Designing a Single Protein-Chain Reporter for Opioid Detection at Cellular Resolution
| Autor: | Wenjing Wang, Kayla E. Kroning |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Green Fluorescent Proteins
Receptors Opioid mu Signal-To-Noise Ratio 010402 general chemistry 01 natural sciences Catalysis In vivo medicine Humans Receptor G protein-coupled receptor 010405 organic chemistry Mechanism (biology) Chemistry HEK 293 cells General Chemistry In vitro 0104 chemical sciences Cell biology Analgesics Opioid Fentanyl HEK293 Cells Opioid Microscopy Fluorescence Cell culture medicine.drug Single-Chain Antibodies |
| Zdroj: | Angewandte Chemie (International ed. in English). 60(24) |
| ISSN: | 1521-3773 |
| Popis: | 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 Single-chain Protein-based Opioid Transmission Indicator Tool for MOR (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 EC50 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Plný text