Cystic fibrosis drug ivacaftor stimulates CFTR channels at picomolar concentrations
- Semmelweis University, Hungary
Figures
Figure 1
Determination of aqueous solubility for Vx-770.
(a) Structural formula of Vx-770 and 3D surface rendering by Molinspiration (https://www.molinspiration.com/cgi-bin/properties). Red arrows identify potentially deprotonatable groups. (b) Absorption …
Figure 2
CFTR current stimulation by Vx-770 is fully reversible.
(a) Macroscopic WT CFTR channel current, elicited in an inside-out patch by exposure to 2 mM ATP (gray bar), is enhanced ~4-fold by application of a 1x saturated (~62 nM) Vx-770 solution (red bar), …
Figure 3
Vx-770 stimulates CFTR currents already at subnanomolar concentrations.
(a-b) Macroscopic WT CFTR currents elicited by 2 mM ATP are reversibly stimulated by exposure (red and brown bars) to indicated concentrations of Vx-770, diluted either from a 1x saturated aqueous …
Figure 4
Delayed activation by low nanomolar Vx-770 suggests multiple sequential slow steps in activation process.
(a) Stimulation of macroscopic WT CFTR currents in 2 mM ATP by exposure to a 1x (black trace; dark gray and red bar) or an 0.01x (gray trace; light gray and purple bar) saturated aqueous solution of …
Figure 5 with 1 supplement
Model fitting of activation/deactivation time courses in response to cytosolic addition/removal of Vx-770.
(a-d) Cartoon representation of schemes with one (a, b) or two (c, d) drug binding sites, and one (a, c) or two (b, d) membrane compartments (cytoplasmic, bottom; extracellular, top). (e) Fit …
Figure 5—figure supplement 1
Scheme 2 adequately fits individual on- and off- time courses but not the global data set.
Pair of normalized macroscopic WT CFTR current activation/deactivation time courses recorded in response to cytosolic addition/removal of 0.01x saturated (gray trace) and 1x saturated (black trace) …
Tables
Table 1
Solubilities of Vx-770 in various solvents at 25°C.
Solubilities were determined as described in detail in Materials and methods. *Aqueous saline with composition described in Materials and methods.
| Solvent | S (mol/L) |
|---|---|
| water (pH=7.1)* | 6.2·10-8 |
| pentane | 1.8·10-5 |
| octane | 2.6·10-3 |
| ethanol | 3.1·10-3 |
| 1-octanol | 3.9·10-3 |
| DMSO | 7.7·10-1 |
Table 2
Model fit parameters for Schemes 3 and 4.
Average fit parameters (mean ± S.E.M.) obtained from global fits of Schemes 3 (left column) and 4 (right column) to three pairs of macroscopic WT CFTR current activation/deactivation time courses …
| Scheme | 3 | 4 |
|---|---|---|
| kin (s-1)* | 4034 ± 464 | 9794 ± 531 |
| kout (s-1)* | 0.06446 ± 0.00742 | 0.1565 ± 0.0085 |
| kon' (M-1s-1) | 870.3 ± 104.0 | 3703 ± 627 |
| koff (s-1) | 0.007451 ± 0.000871 | 0.01075 ± 0.00009 |
| kflip (s-1) | - | 0.04408 ± 0.00583 |
| Po;max/Po;bas | 3.935 ± 0.001 | 3.996 ± 0.007 |
| Kd;mem (µM)** | 8.751 ± 1.160 | 3.106 ± 0.605 |
| K0.5;mem (µM)** | 21.13 ± 2.80 | 7.498 ± 1.460 |
| K0.5;aq (nM)** | 0.6752 ± 0.0895 | 0.6690 ± 0.0955 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (Xenopus laevis) | Xenopus laevis oocytes | African Reptile Park | RRID: NXR_0.0080 | mandyvorster@xsinet.co.za |
| Commercial assay or kit | HiSpeed Plasmid Midi Kit | Qiagen | 12643 | |
| Commercial assay or kit | QuickChange II Mutagenesis Kit | Agilent Technologies | 200524–5 | |
| Commercial assay or kit | mMESSAGE mMACHINE T7 Transcription Kit | ThermoFisher | AM1344 | |
| Chemical compound, drug | Collagenase type II | Gibco | 17107–0125 | |
| Chemical compound, drug | Adenosine 5′-triphosphoribose magnesium (ATP) | Sigma-Aldrich | A9187 | |
| Chemical compound, drug | Protein kinase A catalytic subunit, bovine | Sigma-Aldrich | P2645 | |
| Chemical compound, drug | Vx-770 (solid) | Selleck Chemicals | S1144 | |
| Chemical compound, drug | Vx-770 (10 mM in DMSO) | Selleck Chemicals | S1144 | |
| Software, algorithm | Pclamp9 | Molecular Devices | RRID: SCR_011323 |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.46450.010
