Characterization of the Liposomal Rubidium Uptake Assay

Abstract

The liposomal uptake assay is a useful technique in the study of the ensemble behavior of ion channels. For the study of potassium channels, purified channel protein is reconstituted into liposomes, in which an intra- to extra- liposomal K gradient is created. Uptake of radioactive 86Rb, added to the extra-liposomal solution, is concentrated into liposomes that have K selective channels, and is measured as a surrogate of channel activity. The assay allows one to define experimental conditions that are often difficult to control in other techniques used to study ion channels, such as membrane composition. Baseline characteristics of the assay, such as liposome integrity and K gradient stability, can influence results, and is the main focus of this presentation. Liposomes comprising a 9:1 ratio of POPE:POPG are stable over several hours, and 50% uptake capacity remained for liposomes stored at room temperature for ∼48 hours. The rate of 86 Rb uptake in the presence of valinomycin, a K ionophore used in the measure of maximal uptake, was near maximal at ∼0.1 mcg/ml/mg lipid, with higher concentrations resulting in liposome fragility and lower maximal uptake. The time course of uptake in the presence of valinomycin (0.1 mcg/ml/mg lipid) was on the order of minutes, with a time constant ∼3 minutes. Lipid membrane composition influenced rate of uptake due to valinomycin. Liposomes formed from 100% PE had ∼3 fold decreased rate of uptake compared with 9:1 POPE:POPG liposomes. Further characterizations are ongoing and will be presented.