C/I344C (right) after channel activation with 20 nM PKA and 1 mM ATP. Login to comment
208 In all channel constructs studied, these inhibitory effects of Cu2+ were readily and rapidly reversed by washing Cu2+ from the bath (for example, see right panel for complete reversal of the strong blocking effect on Q98C/I344C). Login to comment
209 (B) Mean fractional current remaining after the addition of different concentrations of Cu2+ for cys-less (), I344C (), and Q98C/I344C (). Login to comment
210 Data are fitted as described in Materials and methods, giving Kd = 129 µM and nH = 1.36 for cys-less, Kd = 19.5 µM and nH = 1.21 for I344C, and Kd = 3.91 µM and nH = 1.65 for Q98C/I344C. Login to comment
228 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). Login to comment
238 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). Login to comment
242 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). Login to comment
244 In contrast, we found no evidence for disulfide bond formation between a pair of introduced cysteine side chains that would be predicted (based on Fig. 9) to be further apart, Q98C in TM1 and M348C in TM6 (Fig. 6, C and D). Login to comment
251 Currently, it is not clear why there are no sites in TM1 that can be accessed from both sides of the membrane in our hands (although it should be noted that Q98C was described as being sensitive to external MTSES in a previous study; Akabas et al., 1994). Login to comment
258 The MTSES modification rate constant for Q98C (440 M1 s1 ; Fig. 3) was somewhat intermediate between these two groups. Login to comment

[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.

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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. Login to comment
72 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. Login to comment
86 A and C are from oocytesinjectedwith wild type CFTR B, Q98C; D,K95C; E, G91C. Login to comment
110 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. Login to comment

[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.

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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). Login to comment

[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.

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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. Login to comment
82 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. Login to comment
108 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). Login to comment
152 For Q98C and K95C mutant channels, the increases in the mean current amplitude following MTSET modification were ~2- and 6-fold, respectively. Login to comment
154 Fig. S2, however, shows that MTSET modification of Q98C-CFTR increases both the open probability and the single-channel amplitude. Login to comment
165 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. Login to comment
174 Similar results were obtained from experiments on L102C (Fig. 6 B) and Q98C mutants. Login to comment
196 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. Login to comment
206 (C) Summary of the modification rates for K95C, Q98C, and L102C in the presence of ATP (solid squares). Login to comment

[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.

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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). Login to comment
111 In contrast to these results with K95C, but in common with TMs 6 and 12 single cysteine mutants, Q98C channels were inhibited by Cd2af9; (Fig. 7). Login to comment
112 Most (18 out of 19) double cysteine mutants including K95C or Q98C were weakly inhibited by Cd2af9; , consistent with Cd2af9; effects on single cysteine mutants in TMs 6 or 12 (or indeed in Q98C) (Fig. 7). Login to comment
183 FIGURE 7. Mean inhibitory effect of Cd2d19; on channels bearing cysteine sidechainsinTM1.Shownisthemeaneffectof100òe;M Cd2af9; onmacroscopic current amplitude for two single cysteine mutants (K95C, Q98C; black bars) and 19 double cysteine mutants that combine the TM1 cysteine listed above the panel with the TMs 6 or 12 cysteine listed below. Login to comment