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Opioids are the most powerful analgesics used clinically; however, severe side effects limit their long-term use. Various concepts involving biased intracellular signaling, partial agonism or multi-receptor targeting have been proposed to identify novel opioids with increased analgesic efficacy but reduced side effects. The search for such 'better opioids' implies screening of huge compound libraries and requires highly reliable, easy to perform and high throughput screening (HTS) assays. Here, we utilize an established membrane potential assay to monitor activation of G protein-coupled inwardly rectifying potassium (GIRK) channels, one of the main effectors of opioid receptor signaling, as readout to determine pharmacological profiles of opioids in a non-invasive manner. Specifically, in this study, we optimize assay conditions and extend the application of this assay to screen all four members of the opioid receptor family, stably expressed in AtT-20 and HEK293 cells. This ultra-sensitive system yielded EC50 values in the nano-molar range. We further validate this system for screening cells stably co-expressing two opioid receptors, which could be a valuable tool for investigating bi-functional ligands and studying interactions between receptors. Additionally, we demonstrate the utility of this assay to study antagonists as well as ligands with varying efficacies. Our results suggest that this assay could easily be up-scaled to HTS assay in order to efficiently study receptor activation and screen for novel opioids.
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