A quantitative flux assay for the study of reconstituted Cl^- channels and transporters

The recent deluge of high-resolution structural information on membrane proteins has not been accompanied by a comparable increase in our ability to functionally interrogate these proteins. Current functional assays often are not quantitative or are performed in conditions that significantly differ from those used in structural experiments, thus limiting the mechanistic correspondence between structural and functional experiments. A flux assay to determine quantitatively the functional properties of purified and reconstituted Cl^- channels and transporters in membranes of defined lipid compositions is described. An ion-sensitive electrode is used to measure the rate of Cl^- efflux from proteoliposomes reconstituted with the desired protein and the fraction of vesicles containing at least one active protein. These measurements enable the quantitative determination of key molecular parameters such as the unitary transport rate, the fraction of proteins that are active, and the molecular mass of the transport protein complex. The approach is illustrated using CLC-ec1, a CLC-type H⁺/Cl^- exchanger as an example. The assay enables the quantitative study of a wide range of Cl^- transporting molecules and proteins whose activity is modulated by ligands, voltage, and membrane composition as well as the investigation of the effects of compounds that directly inhibit or activate the reconstituted transport systems. The present assay is readily adapted to the study of transport systems with diverse substrate specificities and molecular characteristics, and the necessary modifications needed are discussed.