A radioactive uptake assay to measure ion transport across ion channel-containing liposomes.
Here we describe a procedure for incorporating ion channels into lipid vesicles (liposomes) and functional characterization of the channel population by assaying radioactive isotope uptake into these proteoliposomes. The technique as described will work only for potassium channels but can be easily modified, as suggested in the text, for other ion channels and transporters. Purified ion channel proteins in detergent micelles are combined with solubilized lipids. Detergent is subsequently removed from protein-lipid complexes by gel filtration or dialysis into high potassium (high [K+]) buffer. After freezing-thawing and sonication, the resultant larger liposomes are passed over another gel-filtration column to exchange an extraliposomal high [K+] to a low [K+] buffer, thus establishing a large K+ gradient across the liposomal membrane. Trace 86Rb is then added to the extraliposomal space and the reaction begins. If the ion channel is permeable to K+, the K+ inside exits the liposomes down its concentration gradient and the 86Rb outside accumulates in the intraliposomal space until equilibrium is reached. The reaction time course is monitored by measurement of accumulated 86Rb after removal of external 86Rb over an ion-exchange column. The 86Rb flux assay takes 2-5 hours depending on the reaction rate and the number of desired time points.
Nimigean
Crina M
CM
Department of Physiology and Membrane Biology, University of California at Davis, One Shields Avenue, 1315 Tupper Hall, Davis, California 95616, USA. cnimigean@ucdavis.edu
D002850:Chromatography, Gel; D019411:Clinical Laboratory Techniques; D007473:Ion Channels / Q000378:metabolism*; D017136:Ion Transport / Q000502:physiology*; D008081:Liposomes / Q000378:metabolism*; D012414:Rubidium Radioisotopes / Q000378:metabolism*
D007473:Ion Channels; D008081:Liposomes; D012414:Rubidium Radioisotopes