BPS2026 – Quantitative ion channel activity analysis and inhibitor screening by integrated fluorescence and hyperpolarization approaches

Ion channels play essential roles in biological processes, and accurate quantification of their activity is central to biophysical research. Conventional calcium influx assays, such as those using Fura-2, are limited by artifacts from free dye and vesicle variability. We present a vesicle-based assay employing large unilamellar vesicles (LUVs) encapsulated with Fluo-4 and monitored by total internal reflection fluorescence (TIRF) microscopy. Channel activity was quantified through encapsulation ratio analysis, enabling real-time and concentration-dependent kinetics. As a model system, the SARS-CoV-2 envelope protein transmembrane domain (ETM) displayed calcium influx that was effectively inhibited by hexamethylene amiloride (HMA) at substoichiometric levels. This approach provides a robust platform for quantitative studies of viroporins and other channel-forming proteins. Furthermore, the integration with hyperpolarization-enhanced NMR offers a future avenue for ultrasensitive inhibitor screening, combining functional fluorescence imaging with molecular-resolution spectroscopy.