Designing a Single Protein‐Chain Reporter for Opioid Detection at Cellular Resolution
Kayla E. Kroning, Wenjing Wang
Abstract
Abstract Mu‐opioid receptor (MOR) signaling regulates multiple neuronal pathways, including those involved in pain, reward, and respiration. To advance the understanding of MOR's roles in pain modulation, there is a need for high‐throughput screening methods of opioids in vitro and high‐resolution mapping of opioids in the brain. To fill this need, we designed and characterized a genetically encoded fluorescent reporter, called S ingle‐chain P rotein‐based O pioid T ransmission I ndicator T ool for M OR (M‐SPOTIT). M‐SPOTIT represents a new and unique mechanism for fluorescent reporter design and can detect MOR activation, leaving a persistent green fluorescence mark for image analysis. M‐SPOTIT showed an opioid‐dependent signal to noise ratio (S/N) up to 12.5 and was able to detect as fast as a 30‐second opioid exposure in HEK293T cell culture. Additionally, it showed an opioid‐dependent S/N up to 4.6 in neuronal culture and detected fentanyl with an EC 50 of 15 nM. M‐SPOTIT will potentially be useful for high‐throughput detection of opioids in cell cultures and cellular‐resolution detection of opioids in vivo. M‐SPOTIT's novel mechanism can be used as a platform to design other G‐protein‐coupled receptor‐based sensors.