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

Summary

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

Abstract

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl^- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl^- from proteoliposomes following the addition of the K⁺ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H⁺/Cl^- exchanger, as a model system. The efflux assay can be utilized to study any Cl^- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

Introduction

In last two decades the ability to overexpress and purify membrane transport proteins has dramatically increased: ion channels, primary and secondary transporters are now routinely purified from heterologous expression systems as well as natural sources. New approaches to monitor expression, improve and facilitate the extraction and enhance stability of these proteins are constantly being developed ^1-5. These technological breakthroughs have been instrumental in triggering the explosion of atomic-level structural information on membrane proteins which, in turn, enhanced our understanding of the structural bases of their function. In contrast, our ability to....

Protocol

1. Lipid Preparation

1. Aliquot the desired amount lipids into a clear glass tube. Use E. coli polar lipid extract, but most lipid compositions can be used. If the lipids are in powder form, resuspend them in chloroform to a concentration of 20 mg/ml. Dry the lipids at RT under N₂ gas until all solvent has evaporated.
2. Resuspend the lipids in pentane and dry it again a steady stream of gas N₂ to remove leftover traces of chloroform. Dry until all solvent has evaporated. While drying, gently rotate the tube so that the lipid is distributed in a uniform film covering the bottom third of the tube rather than a forming a de....

Results

We describe a detailed and robust protocol to measure Cl^- transport mediated by purified CLC-ec1, a prokaryotic CLC-type H⁺/Cl^- exchanger, reconstituted in liposomes. A schematic representation of the experiment is shown in Figure 3. Proteoliposomes reconstituted with purified CLC-ec1 and containing high internal Cl^- are immersed in a bath solution containing low Cl^-. Under these conditions net Cl^- efflux is prevented by the buildup of pos.......

Discussion

We have described a detailed protocol to measure Cl^- transport mediated by purified anion-selective channels or transporters reconstituted in liposomes. The example used was the prokaryotic H⁺/Cl^- exchanger CLC-ec1. However, the methodology can be readily adapted to study channels gated by ligands ^12,13,15, voltage ^11,12, or sporting different anionic selectivity ^15,16 by replacing the Ag:AgCl electrode with one suitable for the ion under consideration. El.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Liposomicator, Avanti Polar Lipids Inc.	Avanti Polar Lipids Inc.	610200
IEC Centra CL2 Benchtop	Thermo Scientific
Orion Research Model 701A Digital pH-mV meter			These can be found on Ebay.
Non-functional pH probe			Any pH meter probe with silver wires will work. The glass/plastic coating needs to be removed and the wires cleaned.
DI-710 Data Logger	DATAQ instruments
WinDAQ acquisition software	DATAQ instruments
Pierce Disposable Plastic Columns, Gravity-flow, 2ml	Pierce (Thermo Scientific)	29922
KIMAX Culture Tubes, Disposable, Borosilicate Glass	Kimble Chase	73500-13100
Extruder Set With Holder/Heating Block	Avanti Polar Lipids Inc.	610000
Computer