A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl^- Channels and Transporters

The overall goal of this procedure is to quantitatively determine the transport activity of reconstituted chloride channels or transporters. This is accomplished by first reconstituting the pure purified transporter into liposomes at a low density so that each liposome contains either zero or one copy of the active complex after removing the external chloride ions so that there is an outwardly directed ionic gradient, the liposomes are added to the recording chamber where the chloride concentration is monitored using a chloride sensitive electrode. Next chloride flx from the liposomes is initiated by addition of Val Mycin, a potassium ionophore, which aleves the membrane potential.

Once the signal reaches steady state, the experiment is terminated by the addition of detergent to dissolve the liposomes. Ultimately, by measuring the rate of chloride efflux from protea liposomes, reconstituted with a purified channel or transporter, it is possible to quantitatively determine the unitary transport rate and the stoichiometry of the active protein complex. The main advantage of this technique over other existing approaches, such as fluorescence based flux measurements, is that with this technique, it is possible to quantitatively determine key parameters of the reconstituted transporter, such as the unitary turnover rate, and the geometry of the complex.

In addition to providing quantitative measures of these key parameters, this method can be used to rapidly and precisely determine the effect of mutation small molecules, and monitor subunits in the reconstituted transfer protein. The recording setup consists of two chambers, a chloride electrode, a pH meter with an analog or digital electrical output, a digitizer and a computer with appropriate acquisition software. Connect the chloride electrode to the pH meter, then connect the output of the pH meter to the digitizer, which is connected to the computer.

Place the reference electrode in one chamber and the recoding wire in the other. Next place the chambers on a stir plate with the stir bar in the recording chamber. Make sure the stir bar does not touch the electrode.

Connect the chambers using an agar bridge the night before the experiments swell one gram of cidex G 50 beads in 15 milliliters of external buffer with gentle shaking for at least three hours at room temperature before use. Each experiment requires approximately three milliliters of swollen beads while the liposomes are thawing at room temperature. Pour approximately three milliliters of swollen G 50 beads into each column.

Let them dry by gravity. Flow at room temperature. This usually takes one to two minutes.

Next, prepare ular vesicles by extruding the protea liposomes 11 times through a mini extruder using a 0.4 micron Teflon cutoff. Then place the column in a plastic round bottom tube centrifuge for 20 to 30 seconds at 1400 G in a clinical centrifuge to remove excess solution. After this, guarding the flow through, place the column in a 13 by 100 millimeter glass tube and add 100 microliters of the extruded vesicles to the column.

Spin the column for one minute at 500 G.In a clinical centrifuge, collect approximately 200 microliters of the flow through which will be added to the recording chamber. To perform efflux measurement, place two milliliters of 100 millimolar potassium chloride in the reference chamber, and 1.8 milliliters of the external buffer to the recording chamber. Start the acquisition program.

Let the baseline equilibrate. This may take a few minutes. Once the signal reaches a stable baseline, start recording.

Add 15 microliters of a tan molar potassium chloride solution to calibrate the system. Then add liposomes. A small jump might be visible due to incomplete removal of external chloride from the protea liposomes.

Wait until the baseline stabilizes. Next, add CIN to initiate flx. Let the FLX run its time course until it plateaus.

At this point, add 40 microliters of 1.5 molar beta Octa glucocide prepared in the external buffer to dissolve all liposomes. Then wait 10 to 15 seconds. End the recording and proceed to data analysis as described in the text protocol.

When liposomes containing high internal chloride are immersed in low chloride producing a 300 fold chloride gradient, no net ion flux is observed. This is because the protein free liposomes have low intrinsic permeability to ions. No macroscopic FLX is observed from prot liposomes reconstituted with the proton coupled chloride transporter, C-L-C-E-C one.

Since the small amount of chloride exiting from the vesco builds up a net positive charge in the liposome addition of CIN allows potassium ions to move across the membrane relieving the electrical potential and initiating chloride flx. The reaction is terminated and the total amount of chloride ions contained in the vesicles is measured to determine properties of the single reconstituted proteins. The raw data of the efflux time course is first converted from millivolts to a chloride concentration and then normalized the time course is then fitted.

It was determined that the time constant of efflux is 41 seconds and the fraction of liposomes containing zero active copies of C-L-C-E-C one is point 31, indicating that approximately one third of the liposomes contain zero active proteins from these values and measuring the chloride content of vesicles containing at least one copy of C-L-C-E-C one, it is possible to determine a unitary transport rate of C-L-C-E-C one equal to approximately 2, 500 chloride ions per second. After watching this video, you should have a good understanding of how to determine the need to turnover of a transporter. These experiments can be carried out in 10 to 15 minutes when performed carefully.

It is critical to carefully assess the quality of the liposomes as leaks adversely affect the estimates of the turnover rate.