Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

Summary

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.

Abstract

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.

Introduction

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 channels^1,3, 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 activity^6-9. 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 previously^2,11-18, 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 fluorophores^22,23, 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.

Protocol

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 system^17,28. Overexpress CFTR and prepare pellets of Sf9 cells according to this protocol.

1. Crude membrane preparation
   1. Obtain a fresh or thaw a frozen Sf9 cell pellet from a 500 ml culture of cells over-expressing wildtype-, F508del-, or G551D-CFTR protein with a C-terminal His₁₀-tag, grown by standard cell culture methods, as described previously^17,28. Cell pellets will have a volume of approximately 5 ml and a wet weight of approximately 5 g.
   2. Resuspend Sf9 cells in 20 ml of phosphate buffered saline (PBS) pH 7.4 with protease inhibitor cocktail (1 tablet per 50 ml) at 4 °C, ensuring the sample appears visually homogeneous and no clumps of cells remain.
   3. Lyse cells using a high pressure cell disruptor (Table of Materials and Equipment), reaching a minimum of 10,000 psi (preferably 15,000-20,000 psi) for 2 min per passage. Keep the sample at 4 °C during lysis.
      1. Collect the sample in a tube on ice after the lysis period then immediately reload the sample in the cell disruptor. Repeat lysis procedure 3 times. Examine under a microscope to ensure less than 10% remain intact.
         NOTE: other methods to lyse cells, such as glass beads, etc. have not been rigorously examined for this system, however a user may find such an alternate method suitable.
   4. Centrifuge cell lysis material at 4 °C in a high speed centrifuge at 2,000 x g for 20 min. Collect the supernatant and dispose of the pellet. Divide the supernatant among 20 tubes and centrifuge the samples for 1 hr at 4 °C at 125,000 x g in a table top ultracentrifuge.
   5. Remove and discard the supernatant, being careful not to disturb the pellet, and store pellets in the same tubes at -80 °C for less than 2 months until use, or retain on ice and continue with the purification immediately.
2. CFTR solubilization
   1. Obtain 1-4 fresh or frozen Sf9 crude membrane pellets and resuspend each in 1 ml of Buffer 1 (2% fos-choline; see Table 1 for complete recipe) at 4 °C on ice, using a 25 G needle and 1 ml syringe until the solution is homogeneous by eye without visible cell membrane clumps. Where more than one pellet is being solubilized, continue to treat each sample per the instructions for a single pellet below in parallel, pooling the samples at step 1.3.2.
   2. Dissolve 1 mini protease inhibitor tablet in 1 ml of Buffer 2 (10 mM imidazole; see Table 1) which lacks fos-choline 14 detergent (protease inhibitors are 10x more concentrated than their recommended dilution at this point) and add 100 µl to the resuspended crude membrane pellet (protease inhibitors are at their recommended dilution at this point). Set on ice for 45-60 min with mixing by inversion 5 times every 5 min.
   3. Centrifuge resuspended crude membrane pellet in a table top ultracentrifuge for 25 min at 125,000 x g and 4 °C. Retain the supernatant, which contains solubilized CFTR and discard the pellet.
3. Column binding, phosphorylation and elution
   1. Wash 400 µl of nickel-NTA (nitrilotriacetic acid) agarose slurry or equivalent resin with 1 ml of Buffer 2 (10 mM imidazole; see Table 1) which lacks detergent, to remove solvent and buffer at 4 °C. Repeat wash twice more.
   2. Combine solubilized CFTR, remaining protease inhibitor (900 µl) and nickel resin (from 400 µl slurry) to a total of 10 ml with Buffer 2 (10 mM imidazole; see Table 1) which lacks any added detergent. The fos-choline 14 concentration is effectively diluted to 0.2% (w/v) at this step. If there are multiple tubes, pool all samples containing solubilized CFTR, protease inhibitors, nickel-NTA resin and 0.2%(w/v) fos-choline-14 at this point. Incubate for 60 min at 4 °C with gentle shaking.
   3. Pass CFTR-protease inhibitor-nickel NTA solution down a small screening column (Table 1) or an equivalent empty column.
   4. Wash the nickel NTA resin, which is retained in the column, with 4 ml per initial pellet of Buffer 3 (10 mM imidazole+DDM; see Table 1) which lacks fos-choline 14 but contains 1 mM DDM (dodecyl maltoside detergent), at 4 °C . Addition of DDM detergent does not significantly alter the pH of this buffer.
   5. Prepare PKA-MgATP solution by adding 25 µl (5 mM, final concentration) of MgATP, 2,500 U of cAMP-dependent protein kinase A, catalytic subunit (PKA) to 475 µl of Buffer 3 (10 mM imidazole+DDM; see Table 1) to the column. Phosphorylate protein by sealing the bottom end of the column with a cap or Parafilm, and adding 500 µl of PKA-MgATP solution for each initial pellet used.
   6. Incubate at room temperature (20 °C) for 20 min with gentle mixing and resuspension of the resin using a 1 ml pipettor every 2 min to ensure that PKA has come in contact with the entire surface of the nickel NTA resin.
   7. Remove the cap and elute the PKA/MgATP solution from the column. Wash the column with 2 ml of Buffer 3 (10 mM imidazole+DDM; see Table 1) at 4 °C.
   8. Multiply the number of pellets used in the experiment by 4. Wash the column with this number of ml of Buffer 4 (50 mM imidazole+DDM; see Table 1) at 4 °C, by adding 4 ml of buffer to the column, allowing it to flow through and immediately adding the next 4 ml aliquot to the column.
   9. Elute the protein by passing 1 ml of Buffer 5 (600 mM imidazole+DDM; see Table 1) at 4 °C per initial pellet used through the column, 1 ml at a time without a pause to allow the column to dry, and collect the entire eluate containing purified CFTR in a single tube.
   10. Concentrate protein sample to remove imidazole and low molecular weight degradation products using a centrifuge filter device (100,000 molecular weight cut-off) such as described in Table of Materials and Equipment or other similar device, in a total of 10 ml Buffer 6 (75 mM KI+DDM; see Table 1) or other buffer of choice containing 1 mM DDM (such as Buffer 7 for non-iodide efflux experiments, 25 mM HEPES+DDM; see Table 1), by centrifuging at 2,000 x g and 4 °C for about 20 min until the sample concentrates to 200 µl. Dilute sample to 10 ml and repeat the concentration step.
       NOTE: In our experience (unpublished), imidazole significantly inhibits the ATPase activity of CFTR and should be removed at this step by dilution and concentration at least twice if the sample will be used for functional measurements.
   11. Collect concentrated purified CFTR. Keep at 4 °C in the presence of DDM buffer until use within 1-2 days or continue on immediately to the reconstitution step. Do not freeze the purified protein, as in our experience we observe reduced activity.

2. Reconstitution of CFTR

1. Lipid preparation
   NOTE: CFTR has been successfully reconstituted into Egg PC, POPC, 2:1 (w/w) Egg PC:POPC (1:1 w/w) mixture, or a mixture of PE:PS:PC:ergosterol, 5:2:2:1 (w/w).
   1. For iodide efflux experiments, dry 5 mg of Egg PC (200 µl of 25 mg/ml stock, see Table of Materials and Equipment) under a stream of argon gas for 20 min at room temperature in a Pyrex or other sturdy (non-borosilicate) glass tube.
   2. Using absorbance at 280 nm, an ELISA assay or other method, estimate the concentration of the purified CFTR protein sample. For iodide efflux experiments with wildtype CFTR, use a protein:lipid ratio of 1:300-1:3,000 (w/w) to produce a sufficient signal. For G551D- and F508del-CFTR, use a protein:lipid ratio of approximately 1:200-1:1,200 (w/w) in order to detect efflux activity.
   3. Optimize the protein:lipid ratio for each particular system. Calculate the volume of purified protein required. Calculate the volume of buffer with 1 mM DDM required to make a final volume of 1 ml, i.e., 1 ml – (volume of protein solution required) = volume of buffer.
      1. Add the calculated volume of the desired buffer system with 1 mM DDM to the dried lipid (but do not add the CFTR protein at this time) and cover the glass tube with Parafilm. For iodide efflux experiments, resuspend lipids in Buffer 6 (75 mM KI+DDM; see Table 1). For other experiments resuspend in Buffer 8 (25 mM HEPES+DDM, see Table 1) or another desired internal buffer containing 1 mM DDM.
      2. Vortex for 2 min at room temperature to aid in suspension of the lipid in the DDM buffer. Alternately, heat the sample to 37 °C for 1-2 min before vortexing.
   4. Suspend the lipid repeatedly at room temperature with a 1 ml syringe and 25 G needle until no lipid film is visible on the sides of the tube. Avoid injecting air into the solution which would produce fine bubbles and aid in oxidation of iodide and lipids.
   5. Sonicate the suspended lipid in the Parafilm covered tube for a 20 sec burst in a bath sonicator containing ice, and then transfer immediately to ice for 1 min. Repeat 3 times.
      NOTE: Intense sonication turns iodide solutions yellow due to oxidation. Therefore avoid excessive sonication. Monitor the sample for color changes. If a color change is noted, discard the sample and prepare again. The change in color due to oxidation of some of the iodide would result in oxidation of the lipids under the same conditions. Displacing the air from the tube with argon gas before sonicating the solution will help prevent oxidation of lipids and iodide. Intense sonication can break poor quality or scratched glass tubes resulting in loss of the sample.
   6. Add the volume of purified CFTR calculated in step 2.1.3 to the sonicated lipid sample to a achieve a final volume of 1 ml. Mix the protein with the lipid and detergent solution by resuspension 10 times with a 25 G needle and 1 ml syringe.
   7. Set lipid and protein sample on ice for 30 min with swirling of tube to mix 5 times every 5 min.
2. Detergent-binding column preparation
   1. Obtain a single use commercial detergent-binding spin column (see Table of Materials and Equipment) with a 1 ml volume capacity and break the bottom tab to start the column flowing.
   2. Centrifuge the column for 2 min at 2,000 x g to remove the storage buffer, and discard this buffer.
   3. Add 5 ml of Buffer 7 (75 mM KI, see Table 1) to the column and then centrifuge at 200 x g for 4 min to elute the wash buffer. Repeat this step 2 more times for a total of 3 washes to remove all traces of the storage buffer. Discard all flow-through.
      NOTE: It is critical that the buffer used to wash the column DOES NOT contain DDM or any other detergent. The column has a finite binding capacity for detergent and if a buffer containing detergent is used, the column will be ineffective at removing the detergent from the protein-lipid-detergent sample.
   4. Carefully aliquot all 1 ml of the lipid-detergent-protein sample onto the top of the column resin and incubate sample at the top of the column for 2 min at room temperature.
   5. Centrifuge the sample at room temperature for 4 min at 2,000 x g and collect the cloudy proteoliposome sample in a clean 1.5 or 2 ml microfuge tube or a glass tube and place the sample on ice.
   6. Collect remaining proteoliposomes in the column by adding 200 µl of Buffer 7 (75 mM KI, Table 1) or other buffer (without detergent) to the top of the column and repeat the centrifugation step for 2 min.
   7. Add the second eluate to the proteoliposome sample if it looks cloudy.
   8. Store the sample at 4 °C overnight or use immediately for iodide efflux measurements.

3. Iodide Efflux Measurements for Purified, Reconstituted CFTR

1. Generation of an iodide gradient across the proteoliposomal membrane
   1. Pre-swell Sephadex G50 beads in Buffer 9 (75 mM K-Glu; potassium glutamate, see Table 1), (approximately 5 g dry beads in 50 ml buffer) or desired external buffer at room temperature for at least 2 hr or at 4 °C overnight.
   2. Prepare 4 Sephadex G50 columns saturated in Buffer 9 (75 mM K-Glu, see Table 1) or other buffer in small screening columns by loading resin to at least ¾ full in the column.
   3. Centrifuge for 4 min at 2,000 x g to remove excess buffer from the resin. There should be approximately 2.25 ml of packed hydrated resin in the column at the end of the centrifugation step.
      NOTE: Resin should be lightly hydrated but not immersed in liquid and a subsequent brief spin should not elute significant volumes of buffer. It is critical to the successful elution of the proteoliposome sample that the column resin is neither too wet nor too dry and the user may need to experiment with the columns in order to become familiar with the most successful buffer exchange conditions.
   4. Carefully add 125-150 µl of proteoliposome sample to the top of each column and centrifuge for 4 min at 2,000 x g.
   5. Pool proteoliposome eluates together for immediate use in iodide efflux or other experiments.
      NOTE: The eluted volume from each column should be roughly 150 µl and the sample should be somewhat cloudy, but less cloudy than the original sample. Larger volumes or clear eluates suggest that the columns haven’t been dried sufficiently, or have been dried too much, respectively, and will not result in a successful iodide efflux experiment.
2. Iodide efflux assay
   1.  Wash an iodide selective micro electrode (Table of Materials and Equipment) extensively with de-ionized water, and then incubate for 20 min before the experiment in Buffer 10 (15 µM KI, see Table 1). Wash the electrode again extensively with de-ionized water.
   2. Add 150 µl of drug (20 µM typical concentration to produce a final concentration of 10 µM in the assay) in Buffer 9 (75 mM K-Glu, see Table 1) or vehicle in Buffer 9 to one well of a 96-well plate with a small flea stir bar.
      1. Ensure that there are no bubbles present. Use a pipette tip to remove stray bubbles. If a drug solution is being used, ensure that the final DMSO concentration in the well is less than 1% (v/v) (typically 0.1-0.2% (v/v)).
   3. Add 150 µl of reconstituted CFTR protein that was produced in section 3.1 in Buffer 9 (75 mM K-Glu, see Table 1) to the well with drug or buffer, to produce a total volume of 300 µl in the well of the plate.
   4. Place the 96-well plate on a stir plate and stir at 130 rpm (or at a moderate speed of approximately ¼ of the maximum speed).
   5. Insert the tip of the iodide selective electrode carefully into the well with the sample such that the tip is not touching the bottom of the well or being hit by the stir bar. Ensure that the reference electrode, if separate, is also in contact with the solution in the well.
   6. Record tracings using a home-made or commercial computer program that interfaces with the electrode (see Table of Materials and Equipment for details). Use a sampling rate of 2 Hz, with filtering of the signal through a low pass filter.
   7. Allow the sample to equilibrate for 5 min to produce a stable flat, or near flat baseline while recording.
   8. Add 3 µl MgATP (100 mM stock prepared in dH₂O and adjusted to pH 7 with KOH; 1 mM final concentration) to the sample to activate the protein. Allow ~5 min to reach a new stable baseline. Always adjust the pH of the 100 mM MgATP stock to neutral before use to avoid changes in the pH of the external buffer.
   9. Add 3 µl of valinomycin stock (2 µM; 20 nM final concentration) to the sample to shunt changes in potential difference generated by iodide-selective conductance through CFTR. Record the decreasing slope (decrease in mV) due to CFTR-mediated iodide efflux activity for at least 5 min.
   10. To ensure that the trapping of iodide in the proteoliposome lumen was sufficient, add 3 µl of 10% (v/v) Triton X-100 to the sample. Significant and immediate iodide release indicates that sufficient iodide has been trapped in the vesicles such that trapping was not the limiting factor for the changes in slope determined in 3.2.9 but rather the CFTR-mediated activity.
   11. Wash the electrode and stir bar thoroughly with de-ionized water after treatment of samples with drug or with Triton X-100 to ensure all traces of these reagents have been removed to avoid contamination of the next sample.
   12. Prepare a series of dilute KI concentrations in the micromolar to nanomolar range in Buffer 9 (K-Glu buffer, see Table 1). Record the mV response of the electrode to each concentration from 0 to the highest concentration prepared. Construct a standard curve where the probe response (mV) is plotted vs. log of the molar iodide concentration. Use the standard curve to convert the mV response of the probe during the efflux experiment to concentration of iodide released.
       NOTE: Prepare a calibration curve on a weekly basis until a user is sure of how quickly the response of the probe changes. There will be a drift in the response and sensitivity of the probe over time. As the probe ages, the response will degrade. This may be partially abrogated by changing the internal solutions periodically and extensive washing of the probe tip in water after use, which likely limits adherence of molecules to the probe surface.
   13. Determine the average slope of the line of the concentration vs. time plot for each proteoliposome sample for the last 30 sec before addition of MgATP, and after addition of valinomycin but before addition of Triton X-100. Subtract the baseline slope from the valinomycin to determine the CFTR-mediated iodide efflux activity for that sample.

Results

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 exp...

Discussion

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 fun...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
[header]
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