Fluorescence-Based Ion Transport Assays Using Proteoliposomes

Divalent metal ion transporters are conserved across all domains of life and play essential roles in diverse processes such as manganese acquisition during nutritional immunity in bacteria and iron homeostasis in higher eukaryotes [1-3]. Traditional techniques, such as electrophysiological assays, are often unsuitable due to the slow kinetics of many membrane transporters, electroneutral nature of certain transporter types, and the influence of other proteins with similar activity. To overcome these limitations and to investigate both the activity and ion selectivity of transporters, also including those normally expressed intracellularly, we have developed a fluorescence-based transport assay using purified proteins. This in vitro assay uses encapsulated fluorophores to monitor the movement of divalent metal ions (e.g., Mn²⁺, Ca²⁺, Mg²⁺) or protons across liposomal membranes reconstituted with purified transporter proteins. This approach provides detailed functional insight that complements structural and cellular data. Key features • Enables detection of real-time transport activity through precise timing of reagent addition and controlled generation of membrane potential. • Compatible with a wide range of divalent metal ions and ionophores, allowing adaptation to various transporter types. • Applicable to transporters that are naturally expressed in intracellular compartments, but requires a purified protein sample. • Allows detailed analysis of transporter function in a defined lipid environment and testing effects of binders and compounds.