The authors declare that they have no competing financial interests.

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.
<ol>
	<li>Crude membrane preparation
	<ol>
		<li>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 previously17,28. Cell pellets will have a volume of approximately 5 ml and a wet weight of approximately 5 g.</li>
		<li>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 &#176;C, ensuring the sample appears visually homogeneous and no clumps of cells remain.</li>
		<li>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 &#176;C during lysis.
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>Centrifuge cell lysis material at 4 &#176;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 &#176;C at 125,000 x g in a table top ultracentrifuge.</li>
		<li>Remove and discard the supernatant, being careful not to disturb the pellet, and store pellets in the same tubes at -80 &#176;C for less than 2 months until use, or retain on ice and continue with the purification immediately.</li>
	</ol>
	</li>
	<li>CFTR solubilization
	<ol>
		<li>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 &#176;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.</li>
		<li>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 &#181;l 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.</li>
		<li>Centrifuge resuspended crude membrane pellet in a table top ultracentrifuge for 25 min at 125,000 x g and 4 &#176;C. Retain the supernatant, which contains solubilized CFTR and discard the pellet.</li>
	</ol>
	</li>
	<li>Column binding, phosphorylation and elution
	<ol>
		<li>Wash 400 &#181;l 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 &#176;C. Repeat wash twice more.</li>
		<li>Combine solubilized CFTR, remaining protease inhibitor (900 &#181;l) and nickel resin (from 400 &#181;l 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 &#176;C with gentle shaking.</li>
		<li>Pass CFTR-protease inhibitor-nickel NTA solution down a small screening column (Table 1) or an equivalent empty column.</li>
		<li>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 &#176;C . Addition of DDM detergent does not significantly alter the pH of this buffer.</li>
		<li>Prepare PKA-MgATP solution by adding 25 &#181;l (5 mM, final concentration) of MgATP, 2,500 U of cAMP-dependent protein kinase A, catalytic subunit (PKA) to 475 &#181;l 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 &#181;l of PKA-MgATP solution for each initial pellet used.</li>
		<li>Incubate at room temperature (20 &#176;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.</li>
		<li>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 &#176;C.</li>
		<li>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 &#176;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.</li>
		<li>Elute the protein by passing 1 ml of Buffer 5 (600 mM imidazole+DDM; see Table 1) at 4 &#176;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.</li>
		<li>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 &#176;C for about 20 min until the sample concentrates to 200 &#181;l. 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.</li>
		<li>Collect concentrated purified CFTR. Keep at 4 &#176;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.</li>
	</ol>
	</li>
</ol>
2. Reconstitution of CFTR
<ol>
	<li>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).
	<ol>
		<li>For iodide efflux experiments, dry 5 mg of Egg PC (200 &#181;l 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.</li>
		<li>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.</li>
		<li>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 &#8211; (volume of protein solution required) = volume of buffer.
		<ol>
			<li>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.</li>
			<li>Vortex for 2 min at room temperature to aid in suspension of the lipid in the DDM buffer. Alternately, heat the sample to 37 &#176;C for 1-2 min before vortexing.</li>
		</ol>
		</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Set lipid and protein sample on ice for 30 min with swirling of tube to mix 5 times every 5 min.</li>
	</ol>
	</li>
	<li>Detergent-binding column preparation
	<ol>
		<li>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.</li>
		<li>Centrifuge the column for 2 min at 2,000 x g to remove the storage buffer, and discard this buffer.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Collect remaining proteoliposomes in the column by adding 200 &#181;l 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.</li>
		<li>Add the second eluate to the proteoliposome sample if it looks cloudy.</li>
		<li>Store the sample at 4 &#176;C overnight or use immediately for iodide efflux measurements.</li>
	</ol>
	</li>
</ol>
3. Iodide Efflux Measurements for Purified, Reconstituted CFTR
<ol>
	<li>Generation of an iodide gradient across the proteoliposomal membrane
	<ol>
		<li>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 &#176;C overnight.</li>
		<li>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 &#190; full in the column.</li>
		<li>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.</li>
		<li>Carefully add 125-150 &#181;l of proteoliposome sample to the top of each column and centrifuge for 4 min at 2,000 x g.</li>
		<li>Pool proteoliposome eluates together for immediate use in iodide efflux or other experiments. 
		NOTE: The eluted volume from each column should be roughly 150 &#181;l and the sample should be somewhat cloudy, but less cloudy than the original sample. Larger volumes or clear eluates suggest that the columns haven&#8217;t been dried sufficiently, or have been dried too much, respectively, and will not result in a successful iodide efflux experiment.</li>
	</ol>
	</li>
	<li>Iodide efflux assay
	<ol>
		<li>&#160;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 &#181;M KI, see Table 1). Wash the electrode again extensively with de-ionized water.</li>
		<li>Add 150 &#181;l of drug (20 &#181;M typical concentration to produce a final concentration of 10 &#181;M 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.
		<ol>
			<li>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)).</li>
		</ol>
		</li>
		<li>Add 150 &#181;l 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 &#181;l in the well of the plate.</li>
		<li>Place the 96-well plate on a stir plate and stir at 130 rpm (or at a moderate speed of approximately &#188; of the maximum speed).</li>
		<li>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.</li>
		<li>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.</li>
		<li>Allow the sample to equilibrate for 5 min to produce a stable flat, or near flat baseline while recording.</li>
		<li>Add 3 &#181;l 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.</li>
		<li>Add 3 &#181;l of valinomycin stock (2 &#181;M; 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.</li>
		<li>To ensure that the trapping of iodide in the proteoliposome lumen was sufficient, add 3 &#181;l 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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Kim Chiaw, P., Eckford, P. D., Bear, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+the+mechanisms+underlying+CFTR+channel+activity,+the+molecular+basis+for+cystic+fibrosis+and+strategies+for+therapy.">Insights into the mechanisms underlying CFTR channel activity, the molecular basis for cystic fibrosis and strategies for therapy.</a> Essays Biochem. 50 (1), 233-248 (2011).</li><li>Bear, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+functional+reconstitution+of+the+cystic+fibrosis+transmembrane+conductance+regulator+(CFTR).">Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR).</a> Cell. 68 (4), 809-818 (1992).</li><li>Cai, Z., Sohma, Y., Bompadre, S. G., Sheppard, D. N., Hwang, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+high-resolution+single-channel+recording+to+functional+studies+of+cystic+fibrosis+mutants.">Application of high-resolution single-channel recording to functional studies of cystic fibrosis mutants.</a> Methods Mol Biol. 741, 419-441 (2011).</li><li>Kerem, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+cystic+fibrosis+gene:+genetic+analysis.">Identification of the cystic fibrosis gene: genetic analysis.</a> Science. 245 (4922), 1073-1080 (1989).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+defective+anion+transport+in+L+cells+expressing+recombinant+forms+of+CFTR.">Molecular basis of defective anion transport in L cells expressing recombinant forms of CFTR.</a> Hum Mol Genet. 2 (8), 1253-1261 (1993).</li><li>Mendoza, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirements+for+efficient+correction+of+DeltaF508+CFTR+revealed+by+analyses+of+evolved+sequences.">Requirements for efficient correction of DeltaF508 CFTR revealed by analyses of evolved sequences.</a> Cell. 148 (1-2), 164-274 (2012).</li><li>Rabeh, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+both+NBD1+energetics+and+domain+interface+is+required+to+restore+DeltaF508+CFTR+folding+and+function.">Correction of both NBD1 energetics and domain interface is required to restore DeltaF508 CFTR folding and function.</a> Cell. 148 (1-2), 150-163 (2012).</li><li>Aleksandrov, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+modulation+balances+thermodynamic+stability+and+restores+function+of+DeltaF508.">Allosteric modulation balances thermodynamic stability and restores function of DeltaF508.</a> CFTR. J Mol Biol. 419 (1-2), 41-60 (2012).</li><li>Ramsey, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CFTR+potentiator+in+patients+with+cystic+fibrosis+and+the+G551D+mutation.">A CFTR potentiator in patients with cystic fibrosis and the G551D mutation.</a> N Engl J Med. 365 (18), 1663-1672 (2011).</li><li>Riordan, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+recombinant+cystic+fibrosis+transmembrane+conductance+regulator+from+Chinese+hamster+ovary+and+insect+cells.">Purification and characterization of recombinant cystic fibrosis transmembrane conductance regulator from Chinese hamster ovary and insect cells.</a> J. Biol. Chem. 270 (28), 17033-17043 (1995).</li><li>Ryan, L., Rimington, T., Cant, N., Ford, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+purification+of+the+cystic+fibrosis+transmembrane+conductance+regulator+protein+in+Saccharomyces+cerevisiae.">Expression and purification of the cystic fibrosis transmembrane conductance regulator protein in Saccharomyces cerevisiae.</a> JoVE. (61), (2012).</li><li>Ostedgaard, L. S., Welsh, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+purification+of+the+cystic+fibrosis+transmembrane+conductance+regulator.">Partial purification of the cystic fibrosis transmembrane conductance regulator.</a> J Biol Chem. 267 (36), 26142-26149 (1992).</li><li>Peng, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+affinity+isolation+of+recombinant+protein+using+the+baculovirus/insect+cell+expression+system.">One-step affinity isolation of recombinant protein using the baculovirus/insect cell expression system.</a> Protein Expr Purif. 4 (2), 95-100 (1993).</li><li>Ramjeesingh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+procedure+for+the+efficient+purification+of+the+cystic+fibrosis+transmembrane+conductance+regulator+(CFTR).">A novel procedure for the efficient purification of the cystic fibrosis transmembrane conductance regulator (CFTR).</a> Biochem J. 327 (1), 17-21 (1997).</li><li>Rosenberg, M. F., Kamis, A. B., Aleksandrov, L. A., Ford, R. C., Riordan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+crystallization+of+the+cystic+fibrosis+transmembrane+conductance+regulator+(CFTR).">Purification and crystallization of the cystic fibrosis transmembrane conductance regulator (CFTR).</a> J Biol Chem. 279 (37), 39051-39057 (2004).</li><li>Ramjeesingh, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+reconstitution+of+epithelial+chloride+channel+cystic+fibrosis+transmembrane+conductance+regulator.">Purification and reconstitution of epithelial chloride channel cystic fibrosis transmembrane conductance regulator.</a> Methods Enzymol. 294, 227-246 (1999).</li><li>Sorscher, E. J., Sommerfelt, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+recombinant+protein+derived+from+the+baculovirus+expression+system+using+glutathione+affinity+agarose.">Purification of recombinant protein derived from the baculovirus expression system using glutathione affinity agarose.</a> Methods Mol Biol. 3, 337-348 (1995).</li><li>Eckford, P. D., Li, C., Ramjeesingh, M., Bear, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> J Biol Chem. 287 (44), 36639-36649 (2012).</li><li>Alkhouri, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+properties+of+molecular+probes+for+the+rescue+site+on+mutant+cystic+fibrosis+transmembrane+conductance+regulator.">Synthesis and properties of molecular probes for the rescue site on mutant cystic fibrosis transmembrane conductance regulator.</a> J Med Chem. 54 (24), 8693-8701 (2011).</li><li>Eckford, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VX-809+and+Related+Corrector+Compounds+Exhibit+Secondary+Activity+Stabilizing+Active+F508del-CFTR+after+Its+Partial+Rescue+to+the+Cell+Surface.">VX-809 and Related Corrector Compounds Exhibit Secondary Activity Stabilizing Active F508del-CFTR after Its Partial Rescue to the Cell Surface.</a> Chem Biol. 21 (5), 666-678 (2014).</li><li>Kim Chiaw, P., Wellhauser, L., Huan, L. J., Ramjeesingh, M., Bear, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+corrector+modifies+the+channel+function+of+F508del-CFTR.">A chemical corrector modifies the channel function of F508del-CFTR.</a> Mol.Pharmacol. 78 (3), 411-418 (2010).</li><li>Verkman, A. S., Galietta, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride+channels+as+drug+targets.">Chloride channels as drug targets.</a> Nat Rev Drug Discov. 8 (2), 153-171 (2009).</li><li>Pasyk, S., Li, C., Ramjeesingh, M., Bear, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+interaction+of+a+small-molecule+modulator+with+G551D-CFTR,+a+cystic+fibrosis-causing+mutation+associated+with+severe+disease.">Direct interaction of a small-molecule modulator with G551D-CFTR, a cystic fibrosis-causing mutation associated with severe disease.</a> Biochem J. 418 (1), 185-190 (2009).</li><li>Norez, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+CFTR+chloride+channel+activity+and+pharmacology+using+radiotracer+flux+methods.">Determination of CFTR chloride channel activity and pharmacology using radiotracer flux methods.</a> J Cyst Fibros. 3 (2), 119-121 (2004).</li><li>Whitney, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+alginate+secretion+across+the+bacterial+outer+membrane.">Structural basis for alginate secretion across the bacterial outer membrane.</a> Proc Natl Acad Sci U S A. 108 (32), 13083-13088 (2011).</li><li>Islam, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proton-dependent+gating+and+proton+uptake+by+Wzx+support+O-antigen-subunit+antiport+across+the+bacterial+inner+membrane.">Proton-dependent gating and proton uptake by Wzx support O-antigen-subunit antiport across the bacterial inner membrane.</a> mBio. 4 (5), e00678-e00613 (2013).</li><li>Ramjeesingh, M., Conn, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+16.">Ch. 16.</a> Reliable Lab Solutions: Essential Ion Channel MethodsReliable Lab Solutions. , 337-357 (2010).</li><li>Wellhauser, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small-molecule+modulator+interacts+directly+with+deltaPhe508-CFTR+to+modify+its+ATPase+activity+and+conformational+stability.">A small-molecule modulator interacts directly with deltaPhe508-CFTR to modify its ATPase activity and conformational stability.</a> Mol Pharmacol. 75 (6), 1430-1438 (2009).</li><li>Lee, S. Y., Letts, J. A., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+reconstitution+of+purified+human+Hv1+H++channels.">Functional reconstitution of purified human Hv1 H+ channels.</a> J Mol Biol. 387 (5), 1055-1060 (2009).</li><li>Chang, X. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+A+(PKA)+still+activates+CFTR+chloride+channel+after+mutagenesis+of+all+10+PKA+consensus+phosphorylation+sites.">Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites.</a> J. Biol. Chem. 268 (15), 11304-11311 (1993).</li><li>Gadsby, D. C., Nairn, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+CFTR+Cl-+ion+channels+by+phosphorylation+and+dephosphorylation.">Regulation of CFTR Cl- ion channels by phosphorylation and dephosphorylation.</a> Adv.Second Messenger Phosphoprotein Res. 33, 79-106 (1999).</li><li>Li, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATPase+activity+of+the+cystic+fibrosis+transmembrane+conductance+regulator.">ATPase activity of the cystic fibrosis transmembrane conductance regulator.</a> J Biol Chem. 271 (45), 28463-28468 (1996).</li><li>Ma, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiazolidinone+CFTR+inhibitor+identified+by+high-throughput+screening+blocks+cholera+toxin-induced+intestinal+fluid+secretion.">Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion.</a> J Clin Invest. 110 (11), 1651-1658 (2002).</li><li>Kopeikin, Z., Sohma, Y., Li, M., Hwang, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+mechanism+of+CFTR+inhibition+by+a+thiazolidinone+derivative.">On the mechanism of CFTR inhibition by a thiazolidinone derivative.</a> J Gen Physiol. 136 (6), 659-671 (2010).</li><li>Wang, X. F., Reddy, M. M., Quinton, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+new+cystic+fibrosis+transmembrane+conductance+regulator+inhibitor+on+Cl-+conductance+in+human+sweat+ducts.">Effects of a new cystic fibrosis transmembrane conductance regulator inhibitor on Cl- conductance in human sweat ducts.</a> Exp Physiol. 89 (4), 417-425 (2004).</li></ol>

The authors acknowledge the advice of Dr. Mohabir Ramjeesingh during development of the purification methods described here. These studies were supported by Operating Grants to C.E.B. from the Canadian Institutes of Health Research (CIHR) and Cystic Fibrosis Canada (CFC). P.D.W.E. was supported in part by Fellowship awards through the CIHR and CFC. The authors acknowledge the provision of corrector, potentiator and inhibitor compounds from Dr. R. Bridges (Rosalind University of Medicine and Science) and distribution by the Cystic Fibrosis Foundation Therapeutics (CFFT). The authors also acknowledge outstanding service by the Baylor Cell Culture Facility (NIH grant P30 CA125123) in expressing Wt and mutant CFTR protein using the baculovirus system. The inactive VX-770 analog, V-09-1188, was a gift of Vertex Pharmaceuticals.

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&#8217;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.

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 &#62;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.

<table><tbody><tr><td>&lt;em&gt;fos&lt;/em&gt;-choline 14 detergent</td><td>Anatrace Affymetrix (www.anatrace.affymetrix.com)</td><td>F312</td><td>Affymetrix: Anatrace Products; CAS#&amp;nbsp;:77733-28-9</td></tr><tr><td>cOmplete EDTA-free protease inhibitor cocktail tablets</td><td>Roche (www.roche-applied-science.com)</td><td>04 693 132 001</td><td>EDTA-free Protease inhibitor cocktail tablets (1 in 50 ml or mini: 1 in 10 ml) (Roche Diagnostics GmbH; Ref: 11873 580 001)</td></tr><tr><td>cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack&amp;nbsp;</td><td>Roche (www.roche-applied-science.com)</td><td>05 892 791 001</td><td></td></tr><tr><td>Ni-NTA Agarose</td><td>Qaigen GmbH</td><td>1018240</td><td></td></tr><tr><td>Fisherbrand Screening columns</td><td>Fisher Healthcare</td><td>11-387-50</td><td></td></tr><tr><td>Amicon Ultra Centrifugal filters, Ultracel-100K</td><td>Millipore (www.millipore.com)</td><td>UFC910008</td><td></td></tr><tr><td>cAMP-Dependent Protein Kinase A (PKA), Catalytic Subunit</td><td>New England Biolabs (www.neb.com/products/p6000-camp-dependent-protein-kinase-pka-catalytic-subunit)</td><td></td><td>Peirce PKA is also suitable</td></tr><tr><td>PC, Chicken Egg</td><td>Avanti Polar Lipids (www.avantilipids.com)</td><td>840051C</td><td></td></tr><tr><td>POPC</td><td>Avanti Polar Lipids (www.avantilipids.com)</td><td>850457C</td><td></td></tr><tr><td>PS, Porcine Brain</td><td>Avanti Polar Lipids (www.avantilipids.com)</td><td>840032C</td><td>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)</td></tr><tr><td>PE, Chicken Egg</td><td>Avanti Polar Lipids (www.avantilipids.com)</td><td>841118C</td><td></td></tr><tr><td>Ergosterol</td><td>Sigma (www.sigmaaldrich.com)</td><td>45480</td><td></td></tr><tr><td>Pierce Detergent removal spin column</td><td>Thermo Scientific</td><td>87779</td><td>1 ml capacity columns</td></tr><tr><td>valinomycin</td><td>Sigma (www.sigmaaldrich.com)</td><td>V-0627</td><td></td></tr><tr><td>VX-770 (ivacaftor)</td><td>Selleck Chemicals</td><td>S1144</td><td></td></tr><tr><td>iodide selective microelectrode</td><td>Lazar Research Laboratories (www.shelfscientific.com/cgi-bin/tame/newlaz/microionn.tam)</td><td>LIS-146ICM</td><td>&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Clampex 8.1 software</td><td>Axon Instruments (www.axon.com)</td><td></td><td>we use components of the ClampX system with a home made filter to monitor and record the response to our electrode</td></tr><tr><td>Alternate Software: ArrowLabb System&amp;nbsp;</td><td>Lazar Research Laboratories (www.shelfscientific.com/cgi-bin/tame/newlaz/ionsystems.tam)</td><td>LIS-146LICM-XS</td><td>Lazar Research sells a meter that can interface with a computer and software to record the probe response.&amp;nbsp; This software should serve a similar function to our setup.</td></tr><tr><td>small stir bars</td><td>Big Science Inc (www.stirbars.com)</td><td>SBM-0502-CMB</td><td>choose a stir bar small enough to easily fit into a well of a 96-well plate</td></tr><tr><td>Sephadex G50, fine</td><td>GE Health Care (www.gelifesciences.com)</td><td>17-0042-01</td><td></td></tr><tr><td>Sonicator</td><td>Laboratory Supplies Co, Inc.</td><td>G112SP1G</td><td>Bath sonicators from other manufacturers should also be suitable</td></tr></tbody></table>

functional reconstitution and channel activity measurements of purified wildtype and mutant cftr protein

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.

Watch this Scientific Journal Video about Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein at JoVE.com

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

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 ...

functional-reconstitution-channel-activity-measurements-purified

Nanyang Technological University

Research

JoVE Journal

Biology

12.2K Views.  Hospital for Sick Children.  Hier beschreven is een snelle en efficiënte procedure voor de functionele reconstitutie van gezuiverde wildtype en mutante CFTR-eiwit dat activiteit behoudt deze chloride kanaal, die defect in Cystic Fibrosis. Jodide efflux uit opgelost proteoliposomen gemedieerd door CFTR maakt studies van het kanaal en de effecten van kleine moleculen. 

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

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Biochemistry

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

FRET-Based Method to Monitor LRRC8 Channel Activity Without Electrophysiology

Monitoring Leucine-Rich Repeat Containing 8 Channel (LRRC8/VRAC) Activity Using Sensitized-Emission F&#246;rster Resonance Energy Transfer (SE-FRET)

Members of the LRRC8 protein family form heteromeric ion and osmolyte channels with roles in numerous physiological processes. As volume-regulated anion channels (VRACs)/volume-sensitive outwardly rectifying channels (VSORs), they are activated upon osmotic cell swelling and mediate the extrusion of chloride and organic osmolytes, leading to the efflux of water and hence cell shrinkage. Beyond their role in osmotic volume regulation, VRACs have been implicated in cellular processes such as differentiation, migration, and apoptosis. Through their effect on membrane potential and their transport of various signaling molecules, leucine-rich repeat containing 8 (LRRC8) channels play roles in neuron-glia communication, insulin secretion, and immune response. The activation mechanism has remained elusive. LRRC8 channels, like other ion channels, are typically studied using electrophysiological methods. Here, we describe a method to detect LRRC8 channel activation by measuring intra-complex sensitized-emission F&#246;rster resonance energy transfer (SE-FRET) between fluorescent proteins fused to the C-terminal leucine-rich repeat domains of LRRC8 subunits. This method offers the possibility to study channel activation in situ without exchange of the cytosolic environment and during processes such as cell differentiation and apoptosis.

Electrophysiology is the gold standard for investigating ion channel activity. However, there are plenty of alternative approaches, including optical methods. Here, we describe a method to monitor the activity of the leucine-rich repeat containing 8 channel (LRRC8)-formed anion channels using an inter-subunit F&#246;rster resonance energy transfer (FRET)-based method.

Members of the LRRC8 protein family form heteromeric ion and osmolyte channels with roles in numerous physiological processes. As volume-regulated anion ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines

Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery campaigns targeting recombinant ion channel proteins overexpressed in cells such as HEK-293 cells. However, there is increasing emphasis on the use of tissue-relevant cell lines for studying the effects of small molecule interventions. The following protocol describes the adaptation of a fluorescence-based membrane potential assay for the study of ion channels endogenously expressed in epithelial cell lines. The membrane potential assay details a high-throughput assay for chloride channel activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in two commonly studied epithelial cell lines, Caco-2 and Calu-3. In addition, this paper describes a novel application of this system to measure the activity of the Epithelial Sodium Channel (ENaC) in a high-throughput format in the same epithelial cell lines. Together, these fluorescence-based assays provide a robust and flexible platform for studying small molecule modulators, targeting two epithelial channels in a relevant cellular context.

This protocol describes a method for the study of electrogenic membrane proteins by measuring changes in membrane potential. This assay provides a platform for the functional readout of multiple ion channels endogenously expressed in epithelial cell lines.

Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery ...

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

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