ABCMdb

ABCC7 p.Ile344Cys

Predicted by SNAP2:	A: N (66%), C: N (66%), D: D (59%), E: N (57%), F: N (53%), G: D (53%), H: D (53%), K: N (57%), L: N (78%), M: N (82%), N: N (57%), P: N (53%), Q: N (61%), R: N (66%), S: N (72%), T: N (66%), V: N (82%), W: D (66%), Y: D (53%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N,

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

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

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100 The oocytes 750 500 250 0 µS 180012006000 s IBMX MTSEA Cd 2+ DTT 200 100 0 µS 180012006000 s IBMX DTT Cd 2+ MTSEA A B C -100 -80 -60 -40 -20 0 20 40 % Change in conductance Y325C A326C L327C I328C K329C G330C I331C I332C L333C R334C K335C I336C F337C T338C T339C I340C S341C F342C WT I344C V345C R347C M348C A349C V350C T351C Q353C * * * * * Cd 2+ 1mM MTSEA 1mM D FIGURE 1. Login to comment
218 Finally, the MTSEA reactivity was restricted to only five of twenty-six residues in and flanking TM6 in our study, whereas in the earlier study, residues F337C, S341C, I344C, R347C, T351C, R352C, and Q353C were also shown to be accessible to MTS reagents. Login to comment

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

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

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52 We proposed that these spontaneous changes, that are not seen in either wt or Cys-less CFTR, reflect the coordination of trace Table 1: Percent Change in Oocyte Conductance in the Presence of Compounda MTSETþ MTSES- [Ag(CN)2]- [Au(CN)2]- G330C O O O O I331C -51.6 ( 6.3 -28.9 ( 2.1 -63.1 ( 8.8 O I332C O O O O L333C -58.5 ( 4.8 -47.5 ( 7.6 -83.1 ( 2.2 O R334C þ76.9 ( 11.3 -84.4 ( 1.5 -67.4 ( 7.4 -41.4 ( 3.1 K335C þ10.7 ( 2.4 -37.3 ( 1.5 -29.1 ( 6.4 -54.6 ( 4.7 I336C -54.4 ( 7.9 -75.0 ( 0.6 -81.2 ( 10.5 O F337C O O -89.6 ( 1.9 -90.1 ( 1.3 T338C -37.1 ( 3.3 -85.4 ( 2.5 -75.0 ( 5.2 -88.3 ( 1.6 T339C O O -24.5 ( 7.2 O I340C O O -93.8 ( 1.0 O S341C O O -49.3 ( 4.8 O F342C O O -84.7 ( 1.8 O C343 O O O O I344C O O -66.9 ( 9.3 -77.9 ( 2.1 V345C O O -49.1 ( 9.3 O L346C O O O O R347C O O O O M348C O O -47.9 ( 8.8 -50.1 ( 3.3 A349C O O -19.0 ( 2.0 O V350C O O O O T351C O O O O R352C O O -77.5 ( 1.3 O Q353C O O -72.6 ( 4.5 -76.7 ( 2.8 a Values are means ( SE of three or more oocytes. Login to comment
281 Note the lack of consistent results reported for F337C, S341C, I344C, R347C, T351C, R352C, and Q353C (shaded). Login to comment

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

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

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17 For I344C and M348C, the open time was prolonged and the closed time was shortened after modification, suggesting that depositions of positive charges at these positions stabilize the open state but destabilize the closed state. Login to comment
107 Spontaneous ATP-independent gating of cysless/I344C and cysless/M348C was also increased by MTSET because after the removal of ATP, there remained a substantial amount of current, which can be inhibited by CFTR-specific inhibitor, K335C, F337, and T338C at 50 mV membrane potential (0.46 pA for cysless/WT). Login to comment
179 (B) The Po, obtained from the traces in A, of the cysless/I344C channel before (black) and after (blue) modification. Login to comment
181 n = 7 for cysless/ I344C. Login to comment
183 (C) Single-channel amplitude of the cysless/I344C channel before and after modification. Login to comment
186 Instead, we will focus on the four other positive hits (i.e., I344C, V345C, M348C, and Q353C). Login to comment
208 In a representative recording of a patch containing hundreds of cysless/I344C channels (Fig. 10), one can clearly discern the single-channel amplitude from the expanded trace before and after phosphorylation-dependent activation. Login to comment
241 The modification rate by 1 mM MTSES when Figure 10. MTSES decreased single-channel amplitude of cysless/I344C channels. Login to comment
274 (B) A continuous single-channel recording of cysless/I344C showing a dramatic increase of the spontaneous ATP-independent gating after MTSET modification. Login to comment
314 However, our results showing drastic alterations in gating kinetics caused by modulating TM6 in the TMDs (e.g., a long-lasting opening on the order of tens of seconds with the MTSET-modified cysless/I344C channel) raise the possibility that gating motion in the TMDs can also affect ATP binding and hydrolysis in the NBDs (compare Kogan et al. 2001), a subject worth more extensive future explorations. Login to comment
316 In fact, our data show that the Po of spontaneous gating in the absence of ATP (Bompadre et al., 2007; Wang et al., 2010) is visibly increased by MTSET modification (Figs. 4 D and 12 B; Po is 0.19 ± 0.04, n = 5 for cysless/M348C, and 0.63 ± 0.03, n = 4 for cysless/ I344C). Login to comment
317 The single-channel trace of cysless/I344C in Fig. 12 B illustrates a negligible ATP-independent gating before MTSET modification, whereas frequent opening and closing events in the absence of ATP can Locher, 2006) as a template. Login to comment

[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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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]. Login to comment
207 However, charge-conservative mutations in the analgous part of TM6-for example, in I344C, V345C, M348C, and A349C-also failed to significantly alter Cl-conductance [4]. Login to comment

[hide] CFTR: mechanism of anion conduction. Physiol Rev. 1999 Jan;79(1 Suppl):S47-75. Dawson DC, Smith SS, Mansoura MK
CFTR: mechanism of anion conduction.
Physiol Rev. 1999 Jan;79(1 Suppl):S47-75., [PMID:9922376]

Abstract [show]
CFTR: Mechanism of Anion Conduction. Physiol. Rev. 79, Suppl.: S47-S75, 1999. - The purpose of this review is to collect together the results of recent investigations of anion conductance by the cystic fibrosis transmembrane conductance regulator along with some of the basic background that is a prerequisite for developing some physical picture of the conduction process. The review begins with an introduction to the concepts of permeability and conductance and the Nernst-Planck and rate theory models that are used to interpret these parameters. Some of the physical forces that impinge on anion conductance are considered in the context of permeability selectivity and anion binding to proteins. Probes of the conduction process are considered, particularly permeant anions that bind tightly within the pore and block anion flow. Finally, structure-function studies are reviewed in the context of some predictions for the origin of pore properties.

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481 One construct, I344C, exhibited an increase in was elegantly demonstrated by Horn and co-workers (163) who showed that cysteine residues engineered intoconductance when exposed to MTSEA0 . Login to comment

[hide] Nonintegral stoichiometry in CFTR gating revealed ... J Gen Physiol. 2012 Oct;140(4):347-59. Epub 2012 Sep 10. Jih KY, Sohma Y, Hwang TC
Nonintegral stoichiometry in CFTR gating revealed by a pore-lining mutation.
J Gen Physiol. 2012 Oct;140(4):347-59. Epub 2012 Sep 10., [PMID:22966014]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette (ABC) protein superfamily. Unlike most other ABC proteins that function as active transporters, CFTR is an ATP-gated chloride channel. The opening of CFTR's gate is associated with ATP-induced dimerization of its two nucleotide-binding domains (NBD1 and NBD2), whereas gate closure is facilitated by ATP hydrolysis-triggered partial separation of the NBDs. This generally held theme of CFTR gating-a strict coupling between the ATP hydrolysis cycle and the gating cycle-is put to the test by our recent finding of a short-lived, post-hydrolytic state that can bind ATP and reenter the ATP-induced original open state. We accidentally found a mutant CFTR channel that exhibits two distinct open conductance states, the smaller O1 state and the larger O2 state. In the presence of ATP, the transition between the two states follows a preferred O1-->O2 order, a telltale sign of a violation of microscopic reversibility, hence demanding an external energy input likely from ATP hydrolysis, as such preferred gating transition was abolished in a hydrolysis-deficient mutant. Interestingly, we also observed a considerable amount of opening events that contain more than one O1-->O2 transition, indicating that more than one ATP molecule may be hydrolyzed within an opening burst. We thus conclude a nonintegral stoichiometry between the gating cycle and ATP consumption. Our results lead to a six-state gating model conforming to the classical allosteric mechanism: both NBDs and transmembrane domains hold a certain degree of autonomy, whereas the conformational change in one domain will facilitate the conformational change in the other domain.

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164 Events were extracted from traces in Figs. 2 B and 3 A. Chosen events were specified in boxes and numbered in Figs. 2 B and 3 A. in Cysless/I344C-CFTR, these long ATP-independent openings should show up as the larger O2 state, and that, in the presence of ATP, an opening burst could contain numerous O1→O2 transitions. Login to comment
166 We introduced the R352Q mutation into the Cysless/ I344C-CFTR channel. Login to comment
167 Before MESET modification, Cysless/I344C/R352Q mutant channels behaved similarly as Cysless/R352C-CFTR in the presence of ATP (Fig. 5 A). Login to comment
170 After MTSET modification of Cysless/I344C/R352Q, we indeed observed robust ATP-independent openings (Fig. 5 B) with an open lifetime of 1.03 ± 0.30 s (n = 10), the R352C-CFTR (Fig. S2). Login to comment
171 Correspondingly, the percentage of opening bursts encompassing more than one O1→O2 transition is higher in Cysless/R352C (Table 1). Login to comment
174 In Bai et al. (2010), we showed that after Cysless/I344C-CFTR is modified by MTSET, the open probability of this CFTR mutant in the presence of ATP becomes virtually 1 (Bai et al., 2010). Login to comment
177 Representative traces and amplitude histograms for Cysless/ I344C/R352Q-CFTR under these conditions: (A) in the presence of 2.75 mM ATP; (B-C) after MTSET modification, in the absence (B) or presence (C) of 2.75 mM ATP. Login to comment
179 (E) The amplitude of O1 and O2 states of Cysless/I344C/R352Q-CFTR before (the left bar) or after (the right bar) being modified by MTSET. Login to comment
165 Chosen events were specified in boxes and numbered in Figs. 2 B and 3 A. in Cysless/I344C-CFTR, these long ATP-independent openings should show up as the larger O2 state, and that, in the presence of ATP, an opening burst could contain numerous O1࢐O2 transitions. Login to comment
168 Before MESET modification, Cysless/I344C/R352Q mutant channels behaved similarly as Cysless/R352C-CFTR in the presence of ATP (Fig. 5 A). Login to comment
175 In Bai et al. (2010), we showed that after Cysless/I344C-CFTR is modified by MTSET, the open probability of this CFTR mutant in the presence of ATP becomes virtually 1 (Bai et al., 2010). Login to comment
178 Representative traces and amplitude histograms for Cysless/ I344C/R352Q-CFTR under these conditions: (A) in the presence of 2.75 mM ATP; (B-C) after MTSET modification, in the absence (B) or presence (C) of 2.75 mM ATP. Login to comment
180 (E) The amplitude of O1 and O2 states of Cysless/I344C/R352Q-CFTR before (the left bar) or after (the right bar) being modified by MTSET. Login to comment

[hide] Alternating access to the transmembrane domain of ... J Biol Chem. 2012 Mar 23;287(13):10156-65. Epub 2012 Feb 1. Wang W, Linsdell P
Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7).
J Biol Chem. 2012 Mar 23;287(13):10156-65. Epub 2012 Feb 1., [PMID:22303012]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.

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148 Residues closer to the cytoplasmic end of TM6 show regulated access to intracellular MTS reagents without being accessible to extracellular MTS reagents, for example I344C is accessible to intracellular MTSES only in open channels (22) but is not modified by extracellular MTS reagents (14, 17). Login to comment
154 Residues closer to the cytoplasmic end of TM6 show regulated access to intracellular MTS reagents without being accessible to extracellular MTS reagents, for example I344C is accessible to intracellular MTSES only in open channels (22) but is not modified by extracellular MTS reagents (14, 17). 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
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
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
146 Both K95C and I344C were rapidly inhibited by 200 nM Au(CN)2 - (Fig. 6), reflecting a high modification rate constant (Fig. 7). 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] Structural basis for the channel function of a deg... J Gen Physiol. 2011 Nov;138(5):495-507. Bai Y, Li M, Hwang TC
Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7).
J Gen Physiol. 2011 Nov;138(5):495-507., [PMID:22042986]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, but little is known about how this ion channel that harbors an uninterrupted ion permeation pathway evolves from a transporter that works by alternately exposing its substrate conduit to the two sides of the membrane. Here, we assessed reactivity of intracellularly applied thiol-specific probes with cysteine residues substituted into the 12th transmembrane segment (TM12) of CFTR. Our experimental data showing high reaction rates of substituted cysteines toward the probes, strong blocker protection of cysteines against reaction, and reaction-induced alterations in channel conductance support the idea that TM12 of CFTR contributes to the lining of the ion permeation pathway. Together with previous work, these findings raise the possibility that pore-lining elements of CFTR involve structural components resembling those that form the substrate translocation pathway of ABC transporters. In addition, comparison of reaction rates in the open and closed states of the CFTR channel leads us to propose that upon channel opening, the wide cytoplasmic vestibule tightens and the pore-lining TM12 rotates along its helical axis. This simple model for gating conformational changes in the inner pore domain of CFTR argues that the gating transition of CFTR and the transport cycle of ABC proteins share analogous conformational changes. Collectively, our data corroborate the popular hypothesis that degradation of the cytoplasmic-side gate turned an ABC transporter into the CFTR channel.

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198 (C) Second-order rate constants (MTSES  ) of Texas red MTSEA+ modification for cysless/ S1141C-, cysless/N1148C-, cysless/ I344C-, and cysless/M348C-CFTR channels. 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
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
139 Our work concerning intracellular MTS reagent modification in TM6 also identified some cysteines that could be modified in both activated and nonactivated channels (e.g., V345C and M348C), and others that could apparently be modified only after channel activation (e.g., T338C, S341C, and I344C), suggesting a state-dependent conformational change that alters access of internally applied MTS reagents into the pore (El Hiani and Linsdell, 2010). 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
167 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). 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
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
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
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
256 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 M1 s1 ), 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 M1 s1 ). Login to comment

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

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

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107 We did not measure the reaction rate constants for the most extracellular residue, I331C, because we thought that it was unlikely that the reaction rates would be voltage dependent given the absence of voltage dependence at the adjacent, more cytoplasmic residues. We also did not measure the reaction rate constants for the mutants I344C and R347C because, although MTSEAϩ reacted with these residues, MTSES- and MTSETϩ did not react with these k ψ( )( )ln k Ψ 0=( )( ) zFδ RT/( )-ln ψ= t a b l e i Second-order Rate Constants for the Reaction of the MTS Reagents with the Water-exposed Cysteine Mutants k ES (M-1s-1) k EA (M-1s-1) k ET (M-1s-1) mutant -25 mV -50 mV -75 mV -25 mV -50 mV -75 mV -25 mV -50 mV -75 mV L333C 71 Ϯ 3(3) 71 Ϯ 20(2) 71 Ϯ 23(3) 320 Ϯ 89(2) 320 Ϯ 128(2) 333 Ϯ 139(3) 952 Ϯ 136(2) 1,000 Ϯ 350(2) 1,053 Ϯ 443(2) R334C 48 Ϯ 14(2) 48 Ϯ 6(3) 44 Ϯ 8(4) 145 Ϯ 32(2) 163 Ϯ 7(2) 182 Ϯ 21(3) 444 Ϯ 49(2) 454 Ϯ 124(2) 588 Ϯ 95(3) K335C 36 Ϯ 20(3) 23 Ϯ 11(3) 27 Ϯ 16(3) 222 Ϯ 80(3) 121 Ϯ 51(4) 107 Ϯ 30(3) 217 Ϯ 111(3) 235 Ϯ 28(3) 217 Ϯ 95(4) F337C 91 Ϯ 17(2) 80 Ϯ 22(3) 71 Ϯ 20(4) 222 Ϯ 74(2) 222 Ϯ 86(3) 285 Ϯ 81(3) 740 Ϯ 246(3) 740 Ϯ 82(2) 714 Ϯ 51(2) S341C 56 Ϯ 18(3) 56 Ϯ 40(2) 43 Ϯ 12(3) 93 Ϯ 6(3) 110 Ϯ 22(3) 138 Ϯ 34(3) 690 Ϯ 356(3) 556 Ϯ 246(3) 800 Ϯ 224(4) T351C 100 Ϯ 25(5) 57 Ϯ 6(3) 26 Ϯ 9(6) 146 Ϯ 30(4) 195 Ϯ 42(4) 296 Ϯ 18(3) 308 Ϯ 47(10) 392 Ϯ 78(6) 769 Ϯ 89(5) R352C 42 Ϯ 4(3) 26 Ϯ 4(5) 21 Ϯ 6(4) 105 Ϯ 76(3) 137 Ϯ 46(3) 205 Ϯ 58(2) 417 Ϯ 138(4) 800 Ϯ 128(2) 952 Ϯ 408(2) Q353C 125 Ϯ 23(4) 51 Ϯ 12(4) 42 Ϯ 8(4) 83 Ϯ 24(4) 116 Ϯ 42(4) 160 Ϯ 92(3) 189 Ϯ 48(6) 220 Ϯ 48(3) 625 Ϯ 273(4) residues and therefore we could not determine the charge selectivity at these positions.2 The reaction rate constants that we have measured are between 10-and 500-fold slower than the rates of reaction with sulfhydryls in free solution (Table II) (Stauffer and Karlin, 1994). Login to comment

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

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

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114 Another mutant, I344C, reacted with MTSEA+ more slowly, requiring an 8-min application of 2.5 mM MTSEA+ to significantly alter the current (Fig. 5 B). Login to comment
135 The slow rate of reaction of MTSEA' with the I344C mutant and the inability of the larger, anionic MTSES- to react with 1344C suggests that access of the reagents to this residue may be sterically limited, possibly by neighboring residues on other membrane-spanning segments. Login to comment
113 Another mutant, I344C, reacted with MTSEA+ more slowly, requiring an 8-min application of 2.5 mM MTSEA+ to significantly alter the current (Fig. 5 B). Login to comment
133 The slow rate of reaction of MTSEA' with the I344C mutant and the inability of the larger, anionic MTSES- to react with 1344C suggests that access of the reagents to this residue may be sterically limited, possibly by neighboring residues on other membrane-spanning segments. 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] Nonequilibrium gating of CFTR on an equilibrium th... Physiology (Bethesda). 2012 Dec;27(6):351-61. doi: 10.1152/physiol.00026.2012. Jih KY, Hwang TC
Nonequilibrium gating of CFTR on an equilibrium theme.
Physiology (Bethesda). 2012 Dec;27(6):351-61. doi: 10.1152/physiol.00026.2012., [PMID:23223629]

Abstract [show]
Malfunction of cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC protein superfamily that functions as an ATP-gated chloride channel, causes the lethal genetic disease, cystic fibrosis. This review focuses on the most recent findings on the gating mechanism of CFTR. Potential clinical relevance and implications to ABC transporter function are also discussed.

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185 Likewise in Bai et al. (8), chemical modifications of an engineered cysteine (I344C or M348C) in TM6 drastically increase ATP-independent activity to the level of ATP-dependent activity before modifications. 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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164 Similar to what we observed for I344C- and M348C-CFTR (16), this robust ATP-independent gating was seen following modification by MTSET but not by MTS-ethylammonium (MTSEA) (Fig. S4). 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
71 In contrast, the remaining seven double cysteine mutants, namely I344C/S1141C (Fig. 2, C and D), V345C/S1141C, M348C/ S1141C (Fig. 2, C and E), M348C/V1144C, M348C/W1145C, M348C/V1147C, and M348C/N1148C, all showed increased sensitivity to Cd2af9; , leading to a significant decrease in Ki as compared with either of the single cysteine mutants from which they were derived (estimated Ki values b0d; 50 òe;M; Fig. 3). Login to comment
80 In each case, PPi treatment resulted in a weakening of Cd2af9; inhibition (Fig. 4A) and a significant increase in Ki (Fig. 4B) of between 2.3-fold (in I344C/S1141C) and 97-fold (in M348C/ S1141C). Login to comment
83 As shown in Fig. 5, all E1371Q-containing channels tested were only weakly sensitive to inhibition by Cd2af9; , resulting in a significant increase in Ki both in single cysteine (I344C, M348C, S1141C) and in double cysteine (I344C/S1141C, Fig. 5, A-C; M348C/S1141C, Fig. 5, A, D, and E) mutant channels. Login to comment
84 However, the effect of the E1371Q mutation was greater in the double cysteine mutants; this gating mutation increased Ki 30-fold in I344C/S1141C (Fig. 5C) and 2500-fold in M348C/S1141C (Fig. 5E). Login to comment
98 C, sample time courses (upper panels) and I-V curves (lower panels) recorded from similar experiments for the double cysteine mutants I344C/S1141C (left) and M348C/S1141C (right). Login to comment
137 Thus, M348C is able to form Cd2af9; bridges with cysteines at multiple positions in TM12 (S1141C, Q1144C, W1145C, V1147C, N1148C) (Fig. 8B), and S1141C is able to form Cd2af9; bridges with cysteines both in TM1 (K95C) and in TM6 (I344C, V345C, M348C) (Fig. 8C). Login to comment
164 A, sample time courses and I-V curves illustrating the low Cd2af9; sensitivity of constitutively active I344C/S1141C/E1371Q (left panels) and M348C/S1141C/E1371Q (right panels) channels in inside-out patches. Experiments were performed as described in the legend for Fig. 2. Login to comment

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

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

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68 (C) Reaction between I344C-CFTR and 1 mM [Au(CN)2]- in the presence or absence of ATP (see Results for details). Login to comment
70 (D) State-independent reactivity of I344C-CFTR to [Au(CN)2]- . Login to comment
192 [Au(CN)2]- , forskolin, with G1349D, /M/s Outside R334C 189 &#b1; 39 - 403 &#b1; 20 537 &#b1; 56 K335C - - 56 &#b1; 9 1,809 &#b1; 201 F337C 437 &#b1; 49 - 20 &#b1; 3 32 &#b1; 6 T338C 752 &#b1; 59 - 1,135 &#b1; 166 118 &#b1; 18 Inside I344C 32 &#b1; 5 37 &#b1; 4 - - N1148C 437 &#b1; 66 2,089 &#b1; 130 - - Residues located extracellularly (extra.) Login to comment