Cui G , Rahman KS , Infield DT , Kuang C , Prince CZ , McCarty NA .

R01-DK056481 NIDDK NIH HHS , T32 GM008602 NIGMS NIH HHS , R01 DK056481 NIDDK NIH HHS

	Figure 1. ECL1 sequence alignment and homology model. (A) Sequence alignment of ECL1 in CFTR proteins from nine species. Charged residues are indicated in red. Asterisks indicate residues conserved in multiple species. D110, E116, and R117 are disease-related and conserved residues labeled with arrows. (B) Top view of the homology model of CFTR (McCarty laboratory model; Rahman et al., 2013) with charged acidic (green) and basic (red) amino acids shown as spheres; model prepared with PyMOL. (left) Closed state (equivalent to the state where the NBDs are fully dedimerized). (right) Open state. TM1-6 is indicated in brown shades, and TM7-12 is indicated in blue shades.
	Figure 2. Some ECL1 mutants exhibited decreased burst duration. (A) Representative single-channel current traces and their all-points histograms for WT-, D110R-, D112R-, K114D-, E115R-, E116R-, and R117A-CFTR from inside-out membrane patches excised from Xenopus oocytes, with symmetrical 150 mM Cl− solution in the presence of 1 mM MgATP and 50 U/ml PKA. All traces were recorded at VM = −100 mV. c, closed state; f, full open state; s1 and s2, subconductance states 1 and 2. The solid lines in the histograms are fit results to a Gaussian function. (B and C) Single-channel amplitudes of the full open state (B) and mean burst durations (C) of WT-, D110R-, D112R-, K114D-, E115R-, E116R-, and R117A-CFTR. (D) Apparent open probability of WT-, D110R-, E116R-, and R117A-CFTR. *, P < 0.05 compared with WT-CFTR; #, P < 0.001 compared with WT-CFTR. n = 4–7 for all variants. Mean ± SEM is shown. This analysis reflected only openings to the f state.
	Figure 3. R117A-CFTR failed to be locked into a stable open state by AMP-PNP. R117A-CFTR was activated with 1 mM Mg-ATP and PKA and recorded under control conditions with ATP + PKA (−AMP-PNP), followed by addition of 2.75 mM AMP-PNP (+AMP-PNP). Representative traces shown were recorded from the same patch at VM = −100 mV. The bottom panel shows mean burst duration for openings to the full conductance state (left), single-channel amplitude (middle), and apparent open probability (NPo; right). AMP-PNP had no effect on mean burst duration or single-channel amplitude but significantly increased apparent open probability of R117A-CFTR (n = 4). *, P < 0.05 compared with ATP and PKA alone (−AMP-PNP). Mean ± SEM is shown.
	Figure 4. Effects of 1 mM MTSET+ (ET+) and MTSES− (ES−) on WT-, D110C-, E116C-, R117C-, and R334C-CFTR. Representative whole-cell TEVC current traces were recorded at VM = −60 mV in ND96 solution in cells expressing CFTR with the β2AR (see Materials and methods). After activation with 10 µM ISO, oocytes were exposed to 1 mM ET+ or ES− in the continuing presence of ISO. Summary data for all mutants tested under the same conditions are shown below the sample current traces. Some error bars are too small to view. *, P < 0.05 compared with WT-CFTR. n = 4–6. Mean ± SEM is shown.
	Figure 5. Effects of 2.5 µM GlyH-101 on WT-, D110C-, E116C-, and R117C-CFTR. Representative current traces recorded from CFTR expressed in oocytes, at VM = −60 mV. Fractional block of WT-CFTR and mutants with 2.5 µM GlyH is shown below the current traces. **, P < 0.01 compared with WT-CFTR. n = 5–8. Mean ± SEM is shown.
	Figure 6. Some ECL1 mutants exhibit altered rectification. WT-CFTR, K114D-CFTR, and E116R-CFTR currents were generated under a voltage protocol wherein membrane potential was held at 0 mV for 50 ms then ramped from −100 mV to 100 mV over 300 ms with the TEVC technique. Standard ND96 was used as bath solution. Currents are shown after subtraction of background currents (in the absence of 10 µM ISO). Rectification of the I-V relationship (rectification ratio) was quantified as defined in Materials and methods. **, P < 0.01 compared with WT-CFTR. n = 5–7. Mean ± SEM is shown.
	Figure 7. Charge recovery at D110, E116, and R117 partially rescues CFTR to open burst stability. (A–C) Representative single-channel currents of D110R- and D110E- (A), E116R- and E116D- (B), and R117A- and R117K-CFTR (C) recorded under the same conditions as Fig. 2 A. Their all-points amplitude histograms are shown on the right. (D) Mean single-channel amplitude of WT-, D110R-, D110E-, E116R-, E116D-, R117A-, and R117K-CFTR. *, P < 0.05 compared with WT-CFTR. (E) Mean burst duration of WT-, D110R-, D110E-, E116R-, E116D-, R117A-, and R117K-CFTR. #, P < 0.001 indicates differences between D110R- and D110E-CFTR or E116R- and E116D-CFTR; *, P < 0.05 indicates differences between R117A- and R117K-CFTR. n = 4–6 for all mutants. Mean ± SEM is shown.
	Figure 8. R117, E116, and D110 do not interact with each other locally to affect open pore architecture of CFTR. (A) Representative single-channel currents of R117E/E116R-, R117E/D110R-, and R117E/E116R/D110R-CFTR and corresponding all-points amplitude histograms recorded under the same conditions as Fig. 2 A. (B) Mean burst duration of WT-CFTR and the three compound mutants. #, P < 0.001 compared with WT-CFTR. n = 4–6 for all mutants. (C) Mean single-channel amplitudes of WT-, R117E/E116R-, R117E/D110R-, and R117E/E116R/D110R-CFTR. *, P < 0.05 compared with WT-CFTR. Mean ± SEM is shown.
	Figure 9. E116 forms a salt bridge with R104 in both the closed and open states. (A) Representative single-channel current traces of R104E- and R104E/E116R-CFTR recorded with the same experimental conditions as Fig. 2 (left), their all-points amplitude histograms (middle), and mean burst durations (right). **, P < 0.01 indicates a significant difference between E116R- and R104E/E116R-CFTR. (B) Two cysteines engineered at positions 104 and 116 (R104C/E116C) form a spontaneous disulfide bond when CFTR is in the open state. Representative single-channel trace of R104C/E116C-CFTR recorded with the same conditions as A. Control, 150 mM Cl− extracellular solution alone (left, top trace). DTT, dithiothreitol reducing agent. +DTT in pipette: R104C/E116C-CFTR recorded with 1 mM DTT in the extracellular pipette solution (left, middle trace). In the bottom trace, oocytes expressing R104C/E116C-CFTR were incubated in solution containing 5 mM MTSET+ over 10 min before single-channel current recording (+MTSET; left, bottom trace). Their all-points amplitude histograms are shown in the middle panel. Mean fraction of open burst duration is plotted at right for R104C/E116C-CFTR under three different experimental conditions, for each of the open conductance states: s1, dark red; s2, orange; and f, light green. (C) Cross-linking R104C to E116C using MTS-2-MTS locks CFTR channels into the closed state. Representative trace (left) and summary data (right) for macroscopic currents measured from R104C/E116C-CFTR with addition of 1 mM MTS-2-MTS in the absence of ISO at VM = −60 mV. ND96, control bath solution. Current levels in the summary data chart are given relative to control conditions before first exposure to ISO and normalized to maximal current in response to ISO1. #, P < 0.001 compared with ISO1 in n = 4 experiments. Mean ± SEM is shown.
	Figure 10. D110 forms a salt bridge with K892 when CFTR is in the closed state. (A) Representative single-channel current traces of K892E-, D110R/K892E-, and D110C/K892C-CFTR recorded with the same experimental conditions as Fig. 2 (left), their all-points amplitude histograms (middle), and their mean burst durations (right). *, P < 0.05 indicates a significant difference between D110R- and D110R/K892E-CFTR; #, P < 0.001 for D110C/K892C-CFTR compared with D110R alone. (B) Exposure to 1 mM DTT backfilled into the pipette had no effect on WT-CFTR. Two traces recorded from the same inside-out patch from a WT-CFTR–expressing oocyte at VM = −100 mV, with MgATP and PKA in the intracellular solution. −DTT, DTT in the pipette solution but early in the recording; +DTT, late in the recoding after DTT perfused to the tip of pipette. Window current every 1 min was measured with Clampfit 10.2 and normalized to the maximum window current (Imax). I/Imax is plotted with its real time scale shown in the right panel. The data for WT-CFTR were fit with a single-exponential function with τ = 5.33 min (red line). (C) Exposure to 1 mM DTT, backfilled into the pipette, increased the number of active channels in D110C/K892C-CFTR. Two traces recorded from the same inside-out patch at VM = −100 mV, with MgATP and PKA in the intracellular solution. The third trace is a small part of the second trace expanded for viewing. −DTT, pipette solution alone; +DTT, DTT perfused to the tip of pipette. Window current data were fit using nonlinear regression, and no time constant was determined (green line). Mean ± SEM is shown.
	Figure 11. D110C forms a spontaneous disulfide bond with K892C when channels are in the closed state. (A) Representative macropatch currents of WT- and D110C/K892C-CFTR were recorded in inside-out mode with symmetrical 150 mM Cl− solution. Pipette was backfilled with 1 mM DTT solution. A voltage protocol that held at 0 mV then stepped to 100 mV for 30 ms followed by a ramp to −100 mV over 300-ms duration was applied every 5 s. a, control, before ATP + PKA; b, peak current (Imax), with cytoplasmic ATP + PKA; c, current after DTT perfused fully to the tip of pipette, with cytoplasmic ATP + PKA; d, current with DTT perfused to the tip of pipette, wash out of cytoplasmic ATP + PKA. I-V plots of currents at times a–c for both WT- and D110C/K892C-CFTR are shown in the middle panel. Summary data are shown in the right panel. Imax, maximum current activated by ATP and PKA, likely before DTT perfused fully to the tip of pipette; I, current activated by ATP and PKA with DTT perfused to the tip of pipette. **, P < 0.01 indicates a significant difference in the pre- and post-DTT conditions; #, P < 0.001 indicates a significant difference in the pre- and post-DTT conditions. (B) 1 mM DTT further activated D110C/K892C-CFTR current but not WT-CFTR current in TEVC recording condition. Representative traces (left) and summary data (right) for macroscopic currents measured from WT- and D110C/K892C-CFTR with addition of 1 mM DTT in the presence of ISO. ND96, control bath solution. VM = −60 mV. ISO1, 10 µM ISO alone; ISO2 + DTT, 1 mM DTT in 10 µM ISO solution. Current levels in the summary data are given relative to control conditions before first exposure to ISO and normalized to maximal current in response to ISO1 (Imax). **, P < 0.01 compared with ISO1 in n = 4 for WT-CFTR and n = 5 for D110C/K892C-CFTR experiments. (C) ISO plus DTT failed to further activate D110C/K892C-CFTR current in oocytes pretreated with DTT in TEVC recording condition. Representative trace (left) and summary data (right) for macroscopic currents measured from D110C/K892C-CFTR with prior addition of 1 mM DTT for 3 min in ND96 solution. Current levels in the summary data are given relative to control conditions before first exposure to ISO and normalized to maximal current in response to ISO1 (Imax). #, P < 0.001 compared with ISO1 in n = 4 experiments. Mean ± SEM is shown.
	Figure 12. R117 forms a salt bridge with E1126 in the open state. (A) Representative single-channel current traces of E1126R-, R117E/E1126R-, R117C-, and R117C/E1126C-CFTR recorded under the same experimental conditions as Fig. 2 and their all-points amplitude histograms (right). (B) Mean burst durations of WT-CFTR and the above mutants. #, P < 0.01 indicates a significant difference between WT- and R117C-CFTR; **, P < 0.01 indicates a significant difference between WT- and E1126R-CFTR, between R117C and R117E/E1126R-CFTR, and between R117C and R117C/E1126C. n.d., no current detected for R117E-CFTR in Xenopus oocytes. n = 4–6 each. (C) Single-channel amplitudes of WT-CFTR and mutants. *, P < 0.05 compared with WT-CFTR. (D) Mean fraction of open burst duration is plotted for each open conductance state of E1126R- and R117E/E1126R-CFTR. Mean ± SEM is shown.
	Figure 13. CFTR homology model with three pairs of interacting residues labeled in both closed and open states. Top view of the homology model (McCarty laboratory model; Rahman et al., 2013) with salt bridge residues shown as spheres: R117-E1126, red; E116-R104, green; and D110-K892E, magenta. (left) Closed state (equivalent to the state where the NBDs are fully dedimerized). (right) Open state. TM1-6 is indicated in brown shades, and TM7-12 is indicated in blue shades.

Alexander, 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. 2009, Pubmed, Xenbase

Alexander, 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. 2009, Pubmed , Xenbase
Aller, Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. 2009, Pubmed
Bagal, Ion channels as therapeutic targets: a drug discovery perspective. 2013, Pubmed
Bai, Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation. 2010, Pubmed
Cestèle, Structure and function of the voltage sensor of sodium channels probed by a beta-scorpion toxin. 2006, Pubmed
Chang, Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator. 2008, Pubmed
Cotten, Cystic fibrosis-associated mutations at arginine 347 alter the pore architecture of CFTR. Evidence for disruption of a salt bridge. 1999, Pubmed
Csanády, Strict coupling between CFTR's catalytic cycle and gating of its Cl- ion pore revealed by distributions of open channel burst durations. 2010, Pubmed , Xenbase
Cui, Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function. 2013, Pubmed , Xenbase
Cui, Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers. 2012, Pubmed , Xenbase
Cui, Mutations at arginine 352 alter the pore architecture of CFTR. 2008, Pubmed , Xenbase
DeCaen, Disulfide locking a sodium channel voltage sensor reveals ion pair formation during activation. 2008, Pubmed
Delaunay, Human ASIC3 channel dynamically adapts its activity to sense the extracellular pH in both acidic and alkaline directions. 2012, Pubmed
El Hiani, Tuning of CFTR chloride channel function by location of positive charges within the pore. 2012, Pubmed
Fuller, State-dependent inhibition of cystic fibrosis transmembrane conductance regulator chloride channels by a novel peptide toxin. 2007, Pubmed , Xenbase
Fuller, The block of CFTR by scorpion venom is state-dependent. 2005, Pubmed , Xenbase
Gabriel, Cystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse model. 1994, Pubmed
Gao, Cysteine scanning of CFTR's first transmembrane segment reveals its plausible roles in gating and permeation. 2013, Pubmed
Gilbert, Thiol/disulfide exchange equilibria and disulfide bond stability. 1995, Pubmed
Glozman, N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic. 2009, Pubmed
Ha, Electrophysiological characterization of benzofuroindole-induced potentiation of large-conductance Ca2+-activated K+ channels. 2006, Pubmed , Xenbase
Hämmerle, Disease-associated mutations in the extracytoplasmic loops of cystic fibrosis transmembrane conductance regulator do not impede biosynthetic processing but impair chloride channel stability. 2001, Pubmed
Hanrahan, Function and dysfunction of the CFTR chloride channel. 1995, Pubmed
Hingorani, Division of labor--sequential ATP hydrolysis drives assembly of a DNA polymerase sliding clamp around DNA. 1999, Pubmed
Hunt, Cystic fibrosis transmembrane conductance regulator (ABCC7) structure. 2013, Pubmed
Hwang, The CFTR ion channel: gating, regulation, and anion permeation. 2013, Pubmed
Jordan, Evolutionary and functional divergence between the cystic fibrosis transmembrane conductance regulator and related ATP-binding cassette transporters. 2008, Pubmed
Kristidis, Genetic determination of exocrine pancreatic function in cystic fibrosis. 1992, Pubmed
Li, Pseudohalide anions reveal a novel extracellular site for potentiators to increase CFTR function. 2012, Pubmed
Liu, CFTR: covalent modification of cysteine-substituted channels expressed in Xenopus oocytes shows that activation is due to the opening of channels resident in the plasma membrane. 2001, Pubmed , Xenbase
McCarty, Identification of a region of strong discrimination in the pore of CFTR. 2001, Pubmed , Xenbase
McCarty, Voltage-dependent block of the cystic fibrosis transmembrane conductance regulator Cl- channel by two closely related arylaminobenzoates. 1993, Pubmed , Xenbase
McDonough, Novel pore-lining residues in CFTR that govern permeation and open-channel block. 1994, Pubmed
McPhee, A critical role for the S4-S5 intracellular loop in domain IV of the sodium channel alpha-subunit in fast inactivation. 1998, Pubmed , Xenbase
Mornon, Atomic model of human cystic fibrosis transmembrane conductance regulator: membrane-spanning domains and coupling interfaces. 2008, Pubmed
Mornon, Molecular models of the open and closed states of the whole human CFTR protein. 2009, Pubmed
Muanprasat, Discovery of glycine hydrazide pore-occluding CFTR inhibitors: mechanism, structure-activity analysis, and in vivo efficacy. 2004, Pubmed
Norimatsu, Locating a plausible binding site for an open-channel blocker, GlyH-101, in the pore of the cystic fibrosis transmembrane conductance regulator. 2012, Pubmed , Xenbase
Price, Function of Xenopus cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels and use of human-Xenopus chimeras to investigate the pore properties of CFTR. 1996, Pubmed , Xenbase
Rahman, Modeling the conformational changes underlying channel opening in CFTR. 2013, Pubmed
Renauld, Role of the C-terminal part of the extracellular domain of the alpha-ENaC in activation by sulfonylurea glibenclamide. 2009, Pubmed , Xenbase
Sauna, Evidence for a requirement for ATP hydrolysis at two distinct steps during a single turnover of the catalytic cycle of human P-glycoprotein. 2000, Pubmed
Schmidt, Allosteric disulfide bonds. 2006, Pubmed
Sebastian, Origin and evolution of the cystic fibrosis transmembrane regulator protein R domain. 2013, Pubmed
Serohijos, Phenylalanine-508 mediates a cytoplasmic-membrane domain contact in the CFTR 3D structure crucial to assembly and channel function. 2008, Pubmed
Sheppard, Mutations in CFTR associated with mild-disease-form Cl- channels with altered pore properties. 1993, Pubmed
Shi, Base of the thumb domain modulates epithelial sodium channel gating. 2011, Pubmed , Xenbase
Sonawane, Luminally active, nonabsorbable CFTR inhibitors as potential therapy to reduce intestinal fluid loss in cholera. 2006, Pubmed
Springauf, The interaction between two extracellular linker regions controls sustained opening of acid-sensing ion channel 1. 2011, Pubmed , Xenbase
Stockand, Insight toward epithelial Na+ channel mechanism revealed by the acid-sensing ion channel 1 structure. 2008, Pubmed
Sullivan, Epithelial transport in polycystic kidney disease. 1998, Pubmed
Thiagarajah, Chloride channel-targeted therapy for secretory diarrheas. 2013, Pubmed
Van Goor, Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770. 2009, Pubmed
Wang, Activating cystic fibrosis transmembrane conductance regulator channels with pore blocker analogs. 2005, Pubmed
Wang, Mapping the receptor site for alpha-scorpion toxins on a Na+ channel voltage sensor. 2011, Pubmed
Weatherall, Crucial role of a shared extracellular loop in apamin sensitivity and maintenance of pore shape of small-conductance calcium-activated potassium (SK) channels. 2011, Pubmed
Xu, KCNQ1 and KCNE1 in the IKs channel complex make state-dependent contacts in their extracellular domains. 2008, Pubmed , Xenbase
Yu, Ivacaftor potentiation of multiple CFTR channels with gating mutations. 2012, Pubmed
Zhang, Mapping the interaction site for a β-scorpion toxin in the pore module of domain III of voltage-gated Na(+) channels. 2012, Pubmed
Zhang, State-dependent chemical reactivity of an engineered cysteine reveals conformational changes in the outer vestibule of the cystic fibrosis transmembrane conductance regulator. 2005, Pubmed
Zhang, Determination of the functional unit of the cystic fibrosis transmembrane conductance regulator chloride channel. One polypeptide forms one pore. 2005, Pubmed
Zhou, Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore. 2008, Pubmed