Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.
Chloride transport across the apical membranes of epithelial cells in such tissues as the lung, intestine, pancreas and sweat glands is primarily mediated by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), an ATP- and phosphorylation-regulated member of the ABC (ATP-Binding Cassette) C subfamily of membrane proteins (reviewed in1). Like other members of the ABCC subfamily, CFTR is a large, multi-spanning integral membrane protein that binds ATP at two nucleotide binding sites formed at the interface of its nucleotide-binding domains (NBDs), where it possesses modest ATPase activity at a single site. However, unlike other ABCC subfamily members, CFTR has evolved as a unique regulated Cl channel rather than as an active solute transporter.
Mutations in CFTR cause Cystic Fibrosis, a disease affecting multiple organs including the lungs, gastrointestinal tract, pancreatic and reproductive tract, leading to morbidity and mortality in young adults. Lung disease typically accounts for early mortality in Cystic Fibrosis and in most cases is caused by the loss of CFTR function on the surface epithelium of the conducting airways. The lack of CFTR chloride channel activity causes a reduction in both Cl– and water movement across the surface epithelium to modify the fluid layer on the apical surface of the ciliated respiratory epithelium. This results in a viscous airway surface liquid that impairs the ability of ciliated respiratory epithelial cells to effectively clear pathogens out of the airways. As a consequence, most CF patients suffer from recurrent bouts of lung infection and lung damage due to inflammation.
As expected, studies of the mechanism of action of the normal CFTR protein focused primarily on detailed electrophysiological studies of its channel gating activity. Single channel studies have shown directly that CFTR functions as a PKA-dependent Cl– channel which possesses an ATP regulated gate2. Detailed electrophysiological studies provide a great deal of information on single CFTR channels1,3, however there may be concern as to whether the characteristics of any particular single channel that has been studied is reflective of the entire population of CFTR channels and therefore the single channel results should always be considered along with methods to study the macroscopic population. Direct assay of the population channel activity of purified CFTR has the potential to provide insight into the molecular defect associated with disease-causing mutations and to drive discovery of chemical modulators which repair mutant CFTR proteins. To date, there are in excess of 1,900 different mutations in CFTR thought to cause Cystic Fibrosis4. The major mutation, F508del-CFTR, found on at least one allele in approximately 90% of patients in North America and Europe leads to protein misfolding and retention in the endoplasmic reticulum5. F508del-CFTR also has other consequences, including defective channel activity6-9. The resultant absence of CFTR from the cell surface is associated with severe disease. G551D-CFTR, a less common mutation, is thought to be properly folded yet is dysfunctional as a chloride channel at the cell surface6. The development of small molecule correctors and potentiators has the goal of correcting folding and/or trafficking of mutants such as F508del-CFTR to the cell surface, and potentiating or increasing the channel activity of mutations such as G551D when present on the cell surface, respectively. While the correctors VX-809 and VX-661 (are not yet approved for use in patients, the potentiator Kalydeco (ivacaftor; VX-770) is being used at 150 mg every 12 hr in CF patients >6 years with at least one G551D-CFTR mutation, and more recently for patients with one of G178R, S549N, S549R, G551S, G1244E, S1251N, S1255P and G1349D. Kalydeco is both safe and results in improvement of clinical measures of CF disease10, however the mechanism of action of this small molecule was poorly understood at the time of FDA approval for use in patients.
A handful of CFTR purification methods have been described previously2,11-18, many of which require a considerable length of time to complete. In a recent publication19, a unique rapid purification and reconstitution method was described for CFTR overexpressed in the Sf9 cell expression system, and this purified protein in defined lipid systems was used to develop a CFTR halide channel activity assay for a population of CFTR molecules. The assay recapitulates the known effects of phosphorylation, nucleotides and inhibitors on CFTR function. The system was used to interrogate the effects of the potentiator VX-770/Kalydeco on Wt- (wild-type), F508del- and G551D-CFTR and it was shown for the first time that the drug interacts directly with the CFTR protein to potentiate its channel activity in an ATP-independent manner, demonstrating the utility and applicability of these methods to the study of the interaction of CFTR and mutants with nucleotides and small molecules from a population perspective to answer clinically relevant questions about the protein. The methods have also been used to study other potentiator molecules and their derivatives20, as well as the effects of a small molecule corrector on the activity of the protein21.
Efflux assays have been used in many studies previously to investigate the activity of CFTR mutants and the effects of CFTR-modulatory compounds on its activity, including whole cell assays using electrodes, radioactive tracers and fluorophores22,23, membrane vesicles with ion selective electrodes24, and purified reconstituted CFTR with radioactive tracers25. However the use of ion selective electrodes to study purified reconstituted CFTR was first reported recently19. An adaptation of the current method has been used for reconstitution and functional characterization of two membrane proteins in Pseudomonas aeruginosa, a common CF pathogen. Reconstitution of purified AlgE outer membrane protein coupled with iodide efflux measurements were used to support a model for anionic alginate secretion through this transporter26. Reconstitution and iodide efflux measurements were applied to the purified Wzx protein, which allowed a model to be proposed that suggests an H+-dependent antiport mechanism for lipid-linked oligosaccharide translocation across the bacterial inner membrane by this protein27. In both cases iodide was used as a surrogate for the anionic substrate, albeit at lower throughput than one might expect for a native substrate. The method may be suitable for adaptation to other proteins with cationic transport or conduction pathways for anionic substrates.
Here a rapid purification procedure is described for the CFTR protein and its reconstitution into proteoliposomes of defined lipid. The rapid reconstitution procedure can easily be tailored for use with CFTR purified by other methods, provided that the type of detergent used in the purification is amenable to removal by the methods used here or can be exchanged for a suitable detergent before the reconstitution procedure. The iodide efflux method for measurement of channel activity of purified and reconstituted CFTR protein is described in detail and some typical results that can be obtained from this method are presented.
1. Purification of CFTR
NOTE: Please see Table of Materials and Equipment for a list of equipment and materials used in this protocol. A detailed protocol exists for overexpression of human Wt-CFTR and mutants in the Sf9-baculovirus expression system17,28. Overexpress CFTR and prepare pellets of Sf9 cells according to this protocol.
2. Reconstitution of CFTR
3. Iodide Efflux Measurements for Purified, Reconstituted CFTR
Described in this written publication are methods to purify, reconstitute and measure regulated channel activity of the CFTR protein. Figure 1a shows the workflow for the purification, reconstitution and iodide efflux procedures. Methods for reconstitution and channel activity measurements by iodide flux are also shown in further detail in the associated video.
Purification and reconstitution of CFTR into proteoliposomes
CFTR can be functionally expressed at high levels in Sf9 cells using recombinant baculovirus containing Wt or mutant CFTR cDNA2. While not a mammalian expression system, CFTR was expressed using the baculovirus system in Sf9 cells to ensure a high level of expression. CFTR production can be as high as 1% of total cellular protein17,28. The Sf9- baculovirus system has the advantage that the protein is core glycosylated and the quality control machinery in this system is relatively permissive such that CFTR and mutants can be produced on the cell surface. A ten histidine tag was engineered onto the carboxy terminus to allow purification by virtue of affinity of this tag for nickel-NTA. The C-terminal tag helps to prevent co-purification of truncated CFTR proteins and yields functional CFTR protein in ATPase, single channel and iodide efflux experiments17,19,24,28,29. However the purification protocol described here should be suitable for N-terminally tagged CFTR as well.
Previously, it has been shown that CFTR protein can be effectively extracted from Sf9 plasma membrane preparations using NaPFO (sodium pentadecafluoro-octanoate) at room temperature15. However, the susceptibility of NaPFO to precipitate at 4 °C, a temperature conducive to preserving properly folded protein, and the lengthy dialysis required to remove PFO were not amenable to rapid purification and reconstitution methods intended to maximize the activity of the protein in subsequent assays of CFTR function. An evaluation of a panel of detergents (including dodecyl-β-D-maltoside (DDM), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), fos-choline 12 and fos-choline 14, showed that fos-choline 14 was most effective in solubilizing human CFTR-His10 from Sf9 membranes, and that DDM was effective in maintaining the ATPase activity of CFTR in detergent solution (data not shown). Thus a fos-choline 14 detergent extraction step is performed, followed by exchange to DDM detergent to maintain the activity of the protein.
Following purification and concentration, a protein fraction is obtained in DDM detergent that contains a ~150 kDa band as its major component (Figure 2; Silver Stain). This molecular weight corresponds to human CFTR expressed in Sf9cells and analyzed by SDS-PAGE, where it lacks the types of complex glycosylation characteristic of CFTR in mammalian cells. The 150 kDa species appears to be approximately 90% pure following SDS-PAGE analysis and silver staining. Western blotting using the M3A7 anti-CFTR antibody confirms that this mouse monoclonal directed against NBD2 reacts with the 150 kDa band (Figure 2; blot). In certain studies, trace amounts of lower molecular weight components are present as minor bands visible on the gel, at ~60 and ~80 kDa (data not shown). The M3A7 anti-CFTR antibody reacts with the 60 and 80 kDa bands, but does not react with other non-CFTR proteins (data not shown). This suggests that the lower molecular weight components are CFTR degradation products co-purified with CFTR via their C-terminal His tag. The purification is done in the presence of protease inhibitors and it appears that these components are present in the cells before the purification procedure is performed. The concentration step of the procedure removes virtually all of the contaminating lower molecular weight fragments. CFTR can be reconstituted at this point, or protein from another purification procedure can be used in the reconstitution protocol.
A liposomal flux based assay reports the functional reconstitution of a population of purified and reconstituted CFTR molecules as regulated, anion selective channels
In order to determine if a significant proportion of purified CFTR was reconstituted as a regulated chloride channel using the above methods, a proteoliposomal halide flux assay was developed which reports the activity of a population of reconstituted CFTR molecules (shown schematically in Figure 1B). If, in the worst case scenario, the majority of the reconstituted CFTR molecules are misfolded, these molecules will fail to confer a conductance, fail to discriminate between cations and anions and/or the gate will lack regulation by PKA phosphorylation and nucleotides. Lee et al. have published methods that allow estimation of percent functional reconstitution of a membrane protein30.
Empty liposomes (lacking CFTR) or proteoliposomes (bearing reconstituted CFTR) are loaded with KI (75 mM) and extra-liposomal iodide exchanged with potassium glutamate (75 mM) by passing liposomes through a gel filtration column. The KI loaded proteoliposomes are added to a well containing K-Glu (potassium glutamate) to create an outward gradient for iodide and efflux is monitored as increases in extraliposomal iodide over time using an iodide sensitive microelectrode24. The efflux of negatively charged iodide results in a decrease in the mV signal (signal becomes more negative). When the concentration of iodide released over time is plotted, the slope is inverted (as in Figure 3) to show an increase in iodide in the external solution over time.
K-Glu was chosen as the impermeant counterion as it is one example of an anion that does not pass through the CFTR protein, does not appear to cause significant blocking of the pore under the conditions used, and does not cause interference with the iodide selective electrode. An assay could be optimized around another anion, provided that it meets these criteria. Seventy-five mM anion concentrations were selected empirically as concentrations that gave good trapping and sufficient signals for measurement in the assay under the conditions described. There will be essentially no iodide release from iodide-loaded liposomes lacking functionally reconstituted CFTR until the vesicles are permeabilized with detergent (Figure 3; control traces), whereas CFTR-reconstituted, iodide-loaded proteoliposomes mediate iodide release into the external solution after addition of MgATP (1 mM) to open the CFTR channel and valinomycin (20 nM) to prevent the development of a membrane potential (Figure 3A; see trace for protein:lipid mass ratio of 1:3,000 (w/w)).
Valinomycin is an ionophore that shuttles potassium specifically across the membrane, down its concentration gradient and the concentration of 20 nM was chosen empirically as the highest concentration which did not perturb integrity of empty liposome in the present system. This may need to be optimized for each particular system. Without addition of valinomycin, there is an abbreviated iodide release of a few nM that quickly reaches a plateau (data not shown). Although these conditions are known to otherwise maximally activate the anion channel activity of CFTR (as in Figure 3A), it appears that the lack of a significant response reflects the instantaneous increase in intraliposomal positive charge mediated by iodide conduction through the anion selective pore of CFTR, immediately limiting continuous iodide efflux where valinomycin is not included. This valinomycin-mediated response confirms that iodide efflux was limited in the previous experiments because of charge accumulation, supporting the anion selectivity of reconstituted CFTR. If CFTR lacked this selectivity, both potassium and iodide would have been able to efflux from proteoliposomes thereby abrogating the need for valinomycin to initiate flux.
The iodide concentrations measured at these initial time points were empirically chosen to fall within the optimal sensitivity range for the iodide sensing electrode employed. Use of other iodide electrodes may require optimization of these factors. After reaching a plateau, Triton X-100 is added to permeabilize the proteoliposomes and release any remaining trapped iodide. Unlike the initial iodide measurements (obtained between 1 and 2 min after valinomycin addition), the readings at these endpoints are not in the linear range of the electrode calibration curve (data not shown), thereby precluding an accurate evaluation of the fraction of liposomes from which CFTR protein is capable of mediating iodide efflux relative to the total number of liposomes in the assay, or its percent functional reconstitution.
The rate of iodide flux reflects the number of CFTR protein molecules, the open probability and unitary conductance of each CFTR channel. Hence, the rates of iodide efflux should be dependent on the number and open probability of each CFTR molecule. The prediction was tested in this system by increasing the number of CFTR molecules per liposome. The ratio of activated (phosphorylated and MgATP treated) CFTR protein to lipid was varied, with the ratios ranging from 1:300-1:3,000 (w/w) (Figure 3A). The greater the amount of reconstituted CFTR present in the proteoliposomes, the greater the rate of efflux (measured between 1-2 min after valinomycin addition), supporting the prediction that efflux rates are dependent on the number of CFTR molecules in each liposome. The efflux rate obtained using specific protein:lipid ratios are reproducible between experimental days and protein purifications, thus highlighting the rigor of this method.
The rate of iodide efflux appears to relate to the open probability of the CFTR channel in this reconstituted system. As phosphorylation by PKA is required for CFTR activation31 (and review by32), it is expected that there will be a low rate of iodide efflux in vesicles containing CFTR that has not been phosphorylated by exogenous PKA treatment, where the open probability is low. In proteoliposomes that have been PKA treated, the open probability will be much higher, resulting in a much greater anticipated rate of efflux. In practice, for PKA-treated CFTR in the presence of 1 mM MgATP in this system, the rate of iodide efflux measured from one to two minutes after addition of valinomycin is approximately four fold the rate of CFTR protein subjected to MgATP but not PKA (Figure 3B). These findings support the hypothesis that the efflux rate relate to channel open probability. In practice it may be difficult to compare activity in this assay directly to open probability given that other factors such as time dependent changes in the electrochemical driving force will influence the observed efflux activity. The reader is cautioned that this assay is not a replacement for detailed single channel activity recordings.
Interestingly, there is typically observed a low rate of iodide efflux by “unphosphorylated” protein in the presence of MgATP which is significantly greater than efflux from liposomes lacking CFTR (Figure 3B, C). This low rate may reflect basal phosphorylation of CFTR in the Sf9 cell expression system prior to purification33. CFTR purified from other expression systems may have different levels of basal phosphorylation and therefore different residual activity.
This rapid purification protocol yields Wt-CFTR from Sf9 cells at near homogeneity, however for F508del- and G551D-CFTR, the method is better described as an enrichment procedure. Some contaminating bands are observed via SDS-PAGE19, many of which reflect CFTR degradation products that are reactive to CFTR antibodies and appear to be present before cell disruption. The reconstitution and iodide flux methods are suitable for these enriched samples of clinically relevant mutants. As shown in Figure 3D, significant iodide efflux is measured for both F508del- (protein:lipid mass ratio of approximately 1:600) and G551D-CFTR (protein:lipid ratio of approximately 1:300). While both mutants have lower absolute activity than Wt-CFTR (protein:lipid mass ratio of 1:1,200) , it is difficult to compare the samples directly as quantitation cannot be as accurate for the mutants that are contaminated with CFTR degradation products. However both F508del-CFTR and G551D purified by these methods, reconstituted and subjected to efflux measurements respond as expected to small molecule potentiators (Figure 4), and show similar channel function to protein purified by the more rigorous PFO purification method19.
The channel activity of purified and reconstituted CFTR can be modulated by small molecule inhibitors and potentiators
CFTRinh-17234 is the most specific CFTR inhibitor available that has been shown to inhibit the open probability of CFTR chloride channels in single channel conductance measurements35 and to inhibit CFTR activity in other studies22,36. As shown in Figure 3C, the rate of efflux after valinomycin addition is significantly reduced by pretreatment with CFTRinh-172. Hence, this assay of the channel activity of a population of purified and reconstituted CFTR molecules is effective in reporting activity of modulators such as the inhibitory ligand CFTRinh-172.
The assay is sensitive and highly applicable to examination of the effects of small molecule potentiator treatment as well. As shown in Figure 4, all three CFTR genotypes tested are significantly potentiated by active small molecule potentiators such as Kalydeco/VX-770 or VRT-532 (Figure 4C; shown for G551D), while an inactive analog has no effect on the iodide efflux activity of CFTR (shown for F508del-CFTR; Figure 4B). The assay is applicable to the examination of potentiator concentration dependence, and the role of ATP and phosphorylation on potentiator-mediated CFTR activity.
Figure 1: Assay design and workflow. (A) Workflow for the work described in this publication. (B) Schematic representation of the iodide efflux experiment. A modified version of panel B was originally published in The Journal of Biological Chemistry. Eckford, P.D.W.; Li, C.; Ramjeesingh, M.; and Bear, C.E. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Potentiator VX-770 (Ivacaftor) Opens the Defective Channel Gate of Mutant CFTR in a Phosphorylation-dependent but ATP-independent Manner. 2012; 287: 36639-36649. ©The American Society for Biochemistry and Molecular Biology.
Figure 2: The CFTR rapid purification procedure yields purified Wt-CFTR protein. Silver stained SDS-PAGE gel and Western blot using the M3A7 CFTR antibody for purified human Wt-CFTR protein in DDM detergent (1 µg and 0.1 µg for the gel and blot, respectively), isolated from Sf9 cells over-expressing a (His)10-tagged version of the protein. Wt-CFTR (>90% pure) is detected at 150 kDa, appropriate for this protein lacking complex glycosylation. This research was originally published in The Journal of Biological Chemistry. Eckford, P.D.W.; Li, C.; Ramjeesingh, M.; and Bear, C.E. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Potentiator VX-770 (Ivacaftor) Opens the Defective Channel Gate of Mutant CFTR in a Phosphorylation-dependent but ATP-independent Manner. 2012; 287: 36639-36649. ©The American Society for Biochemistry and Molecular Biology.
Figure 3: The iodide efflux assay reports regulated channel activity for the purified and reconstituted CFTR protein. (A) Purified and phosphorylated Wt-CFTR reconstituted at protein:lipid ratios of 1:300 (w/w), 1:1,200 (w/w) and 1:3,000 (w/w) mediates iodide efflux from the vesicle lumen to the bulk bath over time after addition of 1 mM MgATP and 20 nM valinomycin. The addition of 0.1% (w/v) Triton X-100 to permeabilize the proteoliposomes releases trapped iodide in the vesicle lumen at the end of the experiment. (B) Initial iodide efflux time course for reconstituted Wt-CFTR protein that had not been pre-phosphorylated. (C) Initial iodide efflux rates demonstrate the dependence of the observed activity on functional CFTR protein. PKA phosphorylation of the protein is required for significant channel activity, which is inhibited by the addition of the CFTR inhibitor, CTTRinh-172 (20 µM). Data are means ± SEM; *p <0.05, ***p <0.0001. (D) Multiple genotypes produce regulated CFTR signals in the iodide efflux assay, including Wt-, F508del- and G551D-CFTR versus control. Data are means ± SEM; *p<0.05; ***p<0.0004 vs. control. This research (portions of data in panels A, C and D) was originally published in The Journal of Biological Chemistry. Eckford, P.D.W.; Li, C.; Ramjeesingh, M.; and Bear, C.E. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Potentiator VX-770 (Ivacaftor) Opens the Defective Channel Gate of Mutant CFTR in a Phosphorylation-dependent but ATP-independent Manner. 2012; 287: 36639-36649. ©The American Society for Biochemistry and Molecular Biology.
Figure 4: The phosphorylation-dependent potentiation of CFTR by small molecules can be interrogated using the iodide efflux system for Wt-, F508del- and G551D-CFTR. (a) Wt-CFTR is potentiated significantly by the potentiator VX-770 (10 µM), only in the PKA-phosphorylated state. Data are means ± SEM, *p <0.01 vs. +PKA-VX-770. (b) VX-770 (10 µM) but not an inactive analog, V-09-1188 (10 µM), significantly potentiates the iodide efflux activity of phosphorylated F508del-CFTR. Data are means ± SEM, n = 3, ***p <0.001 vs. –VX-770 control. (c) Potentiation of G551D-CFTR by 10 µM VRT-532 or VX-770. Data are means ± SEM, n = 5, **p <0.001, ***p <0.0001 vs. –VX-770 control. This research (panels a-c) was originally published in The Journal of Biological Chemistry. Eckford, P.D.W.; Li, C.; Ramjeesingh, M.; and Bear, C.E. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Potentiator VX-770 (Ivacaftor) Opens the Defective Channel Gate of Mutant CFTR in a Phosphorylation-dependent but ATP-independent Manner. 2012; 287: 36639-36649. ©The American Society for Biochemistry and Molecular Biology.
Buffer | Name | Contents | pH | Adjust pH using |
Buffer 1 | 2% fos-choline | 2% (w/v) fos-choline 14 detergent in 10 mM imidazole, 25 mM HEPES | 7.4 | Imidazole/HEPES |
Buffer 2 | 10 mM imidazole | 10 mM imidazole, 25 mM HEPES (no detergent) | 7.4 | Imidazole/HEPES |
Buffer 3 | 10 mM imidazole+DDM | 10 mM imidazole, 25 mM HEPES, 1 mM dodecyl-β-D-maltoside | 7.4 | Imidazole/HEPES |
Buffer 4 | 50 mM imidazole+DDM | 50 mM imidazole, 25 mM HEPES, 1 mM dodecyl-β-D-maltoside | 7.4 | Imidazole/HEPES |
Buffer 5 | 600 mM imidazole+DDM | 600 mM imidazole, 25 mM HEPES, 1 mM dodecyl-β-D-maltoside | 7.4 | Imidazole/HEPES |
Buffer 6 | 75 mM KI+DDM | 75 mM KI, 20 mM MOPS, 1 mM dodecyl-β-D-maltoside | 7.4 | KOH |
Buffer 7 | 75 mM KI | 75 mM KI, 20 mM MOPS | 7.4 | KOH |
Buffer 8 | 25 mM HEPES+DDM | 25 mM HEPES, 25 mM NaCl, 1 mM dodecyl-β-D-maltoside | 7.4 | HCl/NaOH |
Buffer 9 | 75 mM K-Glu | 75 mM K-Glutamate, 20 mM MOPS | 7.4 | KOH/Glutamic acid |
Buffer 10 | 15 mM KI | 15 mM KI, 20 mM MOPS | 7.4 | KOH |
Table 1: Table of Buffers.
There have been a limited number of purification protocols for full-length, functional CFTR isolation, from a variety of cellular overexpression systems. The method described here is advantageous as it allows rapid purification of Wt-CFTR or high enrichment of F508del- and G551D-CFTR in moderate quantities that is highly functional in assays including ATPase and direct measurements of channel function, including single channel measurements in planar bilayer systems and demonstrated measures of CFTR population channel function that are highly relevant to the study of small molecules19-21. The purification method is integrated into a rapid and effective reconstitution protocol, however purified protein from other methods may be coupled to this reconstitution protocol as well, provided that the purification detergent is amenable to removal similarly.
As opposed to complicated and painstaking single channel experiments from excised membrane patches, the methods described here allow a robust yet straightforward assessment of CFTR channel function, and uniquely this measure reports the function of a larger population of CFTR channels rather than one or a few molecules. Unlike techniques involving membrane patches, this method allows direct assessment of the effects of small molecule modulators on the channel in the near or complete absence of associated proteins, depending on the effectiveness of the coupled purification procedure.
Critical steps within the protocol
Although there are several critical steps in functional reconstitution of CFTR for the anion flux assay, as indicated notes in the Protocol, optimization of the protein:lipid ratio during CFTR reconstitution is key. Such optimization is required for each CFTR genotype.
Modifications and troubleshooting of the technique
The baseline mV response for the probe used here during the iodide efflux assay with samples prepared as described is typically -20 to -50 mV. Significant deviations from this range suggest there may be problems with the sample. Other probes sold by different manufacturers may differ from the guidelines provided here. If a stable baseline cannot be achieved, residual detergent or inactive, denatured, poor quality protein may be permeabilizing the vesicles to iodide.
Failure to detect a significant change in the rate of valinomycin dependent iodide efflux from liposomes containing phosphorylated CFTR in the presence of MgATP may reflect several issues, all of which should be investigated. These issues include: poor quality, partially denatured CFTR or impure CFTR; incomplete removal of detergent during reconstitution; non-optimal protein: lipid ratio in proteoliposomes; or insufficient valinomycin added, rendering charge dissipation incomplete.
The iodide efflux assays offers the flexibility to use pre-phosphorylated CFTR protein (as described in the protocol), or to phosphorylated CFTR during the assay. If the reconstituted protein was not phosphorylated during the purification procedure, the sample can be phosphorylated during the iodide efflux experiment at step 3.2.7, by addition of PKA with the MgATP. In this case ensure that the baseline is recorded for at least 5 min to ensure that the protein has been sufficiently phosphorylated by the PKA enzyme before measuring function with addition of valinomycin. Similar results are obtained by either method.
During the protocol, the modulator molecule is allowed to interact with the CFTR protein for 5 min before the measurement of activity by addition of MgATP and then Valinomycin. As most CFTR modulators are very lipophilic, it may take time between addition to the bath and equilibrium association with the CFTR protein. The assay cannot proceed without the addition of valinomycin, and thus no information is lost by performing the assay in this manner. In situations where the user is not concerned that there may be a lag time before interaction of the small molecule with the protein, the assay could be performed by the acute addition of modulator after the addition of valinomycin, while still in the initial rate phase of the measurement.
Limitations of the technique
As stated previously, this proteoliposomal flux based assay of regulated channel activity by a population of purified and reconstituted full-length CFTR molecules provides a unique method for interrogating ligands that directly modify activity. However, the method is limited in that it doesn’t provide insight into the mechanism through which ligands change the rate of flux through CFTR. Ideally, the proteoliposomal flux assay of purified and reconstituted CFTR should be used in combination with planar lipid bilayer studies of single channel activity. This combination would provide insight into the state of the protein that binds the ligands, i.e. the open and/or the closed state.
Significance with respect to existing methods
There is currently a lack of methods for assessing the direct interaction of small molecules with the CFTR channel versus small molecule modulators that interact with another cellular component to indirectly alter channel activity, e.g., through a modulatory or signaling pathway or disruption of important protein-protein interactions. The iodide efflux method demonstrates direct interaction with the protein of any small molecule that modulates the channel activity of CFTR. Despite the low throughput manner of the measurements, there remains considerable value in these unique methods for studying the direct interaction of CFTR with small molecule potentiators and correctors. Many small molecule modulators are under development by both academic groups and the pharmaceutical industry to treat CF patients clinically, as this is the major thrust of current CF research. It is anticipated that the methods described here will aid in the development and validation of the mechanism of action of the most promising of these molecules.
Future applications of the technique
The methods employed here are also highly amenable to adaption to other proteins of interest. Indeed a modification of the described methods was employed to study two P. aeruginosa membrane proteins implicated in the movement of anionic substrates across the membrane26,27, demonstrating the utility and versatility of these methods. Their use in the study of additional anion channels and transporters is anticipated in the future.
The authors have nothing to disclose.
The authors declare that they have no competing financial interests.
fos-choline 14 detergent | Anatrace Affymetrix (www.anatrace.affymetrix.com) | F312 | Affymetrix: Anatrace Products; CAS# :77733-28-9 |
cOmplete EDTA-free protease inhibitor cocktail tablets | Roche (www.roche-applied-science.com) | 04 693 132 001 | EDTA-free Protease inhibitor cocktail tablets (1 in 50 ml or mini:1 in 10 mL) (Roche Diagnostics GmbH; Ref: 11873 580 001 |
cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack | Roche (www.roche-applied-science.com) | 05 892 791 001 | |
Ni-NTA Agarose | Qaigen GmbH | 1018240 | |
Fisherbrand Screening columns | Fisher Healthcare | 11-387-50 | |
Amicon Ultra Centrifugal filters, Ultracel-100K | Millipore (www.millipore.com) | UFC910008 | |
cAMP-Dependent Protein Kinase A (PKA), Catalytic Subunit | New England Biolabs (www.neb.com/products/p6000-camp-dependent-protein-kinase-pka-catalytic-subunit) | Peirce PKA is also suitable | |
PC, Chicken Egg | Avanti Polar Lipids (www.avantilipids.com) | 840051C | |
POPC | Avanti Polar Lipids (www.avantilipids.com) | 850457C | |
PS, Porcine Brain | Avanti Polar Lipids (www.avantilipids.com) | 840032C | (CFTR has been successfully reconstituted into Egg PC, POPC, 2:1 (w/w) Egg PC:POPC, or a mixture of PE:PS:PC:ergosterol, 5:2:2:1 (w/w)) |
PE, Chicken Egg | Avanti Polar Lipids (www.avantilipids.com) | 841118C | |
Ergosterol | Sigma (www.sigmaaldrich.com) | 45480 | |
Pierce Detergent removal spin column | Thermo Scientific | 87779 | 1 ml capacity columns |
valinomycin | Sigma (www.sigmaaldrich.com) | V-0627 | |
VX-770 (ivacaftor) | Selleck Chemicals | S1144 | |
iodide selective microelectrode | Lazar Research Laboratories (www.shelfscientific.com/cgi-bin/tame/newlaz/microionn.tam) | LIS-146ICM | |
Clampex 8.1 software | Axon Instruments (www.axon.com) | we use components of the ClampX system with a home made filter to monitor and record the response to our electrode | |
Alternate Software: ArrowLabb System | Lazar Research Laboratories (www.shelfscientific.com/cgi-bin/tame/newlaz/ionsystems.tam) | LIS-146LICM-XS | Lazar Research sells a meter that can interface with a computer and software to record the probe response. This software should serve a similar function to our setup |
small stir bars | Big Science Inc (www.stirbars.com) | SBM-0502-CMB | choose a stir bar small enough to easily fit into a well of a 96-well plate |
Sephadex G50, fine | GE Health Care (www.gelifesciences.com) | 17-0042-01 | |
Sonicator | Laboratory Supplies Co, Inc. | G112SP1G | Bath sonicators from other manufacturers should also be suitable |