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

The overall goal of this procedure is to measure the function of a population of CFTR channels in a reconstituted system and to examine the response of the channels to the presence of modulator molecules. This is accomplished by first purifying wild type or mutant CFTR protein from SF nine cells using a rapid purification procedure, which yields functional CFTR protein in detergent solution. The second step is to reconstitute the purified protein into protea liposomes of defined lipid as selected by the experimenter.

Next, a chemical gradient of iodide is developed across the membrane by removing the extra liposomal iodide to generate a driving force for ion movement through the CFTR channel. The final step is to activate the channel and initiate iodide release from the protea liposomes in the presence of the modulator molecule of interest. Ultimately, an iodide selective electrode is used to monitor CFTR channel function in real time in this reconstituted system.

This method enables study of the chloride channel activity of a purified population of CFTR molecules after reconstitution into phospholipid liposomes. Although this method was decided to provide insight into the function of C-F-D-R-N nine channel and the effect of small molecule modulators, this method can also be applied on other systems such as functional characterization of other membrane proteins. To be successful, individuals new to this method need to ensure that they have optimized the ratios of protein to lipid for their system, that they're not working with poorly functional or non-functional protein, and that they have generated a sufficient iodide gradient across well sealed vesicles.

To prepare the lipid first dry five milligrams of egg phosphatidylcholine or egg PC in a non boro silicate glass tube under a stream of argon gas for 20 minutes at room temperature. Next, calculate the volume of buffer six needed as indicated in the text protocol, and then add buffer six directly to the lipid. Seal the glass tube with perfil and then vortex the tube for two minutes at room temperature to suspend the lipid in the buffer.

Next, suspend the lipid at room temperature with a 25 gauge needle attached to a one milliliter syringe until there is no visible lipid film on the side of the tube. The buffer will be cloudy due to the suspended lipid. Sonicate the lipid in the tube for a 22nd burst in a bath, sonicate containing ice, and then place the tube on ice to rest for one minute.

Perform this cycle three times. Next, add the CFTR protein to the lipid, obtaining a final volume of one milliliter. Mix the protein with a lipid suspension 10 times with a 25 gauge needle.

Place the sample on ice for 30 minutes, swirling the tube five times every five minutes to mix while incubating the sample. Break the bottom tab of a single use detergent binding spin column and centrifuge the column for two minutes at 2000 times G.To remove the storage buffer, wash the column with five milliliters of buffer seven and centrifuge the column for four minutes at 2000 times. G to elute the wash buffer.

Wash the column three times, transfer the entire one milliliter protein lipid sample to the top of the column resin, and then incubate the column for two minutes at room temperature. Centrifuge the column at room temperature for four minutes at 2000 times G.Then transfer the cloudy protea liposome sample to a clean micro fuse tube and place the tube on ice. Soak five grams of cidex G 50 beads in 50 milliliters of buffer nine for at least two hours at room temperature.

Then load four small screening columns with the saturated beads until they are at least three fourths full. Centrifuge the columns for four minutes at 2000 times G to remove excess buffer from the resin. Approximately 2.25 milliliters of packed resin should be in the column after centrifugation.

It is critical that the resin is appropriately hydrated for elution with the protea liposomes. Next, transfer 125 microliters of the protea liposome sample to the top of each column and centrifuge for four minutes at 2000 times. G Pool the EITs for use in the iodide FLX experiments.

Place a small F fleece stir bar into one well of a 96 well plate, and then add 150 microliters of VX seven 70 at a concentration of 20 micromolar in buffer nine to the, well. Use a pipette tip to remove any bubbles. Then add 150 microliters of the protea liposome sample to the well for a total volume of 300 microliters.

Place the 96 well plate on a stir plate at 130 revolutions per minute. Insert the tip of the iodide sensitive electrode into the solution without touching the stir bar or the bottom of the well. To record the tracings of the electrode signal, filter the signal through a low pass filter with a sampling rate of two hertz record tracings from the sample for five minutes to allow the sample to equilibrate.

It should produce a stable, nearly flat baseline. Then add three microliters of a 100 millimolar stock solution of magnesium A TP at pH seven. To activate the CFTR channels, allow the sample to equilibrate for another five minutes to reach a new baseline.

Next, add three microliters of a two micromolar stock solution. A valin mycin to eliminate the electrochemical gradient generated by CFTR activity. Record the decrease in voltage due to CFTR mediated iodide FLX activity for at least five minutes.

Then add three microliters of a solution of 10%Triton X 100 to the sample to release iodide from the protea liposome lumen as a control. Significant iodide release indicates that the trapped iodide in the vesicles was not limiting for CFTR activity. After measuring the sample, make a dilution series of potassium iodide from micromolar to nano molar concentrations in buffer nine.

Starting with the most dilute solution, record the response of the electrode in millivolts at each iodide concentration to generate a standard curve. Wild type CFTR was assayed for iodide. F flux activity measured in ano moles per second after reconstitution.

Iodide F flux is stimulated significantly in the presence of CFTR and A TP versus the control lacking CFTR and by the addition of a TP.When CFTR is phosphorylated by the cyclic A MP dependent protein kinase. A catalytic subunit known as PKA, conversely pretreatment with the potent inhibitor CFTR inhibitor 1 72 reduces the iodide FLX rate the ability of VX seven 70 to potentiate iodide F flux of wild type CFTR was tested without and with phosphorylation by PKA VX seven 70 increased the relative iodide FLX only in the presence of phosphorylated CFTR. The effective VX seven 70 was assay using a variant of CFTR called Delta 5 0 8 CFTR and inactive analog V 0 9 11 88 does not potentiate the activity of phosphorylated delta 5 0 8 CFTR while VX seven 70 increase the relative iodide flx Once mastered, this technique can be completed from purification through CFTR activity measurements in a typical eight hour workday, purification of CFTR takes approximately four hours and reconstitution takes another hour before the eFlex assay is started.

Following this procedure, other methods including a T PS assay and the single channel conduct a measurement in the para lipid S can be performed using the same preparation of CFDR protein to further characterize the interaction with the small molecules. Using these methods, a cystic fibrosis investigator will be able to address key questions in the CFTR modulator field, namely, does that modulator interact directly with the CFTR protein and also what is the mechanism of action of that modulator.